%0 Journal Article %J medRxiv %D 2023 %T Inflammatory breast cancer biomarker identification by simultaneous TGIRT-seq profiling of coding and non-coding RNAs in tumors and blood %A Wylie, D %A X Wang %A Yao, J %A Xu, H %A Ferrick-Kiddie, EA %A Iwase, T %A Krishnamurthy, S %A Ueno, NT %A Lambowitz, A M %X Inflammatory breast cancer (IBC) is the most aggressive and lethal breast cancer subtype, but lags in biomarker identification. Here, we used an improved Thermostable Group II Intron Reverse Transcriptase RNA sequencing (TGIRT-seq) method to simultaneously profile coding and non-coding RNAs from tumors, PBMCs, and plasma of IBC and non-IBC patients and healthy donors. Besides RNAs from known IBC-relevant genes, we identified hundreds of other overexpressed coding and non-coding RNAs (p≤0.001) in IBC tumors and PBMCs, including higher proportions with elevated intron-exon depth ratios (IDRs), likely reflecting enhanced transcription resulting in accumulation of intronic RNAs. As a consequence, differentially represented protein-coding gene RNAs in IBC plasma were largely intron RNA fragments, whereas those in healthy donor and non-IBC plasma were largely fragmented mRNAs. Potential IBC biomarkers in plasma included T-cell receptor pre-mRNA fragments traced to IBC tumors and PBMCs; intron RNA fragments correlated with high IDR genes; and LINE-1 and other retroelement RNAs that we found globally up-regulated in IBC and preferentially enriched in plasma. Our findings provide new insights into IBC and demonstrate advantages of broadly analyzing transcriptomes for biomarker identification. The RNA-seq and data analysis methods developed for this study may be broadly applicable to other diseases. %B medRxiv %G eng %U https://www.medrxiv.org/content/10.1101/2023.05.26.23290469v1 %0 Journal Article %J Nature Cell Biology %D 2023 %T Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2 %A Cui, H %A Diedrich, JK %A Wu, DC %A Lim, JJ %A Nottingham, RM %A Moresco, JJ %A Yates, JR %A Blencowe, BJ %A Lambowitz, A M %A Schimmel, P %X Cells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Aminoacyl-tRNA synthetases, enzymes that catalyse the first step of protein synthesis, can also mediate cell signalling. Here we show that depletion of arginine during inflammation decreased levels of nuclear-localized arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts and co-localizes with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal and nuclear speckle protein, and that decreased levels of nuclear ArgRS correlated with changes in condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in the synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a mechanism whereby an aminoacyl-tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery. %B Nature Cell Biology %V 25 %P 592–603 %G eng %U https://www.nature.com/articles/s41556-023-01118-8 %0 Journal Article %J Cell %D 2022 %T Group II-like Reverse Transcriptases Function in Double Strand Break Repair %A S.K. Park %A G. Mohr %A J. Yao %A Russell, R. %A A.M. Lambowitz %X Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase q and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms. %B Cell %V 185 %P 3671-3688 %G eng %U https://www.sciencedirect.com/science/article/pii/S0092867422010637?via%3Dihub %N 20 %0 Journal Article %J Cancer Res %D 2022 %T Abstract P5-07-03: Disease classification modeling of inflammatory breast cancer based on simultaneous profiling of coding and non-coding RNAs in tumor and blood samples by TGIRT-sequencing %A Wylie, DC %A X Wang %A Yao, J %A Xu, H %A Iwase, T %A Krishnamurthy, S %A Ueno, NT %A Lambowitz, A M %X Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal breast cancer subtype but lags in disease-specific RNA biomarkers due in part to its paucity of large discrete tumors. A strategy to overcome this challenge is to identify blood-based RNA biomarkers that are minimally invasive and reflect the state of both the diseased breast tissue and the patient's immune response. Here, we identified IBC-specific RNA biomarkers by thermostable group II intron reverse transcriptase sequencing (TGIRT-seq), a recently developed comprehensive RNA-seq technology that enables simultaneous profiling of all RNA biotypes from small amounts of starting material. We used these biomarkers to develop novel disease classification models for IBC based on coding and non-coding RNAs from FFPE tumor slices, PBMCs, and plasma. Methods: We obtained biological samples including FFPE, PBMC, and plasma from a cohort of ten patients with IBC and compared them to samples from six patients with non-IBC and sixteen healthy donors using TGIRT-seq technology. Results: TGIRT-seq of FFPE tumor slices identified differentially expressed mRNAs and miRNAs found previously to distinguish IBC from non-IBC tumors, as well as numerous additional differentially expressed mRNAs and small non-coding RNAs characteristic of IBC. Surprisingly, TGIRT-seq revealed that the differentially expressed protein-coding gene transcripts fall into two categories: mature mRNAs with reads confined to exons, and pre-mRNAs-derived transcripts with reads distributed across exons and introns, to our knowledge, a distinction not made previously for any cancer type. Differentially expressed miRNAs included both mature miRNAs and other transcripts of miRNA loci. IBC PBMCs showed a characteristic inflammatory response not seen in PBMCs from non-IBC patients, as well as differentially expressed tRNAs, snoRNAs, and other sncRNAs, while plasma samples, although of variable quality, included coding and non-coding RNAs distinctive of IBC. Classification models using panels consisting of sets of 50 selected biomarkers profiled by TGIRT-seq achieved a high degree of accuracy under cross-validation, with models based on PBMCs and plasma RNAs correlating with those based on tumor RNAs, and models using both coding and non-coding RNA biomarkers outperforming those based on either alone. Conclusions: Our findings are the first to define a distinct IBC profile across three different tissue types and advance TGIRT-seq as a promising method for high-resolution RNA biomarker profiling of both primary tumors and liquid biopsies with potentially broad utility for diagnosing and defining treatment response in IBC and other cancers. COI: Thermostable group II intron reverse transcriptase (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas to InGex, LLC. A.M.L., some former and present members of the Lambowitz laboratory, and the University of Texas are minority equity holders in InGex, and receive royalty payments from the sale of TGIRT enzymes and kits and from sublicensing of intellectual property to other companies. %B Cancer Res %V 82 %G eng %U https://aacrjournals.org/cancerres/article/82/4_Supplement/P5-07-03/681245/Abstract-P5-07-03-Disease-classification-modeling %0 Journal Article %J BioRxiv %D 2022 %T Human cells contain myriad excised linear intron RNAs with links to gene regulation and potential utility as biomarkers %A Yao, Jun %A Shelby Winans %A Hengyi Xu %A Elizabeth A. Ferrick-Kiddie %A Manuel Ares Jr. %A Alan M. Lambowitz %X By using TGIRT-seq, we identified >8,500 short full-length excised linear intron (FLEXI) RNAs in human cells. Subsets of FLEXIs accumulated in a cell-type specific manner, and ∼200 corresponded to agotrons or mirtrons or encoded snoRNAs. Analysis of CLIP-seq datasets identified potential interactions between FLEXIs and >100 different RNA-binding proteins (RBPs), 53 of which had binding sites in ≥30 different FLEXIs. In addition to proteins that function in RNA splicing, these 53 RBPs included transcription factors, chromatin remodeling proteins, and cellular growth regulators that impacted FLEXI host gene alternative splicing and/or mRNA levels in knockdown datasets. We computationally identified six groups of RBPs whose binding sites were enriched in different subsets of FLEXIs: AGO1-4 and DICER associated with agotrons and mirtrons; AATF, DKC1, NOLCI, and SMNDC1 associated with snoRNA-encoding FLEXIs; two different combinations of alternative splicing factors found in stress granules; and two novel RBP-intron combinations, one including LARP4 and PABC4, which function together in the cytoplasm to regulate ribosomal protein translation. Our results suggest a model in which proteins involved in transcriptional regulation, alternative splicing, or post-splicing secondary functions bind and stabilize cell-type specific subsets of FLEXIs that perform different biological functions and have potential utility as biomarkers. %B BioRxiv %G eng %U https://www.biorxiv.org/content/10.1101/2020.09.07.285114v4 %0 Journal Article %J NAR Cancer %D 2022 %T High-grade ovarian cancer associated H/ACA snoRNAs promote cancer cell proliferation and survival Laurence Faucher-Giguère, Audrey Roy, Gabrielle Deschamps-Francoeur, Sonia Couture, Ryan M Nottingham, Alan M Lambowitz, Michelle S Scott, Sherif %A Laurence Faucher-Giguère %A Audrey Roy %A Gabrielle Deschamps-Francoeur %A Sonia Couture %A Ryan M. Nottingham %A Alan M. Lambowitz %A Michelle S. Scott %A Sherif Abou Elela %X Small nucleolar RNAs (snoRNAs) are an omnipresent class of non-coding RNAs involved in the modification and processing of ribosomal RNA (rRNA). As snoRNAs are required for ribosome production, the increase of which is a hallmark of cancer development, their expression would be expected to increase in proliferating cancer cells. However, assessing the nature and extent of snoRNAs' contribution to cancer biology has been largely limited by difficulties in detecting highly structured RNA. In this study, we used a dedicated midsize non-coding RNA (mncRNA) sensitive sequencing technique to accurately survey the snoRNA abundance in independently verified high-grade serous ovarian carcinoma (HGSC) and serous borderline tumour (SBT) tissues. The results identified SNORA81, SNORA19 and SNORA56 as an H/ACA snoRNA signature capable of discriminating between independent sets of HGSC, SBT and normal tissues. The expression of the signature SNORA81 correlates with the level of ribosomal RNA (rRNA) modification and its knockdown inhibits 28S rRNA pseudouridylation and accumulation leading to reduced cell proliferation and migration. Together our data indicate that specific subsets of H/ACA snoRNAs may promote tumour aggressiveness by inducing rRNA modification and synthesis. %B NAR Cancer %V 4 %G eng %U https://pubmed.ncbi.nlm.nih.gov/35047824/ %N 1 %0 Journal Article %J bioRxiv %D 2022 %T Group II Intron-Like Reverse Transcriptases Function in Double-Strand Break Repair by Microhomology-Mediated End Joining %A S.K. Park %A G. Mohr %A J. Yao %A Russell, R. %A A.M. Lambowitz %X Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase q and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms. %B bioRxiv %V 484287 %G eng %U https://www.biorxiv.org/content/10.1101/2022.03.14.484287v1 %0 Journal Article %J BioRxiv %D 2021 %T Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2 View ORCID Profile, , View ORCID Profile, Justin J. Lim, Ryan M. Nottingham, James J. Moresco, John R. Yates III, Benjamin J. Blencowe, Alan M. L %A Haissi Cui %A Jolene K. Diedrich %A Douglas C. Wu %A Justin J. Lim %A Ryan M. Nottingham %A James J. Moresco %A John R. Yates III %A Benjamin J. Blencowe %A Alan M. Lambowitz %A Paul Schimmel %X Cells respond to perturbations like inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Here, we found that depletion of arginine during inflammation decreased levels of a nuclear form of arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal protein and nuclear speckle component and that arginine depletion impacted both condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a novel mechanism whereby a tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery. %B BioRxiv %G eng %U https://www.biorxiv.org/content/10.1101/2021.09.07.459304v2 %0 Journal Article %J J. Biol. Chem. %D 2021 %T Structural basis for template switching by a group II intron–encoded non-LTR-retroelement reverse transcriptase %A Alfred M. Lentzsch %A Jennifer L. Stamos %A Yao, Jun %A Russell, Rick %A Alan M. Lambowitz %X Reverse transcriptases (RTs) can switch template strands during complementary DNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching (TS) plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-Seq, and may contribute to retroelement fitness by increasing evolutionary diversity and enabling continuous complementary DNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a TS complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template– DNA primer heteroduplex with a 1-nt 30 -DNA overhang. The structure showed that the 30 end of the acceptor RNA binds in a pocket formed by an N-terminal extension present in non–long terminal repeat–retroelement RTs and the RT fingertips loop, with the 30 nucleotide of the acceptor base paired to the 1-nt 30 - DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structureguided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine residue near the RT active site that mediates nontemplated nucleotide addition. Mutation of the latter residue decreased multiple sequential template switches in RNA-Seq. Our results provide new insights into the mechanisms of TS and nontemplated nucleotide addition by RTs, suggest how these reactions could be improved for RNA-Seq, and reveal common structural features for TS by non–long terminal repeat–retroelement RTs and viral RNA–dependent RNA polymerases. %B J. Biol. Chem. %V 297 %P 100971 %G eng %U https://www.jbc.org/article/S0021-9258(21)00773-0/fulltext %N 2 %0 Journal Article %J Bio-protocol %D 2021 %T TGIRT-seq Protocol for the Comprehensive Profiling of Coding and Non-coding RNA Biotypes in Cellular, Extracellular Vesicle, and Plasma RNAs %A Xu, H %A Nottingham, Ryan M %A Lambowitz, A M %X High-throughput RNA sequencing (RNA-seq) has extraordinarily advanced our understanding of gene expression and disease etiology, and is a powerful tool for the identification of biomarkers in a wide range of organisms. However, most RNA-seq methods rely on retroviral reverse transcriptases (RTs), enzymes that have inherently low fidelity and processivity, to convert RNAs into cDNAs for sequencing. Here, we describe an RNA-seq protocol using Thermostable Group II Intron Reverse Transcriptases (TGIRTs), which have high fidelity, processivity, and strand-displacement activity, as well as a proficient template-switching activity that enables efficient and seamless RNA-seq adapter addition. By combining these activities, TGIRT-seq enables the simultaneous profiling of all RNA biotypes from small amounts of starting material, with superior RNA-seq metrics, and unprecedented ability to sequence structured RNAs. The TGIRT-seq protocol for Illumina sequencing consists of three steps: (i) addition of a 3' RNA-seq adapter, coupled to the initiation of cDNA synthesis at the 3' end of a target RNA, via template switching from a synthetic adapter RNA/DNA starter duplex; (ii) addition of a 5' RNA-seq adapter, by using thermostable 5' App DNA/RNA ligase to ligate an adapter oligonucleotide to the 3' end of the completed cDNA; (iii) minimal PCR amplification, to add capture sites and indices for Illumina sequencing. TGIRT-seq for the Illumina sequencing platform has been used for comprehensive profiling of coding and non-coding RNAs in ribodepleted, chemically fragmented cellular RNAs, and for the analysis of intact (non-chemically fragmented) cellular, extracellular vesicle (EV), and plasma RNAs, where it yields continuous full-length end-to-end sequences of structured small noncoding RNAs (sncRNAs), including tRNAs, snoRNAs, snRNAs, pre-miRNAs, and full-length excised linear intron (FLEXI) RNAs. %B Bio-protocol %V 11 %G eng %N 23 %0 Journal Article %J eLife %D 2020 %T Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling %A Yao, Jun %A Douglas C. Wu %A Nottingham, Ryan M %A Lambowitz, Alan M %X Human plasma contains >40,000 different coding and non-coding RNAs that are potential biomarkers for human diseases. Here, we used thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) combined with peak calling to simultaneously profile all RNA biotypes in apheresis-prepared human plasma pooled from healthy individuals. Extending previous TGIRT-seq analysis, we found that human plasma contains largely fragmented mRNAs from >19,000 protein-coding genes, abundant full-length, mature tRNAs and other structured small non-coding RNAs, and less abundant tRNA fragments and mature and pre-miRNAs. Many of the mRNA fragments identified by peak calling correspond to annotated protein-binding sites and/or have stable predicted secondary structures that could afford protection from plasma nucleases. Peak calling also identified novel repeat RNAs, miRNA-sized RNAs, and putatively structured intron RNAs of potential biological, evolutionary, and biomarker significance, including a family of full-length excised introns RNAs, subsets of which correspond to mirtron pre-miRNAs or agotrons. %B eLife %G eng %U https://elifesciences.org/articles/60743 %0 Journal Article %J Journal of Biological Chemistry %D 2019 %T Template switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq %A A.M. Lentzsch %A J. Yao %A Russell, R. %A A.M. Lambowitz %X

The reverse transcriptases (RTs) encoded by mobile group II introns and other non-LTR retroelements differ from retroviral RTs in being able to template-switch efficiently from the 5 end of one template to the 3 end of another with little or no complementarity between the donor and acceptor templates. Here, to establish a complete kinetic framework for the reaction and to identify conditions that more efficiently capture acceptor RNAs or DNAs, we used a thermostable group II intron RT (TGIRT; GsI–IIC RT) that can template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3-DNA overhang end. We found that the rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3-DNA overhang complementary to the 3 nucleotide of the acceptor RNA, suggesting a role for nontemplated nucleotide addition of a complementary nucleotide to the 3 end of cDNAs synthesized from natural templates. Longer 3-DNA overhangs progressively decreased the templateswitching rate, even when complementary to the 3 end of the acceptor template. The reliance on only a single bp with the 3 nucleotide of the acceptor together with discrimination against mismatches and the high processivity of group II intron RTs enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3 end. We discuss the possible biological functions of the template-switching activity of group II intron- and other non-LTR retroelement– encoded RTs, as well as the optimization of this activity for adapter addition in RNAand DNA-Seq protocols.

%B Journal of Biological Chemistry %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/31712313 %0 Journal Article %J eLife %D 2019 %T Distinct mechanisms of microRNA sorting into cancer cell-derived extracellular vesicle subtypes. %A Temoche-Diaz, M. M. %A M.J. Shurtleff %A Nottingham, R .M. %A J. Yao %A Fadadu, R. P. %A A.M. Lambowitz %A R. Schekman %X Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown. We report the separation of two small EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. These EV sub-types use different mechanisms of miRNA sorting (selective and non-selective), suggesting that sorting occurs via fundamentally distinct processes, possibly dependent on EV origin. Using biochemical and genetic tools, we identified the Lupus La protein as mediating sorting of selectively packaged miRNAs. We found that two motifs embedded in miR-122 are responsible for high-affinity binding to Lupus La and sorting into vesicles formed in a cell-free reaction. Thus, tumor cells can simultaneously deploy multiple EV species using distinct sorting mechanisms that may enable diverse functions in normal and cancer biology. %B eLife %V 8 %G eng %0 Journal Article %J PLoS Genetics %D 2019 %T BCDIN3D regulates tRNAHis 3’ fragment processing. %A Reinsborough, C. W. %A Ipas, H. %A Abell, N. S. %A R.M. Nottingham %A J. Yao %A Devanathan, S. K. %A Shelton, S. B. %A A.M. Lambowitz %A Xhemalce, B. %X 5’ ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5’ monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq). Our analyses showed that BCDIN3D interacts with full-length phospho-methylated tRNAHis and miR-4454. Interestingly, we found that miR-4454 is not synthesized from its annotated genomic locus, which is a primer-binding site for an endogenous retrovirus, but rather by Dicer cleavage of mature tRNAHis. Sequence analysis revealed that miR-4454 is identical to the 3’ end of tRNAHis. Moreover, we were able to generate this ‘miRNA’ in vitro through incubation of mature tRNAHis with Dicer. As found previously for several pre-miRNAs, a 5’P-tRNAHis appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNAHis. Moreover, tRNAHis 3’-fragment/‘miR-4454’ levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNAHis 3’-fragment processing without negatively affecting tRNAHis’s canonical function of aminoacylation. %B PLoS Genetics %V 15 %G eng %N 7 %0 Journal Article %J Cold Spring Harbor Perspective in Biology %D 2019 %T Group II Intron RNPs and ReverseTranscriptases: From Retroelements to Research Tools %A Belfort, M %A Lambowitz, A M %X Group II introns, self-splicing retrotransposons, serve as both targets of investigation into their structure, splicing, and retromobility and a source of tools for genome editing and RNA analysis. Here, we describe the first cryo-electron microscopy (cryo-EM) structure determination, at 3.8–4.5 Å, of a group II intron ribozyme complexed with its encoded protein, containing a reverse transcriptase (RT), required for RNA splicing and retromobility. We also describe a method called RIG-seq using a retrotransposon indicator gene for high-throughput integration profiling of group II introns and other retrotransposons. Targetrons, RNA-guided gene targeting agents widely used for bacterial genome engineering, are described next. Finally, we detail thermostable group II intron RTs, which synthesize cDNAs with high accuracy and processivity, for use in various RNA-seq applications and relate their properties to a 3.0-Å crystal structure of the protein poised for reverse transcription. Biological insights from these group II intron revelations are discussed. %B Cold Spring Harbor Perspective in Biology %I Cold Spring Harb Perspect Biol %G eng %U https://cshperspectives.cshlp.org/content/11/4/a032375.full %0 Journal Article %J Scientific Reports %D 2019 %T Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer-formation and bias correction. %A Xu, H. %A J. Yao %A D.C. Wu %A Lambowitz, A M %X Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNA-seq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by rationally designing RNA-seq adapters that minimize adapter dimer formation. Additionally, we developed biochemical and computational methods for remediating 5′- and 3′-end biases, the latter based on a random forest regression model that provides insight into the contribution of different factors to these biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5′- and 3′-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation. %B Scientific Reports %V 9 %G eng %U https://www.researchgate.net/publication/333428625_Improved_TGIRT-seq_methods_for_comprehensive_transcriptome_profiling_with_decreased_adapter_dimer_formation_and_bias_correction %N 1 %0 Journal Article %J Mol. Cell %D 2018 %T A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition. %A Mohr, G %A Silas, Sukrit %A J. Stamos %A K.S. Makarova %A Markham, Laura M %A J. Yao %A Lucas-Elio, P. %A Sanchez-Amat, Antonio %A A.Z. Fire %A E.V. Koonin %A Lambowitz, A M %X Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion. %B Mol. Cell %P 700-714 %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/30344094 %N 72 %0 Journal Article %J RNA %D 2018 %T Simultaneous sequencing of coding and noncoding RNA reveals a human transcriptome dominated by a small number of highly expressed noncoding genes. %A V. Boivin %A G. Deschamps-Francoeur %A S. Couture %A R.M. Nottingham %A P. Bouchard-Bourelle %A A.M. Lambowitz %X Comparing the abundance of one RNA molecule to another is crucial for understanding cellular functions but most sequencing techniques can target only specific subsets of RNA. In this study, we used a new fragmented ribodepleted TGIRT sequencing method that uses a thermostable group II intron reverse transcriptase (TGIRT) to generate a portrait of the human transcriptome depicting the quantitative relationship of all classes of nonribosomal RNA longer than 60 nt. Comparison between different sequencing methods indicated that FRT is more accurate in ranking both mRNA and noncoding RNA than viral reverse transcriptase-based sequencing methods, even those that specifically target these species. Measurements of RNA abundance in different cell lines using this method correlate with biochemical estimates, confirming tRNA as the most abundant nonribosomal RNA biotype. However, the single most abundant transcript is 7SL RNA, a component of the signal recognition particle. Structured noncoding RNAs (sncRNAs) associated with the same biological process are expressed at similar levels, with the exception of RNAs with multiple functions like U1 snRNA. In general, sncRNAs forming RNPs are hundreds to thousands of times more abundant than their mRNA counterparts. Surprisingly, only 50 sncRNA genes produce half of the non-rRNA transcripts detected in two different cell lines. Together the results indicate that the human transcriptome is dominated by a small number of highly expressed sncRNAs specializing in functions related to translation and splicing. %B RNA %V 24 %P 950-965 %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/29703781 %N 7 %0 Journal Article %J Journal of Molecular Biology %D 2018 %T A Highly Proliferative Group IIC Intron from Geobacillus stearothermophilus Reveals New Features of Group II Intron Mobility and Splicing. %A G. Mohr %A S.Y. Kang %A S.K. Park %A Y. Qin %A J. Grohman %A J. Yao %A J.L. Stamos %A A.M. Lambowitz %X The thermostable Geobacillus stearothermophilus GsI-IIC intron is among the few bacterial group II introns found to proliferate to high copy number in its host genome. Here, we developed a bacterial genetic assay for retrohoming and biochemical assays for protein-dependent and self-splicing of GsI-IIC. We found that GsI-IIC, like other group IIC introns, retrohomes into sites having a 5'-exon DNA hairpin, typically from a bacterial transcription terminator, followed by short intron-binding sequences (IBSs) recognized by base pairing of exon-binding sequences (EBSs) in the intron RNA. Intron RNA insertion occurs preferentially but not exclusively into the parental lagging strand at DNA replication forks, using a nascent lagging strand DNA as a primer for reverse transcription. In vivo mobility assays, selections, and mutagenesis indicated that a variety of GC-rich DNA hairpins of 7-19 bp with continuous base pairs or internal elbow regions support efficient intron mobility and identified a critically recognized nucleotide (T-5) between the hairpin and IBS1, a feature not reported previously for group IIC introns. Neither the hairpin nor T-5 is required for intron excision or lariat formation during RNA splicing, but the 5'-exon sequence can affect the efficiency of exon ligation. Structural modeling suggests that the 5'-exon DNA hairpin and T-5 bind to the thumb and DNA-binding domains of GsI-IIC reverse transcriptase. This mode of DNA target site recognition enables the intron to proliferate to high copy number by recognizing numerous transcription terminators and then finding the best match for the EBS/IBS interactions within a short distance downstream. %B Journal of Molecular Biology %V 430 %P 2760-2783 %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/29913158 %N 17 %0 Journal Article %J BMC Genomics %D 2018 %T Limitations of alignment-free tools in total RNA-seq quantification. %A D.C. Wu %A J. Yao %A K.S. Ho %A A.M. Lambowitz %A C.O. Wilke %X

BACKGROUND:

Alignment-free RNA quantification tools have significantly increased the speed of RNA-seq analysis. However, it is unclear whether these state-of-the-art RNA-seq analysis pipelines can quantify small RNAs as accurately as they do with long RNAs in the context of total RNA quantification.

RESULT:

We comprehensively tested and compared four RNA-seq pipelines for accuracy of gene quantification and fold-change estimation. We used a novel total RNA benchmarking dataset in which small non-coding RNAs are highly represented along with other long RNAs. The four RNA-seq pipelines consisted of two commonly-used alignment-free pipelines and two variants of alignment-based pipelines. We found that all pipelines showed high accuracy for quantifying the expression of long and highly-abundant genes. However, alignment-free pipelines showed systematically poorer performance in quantifying lowly-abundant and small RNAs.

CONCLUSION:

We have shown that alignment-free and traditional alignment-based quantification methods perform similarly for common gene targets, such as protein-coding genes. However, we have identified a potential pitfall in analyzing and quantifying lowly-expressed genes and small RNAs with alignment-free pipelines, especially when these small RNAs contain biological variations.

%B BMC Genomics %V 19 %P 510 %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/29969991 %N 1 %0 Journal Article %J Molecular Cell %D 2017 %T Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications. %A J. Stamos %A A.M. Lentzsch %A A.M. Lambowitz %X Bacterial group II intron reverse transcriptases (RTs) function in both intron mobility and RNA splicing and are evolutionary predecessors of retrotransposon, telomerase, and retroviral RTs as well as the spliceosomal protein Prp8 in eukaryotes. Here we determined a crystal structure of a full-length thermostable group II intron RT in complex with an RNA template-DNA primer duplex and incoming deoxynucleotide triphosphate (dNTP) at 3.0-A˚ resolution. We find that the binding of template-primer and key aspects of the RT active site are surprisingly different from retroviral RTs but remarkably similar to viral RNA-dependent RNA polymerases. The structure reveals a host of features not seen previously in RTs that may contribute to distinctive biochemical properties of group II intron RTs, and it provides a prototype for many related bacterial and eukaryotic non-LTR retroelement RTs. It also reveals how protein structural features used for reverse transcription evolved to promote the splicing of both group II and spliceosomal introns. %B Molecular Cell %V 68 %P 926–939 %G eng %U http://www.cell.com/molecular-cell/fulltext/S1097-2765(17)30799-2 %0 Journal Article %J Nucleic Acids Research %D 2017 %T Detection of expanded RNA repeats using thermostable group II intron reverse transcriptase. %A S.T. Carrell %A Z. Tang %A S. Mohr %A A.M. Lambowitz %A C.A. Thorton %X Cellular accumulation of repetitive RNA occurs in several dominantly-inherited genetic disorders. Expanded CUG, CCUG or GGGGCC repeats are expressed in myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), or familial amyotrophic lateral sclerosis, respectively. Expanded repeat RNAs (ER-RNAs) exert a toxic gain-of-function and are prime therapeutic targets in these diseases. However, efforts to quantify ER-RNA levels or monitor knockdown are confounded by stable structure and heterogeneity of the ER-RNA tract and background signal from non-expanded repeats. Here, we used a thermostable group II intron reverse transcriptase (TGIRT-III) to convert ER-RNA to cDNA, followed by quantification on slot blots. We found that TGIRT-III was capable of reverse transcription (RTn) on enzymatically synthesized ER-RNAs. By using conditions that limit cDNA synthesis from off-target sequences, we observed hybridization signals on cDNA slot blots from DM1 and DM2 muscle samples but not from healthy controls. In transgenic mouse models of DM1 the cDNA slot blots accurately reflected the differences of ER-RNA expression across different transgenic lines, and showed therapeutic reductions in skeletal and cardiac muscle, accompanied by improvements of the DM1-associated splicing defects. TGIRT-III was also active on CCCCGG- and GGGGCC-repeats, suggesting that ER-RNA analysis is feasible for several repeat expansion disorders. %B Nucleic Acids Research %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/29036654 %0 Journal Article %J National Academy of Sciences %D 2017 %T Broad role for YBX1 in defining the small noncoding RNA composition of exosomes. %A M.J. Shurtleff %A J. Yao %A Y. Qin %A R.M. Nottingham %A M. Temoche-Diaz %A R. Schekman %A A.M. Lambowitz %X RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their coisolation with contaminants, lack of knowledge of the mechanisms of RNA sorting into EVs, and limitations of conventional RNA-sequencing methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspanin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature transfer RNAs (tRNAs) and other small noncoding RNAs (ncRNAs) encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long ncRNAs, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5′ terminal oligopyrimidine (5′ TOP) motif. Our results indicate that the RNA-binding protein YBX1, which is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small ncRNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for posttranscriptional modification in the sorting of some RNA species into EVs. Our results suggest that EVs and exosomes could play a role in the purging and intercellular transfer of excess free RNAs, including full-length tRNAs and other small ncRNAs. %B National Academy of Sciences %V 114 %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/29073095 %N 43 %0 Journal Article %J American Society for Microbiology %D 2017 %T On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires. %A S. Silas %A K.S. Makarova %A S. Shmakov %A D. Páez-Espino %A G. Mohr %A Y. Liu %A M. Davidson %A S. Roux %A S.R. Krishnamurthy %A B. Fu %A L.L. Hansen %A D. Wang %A M.B. Sullivan %A A. Millard %A M.R. Clokie %A B. Devaki %A A.M. Lambowitz %A N.C. Kyrpides %A E.V. Koonin %A A.Z. Fire %X Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPRassociated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium—Arthrospira platensis. Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1- associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. %B American Society for Microbiology %V 8 %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/28698278 %N 4 %0 Journal Article %J Scientific Reports %D 2017 %T Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching. %A Douglas C. Wu %A Alan M. Lambowitz %X High-throughput single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA from plasma and other bodily fluids is a powerful method for non-invasive prenatal testing, and diagnosis of cancers and other diseases. Here, we developed a facile ssDNA-seq method, which exploits a novel template-switching activity of thermostable group II intron reverse transcriptases (TGIRTs) for DNA-seq library construction. This activity enables TGIRT enzymes to initiate DNA synthesis directly at the 3′ end of a DNA strand while simultaneously attaching a DNA-seq adapter without end repair, tailing, or ligation. Initial experiments using this method to sequence E. coli genomic DNA showed that the TGIRT enzyme has surprisingly robust DNA polymerase activity. Further experiments showed that TGIRT-seq of plasma DNA from a healthy individual enables analysis of nucleosome positioning, transcription factor-binding sites, DNA methylation sites, and tissues-of-origin comparably to established methods, but with a simpler workflow that captures precise DNA ends. %B Scientific Reports %V 7 %G eng %U https://www.nature.com/articles/s41598-017-09064-w %N 8421 %0 Journal Article %J Nat Methods %D 2017 %T DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. %A Zubradt, Meghan %A Gupta, Paromita %A Persad, Sitara %A Lambowitz, Alan M %A Weissman, Jonathan S %A Rouskin, Silvi %X Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure. %B Nat Methods %V 14 %P 75-82 %8 2017 Jan %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/27819661 %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/27819661?dopt=Abstract %R 10.1038/nmeth.4057 %0 Journal Article %J Genes Dev %D 2016 %T DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs. %A Burke, James M %A Kincaid, Rodney P %A Nottingham, Ryan M %A Lambowitz, Alan M %A Sullivan, Christopher S %K Acid Anhydride Hydrolases %K Adenoviridae %K Argonaute Proteins %K Dual-Specificity Phosphatases %K Gene Knockout Techniques %K HEK293 Cells %K Humans %K Leukemia Virus, Bovine %K MicroRNAs %K Phosphorylation %K RNA Polymerase III %K RNA, Untranslated %K RNA, Viral %X RNA silencing is a conserved eukaryotic gene expression regulatory mechanism mediated by small RNAs. In Caenorhabditis elegans, the accumulation of a distinct class of siRNAs synthesized by an RNA-dependent RNA polymerase (RdRP) requires the PIR-1 phosphatase. However, the function of PIR-1 in RNAi has remained unclear. Since mammals lack an analogous siRNA biogenesis pathway, an RNA silencing role for the mammalian PIR-1 homolog (dual specificity phosphatase 11 [DUSP11]) was unexpected. Here, we show that the RNA triphosphatase activity of DUSP11 promotes the RNA silencing activity of viral microRNAs (miRNAs) derived from RNA polymerase III (RNAP III) transcribed precursors. Our results demonstrate that DUSP11 converts the 5' triphosphate of miRNA precursors to a 5' monophosphate, promoting loading of derivative 5p miRNAs into Argonaute proteins via a Dicer-coupled 5' monophosphate-dependent strand selection mechanism. This mechanistic insight supports a likely shared function for PIR-1 in C. elegans Furthermore, we show that DUSP11 modulates the 5' end phosphate group and/or steady-state level of several host RNAP III transcripts, including vault RNAs and Alu transcripts. This study shows that steady-state levels of select noncoding RNAs are regulated by DUSP11 and defines a previously unknown portal for small RNA-mediated silencing in mammals, revealing that DUSP11-dependent RNA silencing activities are shared among diverse metazoans. %B Genes Dev %V 30 %P 2076-2092 %8 2016 Sep 15 %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/27798849 %N 18 %1 http://www.ncbi.nlm.nih.gov/pubmed/27798849?dopt=Abstract %R 10.1101/gad.282616.116 %0 Journal Article %J J Biol Chem %D 2016 %T Structural Divergence of the Group I Intron Binding Surface in Fungal Mitochondrial Tyrosyl-tRNA Synthetases That Function in RNA Splicing. %A Lamech, Lilian T %A Saoji, Maithili %A Paukstelis, Paul J %A Lambowitz, Alan M %X The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mitochondrial tRNA(Tyr) and act as splicing cofactors for autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon binding domains and that the catalytic domain uses a newly evolved group I intron binding surface that includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined x-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the Aspergillus fumigatus mtTyrRS showed that the overall topology of the group I intron binding surface is conserved but with variations in key intron binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions, which also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi. %B J Biol Chem %V 291 %P 11911-27 %8 2016 May 27 %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/27036943 %N 22 %1 http://www.ncbi.nlm.nih.gov/pubmed/27036943?dopt=Abstract %R 10.1074/jbc.M116.725390 %0 Journal Article %J RNA %D 2016 %T RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase. %A Nottingham, Ryan M %A Wu, Douglas C %A Qin, Yidan %A Yao, Jun %A Hunicke-Smith, Scott %A Lambowitz, Alan M %X Next-generation RNA sequencing (RNA-seq) has revolutionized our ability to analyze transcriptomes. Current RNA-seq methods are highly reproducible, but each has biases resulting from different modes of RNA sample preparation, reverse transcription, and adapter addition, leading to variability between methods. Moreover, the transcriptome cannot be profiled comprehensively because highly structured RNAs, such as tRNAs and snoRNAs, are refractory to conventional RNA-seq methods. Recently, we developed a new method for strand-specific RNA-seq using thermostable group II intron reverse transcriptases (TGIRTs). TGIRT enzymes have higher processivity and fidelity than conventional retroviral reverse transcriptases plus a novel template-switching activity that enables RNA-seq adapter addition during cDNA synthesis without using RNA ligase. Here, we obtained TGIRT-seq data sets for well-characterized human RNA reference samples and compared them to previous data sets obtained for these RNAs by the Illumina TruSeq v2 and v3 methods. We find that TGIRT-seq recapitulates the relative abundance of human transcripts and RNA spike-ins in ribo-depleted, fragmented RNA samples comparably to non-strand-specific TruSeq v2 and better than strand-specific TruSeq v3. Moreover, TGIRT-seq is more strand specific than TruSeq v3 and eliminates sampling biases from random hexamer priming, which are inherent to TruSeq. The TGIRT-seq data sets also show more uniform 5' to 3' gene coverage and identify more splice junctions, particularly near the 5' ends of mRNAs, than do the TruSeq data sets. Finally, TGIRT-seq enables the simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq experiment as structured small ncRNAs, including tRNAs, which are essentially absent with TruSeq. %B RNA %V 22 %P 597-613 %8 2016 Apr %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/26826130 %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/26826130?dopt=Abstract %R 10.1261/rna.055558.115 %0 Journal Article %J RNA %D 2016 %T High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases. %A Qin, Yidan %A Yao, Jun %A Wu, Douglas C %A Nottingham, Ryan M %A Mohr, Sabine %A Hunicke-Smith, Scott %A Lambowitz, Alan M %X Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from <1 ng of plasma RNA in <5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. %B RNA %V 22 %P 111-28 %8 2016 Jan %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/26554030 %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/26554030?dopt=Abstract %R 10.1261/rna.054809.115 %0 Journal Article %J Science %D 2016 %T Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein. %A Silas, Sukrit %A Mohr, Georg %A Sidote, David J %A Markham, Laura M %A Sanchez-Amat, Antonio %A Bhaya, Devaki %A Lambowitz, Alan M %A Fire, Andrew Z %X CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. %B Science %V 351 %P aad4234 %8 2016 Feb 26 %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/26917774 %N 6276 %1 http://www.ncbi.nlm.nih.gov/pubmed/26917774?dopt=Abstract %R 10.1126/science.aad4234 %0 Journal Article %J Nat Methods %D 2015 %T Efficient and quantitative high-throughput tRNA sequencing. %A Zheng, Guanqun %A Qin, Yidan %A Clark, Wesley C %A Dai, Qing %A Yi, Chengqi %A He, Chuan %A Lambowitz, Alan M %A Pan, Tao %K Algorithms %K Base Sequence %K Gene Library %K HEK293 Cells %K High-Throughput Nucleotide Sequencing %K Humans %K Molecular Sequence Data %K RNA, Transfer %X Despite its biological importance, tRNA has not been adequately sequenced by standard methods because of its abundant post-transcriptional modifications and stable structure, which interfere with cDNA synthesis. We achieved efficient and quantitative tRNA sequencing in HEK293T cells by using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase to overcome these obstacles. Our method, DM-tRNA-seq, should be applicable to investigations of tRNA in all organisms. %B Nat Methods %V 12 %P 835-7 %8 2015 Sep %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/26214130 %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/26214130?dopt=Abstract %R 10.1038/nmeth.3478 %0 Journal Article %J PLoS Genet %D 2015 %T Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation. %A Truong, David M %A Hewitt, F Curtis %A Hanson, Joseph H %A Cui, Xiaoxia %A Lambowitz, Alan M %X Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms. %B PLoS Genet %V 11 %P e1005422 %8 2015 Aug %G eng %U https://www.ncbi.nlm.nih.gov/pubmed/26241656 %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/26241656?dopt=Abstract %R 10.1371/journal.pgen.1005422 %0 Journal Article %J Microbiol Spectr %D 2015 %T Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution. %A Lambowitz, Alan M %A Belfort, Marlene %X

This review focuses on recent developments in our understanding of group II intron function, the relationships of these introns to retrotransposons and spliceosomes, and how their common features have informed thinking about bacterial group II introns as key elements in eukaryotic evolution. Reverse transcriptase-mediated and host factor-aided intron retrohoming pathways are considered along with retrotransposition mechanisms to novel sites in bacteria, where group II introns are thought to have originated. DNA target recognition and movement by target-primed reverse transcription infer an evolutionary relationship among group II introns, non-LTR retrotransposons, such as LINE elements, and telomerase. Additionally, group II introns are almost certainly the progenitors of spliceosomal introns. Their profound similarities include splicing chemistry extending to RNA catalysis, reaction stereochemistry, and the position of two divalent metals that perform catalysis at the RNA active site. There are also sequence and structural similarities between group II introns and the spliceosome's small nuclear RNAs (snRNAs) and between a highly conserved core spliceosomal protein Prp8 and a group II intron-like reverse transcriptase. It has been proposed that group II introns entered eukaryotes during bacterial endosymbiosis or bacterial-archaeal fusion, proliferated within the nuclear genome, necessitating evolution of the nuclear envelope, and fragmented giving rise to spliceosomal introns. Thus, these bacterial self-splicing mobile elements have fundamentally impacted the composition of extant eukaryotic genomes, including the human genome, most of which is derived from close relatives of mobile group II introns.

%B Microbiol Spectr %V 3 %8 2015 Feb %G ENG %U http://www.asmscience.org/content/journal/microbiolspec/10.1128/microbiolspec.MDNA3-0050-2014 %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25878921?dopt=Abstract %0 Journal Article %J Science %D 2015 %T Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. %A Shen, Peter S %A Park, Joseph %A Qin, Yidan %A Li, Xueming %A Parsawar, Krishna %A Larson, Matthew H %A Cox, James %A Cheng, Yifan %A Lambowitz, Alan M %A Weissman, Jonathan S %A Brandman, Onn %A Frost, Adam %K Cryoelectron Microscopy %K Nucleic Acid Conformation %K Peptide Biosynthesis, Nucleic Acid-Independent %K Protein Conformation %K Ribosome Subunits, Large, Eukaryotic %K RNA, Messenger %K RNA, Transfer, Ala %K RNA, Transfer, Thr %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Ubiquitin-Protein Ligases %X

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo–electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40Sdissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions (“CAT tails”).

%B Science %V 347 %P 75-8 %8 2015 Jan 2 %G eng %U http://www.sciencemag.org/content/347/6217/75.long %N 6217 %1 http://www.ncbi.nlm.nih.gov/pubmed/25554787?dopt=Abstract %R 10.1126/science.1259724 %0 Journal Article %J Mob DNA %D 2014 %T Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis. %A Enyeart, Peter J %A Mohr, Georg %A Ellington, Andrew D %A Lambowitz, Alan M %X

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

%B Mob DNA %V 5 %P 2 %8 2014 %G eng %U http://www.mobilednajournal.com/content/5/1/2 %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/24410776?dopt=Abstract %R 10.1186/1759-8753-5-2 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2014 %T Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5. %A Katibah, George E %A Qin, Yidan %A Sidote, David J %A Yao, Jun %A Lambowitz, Alan M %A Collins, Kathleen %K Humans %K Models, Molecular %K Neoplasm Proteins %K Nucleic Acid Conformation %K Protein Binding %K RNA %X

Interferon (IFN) responses play key roles in cellular defense against pathogens. Highly expressed IFN-induced proteins with tetratricopeptide repeats (IFITs) are proposed to function as RNA binding proteins, but the RNA binding and discrimination specificities of IFIT proteins remain unclear. Here we show that human IFIT5 has comparable affinity for RNAs with diverse phosphate-containing 5′-ends, excluding the higher eukaryotic mRNA cap. Systematic mutagenesis revealed that sequence substitutions in IFIT5 can alternatively expand or introduce bias in protein binding to RNAs with 5′ monophosphate, triphosphate, cap0 (triphosphate-bridged N7-methylguanosine), or cap1 (cap0 with RNA 2′-O-methylation). We defined the breadth of cellular ligands for IFIT5 by using a thermostable group II intron reverse transcriptase for RNA sequencing. We show that IFIT5 binds precursor and processed tRNAs, as well as other RNA polymerase III transcripts. Our findings establish the RNA recognition specificity of the human innate immune response protein IFIT5.

%B Proc Natl Acad Sci U S A %V 111 %P 12025-30 %8 2014 Aug 19 %G eng %U http://www.pnas.org/content/111/33/12025.long %N 33 %1 http://www.ncbi.nlm.nih.gov/pubmed/25092312?dopt=Abstract %R 10.1073/pnas.1412842111 %0 Journal Article %J Biotechnol Biofuels %D 2014 %T The contribution of cellulosomal scaffoldins to cellulose hydrolysis by Clostridium thermocellum analyzed by using thermotargetrons. %A Hong, Wei %A Zhang, Jie %A Feng, Yingang %A Mohr, Georg %A Lambowitz, Alan M %A Cui, Gu-Zhen %A Liu, Ya-Jun %A Cui, Qiu %X

Clostridium thermocellum is a thermophilic anaerobic bacterium that degrades cellulose by using a highly effective cellulosome, a macromolecular complex consisting of multiple cellulose degrading enzymes organized and attached to the cell surface by non-catalytic scaffoldins. However, due largely to lack of efficient methods for genetic manipulation of C. thermocellum, it is still unclear how the different scaffoldins and their functional modules contribute to cellulose hydrolysis.

%B Biotechnol Biofuels %V 7 %P 80 %8 2014 %G eng %U http://www.biotechnologyforbiofuels.com/content/7/1/80 %1 http://www.ncbi.nlm.nih.gov/pubmed/24955112?dopt=Abstract %R 10.1186/1754-6834-7-80 %0 Journal Article %J PLoS Biol %D 2014 %T Evolution of RNA-protein interactions: non-specific binding led to RNA splicing activity of fungal mitochondrial tyrosyl-tRNA synthetases. %A Lamech, Lilian T %A Mallam, Anna L %A Lambowitz, Alan M %X

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was “fixed” by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins.

%B PLoS Biol %V 12 %P e1002028 %8 2014 Dec %G eng %U http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002028 %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/25536042?dopt=Abstract %R 10.1371/journal.pbio.1002028 %0 Journal Article %J Elife %D 2014 %T Molecular insights into RNA and DNA helicase evolution from the determinants of specificity for a DEAD-box RNA helicase. %A Mallam, Anna L %A Sidote, David J %A Lambowitz, Alan M %X

How different helicase families with a conserved catalytic ‘helicase core’ evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. In this study, we used Mss116, a yeast DEAD-box protein that utilizes ATP to locally unwind dsRNA, to investigate helicase specificity and mechanism. Our results define the molecular basis for the substrate specificity of a DEAD-box protein. Additionally, they show that Mss116 has ambiguous substrate-binding properties and interacts with all four NTPs and both RNA and DNA. The efficiency of unwinding correlates with the stability of the ‘closed-state’ helicase core, a complex with nucleotide and nucleic acid that forms as duplexes are unwound. Crystal structures reveal that core stability is modulated by family-specific interactions that favor certain substrates. This suggests how present-day helicases diversified from an ancestral core with broad specificity by retaining core closure as a common catalytic mechanism while optimizing substrate-binding interactions for different cellular functions. 

%B Elife %V 4 %8 2014 %G eng %U http://elifesciences.org/content/3/e04630 %1 http://www.ncbi.nlm.nih.gov/pubmed/25497230?dopt=Abstract %R 10.7554/eLife.04630 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2013 %T Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core. %A Truong, David M %A Sidote, David J %A Russell, Rick %A Lambowitz, Alan M %K Biotechnology %K Blotting, Northern %K Catalytic Domain %K Directed Molecular Evolution %K DNA Primers %K Escherichia coli %K Magnesium %K Models, Molecular %K Protein Conformation %K Protein Engineering %K Retroelements %K RNA, Catalytic %X

Mobile group II introns are bacterial retrotransposons thought to be evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of a catalytically active intron RNA ("ribozyme") and an intron-encoded reverse transcriptase, which function together to promote RNA splicing and intron mobility via reverse splicing of the intron RNA into new DNA sites ("retrohoming"). Although group II introns are active in bacteria, their natural hosts, they function inefficiently in eukaryotes, where lower free Mg(2+) concentrations decrease their ribozyme activity and constitute a natural barrier to group II intron proliferation within nuclear genomes. Here, we show that retrohoming of the Ll.LtrB group II intron is strongly inhibited in an Escherichia coli mutant lacking the Mg(2+) transporter MgtA, and we use this system to select mutations in catalytic core domain V (DV) that partially rescue retrohoming at low Mg(2+) concentrations. We thus identified mutations in the distal stem of DV that increase retrohoming efficiency in the MgtA mutant up to 22-fold. Biochemical assays of splicing and reverse splicing indicate that the mutations increase the fraction of intron RNA that folds into an active conformation at low Mg(2+) concentrations, and terbium-cleavage assays suggest that this increase is due to enhanced Mg(2+) binding to the distal stem of DV. Our findings indicate that DV is involved in a critical Mg(2+)-dependent RNA folding step in group II introns and demonstrate the feasibility of selecting intron variants that function more efficiently at low Mg(2+) concentrations, with implications for evolution and potential applications in gene targeting.

%B Proc Natl Acad Sci U S A %V 110 %P E3800-9 %8 2013 Oct 1 %G eng %U http://www.pnas.org/content/110/40/E3800.long %N 40 %1 http://www.ncbi.nlm.nih.gov/pubmed/24043808?dopt=Abstract %R 10.1073/pnas.1315742110 %0 Journal Article %J Mol Syst Biol %D 2013 %T Generalized bacterial genome editing using mobile group II introns and Cre-lox. %A Enyeart, Peter J %A Chirieleison, Steven M %A Dao, Mai N %A Perutka, Jiri %A Quandt, Erik M %A Yao, Jun %A Whitt, Jacob T %A Keatinge-Clay, Adrian T %A Lambowitz, Alan M %A Ellington, Andrew D %K Bacillus subtilis %K Base Sequence %K Escherichia coli %K Genetic Engineering %K Genetic Loci %K Genome, Bacterial %K Integrases %K Introns %K Lac Operon %K Molecular Sequence Data %K Mutagenesis, Insertional %K Nucleic Acid Conformation %K Recombination, Genetic %K Sequence Deletion %K Sequence Inversion %K Shewanella %K Staphylococcus aureus %X

Efficient bacterial genetic engineering approaches with broad-host applicability are rare. We combine two systems, mobile group II introns ('targetrons') and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one-step cut-and-paste operations. We demonstrate insertion of a 12-kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one-step cut-and-pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre-mediated manipulations typically approaches 100%.

%B Mol Syst Biol %V 9 %P 685 %8 2013 %G eng %U http://msb.embopress.org/content/9/1/685.long %1 http://www.ncbi.nlm.nih.gov/pubmed/24002656?dopt=Abstract %R 10.1038/msb.2013.41 %0 Journal Article %J PLoS Genet %D 2013 %T Genetic and biochemical assays reveal a key role for replication restart proteins in group II intron retrohoming. %A Yao, Jun %A Truong, David M %A Lambowitz, Alan M %X

Mobile group II introns retrohome by an RNP-based mechanism in which the intron RNA reverse splices into a DNA site and is reverse transcribed by the associated intron-encoded protein. The resulting intron cDNA is then integrated into the genome by cellular mechanisms that have remained unclear. Here, we used an Escherichia coli genetic screen and Taqman qPCR assay that mitigate indirect effects to identify host factors that function in retrohoming. We then analyzed mutants identified in these and previous genetic screens by using a new biochemical assay that combines group II intron RNPs with cellular extracts to reconstitute the complete retrohoming reaction in vitro. The genetic and biochemical analyses indicate a retrohoming pathway involving degradation of the intron RNA template by a host RNase H and second-strand DNA synthesis by the host replicative DNA polymerase. Our results reveal ATP-dependent steps in both cDNA and second-strand synthesis and a surprising role for replication restart proteins in initiating second-strand synthesis in the absence of DNA replication. We also find an unsuspected requirement for host factors in initiating reverse transcription and a new RNA degradation pathway that suppresses retrohoming. Key features of the retrohoming mechanism may be used by human LINEs and other non-LTR-retrotransposons, which are related evolutionarily to mobile group II introns. Our findings highlight a new role for replication restart proteins, which function not only to repair DNA damage caused by mobile element insertion, but have also been co-opted to become an integral part of the group II intron retrohoming mechanism.

%B PLoS Genet %V 9 %P e1003469 %8 2013 Apr %G eng %U http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1003469 %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/23637634?dopt=Abstract %R 10.1371/journal.pgen.1003469 %0 Journal Article %J BMC Biotechnol %D 2013 %T Rapid targeted gene disruption in Bacillus anthracis. %A Saldanha, Roland J %A Pemberton, Adin %A Shiflett, Patrick %A Perutka, Jiri %A Whitt, Jacob T %A Ellington, Andrew %A Lambowitz, Alan M %A Kramer, Ryan %A Taylor, Deborah %A Lamkin, Thomas J %K Bacillus anthracis %K Chromosomes, Bacterial %K Cloning, Molecular %K DNA, Bacterial %K Escherichia coli %K Gene Targeting %K Genes, Bacterial %K Genetic Vectors %K Introns %K Mutagenesis, Insertional %K Nucleic Acid Conformation %K Plasmids %K Selection, Genetic %X

BACKGROUND: Anthrax is a zoonotic disease recognized to affect herbivores since Biblical times and has the widest range of susceptible host species of any known pathogen. The ease with which the bacterium can be weaponized and its recent deliberate use as an agent of terror, have highlighted the importance of gaining a deeper understanding and effective countermeasures for this important pathogen. High quality sequence data has opened the possibility of systematic dissection of how genes distributed on both the bacterial chromosome and associated plasmids have made it such a successful pathogen. However, low transformation efficiency and relatively few genetic tools for chromosomal manipulation have hampered full interrogation of its genome. RESULTS: Group II introns have been developed into an efficient tool for site-specific gene inactivation in several organisms. We have adapted group II intron targeting technology for application in Bacillus anthracis and generated vectors that permit gene inactivation through group II intron insertion. The vectors developed permit screening for the desired insertion through PCR or direct selection of intron insertions using a selection scheme that activates a kanamycin resistance marker upon successful intron insertion. CONCLUSIONS: The design and vector construction described here provides a useful tool for high throughput experimental interrogation of the Bacillus anthracis genome and will benefit efforts to develop improved vaccines and therapeutics.

%B BMC Biotechnol %V 13 %P 72 %8 2013 %G eng %U http://www.biomedcentral.com/1472-6750/13/72 %1 http://www.ncbi.nlm.nih.gov/pubmed/24047152?dopt=Abstract %R 10.1186/1472-6750-13-72 %0 Journal Article %J PLoS One %D 2013 %T A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum. %A Mohr, Georg %A Hong, Wei %A Zhang, Jie %A Cui, Gu-Zhen %A Yang, Yunfeng %A Cui, Qiu %A Liu, Ya-Jun %A Lambowitz, Alan M %K Base Pairing %K Base Sequence %K Binding Sites %K Chromosomes, Bacterial %K Clostridium thermocellum %K Cyanobacteria %K Escherichia coli %K Gene Order %K Gene Targeting %K Genetic Vectors %K Introns %K L-Lactate Dehydrogenase %K Lac Operon %K Metabolome %K Metabolomics %K Mutagenesis, Insertional %K Nucleic Acid Conformation %K Phosphate Acetyltransferase %K Plasmids %K RNA, Bacterial %K RNA, Catalytic %K Temperature %X

BACKGROUND: Targetrons are gene targeting vectors derived from mobile group II introns. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which use their combined activities to achieve highly efficient site-specific DNA integration with readily programmable DNA target specificity. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used a mobile group II intron from the thermophilic cyanobacterium Thermosynechococcus elongatus to construct a thermotargetron for gene targeting in thermophiles. After determining its DNA targeting rules by intron mobility assays in Escherichia coli at elevated temperatures, we used this thermotargetron in Clostridium thermocellum, a thermophile employed in biofuels production, to disrupt six different chromosomal genes (cipA, hfat, hyd, ldh, pta, and pyrF). High integration efficiencies (67-100% without selection) were achieved, enabling detection of disruptants by colony PCR screening of a small number of transformants. Because the thermotargetron functions at high temperatures that promote DNA melting, it can recognize DNA target sequences almost entirely by base pairing of the intron RNA with less contribution from the intron-encoded protein than for mesophilic targetrons. This feature increases the number of potential targetron-insertion sites, while only moderately decreasing DNA target specificity. Phenotypic analysis showed that thermotargetron disruption of the genes encoding lactate dehydrogenase (ldh; Clo1313_1160) and phosphotransacetylase (pta; Clo1313_1185) increased ethanol production in C. thermocellum by decreasing carbon flux toward lactate and acetate. CONCLUSIONS/SIGNIFICANCE: Thermotargetron provides a new, rapid method for gene targeting and genetic engineering of C. thermocellum, an industrially important microbe, and should be readily adaptable for gene targeting in other thermophiles.

%B PLoS One %V 8 %P e69032 %8 2013 %G eng %U http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069032 %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/23874856?dopt=Abstract %R 10.1371/journal.pone.0069032 %0 Journal Article %J RNA %D 2013 %T Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. %A Mohr, Sabine %A Ghanem, Eman %A Smith, Whitney %A Sheeter, Dennis %A Qin, Yidan %A King, Olga %A Polioudakis, Damon %A Iyer, Vishwanath R %A Hunicke-Smith, Scott %A Swamy, Sajani %A Kuersten, Scott %A Lambowitz, Alan M %K Base Sequence %K Cloning, Molecular %K Conserved Sequence %K DNA, Complementary %K Escherichia coli %K Escherichia coli Proteins %K Gene Expression Profiling %K Gene Library %K Geobacillus stearothermophilus %K HeLa Cells %K Humans %K Introns %K MCF-7 Cells %K MicroRNAs %K Molecular Sequence Data %K Open Reading Frames %K Periplasmic Binding Proteins %K Plasmids %K Protein Stability %K Recombinant Fusion Proteins %K Reverse Transcriptase Polymerase Chain Reaction %K RNA-Directed DNA Polymerase %K Sequence Analysis, RNA %K Temperature %X

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

%B RNA %V 19 %P 958-70 %8 2013 Jul %G eng %U http://rnajournal.cshlp.org/content/19/7/958.long %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/23697550?dopt=Abstract %R 10.1261/rna.039743.113 %0 Journal Article %J RNA Biol %D 2013 %T Toward a molecular understanding of RNA remodeling by DEAD-box proteins. %A Russell, Rick %A Jarmoskaite, Inga %A Lambowitz, Alan M %K DEAD-box RNA Helicases %K RNA %X

DEAD-box proteins are superfamily 2 helicases that function in all aspects of RNA metabolism. They employ ATP binding and hydrolysis to generate tight, yet regulated RNA binding, which is used to unwind short RNA helices non-processively and promote structural transitions of RNA and RNA-protein substrates. In the last few years, substantial progress has been made toward a detailed, quantitative understanding of the structural and biochemical properties of DEAD-box proteins. Concurrently, progress has been made toward a physical understanding of the RNA rearrangements and folding steps that are accelerated by DEAD-box proteins in model systems. Here, we review the recent progress on both of these fronts, focusing on the mitochondrial DEAD-box proteins Mss116 and CYT-19 and their mechanisms in promoting the splicing of group I and group II introns.

%B RNA Biol %V 10 %P 44-55 %8 2013 Jan %G eng %U http://www.tandfonline.com/doi/abs/10.4161/rna.22210?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub%3Dpubmed&#.VN0CjbDF990 %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/22995827?dopt=Abstract %R 10.4161/rna.22210 %0 Journal Article %J PLoS Genet %D 2012 %T The retrohoming of linear group II intron RNAs in Drosophila melanogaster occurs by both DNA ligase 4-dependent and -independent mechanisms. %A White, Travis B %A Lambowitz, Alan M %K Animals %K DNA Damage %K DNA End-Joining Repair %K DNA Ligases %K Drosophila melanogaster %K Introns %K Mutation %K Retroelements %K RNA %K RNA Splicing %K RNA-Directed DNA Polymerase %X Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3' end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications. %B PLoS Genet %V 8 %P e1002534 %8 2012 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/22359518?dopt=Abstract %R 10.1371/journal.pgen.1002534 %0 Journal Article %J Nature %D 2012 %T Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p. %A Mallam, Anna L %A Del Campo, Mark %A Gilman, Benjamin %A Sidote, David J %A Lambowitz, Alan M %K Adenosine Triphosphatases %K Adenosine Triphosphate %K Amino Acid Motifs %K Base Sequence %K Catalytic Domain %K Conserved Sequence %K Crystallography, X-Ray %K DEAD-box RNA Helicases %K Evolution, Molecular %K GC Rich Sequence %K Models, Molecular %K Nucleic Acid Conformation %K Protein Structure, Tertiary %K RNA, Double-Stranded %K RNA-Binding Proteins %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Structure-Activity Relationship %K Substrate Specificity %X DEAD-box proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life. They contain a conserved 'helicase core' of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA-protein complexes without globally disrupting RNA structure. However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a 'closed state' with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms. %B Nature %V 490 %P 121-5 %8 2012 Oct 4 %G eng %N 7418 %1 http://www.ncbi.nlm.nih.gov/pubmed/22940866?dopt=Abstract %R 10.1038/nature11402 %0 Journal Article %J J Mol Biol %D 2011 %T ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo. %A Potratz, Jeffrey P %A Del Campo, Mark %A Wolf, Rachel Z %A Lambowitz, Alan M %A Russell, Rick %K Adenosine Triphosphate %K Blotting, Northern %K DEAD-box RNA Helicases %K Introns %K Mitochondria %K Molecular Chaperones %K Polymerase Chain Reaction %K RNA Helicases %K RNA Splicing %K RNA, Catalytic %K RNA, Fungal %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %X The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist in the folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize nonnative intronic structures. Nevertheless, Mss116p acceleration of all three constructs depends on ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mitochondrial DNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing the conversion of nonfunctional RNA structure into functional RNA structure. %B J Mol Biol %V 411 %P 661-79 %8 2011 Aug 19 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/21679717?dopt=Abstract %R 10.1016/j.jmb.2011.05.047 %0 Journal Article %J Cold Spring Harb Perspect Biol %D 2011 %T Group II introns: mobile ribozymes that invade DNA. %A Lambowitz, Alan M %A Zimmerly, Steven %K Animals %K DNA %K Evolution, Molecular %K Humans %K Introns %K Nucleic Acid Conformation %K RNA Splicing %K RNA, Catalytic %K RNA-Directed DNA Polymerase %X Group II introns are mobile ribozymes that self-splice from precursor RNAs to yield excised intron lariat RNAs, which then invade new genomic DNA sites by reverse splicing. The introns encode a reverse transcriptase that stabilizes the catalytically active RNA structure for forward and reverse splicing, and afterwards converts the integrated intron RNA back into DNA. The characteristics of group II introns suggest that they or their close relatives were evolutionary ancestors of spliceosomal introns, the spliceosome, and retrotransposons in eukaryotes. Further, their ribozyme-based DNA integration mechanism enabled the development of group II introns into gene targeting vectors ("targetrons"), which have the unique feature of readily programmable DNA target specificity. %B Cold Spring Harb Perspect Biol %V 3 %P a003616 %8 2011 Aug %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/20463000?dopt=Abstract %R 10.1101/cshperspect.a003616 %0 Journal Article %J J Mol Biol %D 2011 %T High-throughput genetic identification of functionally important regions of the yeast DEAD-box protein Mss116p. %A Mohr, Georg %A Del Campo, Mark %A Turner, Kathryn G %A Gilman, Benjamin %A Wolf, Rachel Z %A Lambowitz, Alan M %K Amino Acid Motifs %K Amino Acid Sequence %K Binding Sites %K Blotting, Northern %K Conserved Sequence %K Crystallography, X-Ray %K DEAD-box RNA Helicases %K Evolution, Molecular %K Immunoblotting %K Molecular Sequence Data %K Mutagenesis, Site-Directed %K Mutation %K Protein Binding %K Protein Conformation %K RNA Splicing %K RNA, Fungal %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Sequence Homology, Amino Acid %X The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions-one the previously noted post II region in the helicase core and the other in the CTE-that may help displace or sequester the opposite RNA strand during RNA unwinding. %B J Mol Biol %V 413 %P 952-72 %8 2011 Nov 11 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/21945532?dopt=Abstract %R 10.1016/j.jmb.2011.09.015 %0 Journal Article %J Biochemistry %D 2011 %T NMR Structure of the C-terminal domain of a tyrosyl-tRNA synthetase that functions in group I intron splicing. %A Paukstelis, Paul J %A Chari, Nandini %A Lambowitz, Alan M %A Hoffman, David %K Alternative Splicing %K Aspergillus nidulans %K Catalytic Domain %K Crystallography, X-Ray %K DNA %K Geobacillus stearothermophilus %K Introns %K Magnetic Resonance Spectroscopy %K Models, Molecular %K Models, Theoretical %K Molecular Conformation %K Protein Structure, Secondary %K Protein Structure, Tertiary %K Solvents %K Tyrosine-tRNA Ligase %X The mitochondrial tyrosyl-tRNA synthetases (mt TyrRSs) of Pezizomycotina fungi are bifunctional proteins that aminoacylate mitochondrial tRNA(Tyr) and are structure-stabilizing splicing cofactors for group I introns. Studies with the Neurospora crassa synthetase (CYT-18 protein) showed that splicing activity is dependent upon Pezizomycotina-specific structural adaptations that form a distinct group I intron-binding site in the N-terminal catalytic domain. Although CYT-18's C-terminal domain also binds group I introns, it has been intractable to X-ray crystallography in the full-length protein. Here, we determined an NMR structure of the isolated C-terminal domain of the Aspergillus nidulans mt TyrRS, which is closely related to but smaller than CYT-18's. The structure shows an S4 fold like that of bacterial TyrRSs, but with novel features, including three Pezizomycontia-specific insertions. (15)N-(1)H two-dimensional NMR showed that C-terminal domains of the full-length A. nidulans and Geobacillus stearothermophilus synthetases do not tumble independently in solution, suggesting restricted orientations. Modeling onto a CYT-18/group I intron cocrystal structure indicates that the C-terminal domains of both subunits of the homodimeric protein bind different ends of the intron RNA, with one C-terminal domain having to undergo a large shift on its flexible linker to bind tRNA(Tyr) or the intron RNA on either side of the catalytic domain. The modeling suggests that the C-terminal domain acts together with the N-terminal domain to clamp parts of the intron's catalytic core, that at least one C-terminal domain insertion functions in group I intron binding, and that some C-terminal domain regions bind both tRNA(Tyr) and group I intron RNAs. %B Biochemistry %V 50 %P 3816-26 %8 2011 May 10 %G eng %N 18 %1 http://www.ncbi.nlm.nih.gov/pubmed/21438536?dopt=Abstract %R 10.1021/bi200189u %0 Journal Article %J Proc Natl Acad Sci U S A %D 2011 %T Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail. %A Mallam, Anna L %A Jarmoskaite, Inga %A Tijerina, Pilar %A Del Campo, Mark %A Seifert, Soenke %A Guo, Liang %A Russell, Rick %A Lambowitz, Alan M %K Binding Sites %K Circular Dichroism %K DEAD-box RNA Helicases %K Fungal Proteins %K Models, Molecular %K Neurospora crassa %K Nucleic Acid Conformation %K Protein Conformation %K Protein Structure, Secondary %K Protein Structure, Tertiary %K Recombinant Proteins %K RNA, Fungal %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Scattering, Small Angle %K Structural Homology, Protein %K X-Ray Diffraction %X The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes. %B Proc Natl Acad Sci U S A %V 108 %P 12254-9 %8 2011 Jul 26 %G eng %N 30 %1 http://www.ncbi.nlm.nih.gov/pubmed/21746911?dopt=Abstract %R 10.1073/pnas.1109566108 %0 Journal Article %J RNA %D 2010 %T Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase. %A Gu, Shan-Qing %A Cui, Xiaoxia %A Mou, Sijiong %A Mohr, Sabine %A Yao, Jun %A Lambowitz, Alan M %K Bacterial Proteins %K Binding Sites %K Down-Regulation %K Escherichia coli %K Introns %K Lactococcus lactis %K Models, Genetic %K Retroelements %K RNA, Bacterial %K RNA-Binding Proteins %K RNA-Directed DNA Polymerase %X Mobile group II introns encode a reverse transcriptase that binds the intron RNA to promote RNA splicing and intron mobility, the latter via reverse splicing of the excised intron into DNA sites, followed by reverse transcription. Previous work showed that the Lactococcus lactis Ll.LtrB intron reverse transcriptase, denoted LtrA protein, binds with high affinity to DIVa, a stem-loop structure at the beginning of the LtrA open reading frame and makes additional contacts with intron core regions that stabilize the active RNA structure for forward and reverse splicing. LtrA's binding to DIVa down-regulates its translation and is critical for initiation of reverse transcription. Here, by using high-throughput unigenic evolution analysis with a genetic assay in which LtrA binding to DIVa down-regulates translation of GFP, we identified regions at LtrA's N terminus that are required for DIVa binding. Then, by similar analysis with a reciprocal genetic assay, we confirmed that residual splicing of a mutant intron lacking DIVa does not require these N-terminal regions, but does require other reverse transcriptase (RT) and X/thumb domain regions that bind the intron core. We also show that N-terminal fragments of LtrA by themselves bind specifically to DIVa in vivo and in vitro. Our results suggest a model in which the N terminus of nascent LtrA binds DIVa of the intron RNA that encoded it and nucleates further interactions with core regions that promote RNP assembly for RNA splicing and intron mobility. Features of this model may be relevant to evolutionarily related non-long-terminal-repeat (non-LTR)-retrotransposon RTs. %B RNA %V 16 %P 732-47 %8 2010 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/20179150?dopt=Abstract %R 10.1261/rna.2007310 %0 Journal Article %J PLoS Biol %D 2010 %T Mechanisms used for genomic proliferation by thermophilic group II introns. %A Mohr, Georg %A Ghanem, Eman %A Lambowitz, Alan M %K Base Sequence %K Cyanobacteria %K DNA, Bacterial %K Genome, Bacterial %K Introns %K Molecular Sequence Data %K Open Reading Frames %K Sequence Homology, Nucleic Acid %X Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelements hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting approximately 1.3% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases. %B PLoS Biol %V 8 %P e1000391 %8 2010 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/20543989?dopt=Abstract %R 10.1371/journal.pbio.1000391 %0 Journal Article %J Acta Crystallogr Sect F Struct Biol Cryst Commun %D 2009 %T Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP. %A Del Campo, Mark %A Lambowitz, Alan M %K Adenylyl Imidodiphosphate %K Cloning, Molecular %K Crystallization %K Crystallography, X-Ray %K DEAD-box RNA Helicases %K Molecular Conformation %K Recombinant Proteins %K RNA %K Saccharomyces cerevisiae Proteins %X The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/Delta598-664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions (< or =1 mg ml(-1)), but its solubility could be increased by adding 50 mM L-arginine plus 50 mM L-glutamate and 50% glycerol to achieve concentrations of approximately 10 mg ml(-1). Initial crystals were obtained by the microbatch method in the presence of a U(10) RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/Delta598-664 in complex with AMP-PNP and U(10) belonged to space group P2(1)2(1)2, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 A, and diffracted X-rays to beyond 1.9 A resolution using synchrotron radiation from sector 21 at the Advanced Photon Source. %B Acta Crystallogr Sect F Struct Biol Cryst Commun %V 65 %P 832-5 %8 2009 Aug 1 %G eng %N Pt 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/19652352?dopt=Abstract %R 10.1107/S1744309109027225 %0 Journal Article %J RNA %D 2009 %T EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting. %A Zhuang, Fanglei %A Karberg, Michael %A Perutka, Jiri %A Lambowitz, Alan M %K Escherichia coli %K Gene Targeting %K Interspersed Repetitive Sequences %K Introns %K Plasmids %X We find that group II intron EcI5, a subclass CL/IIB1 intron from an Escherichia coli virulence plasmid, is highly active in retrohoming in E. coli. Both full-length EcI5 and an EcI5-DeltaORF intron with the intron-encoded protein expressed separately from the same donor plasmid retrohome into a recipient plasmid target site at substantially higher frequencies than do similarly configured Lactococcus lactis Ll.LtrB introns. A comprehensive view of DNA target site recognition by EcI5 was obtained from selection experiments with donor and recipient plasmid libraries in which different recognition elements were randomized. These experiments suggest that EcI5, like other mobile group II introns, recognizes DNA target sequences by using both the intron-encoded protein and base-pairing of the intron RNA, with the latter involving EBS1, EBS2, and EBS3 sequences characteristic of class IIB introns. The intron-encoded protein appears to recognize a small number of bases flanking those recognized by the intron RNA, but their identity is different than in previously characterized group II introns. A computer algorithm based on the empirically determined DNA recognition rules enabled retargeting of EcI5 to integrate specifically at 10 different sites in the chromosomal lacZ gene at frequencies up to 98% without selection. Our findings provide insight into modes of DNA target site recognition used by mobile group II introns. More generally, they show how the diversity of mobile group II introns can be exploited to provide a large variety of different target specificities and potentially other useful properties for gene targeting. %B RNA %V 15 %P 432-49 %8 2009 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/19155322?dopt=Abstract %R 10.1261/rna.1378909 %0 Journal Article %J Yeast %D 2009 %T Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification. %A Markov, Dmitriy A %A Savkina, Maria %A Anikin, Michael %A Del Campo, Mark %A Ecker, Karen %A Lambowitz, Alan M %A De Gnore, Jon P %A McAllister, William T %K Amino Acid Sequence %K Chromatography, Affinity %K Chromatography, Liquid %K DNA-Directed RNA Polymerases %K GTP-Binding Proteins %K Mitochondria %K Mitochondrial Proteins %K Models, Molecular %K Molecular Sequence Data %K Protein Structure, Tertiary %K RNA %K RNA, Fungal %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Sequence Homology, Amino Acid %K Tandem Mass Spectrometry %X The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP-protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP-TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP-mtRNAP fusion, pulled down associated proteins, and identified them by LC-MS-MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. %B Yeast %V 26 %P 423-40 %8 2009 Aug %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/19536766?dopt=Abstract %R 10.1002/yea.1672 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2009 %T Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation. %A Zhuang, Fanglei %A Mastroianni, Marta %A White, Travis B %A Lambowitz, Alan M %K Animals %K Base Sequence %K Cell Nucleus %K DNA, Complementary %K Drosophila melanogaster %K Introns %K Oocytes %K Retroelements %K RNA %K Xenopus laevis %X Mobile group II introns retrohome by an RNP-based mechanism in which the excised intron lariat RNA fully reverse splices into a DNA site via 2 sequential transesterification reactions and is reverse transcribed by the associated intron-encoded protein. However, linear group II intron RNAs, which can arise by either hydrolytic splicing or debranching of lariat RNA, cannot carry out both reverse-splicing steps and were thus expected to be immobile. Here, we used facile microinjection assays in 2 eukaryotic systems, Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, to show that group II intron RNPs containing linear intron RNA can retrohome by carrying out the first step of reverse splicing into a DNA site, thereby ligating the 3' end of the intron RNA to the 5' end of the downstream exon DNA. The attached linear intron RNA is then reverse transcribed, yielding an intron cDNA whose free end is linked to the upstream exon DNA. Some of these retrohoming events result in the precise insertion of full-length intron. Most, however, yield aberrant 5' junctions with 5' exon resections, 5' intron truncations, and/or extra nucleotide residues, hallmarks of nonhomologous end-joining. Our findings reveal a mobility mechanism for linear group II intron RNAs, show how group II introns can co-opt different DNA repair pathways for retrohoming, and suggest that linear group II intron RNAs might be used for site-specific DNA integration in gene targeting. %B Proc Natl Acad Sci U S A %V 106 %P 18189-94 %8 2009 Oct 27 %G eng %N 43 %1 http://www.ncbi.nlm.nih.gov/pubmed/19833873?dopt=Abstract %R 10.1073/pnas.0910277106 %0 Journal Article %J Mol Cell %D 2009 %T Structure of the Yeast DEAD box protein Mss116p reveals two wedges that crimp RNA. %A Del Campo, Mark %A Lambowitz, Alan M %K Adenosine Diphosphate %K Adenosine Triphosphate %K Adenylyl Imidodiphosphate %K Binding Sites %K Crystallography, X-Ray %K DEAD-box RNA Helicases %K Models, Molecular %K Nucleic Acid Conformation %K Organometallic Compounds %K Poly U %K Protein Conformation %K Protein Structure, Tertiary %K RNA %K Saccharomyces cerevisiae Proteins %K Structure-Activity Relationship %X The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF(3)(-), or ADP-AlF(4)(-). The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge alpha helix bending RNA 3' of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5' of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper. %B Mol Cell %V 35 %P 598-609 %8 2009 Sep 11 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/19748356?dopt=Abstract %R 10.1016/j.molcel.2009.07.032 %0 Journal Article %J J Mol Biol %D 2009 %T Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones. %A Del Campo, Mark %A Mohr, Sabine %A Jiang, Yue %A Jia, Huijue %A Jankowsky, Eckhard %A Lambowitz, Alan M %K Base Sequence %K DEAD-box RNA Helicases %K DNA, Mitochondrial %K Genetic Complementation Test %K Introns %K Molecular Chaperones %K Molecular Sequence Data %K Neurospora crassa %K Nucleic Acid Conformation %K Protein Biosynthesis %K RNA %K RNA Splicing %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %X The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Delta strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-DeltaP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure. %B J Mol Biol %V 389 %P 674-93 %8 2009 Jun 19 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/19393667?dopt=Abstract %R 10.1016/j.jmb.2009.04.043 %0 Journal Article %J J Mol Biol %D 2008 %T Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro. %A Mohr, Georg %A Del Campo, Mark %A Mohr, Sabine %A Yang, Quansheng %A Jia, Huijue %A Jankowsky, Eckhard %A Lambowitz, Alan M %K Adenosine Triphosphatases %K Amino Acid Motifs %K Amino Acid Sequence %K Amino Acids, Basic %K Binding Sites %K Computational Biology %K Conserved Sequence %K Crystallography, X-Ray %K DEAD-box RNA Helicases %K Drosophila Proteins %K Escherichia coli %K Evolution, Molecular %K Gene Silencing %K Genetic Complementation Test %K Hydrophobic and Hydrophilic Interactions %K In Vitro Techniques %K Introns %K Isoelectric Point %K Kinetics %K Mitochondria %K Molecular Chaperones %K Molecular Sequence Data %K Mutation, Missense %K Plasmids %K Protein Binding %K Protein Biosynthesis %K Protein Structure, Secondary %K Protein Structure, Tertiary %K RNA %K RNA Splicing %K RNA, Catalytic %K RNA, Fungal %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Sequence Homology, Amino Acid %K Temperature %X The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended alpha-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted alpha-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the alpha-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p's C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions. %B J Mol Biol %V 375 %P 1344-64 %8 2008 Feb 1 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/18096186?dopt=Abstract %R 10.1016/j.jmb.2007.11.041 %0 Journal Article %J PLoS Biol %D 2008 %T Group II intron protein localization and insertion sites are affected by polyphosphate. %A Zhao, Junhua %A Niu, Wei %A Yao, Jun %A Mohr, Sabine %A Marcotte, Edward M %A Lambowitz, Alan M %K Bacterial Proteins %K DNA Transposable Elements %K Escherichia coli %K Introns %K Microarray Analysis %K Polyphosphates %K RNA-Directed DNA Polymerase %X Mobile group II introns consist of a catalytic intron RNA and an intron-encoded protein with reverse transcriptase activity, which act together in a ribonucleoprotein particle to promote DNA integration during intron mobility. Previously, we found that the Lactococcus lactis Ll.LtrB intron-encoded protein (LtrA) expressed alone or with the intron RNA to form ribonucleoprotein particles localizes to bacterial cellular poles, potentially accounting for the intron's preferential insertion in the oriC and ter regions of the Escherichia coli chromosome. Here, by using cell microarrays and automated fluorescence microscopy to screen a transposon-insertion library, we identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) whose disruption results in both an increased proportion of cells with more diffuse LtrA localization and a more uniform genomic distribution of Ll.LtrB-insertion sites. Surprisingly, we find that a common factor affecting LtrA localization in these and other disruptants is the accumulation of intracellular polyphosphate, which appears to bind LtrA and other basic proteins and delocalize them away from the poles. Our findings show that the intracellular localization of a group II intron-encoded protein is a major determinant of insertion-site preference. More generally, our results suggest that polyphosphate accumulation may provide a means of localizing proteins to different sites of action during cellular stress or entry into stationary phase, with potentially wide physiological consequences. %B PLoS Biol %V 6 %P e150 %8 2008 Jun 24 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/18593213?dopt=Abstract %R 10.1371/journal.pbio.0060150 %0 Journal Article %J PLoS One %D 2008 %T Group II intron-based gene targeting reactions in eukaryotes. %A Mastroianni, Marta %A Watanabe, Kazuo %A White, Travis B %A Zhuang, Fanglei %A Vernon, Jamie %A Matsuura, Manabu %A Wallingford, John %A Lambowitz, Alan M %K Animals %K DNA %K Drosophila melanogaster %K Eukaryotic Cells %K Gene Targeting %K Genetic Vectors %K Introns %K Magnesium %K Models, Biological %K Models, Genetic %K Oocytes %K Plasmids %K Recombination, Genetic %K RNA %K Xenopus laevis %X BACKGROUND: Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors ("targetrons") with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg(2+) concentrations. By supplying additional Mg(2+), site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg(2+)-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization. CONCLUSIONS/SIGNIFICANCE: Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms. %B PLoS One %V 3 %P e3121 %8 2008 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/18769669?dopt=Abstract %R 10.1371/journal.pone.0003121 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2008 %T Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity. %A Paukstelis, Paul J %A Lambowitz, Alan M %K Alternative Splicing %K Amino Acid Sequence %K Antifungal Agents %K Aspergillus nidulans %K Coccidioides %K Conserved Sequence %K Evolution, Molecular %K Fungal Proteins %K Histoplasma %K Introns %K Mitochondria %K Mitochondrial Proteins %K Molecular Sequence Data %K Mutagenesis, Insertional %K Neurospora crassa %K Protein Conformation %K RNA, Fungal %K Sequence Alignment %K Sequence Analysis, Protein %K Tyrosine %K Tyrosine-tRNA Ligase %X The bifunctional Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) both aminoacylates mitochondrial tRNA(Tyr) and acts as a structure-stabilizing splicing cofactor for group I introns. Previous studies showed that CYT-18 has distinct tRNA(Tyr) and group I intron-binding sites, with the latter formed by three small "insertions" in the nucleotide-binding fold and other structural adaptations compared with nonsplicing bacterial tyrosyl-tRNA synthetases. Here, analysis of genomic sequences shows that mitochondrial tyrosyl-tRNA synthetases with structural adaptations similar to CYT-18's are uniquely characteristic of fungi belonging to the subphylum Pezizomycotina, and biochemical assays confirm group I intron splicing activity for the enzymes from several of these organisms, including Aspergillus nidulans and the human pathogens Coccidioides posadasii and Histoplasma capsulatum. By combining multiple sequence alignments with a previously determined cocrystal structure of a CYT-18/group I intron RNA complex, we identify conserved features of the Pezizomycotina enzymes related to group I intron and tRNA interactions. Our results suggest that mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity evolved during or after the divergence of the fungal subphyla Pezizomycotina and Saccharomycotina by a mechanism involving the concerted differentiation of preexisting protein loop regions. The unique group I intron splicing activity of these fungal enzymes may provide a new target for antifungal drugs. %B Proc Natl Acad Sci U S A %V 105 %P 6010-5 %8 2008 Apr 22 %G eng %N 16 %1 http://www.ncbi.nlm.nih.gov/pubmed/18413600?dopt=Abstract %R 10.1073/pnas.0801722105 %0 Journal Article %J Nature %D 2008 %T Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA. %A Paukstelis, Paul J %A Chen, Jui-Hui %A Chase, Elaine %A Lambowitz, Alan M %A Golden, Barbara L %K Crystallography, X-Ray %K Introns %K Models, Molecular %K Molecular Conformation %K Neurospora crassa %K Protein Binding %K RNA %K RNA Splicing %K RNA, Catalytic %K RNA-Binding Proteins %K Staphylococcus Phages %K Tyrosine-tRNA Ligase %X The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world. %B Nature %V 451 %P 94-7 %8 2008 Jan 3 %G eng %N 7174 %1 http://www.ncbi.nlm.nih.gov/pubmed/18172503?dopt=Abstract %R 10.1038/nature06413 %0 Journal Article %J Mol Cell %D 2008 %T A three-dimensional model of a group II intron RNA and its interaction with the intron-encoded reverse transcriptase. %A Dai, Lixin %A Chai, Dinggeng %A Gu, Shan-Qing %A Gabel, Jesse %A Noskov, Sergei Y %A Blocker, Forrest J H %A Lambowitz, Alan M %A Zimmerly, Steven %K Amino Acid Sequence %K Bacterial Proteins %K Binding Sites %K Cross-Linking Reagents %K Introns %K Models, Molecular %K Molecular Sequence Data %K Nucleic Acid Conformation %K Phylogeny %K Protein Conformation %K RNA %K RNA Splicing %K RNA, Catalytic %K RNA-Directed DNA Polymerase %X Group II introns are self-splicing ribozymes believed to be the ancestors of spliceosomal introns. Many group II introns encode reverse transcriptases that promote both RNA splicing and intron mobility to new genomic sites. Here we used a circular permutation and crosslinking method to establish 16 intramolecular distance relationships within the mobile Lactococcus lactis Ll.LtrB-DeltaORF intron. Using these new constraints together with 13 established tertiary interactions and eight published crosslinks, we modeled a complete three-dimensional structure of the intron. We also used the circular permutation strategy to map RNA-protein interaction sites through fluorescence quenching and crosslinking assays. Our model provides a comprehensive structural framework for understanding the function of group II ribozymes, their natural structural variations, and the mechanisms by which the intron-encoded protein promotes RNA splicing and intron mobility. The model also suggests an arrangement of active site elements that may be conserved in the spliceosome. %B Mol Cell %V 30 %P 472-85 %8 2008 May 23 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/18424209?dopt=Abstract %R 10.1016/j.molcel.2008.04.001 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2008 %T DEAD-box proteins can completely separate an RNA duplex using a single ATP. %A Chen, Yingfeng %A Potratz, Jeffrey P %A Tijerina, Pilar %A Del Campo, Mark %A Lambowitz, Alan M %A Russell, Rick %K Adenosine Triphosphate %K DEAD-box RNA Helicases %K Models, Chemical %K Nucleic Acid Conformation %K Protein Conformation %K RNA, Double-Stranded %K Saccharomyces cerevisiae Proteins %X DEAD-box proteins are ubiquitous in RNA metabolism and use ATP to mediate RNA conformational changes. These proteins have been suggested to use a fundamentally different mechanism from the related DNA and RNA helicases, generating local strand separation while remaining tethered through additional interactions with structured RNAs and RNA-protein (RNP) complexes. Here, we provide a critical test of this model by measuring the number of ATP molecules hydrolyzed by DEAD-box proteins as they separate short RNA helices characteristic of structured RNAs (6-11 bp). We show that the DEAD-box protein CYT-19 can achieve complete strand separation using a single ATP, and that 2 related proteins, Mss116p and Ded1p, display similar behavior. Under some conditions, considerably <1 ATP is hydrolyzed per separation event, even though strand separation is strongly dependent on ATP and is not supported by the nucleotide analog AMP-PNP. Thus, ATP strongly enhances strand separation activity even without being hydrolyzed, most likely by eliciting or stabilizing a protein conformation that promotes strand separation, and AMP-PNP does not mimic ATP in this regard. Together, our results show that DEAD-box proteins can disrupt short duplexes by using a single cycle of ATP-dependent conformational changes, strongly supporting and extending models in which DEAD-box proteins perform local rearrangements while remaining tethered to their target RNAs or RNP complexes. This mechanism may underlie the functions of DEAD-box proteins by allowing them to generate local rearrangements without disrupting the global structures of their targets. %B Proc Natl Acad Sci U S A %V 105 %P 20203-8 %8 2008 Dec 23 %G eng %N 51 %1 http://www.ncbi.nlm.nih.gov/pubmed/19088196?dopt=Abstract %R 10.1073/pnas.0811075106 %0 Journal Article %J RNA %D 2008 %T Toward predicting self-splicing and protein-facilitated splicing of group I introns. %A Vicens, Quentin %A Paukstelis, Paul J %A Westhof, Eric %A Lambowitz, Alan M %A Cech, Thomas R %K Base Composition %K Base Sequence %K Catalysis %K Introns %K Neurospora crassa %K Nucleic Acid Conformation %K RNA Splicing %K RNA, Catalytic %K RNA, Messenger %K Tyrosine-tRNA Ligase %X In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence. %B RNA %V 14 %P 2013-29 %8 2008 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/18768647?dopt=Abstract %R 10.1261/rna.1027208 %0 Journal Article %J J Mol Biol %D 2007 %T Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity. %A Halls, Coralie %A Mohr, Sabine %A Del Campo, Mark %A Yang, Quansheng %A Jankowsky, Eckhard %A Lambowitz, Alan M %K Adenosine Triphosphate %K Animals %K DEAD-box RNA Helicases %K Hydrolysis %K Introns %K Magnesium %K Mitochondria %K Molecular Chaperones %K Neurospora crassa %K Nucleic Acid Denaturation %K Open Reading Frames %K Protein Binding %K RNA Splicing %K RNA, Catalytic %K RNA, Fungal %K RNA-Binding Proteins %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Substrate Specificity %K Tetrahymena thermophila %X The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates. %B J Mol Biol %V 365 %P 835-55 %8 2007 Jan 19 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/17081564?dopt=Abstract %R 10.1016/j.jmb.2006.09.083 %0 Journal Article %J Mol Cell %D 2007 %T DEAD-box proteins unwind duplexes by local strand separation. %A Yang, Quansheng %A Del Campo, Mark %A Lambowitz, Alan M %A Jankowsky, Eckhard %K Adenosine Triphosphate %K DEAD-box RNA Helicases %K Kinetics %K Models, Genetic %K Nucleic Acid Conformation %K RNA Stability %K RNA, Double-Stranded %K RNA, Fungal %K RNA-Binding Proteins %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Substrate Specificity %X DEAD-box proteins catalyze ATP-driven, local structural changes in RNA or RNA-protein complexes (RNP) during which only few RNA base pairs are separated. It is unclear how duplex unwinding by DEAD-box proteins differs from unwinding by canonical helicases, which can separate many base pairs by directional and processive translocation on the nucleic acid, starting from a helical end. Here, we show that two different DEAD-box proteins, Ded1p and Mss116p, can unwind RNA duplexes from internal as well as terminal helical regions and act on RNA segments as small as two nucleotides flanked by DNA. The data indicate that duplex unwinding by DEAD-box proteins is based on local destabilization of RNA helical regions. No directional movement of the enzymes through the duplex is involved. We propose a three-step mechanism in which DEAD-box proteins unwind duplexes as "local strand separators." This unwinding mode is well-suited for local structural changes in complex RNA or RNP assemblies. %B Mol Cell %V 28 %P 253-63 %8 2007 Oct 26 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/17964264?dopt=Abstract %R 10.1016/j.molcel.2007.08.016 %0 Journal Article %J Mol Cell %D 2007 %T Do DEAD-box proteins promote group II intron splicing without unwinding RNA? %A Del Campo, Mark %A Tijerina, Pilar %A Bhaskaran, Hari %A Mohr, Sabine %A Yang, Quansheng %A Jankowsky, Eckhard %A Russell, Rick %A Lambowitz, Alan M %K DEAD-box RNA Helicases %K Introns %K Mutation %K Nucleic Acid Conformation %K Nucleic Acid Denaturation %K Recombinant Fusion Proteins %K RNA %K RNA Splicing %K Saccharomyces cerevisiae Proteins %X The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones. %B Mol Cell %V 28 %P 159-66 %8 2007 Oct 12 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/17936712?dopt=Abstract %R 10.1016/j.molcel.2007.07.028 %0 Journal Article %J Appl Environ Microbiol %D 2007 %T Gene targeting in gram-negative bacteria by use of a mobile group II intron ("Targetron") expressed from a broad-host-range vector. %A Yao, Jun %A Lambowitz, Alan M %K Agrobacterium tumefaciens %K Benzoates %K Escherichia coli %K Gene Deletion %K Gene Targeting %K Genetic Vectors %K Gram-Negative Bacteria %K Introns %K Plasmids %K Promoter Regions, Genetic %K Pseudomonas aeruginosa %X Mobile group II introns ("targetrons") can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria. %B Appl Environ Microbiol %V 73 %P 2735-43 %8 2007 Apr %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/17322321?dopt=Abstract %R 10.1128/AEM.02829-06 %0 Journal Article %J Biochemistry %D 2007 %T Probing the mechanisms of DEAD-box proteins as general RNA chaperones: the C-terminal domain of CYT-19 mediates general recognition of RNA. %A Grohman, Jacob K %A Del Campo, Mark %A Bhaskaran, Hari %A Tijerina, Pilar %A Lambowitz, Alan M %A Russell, Rick %K Animals %K Base Sequence %K Binding Sites %K DEAD-box RNA Helicases %K Molecular Chaperones %K Nucleic Acid Conformation %K Papain %K Protozoan Proteins %K RNA %K RNA, Catalytic %K Tetrahymena thermophila %X The DEAD-box protein CYT-19 functions in the folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA-binding site, distinct from the unwinding active site, which enhances unwinding activity by binding nonspecifically to the adjacent RNA structure. Here, we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds the adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, because the truncated protein is less active in facilitating the folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs, so that it can efficiently disrupt exposed, non-native structural elements, allowing them to refold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of these domains may function similarly as nonspecific RNA-binding elements that enhance general RNA chaperone activity. %B Biochemistry %V 46 %P 3013-22 %8 2007 Mar 20 %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/17311413?dopt=Abstract %R 10.1021/bi0619472 %0 Journal Article %J Biochemistry %D 2006 %T Atomic force microscopy reveals DNA bending during group II intron ribonucleoprotein particle integration into double-stranded DNA. %A Noah, James W %A Park, Soyeun %A Whitt, Jacob T %A Perutka, Jiri %A Frey, Wolfgang %A Lambowitz, Alan M %K Bacterial Proteins %K Base Sequence %K Binding Sites %K DNA, Bacterial %K Escherichia coli %K Escherichia coli Proteins %K Introns %K Lactococcus lactis %K Macromolecular Substances %K Microscopy, Atomic Force %K Models, Biological %K Mutation %K Nucleic Acid Conformation %K Protein Binding %K Ribonucleoproteins %X The mobile Lactococcus lactis Ll.LtrB group II intron integrates into DNA target sites by a mechanism in which the intron RNA reverse splices into one DNA strand while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3' end to prime reverse transcription of the inserted intron RNA. These reactions are mediated by an RNP particle that contains the intron-encoded protein and the excised intron lariat RNA, with both the protein and base pairing of the intron RNA used to recognize DNA target sequences. Here, computational analysis indicates that Escherichia coli DNA target sequences that support Ll.LtrB integration have greater predicted bendability than do random E. coli genomic sequences, and atomic force microscopy shows that target DNA is bent during the reaction with Ll.LtrB RNPs. Time course and mutational analyses show that DNA bending occurs after reverse splicing and requires subsequent interactions between the intron-encoded protein and the 3' exon, which lead to two progressively larger bend angles. Our results suggest a model in which RNPs bend the target DNA by maintaining initial contacts with the 5' exon while engaging in subsequent 3' exon interactions that successively position the scissile phosphate for bottom-strand cleavage at the DNA endonuclease active site and then reposition the 3' end of the cleaved bottom strand to the reverse transcriptase active site for initiation of cDNA synthesis. Our findings indicate that bendability of the DNA target site is a significant factor for Ll.LtrB RNP integration. %B Biochemistry %V 45 %P 12424-35 %8 2006 Oct 17 %G eng %N 41 %1 http://www.ncbi.nlm.nih.gov/pubmed/17029398?dopt=Abstract %R 10.1021/bi060612h %0 Journal Article %J Proc Natl Acad Sci U S A %D 2006 %T A DEAD-box protein alone promotes group II intron splicing and reverse splicing by acting as an RNA chaperone. %A Mohr, Sabine %A Matsuura, Manabu %A Perlman, Philip S %A Lambowitz, Alan M %K Adenosine Triphosphate %K Base Sequence %K Introns %K Kinetics %K Magnesium %K Molecular Chaperones %K Nucleic Acid Conformation %K Recombinant Proteins %K RNA Helicases %K RNA Splicing %K RNA, Fungal %K RNA-Directed DNA Polymerase %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Salts %K Temperature %X Group II intron RNAs self-splice in vitro but only at high salt and/or Mg2+ concentrations and have been thought to require proteins to stabilize their active structure for efficient splicing in vivo. Here, we show that a DEAD-box protein, CYT-19, can by itself promote the splicing and reverse splicing of the yeast aI5gamma and bI1 group II introns under near-physiological conditions by acting as an ATP-dependent RNA chaperone, whose continued presence is not required after RNA folding. Our results suggest that the folding of some group II introns may be limited by kinetic traps and that their active structures, once formed, do not require proteins or high Mg2+ concentrations for structural stabilization. Thus, during evolution, group II introns could have spliced and transposed by reverse splicing by using ubiquitous RNA chaperones before acquiring more specific protein partners to promote their splicing and mobility. More generally, our results provide additional evidence for the widespread role of RNA chaperones in folding cellular RNAs. %B Proc Natl Acad Sci U S A %V 103 %P 3569-74 %8 2006 Mar 7 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/16505350?dopt=Abstract %R 10.1073/pnas.0600332103 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2006 %T Profile of Alan M. Lambowitz. %A Zagorski, Nick %K Evolution, Molecular %K History, 20th Century %K History, 21st Century %K Humans %K Microbiology %K Mitochondria %K Mycology %K United States %B Proc Natl Acad Sci U S A %V 103 %P 1669-71 %8 2006 Feb 7 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/16449389?dopt=Abstract %R 10.1073/pnas.0508183103 %0 Journal Article %J RNA %D 2006 %T Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing. %A Yao, Jun %A Zhong, Jin %A Fang, Yuan %A Geisinger, Edward %A Novick, Richard P %A Lambowitz, Alan M %K Base Sequence %K DNA, Bacterial %K Escherichia coli %K Genes, Bacterial %K Genes, Essential %K Humans %K Introns %K Mutagenesis, Insertional %K Operon %K Reverse Transcriptase Polymerase Chain Reaction %K RNA Splicing %K RNA, Bacterial %K Staphylococcus aureus %K Thermodynamics %X We show that a targetron based on the Lactococcus lactis Ll.LtrB group II intron can be used for efficient chromosomal gene disruption in the human pathogen Staphylococcus aureus. Targetrons expressed from derivatives of vector pCN37, which uses a cadmium-inducible promoter, or pCN39, a derivative of pCN37 with a temperature-sensitive replicon, gave site-specific disruptants of the hsa and seb genes in 37%-100% of plated colonies without selection. To disrupt hsa, an essential gene, we used a group II intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, enabling the production of functional HSa protein. We show that because splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32 degrees C but not 43 degrees C. The temperature sensitivity of the splicing reaction suggests a general means of obtaining one-step conditional disruptions in any organism. In nature, temperature sensitivity of group II intron splicing could limit the temperature range of an organism containing a group II intron inserted in an essential gene. %B RNA %V 12 %P 1271-81 %8 2006 Jul %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/16741231?dopt=Abstract %R 10.1261/rna.68706 %0 Journal Article %J RNA %D 2005 %T Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase. %A Blocker, Forrest J H %A Mohr, Georg %A Conlan, Lori H %A Qi, Li %A Belfort, Marlene %A Lambowitz, Alan M %K Amino Acid Sequence %K Bacterial Proteins %K Base Sequence %K Binding Sites %K Genes, Bacterial %K HIV Reverse Transcriptase %K HIV-1 %K Introns %K Lactococcus lactis %K Models, Molecular %K Molecular Sequence Data %K Protein Conformation %K Protein Structure, Secondary %K Protein Structure, Tertiary %K Retroelements %K RNA, Bacterial %K RNA-Directed DNA Polymerase %K Sequence Homology, Amino Acid %K Static Electricity %X Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure. The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains. Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains. Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance. Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs. Three-dimensional models of LtrA constructed by "threading" the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template-primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template-primer binding track, distal regions of the fingers, and patches on the protein's back surface. These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription. %B RNA %V 11 %P 14-28 %8 2005 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/15574519?dopt=Abstract %R 10.1261/rna.7181105 %0 Journal Article %J RNA %D 2005 %T Number, position, and significance of the pseudouridines in the large subunit ribosomal RNA of Haloarcula marismortui and Deinococcus radiodurans. %A Del Campo, Mark %A Recinos, Claudia %A Yanez, Giscard %A Pomerantz, Steven C %A Guymon, Rebecca %A Crain, Pamela F %A McCloskey, James A %A Ofengand, James %K Base Sequence %K Deinococcus %K Escherichia coli %K Haloarcula marismortui %K Hydro-Lyases %K Nucleic Acid Conformation %K Pseudouridine %K RNA, Archaeal %K RNA, Bacterial %K RNA, Ribosomal, 23S %X The number and position of the pseudouridines of Haloarcula marismortui and Deinococcus radiodurans large subunit RNA have been determined by a combination of total nucleoside analysis by HPLC-mass spectrometry and pseudouridine sequencing by the reverse transcriptase method and by LC/MS/MS. Three pseudouridines were found in H. marismortui, located at positions 1956, 1958, and 2621 corresponding to Escherichia coli positions 1915, 1917, and 2586, respectively. The three pseudouridines are all in locations found in other organisms. Previous reports of a larger number of pseudouridines in this organism were incorrect. Three pseudouridines and one 3-methyl pseudouridine (m3Psi) were found in D. radiodurans 23S RNA at positions 1894, 1898 (m3Psi), 1900, and 2584, the m3Psi site being determined by a novel application of mass spectrometry. These positions correspond to E. coli positions 1911, 1915, 1917, and 2605, which are also pseudouridines in E. coli (1915 is m3Psi). The pseudouridines in the helix 69 loop, residues 1911, 1915, and 1917, are in positions highly conserved among all phyla. Pseudouridine 2584 in D. radiodurans is conserved in eubacteria and a chloroplast but is not found in archaea or eukaryotes, whereas pseudouridine 2621 in H. marismortui is more conserved in eukaryotes and is not found in eubacteria. All the pseudoridines are near, but not exactly at, nucleotides directly involved in various aspects of ribosome function. In addition, two D. radiodurans Psi synthases responsible for the four Psi were identified. %B RNA %V 11 %P 210-9 %8 2005 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/15659360?dopt=Abstract %R 10.1261/rna.7209905 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2005 %T The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function. %A Huang, Hon-Ren %A Rowe, Claire E %A Mohr, Sabine %A Jiang, Yue %A Lambowitz, Alan M %A Perlman, Philip S %K Amino Acid Motifs %K DEAD-box RNA Helicases %K Introns %K Mitochondria %K Molecular Chaperones %K Mutation %K Protein Biosynthesis %K RNA %K RNA Helicases %K RNA Processing, Post-Transcriptional %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %X Group I and II introns self-splice in vitro, but require proteins for efficient splicing in vivo, to stabilize the catalytically active RNA structure. Recent studies showed that the splicing of some Neurospora crassa mitochondrial group I introns additionally requires a DEAD-box protein, CYT-19, which acts as an RNA chaperone to resolve nonnative structures formed during RNA folding. Here we show that, in Saccharomyces cerevisiae mitochondria, a related DEAD-box protein, Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs, and that all these defects can be partially or completely suppressed by the expression of CYT-19. Results for the aI2 group II intron indicate that Mss116p is needed after binding the intron-encoded maturase, likely for the disruption of stable but inactive RNA structures. Our results suggest that both group I and II introns are prone to kinetic traps in RNA folding in vivo and that the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones. %B Proc Natl Acad Sci U S A %V 102 %P 163-8 %8 2005 Jan 4 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/15618406?dopt=Abstract %R 10.1073/pnas.0407896101 %0 Journal Article %J Mol Cell %D 2005 %T A tyrosyl-tRNA synthetase adapted to function in group I intron splicing by acquiring a new RNA binding surface. %A Paukstelis, Paul J %A Coon, Robert %A Madabusi, Lakshmi %A Nowakowski, Jacek %A Monzingo, Arthur %A Robertus, Jon %A Lambowitz, Alan M %K Amino Acid Sequence %K Amino Acid Substitution %K Catalytic Domain %K Crystallography, X-Ray %K Genes, Fungal %K Hydroxyl Radical %K Introns %K Models, Molecular %K Molecular Sequence Data %K Mutagenesis, Insertional %K Mutagenesis, Site-Directed %K Neurospora crassa %K Nucleic Acid Conformation %K Protein Structure, Tertiary %K RNA Splicing %K RNA, Fungal %K RNA, Transfer, Amino Acyl %K Sequence Homology, Amino Acid %K Static Electricity %K Tyrosine-tRNA Ligase %X We determined a 1.95 A X-ray crystal structure of a C-terminally truncated Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) that functions in splicing group I introns. CYT-18's nucleotide binding fold and intermediate alpha-helical domains superimpose on those of bacterial TyrRSs, except for an N-terminal extension and two small insertions not found in nonsplicing bacterial enzymes. These additions surround the cyt-18-1 mutation site and are sites of suppressor mutations that restore splicing, but not synthetase activity. Highly constrained models based on directed hydroxyl radical cleavage assays show that the group I intron binds at a site formed in part by the three additions on the nucleotide binding fold surface opposite that which binds tRNATyr. Our results show how essential proteins can progressively evolve new functions. %B Mol Cell %V 17 %P 417-28 %8 2005 Feb 4 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/15694342?dopt=Abstract %R 10.1016/j.molcel.2004.12.026 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2005 %T A bacterial group II intron-encoded reverse transcriptase localizes to cellular poles. %A Zhao, Junhua %A Lambowitz, Alan M %K Bacterial Proteins %K DNA Replication %K DNA-Binding Proteins %K Escherichia coli %K Green Fluorescent Proteins %K Introns %K Lactococcus lactis %K Microscopy, Fluorescence %K Recombinant Fusion Proteins %K RNA-Directed DNA Polymerase %K Transcription Factors %X The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein) that binds the intron RNA to promote RNA splicing and intron mobility. Here, we used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to cellular poles in Escherichia coli and Lactococcus lactis. This polar localization occurs with or without coexpression of Ll.LtrB intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three nonoverlapping LtrA subsegments, possibly reflecting dependence on common redundant signals and/or protein physical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. The polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in the origin and terminus regions of the E. coli chromosome, may facilitate access to exposed DNA in these regions, and could potentially link group II intron mobility to the host DNA replication and/or cell division machinery. %B Proc Natl Acad Sci U S A %V 102 %P 16133-40 %8 2005 Nov 8 %G eng %N 45 %1 http://www.ncbi.nlm.nih.gov/pubmed/16186487?dopt=Abstract %R 10.1073/pnas.0507057102 %0 Journal Article %J Nucleic Acids Res %D 2005 %T Gene targeting using randomly inserted group II introns (targetrons) recovered from an Escherichia coli gene disruption library. %A Yao, Jun %A Zhong, Jin %A Lambowitz, Alan M %K Base Pairing %K DNA %K Escherichia coli %K Gene Library %K Gene Targeting %K Genes, Bacterial %K Introns %K Lactococcus lactis %K Plasmids %K Polymerase Chain Reaction %K Retroelements %X The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse-splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it to prime reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base-pairing of the intron RNA. Consequently, group II introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, we show that an Escherichia coli gene disruption library obtained using such randomly inserting Ll.LtrB introns contains most viable E.coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. Additionally, we identified a subset of introns that insert at sites lacking T+5, a nucleotide residue critical for second-strand cleavage. All such introns tested individually gave the desired specific disruption, some by switching to an alternate retrohoming mechanism targeting single-stranded DNA and using a nascent lagging DNA strand to prime reverse transcription. %B Nucleic Acids Res %V 33 %P 3351-62 %8 2005 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/15947133?dopt=Abstract %R 10.1093/nar/gki649 %0 Journal Article %J Genes Dev %D 2005 %T Recruitment of host functions suggests a repair pathway for late steps in group II intron retrohoming. %A Smith, Dorie %A Zhong, Jin %A Matsuura, Manabu %A Lambowitz, Alan M %A Belfort, Marlene %K Bacterial Proteins %K DNA Ligases %K DNA Repair %K DNA Replication %K DNA Transposable Elements %K DNA, Bacterial %K Escherichia coli %K Introns %K Retroelements %K RNA Splicing %K RNA, Bacterial %X Retrohoming of group II introns occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Host repair enzymes are predicted to complete this process. Here, we screened a battery of Escherichia coli mutants defective in host functions that are potentially involved in retrohoming of the Lactococcus lactis Ll.LtrB intron. We found strong (greater than threefold) effects for several enzymes, including nucleases directed against RNA and DNA, replicative and repair polymerases, and DNA ligase. A model including the presumptive roles of these enzymes in resection of DNA, degradation of the intron RNA template, traversion of RNA-DNA junctions, and second-strand DNA synthesis is described. The completion of retrohoming is viewed as a DNA repair process, with features that may be shared by other non-LTR retroelements. %B Genes Dev %V 19 %P 2477-87 %8 2005 Oct 15 %G eng %N 20 %1 http://www.ncbi.nlm.nih.gov/pubmed/16230535?dopt=Abstract %R 10.1101/gad.1345105 %0 Journal Article %J Mol Ther %D 2005 %T Retargeting mobile group II introns to repair mutant genes. %A Jones, John Patrick %A Kierlin, Monique N %A Coon, Robert G %A Perutka, Jiri %A Lambowitz, Alan M %A Sullenger, Bruce A %K Animals %K Base Sequence %K Cell Line %K DNA Repair %K Globins %K Humans %K Introns %K Lac Operon %K Molecular Sequence Data %K Mutation %K Nucleic Acid Conformation %K RNA, Catalytic %K Sensitivity and Specificity %K Substrate Specificity %X Retroposable elements such as retroviral and lentiviral vectors have been employed for many gene therapy applications. Unfortunately, such gene transfer vectors integrate genes into many different DNA sequences and unintended integration of the vector near a growth-promoting gene can engender pathological consequences. For example, retroviral vector-mediated gene transfer induced leukemia in 2 of 11 children treated for severe combined immunodeficiency, raising significant safety issues for gene transfer strategies that cannot be targeted to specific sequences. Here, we examine the use of a mobile retroposable genetic element that can be targeted to introduce therapeutic sequences site specifically into mutant genes. The data demonstrate that the mobile group II intron from Lactococcus lactis can be targeted to insert into and repair mutant lacZ (approved gene symbol GLB1) and beta-globin (approved gene symbol HBB) genes with high efficiency and fidelity in model systems in bacteria. These results suggest that these mobile genetic elements represent a novel class of agents for performing targeted genetic repair. %B Mol Ther %V 11 %P 687-94 %8 2005 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/15851007?dopt=Abstract %R 10.1016/j.ymthe.2005.01.014 %0 Journal Article %J Mol Microbiol %D 2005 %T Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment. %A Coros, Colin J %A Landthaler, Markus %A Piazza, Carol Lyn %A Beauregard, Arthur %A Esposito, Donna %A Perutka, Jiri %A Lambowitz, Alan M %A Belfort, Marlene %K Bacterial Proteins %K DNA Replication %K DNA Transposable Elements %K DNA, Bacterial %K DNA, Single-Stranded %K Escherichia coli Proteins %K Introns %K Lactococcus lactis %K Models, Biological %K Models, Genetic %K Retroelements %X Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli, retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli, using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis, in E. coli, Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference. %B Mol Microbiol %V 56 %P 509-24 %8 2005 Apr %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/15813740?dopt=Abstract %R 10.1111/j.1365-2958.2005.04554.x %0 Journal Article %J J Mol Biol %D 2004 %T A group II intron-encoded maturase functions preferentially in cis and requires both the reverse transcriptase and X domains to promote RNA splicing. %A Cui, Xiaoxia %A Matsuura, Manabu %A Wang, Qin %A Ma, Hongwen %A Lambowitz, Alan M %K Base Sequence %K Escherichia coli %K Introns %K Molecular Sequence Data %K Nucleic Acid Conformation %K Reverse Transcriptase Polymerase Chain Reaction %K RNA Splicing %K RNA, Bacterial %K RNA-Directed DNA Polymerase %X Mobile group II introns encode proteins with both reverse transcriptase activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active structure of the intron RNA. Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein binds first to a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of its own coding region, and then makes additional contacts with conserved catalytic core regions to stabilize the active RNA structure. Here, we developed an Escherichia coli genetic assay that links the splicing of the Ll.LtrB intron to the expression of green fluorescent protein and used it to study the in vivo splicing of wild-type and mutant introns and to delineate regions of the maturase required for splicing. Our results show that the maturase functions most efficiently when expressed in cis from the same transcript as the intron RNA. In agreement with previous in vitro assays, we find that the high-affinity binding site in DIVa is required for efficient splicing of the Ll.LtrB intron in vivo, but in the absence of DIVa, 6-10% residual splicing occurs by the direct binding of the maturase to the catalytic core. Critical regions of the maturase were identified by statistically analyzing ratios of missense to silent mutations in functional LtrA variants isolated from a library generated by mutagenic PCR ("unigenic evolution"). This analysis shows that both the reverse transcriptase domain and domain X, which likely corresponds to the reverse transcriptase thumb, are required for RNA splicing, while the C-terminal DNA-binding and DNA endonuclease domains are not required. Within the reverse transcriptase domain, the most critical regions for maturase activity include parts of the fingers and palm that function in template and primer binding in HIV-1 reverse transcriptase, but the integrity of the reverse transcriptase active site is not required. Biochemical analysis of LtrA mutants indicates that the N terminus of the reverse transcriptase domain is required for high-affinity binding of the intron RNA, possibly via direct interaction with DIVa, while parts of domain X interact with conserved regions of the catalytic core. Our results support the hypothesis that the intron-encoded protein adapted to function in splicing by using, at least in part, interactions used initially to recognize the intron RNA as a template for reverse transcription. %B J Mol Biol %V 340 %P 211-31 %8 2004 Jul 2 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/15201048?dopt=Abstract %R 10.1016/j.jmb.2004.05.004 %0 Journal Article %J RNA %D 2004 %T High-affinity binding site for a group II intron-encoded reverse transcriptase/maturase within a stem-loop structure in the intron RNA. %A Watanabe, Kazuo %A Lambowitz, Alan M %K Bacterial Proteins %K Base Pairing %K Base Sequence %K Binding Sites %K Introns %K Lactococcus lactis %K Molecular Sequence Data %K Mutation %K Nucleic Acid Conformation %K Protein Biosynthesis %K RNA Splicing %K RNA, Bacterial %K RNA-Directed DNA Polymerase %K Sequence Deletion %K Sequence Homology, Nucleic Acid %X Mobile group II introns encode proteins that have reverse transcriptase and maturase activities and bind specifically to the intron RNA to promote both RNA splicing and intron mobility. Previous studies with the Lactococcus lactis Ll.LtrB intron showed that the intron-encoded protein (LtrA) has a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure containing the translation initiation region of the LtrA open reading frame, and that this binding site consists of a small stem-loop emanating from a purine-rich internal loop. The binding of LtrA to DIVa is important for translational regulation, RNA splicing, and intron mobility. Here, we show by in vitro selection that part of the purine-rich internal loop can be closed by base pairing, enabling the LtrA binding site to be represented as an extended stem-loop structure with a bulged A (A556) required for tight binding of LtrA. The deletion or pairing of A556 has relatively little effect on maturase-promoted RNA splicing, but significantly inhibits intron mobility. The wild-type DIVa structure has a second bulged A (A553), which is selected against in tightly binding variants. As expected from the selection, the deletion or pairing of A553 results in tighter binding of LtrA, but surprisingly, also inhibits intron mobility. These findings suggest that the binding of LtrA to DIVa is delicately balanced, so that either too weak or too tight binding can be deleterious. The nature of the maturase/DIVa interaction and its role in translational regulation are reminiscent of the coat protein/RNA hairpin interactions of single-stranded RNA phages. %B RNA %V 10 %P 1433-43 %8 2004 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/15273321?dopt=Abstract %R 10.1261/rna.7730104 %0 Journal Article %J Annu Rev Genet %D 2004 %T Mobile group II introns. %A Lambowitz, Alan M %A Zimmerly, Steven %K Bacteria %K Base Sequence %K DNA Replication %K DNA, Mitochondrial %K Evolution, Molecular %K Gene Targeting %K Introns %K Models, Genetic %K Models, Molecular %K Molecular Sequence Data %K Molecular Structure %K Phylogeny %K Retroelements %K RNA Splicing %K RNA, Catalytic %K Spliceosomes %K Yeasts %X Mobile group II introns, found in bacterial and organellar genomes, are both catalytic RNAs and retrotransposable elements. They use an extraordinary mobility mechanism in which the excised intron RNA reverse splices directly into a DNA target site and is then reverse transcribed by the intron-encoded protein. After DNA insertion, the introns remove themselves by protein-assisted, autocatalytic RNA splicing, thereby minimizing host damage. Here we discuss the experimental basis for our current understanding of group II intron mobility mechanisms, beginning with genetic observations in yeast mitochondria, and culminating with a detailed understanding of molecular mechanisms shared by organellar and bacterial group II introns. We also discuss recently discovered links between group II intron mobility and DNA replication, new insights into group II intron evolution arising from bacterial genome sequencing, and the evolutionary relationship between group II introns and both eukaryotic spliceosomal introns and non-LTR-retrotransposons. Finally, we describe the development of mobile group II introns into gene-targeting vectors, "targetrons," which have programmable target specificity. %B Annu Rev Genet %V 38 %P 1-35 %8 2004 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/15568970?dopt=Abstract %R 10.1146/annurev.genet.38.072902.091600 %0 Journal Article %J RNA %D 2004 %T The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns. %A Chen, Xin %A Mohr, Georg %A Lambowitz, Alan M %K Introns %K Mitochondria %K Mutation %K Neurospora crassa %K Nucleic Acid Conformation %K Protein Structure, Tertiary %K Saccharomyces cerevisiae %K Sequence Analysis, Protein %K Tyrosine-tRNA Ligase %X The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by stabilizing the catalytically active RNA structure. To accomplish this, CYT-18 recognizes conserved structural features of group I intron RNAs using regions of the N-terminal nucleotide-binding fold, intermediate alpha-helical, and C-terminal RNA-binding domains that also function in binding tRNA(Tyr). Curiously, whereas the splicing of the N. crassa mitochondrial large subunit rRNA intron is completely dependent on CYT-18's C-terminal RNA-binding domain, all other group I introns tested thus far are spliced efficiently by a truncated protein lacking this domain. To investigate the function of the C-terminal domain, we used an Escherichia coli genetic assay to isolate mutants of the Saccharomyces cerevisiae mitochondrial large subunit rRNA and phage T4 td introns that can be spliced in vivo by the wild-type CYT-18 protein, but not by the C-terminally truncated protein. Mutations that result in dependence on CYT-18's C-terminal domain include those disrupting two long-range GNRA tetraloop/receptor interactions: L2-P8, which helps position the P1 helix containing the 5'-splice site, and L9-P5, which helps establish the correct relative orientation of the P4-P6 and P3-P9 domains of the group I intron catalytic core. Our results indicate that different structural mutations in group I intron RNAs can result in dependence on different regions of CYT-18 for RNA splicing. %B RNA %V 10 %P 634-44 %8 2004 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/15037773?dopt=Abstract %0 Journal Article %J J Mol Biol %D 2004 %T Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. %A Perutka, Jiri %A Wang, Wenjun %A Goerlitz, David %A Lambowitz, Alan M %K Algorithms %K Amino Acid Motifs %K Base Pairing %K Base Sequence %K Blotting, Southern %K Computational Biology %K DNA Helicases %K Escherichia coli Proteins %K Introns %K Lactococcus lactis %K Mutagenesis, Site-Directed %X Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small number of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1-80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E.coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational analysis provides new insight into group II intron target site recognition, and the set of E.coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins. %B J Mol Biol %V 336 %P 421-39 %8 2004 Feb 13 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/14757055?dopt=Abstract %0 Journal Article %J Biochemistry %D 2003 %T Effects of maturase binding and Mg2+ concentration on group II intron RNA folding investigated by UV cross-linking. %A Noah, James W %A Lambowitz, Alan M %K Base Sequence %K DNA Primers %K Introns %K Kinetics %K Magnesium %K Molecular Sequence Data %K Nucleic Acid Conformation %K Protein Binding %K Protein Structure, Tertiary %K RNA Splicing %K RNA, Bacterial %K RNA-Directed DNA Polymerase %K Ultraviolet Rays %X The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase/maturase (LtrA protein) that promotes RNA splicing by stabilizing the catalytically active RNA structure. Here, we mapped 17 UV cross-links induced in both wild-type Ll.LtrB RNA and Ll.LtrB-Delta2486 RNA, which has a branch-point deletion that prevents splicing, and we used these cross-links to follow tertiary structure formation under different conditions in the presence or absence of the LtrA protein. Twelve of the cross-links are long-range, with six near known tertiary interaction sites in the active RNA structure. In a reaction medium containing 0.5 M NH(4)Cl, eight of the 17 cross-links were detected in the absence of Mg(2+) or the presence of EDTA, and all were detected at 5 mM Mg(2+), where efficient splicing requires the LtrA protein. The frequencies of all but four cross-links increased with increasing Mg(2+) concentrations, becoming maximal between 4 and 50 mM Mg(2+), where the intron is self-splicing. These findings suggest that a high Mg(2+) concentration induces self-splicing by globally stabilizing tertiary structure, including key tertiary interactions that are required for catalytic activity. Significantly, the binding of the maturase under protein-dependent splicing conditions (0.5 M NH(4)Cl and 5 mM Mg(2+)) increased the frequency of only nine cross-links, seven of which are long-range, suggesting that, in contrast to a high Mg(2+) concentration, LtrA promotes splicing by stabilizing critical tertiary structure interactions, while leaving other regions of the intron relatively flexible. This difference may contribute to the high rate of protein-dependent splicing, relative to the rate of self-splicing. The propensity of the intron RNA to form tertiary structure even at relatively low Mg(2+) concentrations raises the possibility that the maturase functions at least in part by tertiary structure capture. Finally, an abundant central wheel cross-link, present in >50% of the molecules at 5 mM Mg(2+), suggests models in which group II intron domains I and II are either coaxially stacked or aligned in parallel, bringing the 5'-splice site together with the 3'-splice site and catalytic core elements at JII/III. This and other cross-links provide new constraints for three-dimensional structural modeling of the group II intron catalytic core. %B Biochemistry %V 42 %P 12466-80 %8 2003 Nov 4 %G eng %N 43 %1 http://www.ncbi.nlm.nih.gov/pubmed/14580192?dopt=Abstract %R 10.1021/bi035339n %0 Journal Article %J Appl Environ Microbiol %D 2003 %T Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection. %A Frazier, Courtney L %A San Filippo, Joseph %A Lambowitz, Alan M %A Mills, David A %K Bacterial Proteins %K Base Sequence %K Carboxy-Lyases %K Conjugation, Genetic %K DNA Transposable Elements %K Genetic Engineering %K Introns %K Lactococcus lactis %K Molecular Sequence Data %K Recombination, Genetic %K RNA Splicing %K Tetracycline Resistance %X Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci. %B Appl Environ Microbiol %V 69 %P 1121-8 %8 2003 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/12571038?dopt=Abstract %0 Journal Article %J EMBO J %D 2003 %T Group II intron mobility using nascent strands at DNA replication forks to prime reverse transcription. %A Zhong, Jin %A Lambowitz, Alan M %K Bacterial Proteins %K Base Sequence %K Binding Sites %K DNA Replication %K DNA Transposable Elements %K DNA, Bacterial %K Genes, Bacterial %K Introns %K Lactococcus lactis %K Models, Biological %K Mutation %K RNA Splicing %K RNA, Bacterial %K Transcription, Genetic %X The Lactococcus lactis Ll.LtrB group II intron uses a major retrohoming mechanism in which the excised intron RNA reverse splices into one strand of a DNA target site, while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. Here, experiments with mutant introns and target sites indicate that Ll.LtrB can retrohome without second-strand cleavage by using a nascent strand at a DNA replication fork as the primer for reverse transcription. This mechanism connecting intron mobility to target DNA replication may be used by group II intron species that encode proteins lacking the C-terminal DNA endonuclease domain and for group II intron retrotransposition to ectopic sites. %B EMBO J %V 22 %P 4555-65 %8 2003 Sep 1 %G eng %N 17 %1 http://www.ncbi.nlm.nih.gov/pubmed/12941706?dopt=Abstract %R 10.1093/emboj/cdg433 %0 Journal Article %J J Mol Biol %D 2003 %T Mobility of the Sinorhizobium meliloti group II intron RmInt1 occurs by reverse splicing into DNA, but requires an unknown reverse transcriptase priming mechanism. %A Muñoz-Adelantado, Estefanía %A San Filippo, Joseph %A Martínez-Abarca, Francisco %A García-Rodríguez, Fernando M %A Lambowitz, Alan M %A Toro, Nicolás %K Base Sequence %K DNA, Bacterial %K Introns %K Molecular Sequence Data %K RNA Splicing %K RNA-Directed DNA Polymerase %K Sinorhizobium meliloti %X The mobile group II introns characterized to date encode ribonucleoprotein complexes that promote mobility by a major retrohoming mechanism in which the intron RNA reverse splices directly into the sense strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase/maturase cleaves the antisense strand and uses it as primer for reverse transcription of the inserted intron RNA. Here, we show that the Sinorhizobium meliloti group II intron RmInt1, which encodes a protein lacking a DNA endonuclease domain, similarly uses both the intron RNA and an intron-encoded protein with reverse transcriptase and maturase activities for mobility. However, while RmInt1 reverse splices into both single-stranded and double-stranded DNA target sites, it is unable to carry out site-specific antisense-strand cleavage due to the lack of a DNA endonuclease domain. Our results suggest that RmInt1 mobility involves reverse splicing into double-stranded or single-stranded DNA target sites, but due to the lack of DNA endonuclease function, it requires an alternate means of procuring a primer for target DNA-primed reverse transcription. %B J Mol Biol %V 327 %P 931-43 %8 2003 Apr 11 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/12662921?dopt=Abstract %0 Journal Article %J Mol Cell %D 2003 %T The pathway for DNA recognition and RNA integration by a group II intron retrotransposon. %A Aizawa, Yasunori %A Xiang, Qing %A Lambowitz, Alan M %A Pyle, Anna Marie %K Bacterial Proteins %K Base Sequence %K Binding Sites %K DNA %K Dose-Response Relationship, Drug %K Introns %K Kinetics %K Models, Biological %K Molecular Sequence Data %K Oligonucleotides %K Retroelements %K RNA %K RNA-Directed DNA Polymerase %K Time Factors %X Group II intron RNPs are mobile genetic elements that attack and invade duplex DNA. In this work, we monitor the invasion reaction in vitro and establish a quantitative kinetic framework for the steps of this complex cascade. We find that target site specificity is achieved after DNA binding, which occurs nonspecifically. RNP searches the bound DNA before undergoing a conformational change that is associated with identification of its specific binding site. The study reveals a facile equilibrium between intron invasion and splicing, indicating that RNP invasion of top strand DNA is a relatively unfavorable event. Group II mobility must therefore depend on the trapping of invasion products, potentially through interaction of the intron-encoded protein with the DNA target and/or initiation of reverse transcription. %B Mol Cell %V 11 %P 795-805 %8 2003 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/12667460?dopt=Abstract %0 Journal Article %J Nucleic Acids Res %D 2003 %T Putative proteins related to group II intron reverse transcriptase/maturases are encoded by nuclear genes in higher plants. %A Mohr, Georg %A Lambowitz, Alan M %K Amino Acid Sequence %K Arabidopsis %K Cell Nucleus %K Chromosome Mapping %K Chromosomes, Plant %K Endoribonucleases %K Genome, Plant %K Introns %K Molecular Sequence Data %K Nucleotidyltransferases %K Open Reading Frames %K Oryza sativa %K Phylogeny %K Plant Proteins %K RNA-Directed DNA Polymerase %K Sequence Homology, Amino Acid %X The Arabidopsis thaliana nuclear genome sequence revealed several open reading frames encoding proteins related to group II intron-encoded reverse transcriptase/maturases. Here, we show via sequence alignments that at least four such open reading frames are conserved in the nuclear genomes of A.thaliana and Oryza sativa (rice) and that they encode putative proteins belonging to two different classes (nMat-1 and nMat-2), neither of which is associated with a group II intron RNA structure. The two nMat-1 proteins have reverse transcriptase, maturase and DNA endonuclease domains characteristic of canonical group II intron-encoded proteins, while the two nMat-2 proteins have reverse transcriptase and maturase domains linked to a novel C-terminal domain. Although some nMat proteins have mutations expected to inactivate intron mobility functions, all could potentially retain the RNA splicing function. These nuclear maturase-like proteins may be imported into organelles to function in group II intron splicing and/or they may have assumed other cellular functions. Nuclear-encoded maturases could regulate organellar gene expression and may reflect a step in the evolution of mobile group II introns into spliceosomal introns. %B Nucleic Acids Res %V 31 %P 647-52 %8 2003 Jan 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/12527773?dopt=Abstract %0 Journal Article %J Nucleic Acids Res %D 2003 %T Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker. %A Zhong, Jin %A Karberg, Michael %A Lambowitz, Alan M %K Base Sequence %K Binding Sites %K Chromosomes, Bacterial %K DNA, Bacterial %K Gene Targeting %K Genes, Bacterial %K Genetic Markers %K Genetic Vectors %K Genome, Bacterial %K Introns %K Mutagenesis, Insertional %K Polymerase Chain Reaction %K Recombination, Genetic %K Retroelements %X Mobile group II introns have been used to develop a novel class of gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron containing a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial gene disruption at near 100% efficiency after selection. The targetron can be generated via PCR without cloning, and after intron integration, the marker gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a gene knockout library. Analysis of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy number of origin proximal genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute. %B Nucleic Acids Res %V 31 %P 1656-64 %8 2003 Mar 15 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/12626707?dopt=Abstract %0 Journal Article %J Mol Cell Biol %D 2003 %T The DIVa maturase binding site in the yeast group II intron aI2 is essential for intron homing but not for in vivo splicing. %A Huang, Hon-Ren %A Chao, Michael Y %A Armstrong, Barbara %A Wang, Yong %A Lambowitz, Alan M %A Perlman, Philip S %K Base Sequence %K Binding Sites %K DNA, Fungal %K DNA, Mitochondrial %K Genetic Complementation Test %K Introns %K Molecular Sequence Data %K Mutation %K Nucleic Acid Conformation %K Open Reading Frames %K RNA %K RNA Splicing %K RNA, Fungal %K RNA-Directed DNA Polymerase %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %X Splicing of the Saccharomyces cerevisiae mitochondrial DNA group II intron aI2 depends on the intron-encoded 62-kDa reverse transcriptase-maturase protein (p62). In wild-type strains, p62 remains associated with the excised intron lariat RNA in ribonucleoprotein (RNP) particles that are essential for intron homing. Studies of a bacterial group II intron showed that the DIVa substructure of intron domain IV is a high-affinity binding site for its maturase. Here we first present in vitro evidence extending that conclusion to aI2. Then, experiments with aI2 DIVa mutant strains show that the binding of p62 to DIVa is not essential for aI2 splicing in vivo but is essential for homing. Because aI2 splicing in the DIVa mutant strains remains maturase dependent, splicing must rely on other RNA-protein contacts. The p62 that accumulates in the mutant strains has reverse transcriptase activity, but fractionation experiments at high and low salt concentrations show that it associates more weakly than the wild-type protein with endogenous mitochondrial RNAs, and that phenotype probably explains the homing defect. Replacing the DIVa of aI2 with that of the closely related intron aI1 improves in vivo splicing but not homing, indicating that DIVa contributes to the specificity of the maturase-RNA interaction needed for homing. %B Mol Cell Biol %V 23 %P 8809-19 %8 2003 Dec %G eng %N 23 %1 http://www.ncbi.nlm.nih.gov/pubmed/14612420?dopt=Abstract %0 Journal Article %J Proc Natl Acad Sci U S A %D 2002 %T tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase. %A Myers, Christopher A %A Kuhla, Birte %A Cusack, Stephen %A Lambowitz, Alan M %K Cloning, Molecular %K Cysteine %K Hydroxyl Radical %K Introns %K Models, Molecular %K Neurospora crassa %K Nucleic Acid Conformation %K Protein Structure, Quaternary %K RNA, Transfer %K Tyrosine-tRNA Ligase %X The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs. %B Proc Natl Acad Sci U S A %V 99 %P 2630-5 %8 2002 Mar 5 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/11854463?dopt=Abstract %R 10.1073/pnas.052596299 %0 Journal Article %J J Mol Biol %D 2002 %T Binding of a group II intron-encoded reverse transcriptase/maturase to its high affinity intron RNA binding site involves sequence-specific recognition and autoregulates translation. %A Singh, Ravindra N %A Saldanha, Roland J %A D'Souza, Lisa M %A Lambowitz, Alan M %K Bacterial Proteins %K Base Sequence %K Binding Sites %K Escherichia coli %K Genes, Reporter %K Genetic Variation %K Introns %K Lac Operon %K Lactococcus lactis %K Models, Genetic %K Molecular Sequence Data %K Mutation %K Nucleic Acid Conformation %K Protein Biosynthesis %K RNA Splicing %K RNA, Bacterial %K RNA-Directed DNA Polymerase %K Saccharomyces cerevisiae Proteins %X Mobile group II introns encode reverse transcriptases that bind specifically to the intron RNAs to promote both intron mobility and RNA splicing (maturase activity). Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein (LtrA) binds first to a primary high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of the LtrA coding sequence, and then makes additional contacts with conserved regions of the intron to fold the RNA into the catalytically active structure. Here, we analyzed the DIVa binding site by iterative in vitro selection and in vitro mutagenesis. Our results show that LtrA binds to a small region at the distal end of DIVa that contains the ribosome-binding site and initiation codon of the LtrA open reading frame. The critical elements are in a small stem-loop structure emanating from a purine-rich internal loop, with both sequence and structure playing a role in LtrA recognition. The ribosome-binding site falls squarely within the LtrA-binding region and is sequestered directly by the binding of LtrA or by stabilization of the small stem-loop or both. Finally, by using LacZ fusions in Escherichia coli, we show that the binding of LtrA to DIVa down-regulates translation. This mode of regulation limits accumulation of the potentially deleterious intron-encoded protein and may facilitate splicing by halting ribosome entry into the intron. The recognition of the DIVa loop-stem-loop structure accounts, in part, for the intron specificity of group II intron maturases and has parallels in template-recognition mechanisms used by other reverse transcriptases. %B J Mol Biol %V 318 %P 287-303 %8 2002 Apr 26 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/12051838?dopt=Abstract %R 10.1016/S0022-2836(02)00054-2 %0 Journal Article %J J Mol Biol %D 2002 %T Characterization of the C-terminal DNA-binding/DNA endonuclease region of a group II intron-encoded protein. %A San Filippo, Joseph %A Lambowitz, Alan M %K Amino Acid Motifs %K Amino Acid Sequence %K Bacterial Proteins %K Binding Sites %K Catalysis %K Cations, Divalent %K Colicins %K Conserved Sequence %K DNA Primers %K DNA-Binding Proteins %K Endodeoxyribonucleases %K Introns %K Lactococcus lactis %K Magnesium %K Models, Molecular %K Molecular Sequence Data %K Mutation %K Protein Binding %K Protein Structure, Tertiary %K RNA-Directed DNA Polymerase %K Sequence Alignment %K Zinc %X Group II intron retrohoming occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase uses a C-terminal DNA endonuclease activity to cleave the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. Here, we characterized the C-terminal DNA-binding/DNA endonuclease region of the LtrA protein encoded by the Lactococcus lactis Ll.LtrB intron. This C-terminal region consists of an upstream segment that contributes to DNA binding, followed by a DNA endonuclease domain that contains conserved sequence motifs characteristic of H-N-H DNA endonucleases, interspersed with two pairs of conserved cysteine residues. Atomic emission spectroscopy of wild-type and mutant LtrA proteins showed that the DNA endonuclease domain contains a single tightly bound Mg(2+) ion at the H-N-H active site. Although the conserved cysteine residue pairs could potentially bind Zn(2+), the purified LtrA protein is active despite the presence of only sub-stoichiometric amounts of Zn(2+), and the addition of exogenous Zn(2+) inhibits the DNA endonuclease activity. Multiple sequence alignments identified features of the DNA-binding region and DNA endonuclease domain that are conserved in LtrA and related group II intron proteins, and their functional importance was demonstrated by unigenic evolution analysis and biochemical assays of mutant LtrA protein with alterations in key amino acid residues. Notably, deletion of the DNA endonuclease domain or mutations in its conserved sequence motifs strongly inhibit reverse transcriptase activity, as well as bottom-strand cleavage, while retaining other activities of the LtrA protein. A UV-cross-linking assay showed that these DNA endonuclease domain mutations do not block DNA primer binding and thus likely inhibit reverse transcriptase activity either by affecting the positioning of the primer or the conformation of the reverse transcriptase domain. %B J Mol Biol %V 324 %P 933-51 %8 2002 Dec 13 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/12470950?dopt=Abstract %0 Journal Article %J Cell %D 2002 %T A DEAD-box protein functions as an ATP-dependent RNA chaperone in group I intron splicing. %A Mohr, Sabine %A Stryker, John M %A Lambowitz, Alan M %K Adenosine Triphosphatases %K Adenosine Triphosphate %K Alternative Splicing %K Base Sequence %K Blotting, Northern %K Cloning, Molecular %K Dose-Response Relationship, Drug %K Fungal Proteins %K Immunoblotting %K Introns %K Kinetics %K Models, Genetic %K Molecular Sequence Data %K Mutation %K Neurospora crassa %K Nucleic Acid Conformation %K Open Reading Frames %K Phenotype %K RNA %K Substrate Specificity %K Temperature %K Time Factors %X The Neurospora crassa CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in splicing group I introns by inducing formation of the catalytically active RNA structure. Here, we identified a DEAD-box protein (CYT-19) that functions in concert with CYT-18 to promote group I intron splicing in vivo and vitro. CYT-19 does not bind specifically to group I intron RNAs and instead functions as an ATP-dependent RNA chaperone to destabilize nonnative RNA structures that constitute kinetic traps in the CYT-18-assisted RNA-folding pathway. Our results demonstrate that a DExH/D-box protein has a specific, physiologically relevant chaperone function in the folding of a natural RNA substrate. %B Cell %V 109 %P 769-79 %8 2002 Jun 14 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/12086675?dopt=Abstract %0 Journal Article %J J Mol Biol %D 2001 %T Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing. Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns. %A Mohr, G %A Rennard, R %A Cherniack, A D %A Stryker, J %A Lambowitz, A M %K Adenosine Monophosphate %K Amino Acid Sequence %K Gene Deletion %K Introns %K Kinetics %K Molecular Sequence Data %K Neurospora crassa %K Protein Conformation %K Protein Structure, Tertiary %K Recombinant Proteins %K RNA %K RNA Splicing %K Sequence Homology, Amino Acid %K Tyrosine %K Tyrosine-tRNA Ligase %X The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by helping the intron RNA fold into the catalytically active structure. The regions required for splicing include an idiosyncratic N-terminal extension, the nucleotide-binding fold domain, and the C-terminal RNA-binding domain. Here, we show that the idiosyncratic N-terminal region is in fact comprised of two functionally distinct parts: an upstream region consisting predominantly of a predicted amphipathic alpha-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted alpha-helices H1 and H2, corresponding to features in the X-ray crystal structure of the Bacillus stearothermophilus TyrRS. Bacterial genetic assays with libraries of CYT-18 mutants having random mutations in the N-terminal region identified functionally important amino acid residues and supported the predicted structures of the H0 and H1 alpha-helices. The function of N and C-terminal domains of CYT-18 was investigated by detailed biochemical analysis of deletion mutants. The results confirmed that the N-terminal extension is required only for splicing activity, but surprisingly, at least in the case of the N. crassa mitochondrial (mt) large ribosomal subunit (LSU) intron, it appears to act primarily by stabilizing the structure of another region that interacts directly with the intron RNA. The H1/H2 region is required for splicing activity and TyrRS activity with the N. crassa mt tRNA(Tyr), but not for TyrRS activity with Escherichia coli tRNA(Tyr), implying a somewhat different mode of recognition of the two tyrosyl-tRNAs. Finally, a CYT-18 mutant lacking the N-terminal H0 region is totally defective in binding or splicing the N. crassa ND1 intron, but retains substantial residual activity with the mt LSU intron, and conversely, a CYT-18 mutant lacking the C-terminal RNA-binding domain is totally defective in binding or splicing the mt LSU intron, but retains substantial residual activity with the ND1 intron. These findings lead to the surprising conclusion that CYT-18 promotes splicing via different sets of interactions with different group I introns. We suggest that these different modes of promoting splicing evolved from an initial interaction based on the recognition of conserved tRNA-like structural features of the group I intron catalytic core. %B J Mol Biol %V 307 %P 75-92 %8 2001 Mar 16 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/11243805?dopt=Abstract %R 10.1006/jmbi.2000.4460 %0 Journal Article %J Nat Biotechnol %D 2001 %T Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. %A Karberg, M %A Guo, H %A Zhong, J %A Coon, R %A Perutka, J %A Lambowitz, A M %K Base Sequence %K Blotting, Southern %K Chromosomes %K Codon, Terminator %K Escherichia coli %K Gene Library %K Gene Transfer Techniques %K Genes, Bacterial %K Genetic Engineering %K Genetic Vectors %K Introns %K Molecular Sequence Data %K Plasmids %K Point Mutation %K Polymerase Chain Reaction %K Recombination, Genetic %X Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria. %B Nat Biotechnol %V 19 %P 1162-7 %8 2001 Dec %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/11731786?dopt=Abstract %R 10.1038/nbt1201-1162 %0 Journal Article %J J Mol Biol %D 2001 %T Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference. %A Singh, N N %A Lambowitz, A M %K Adenine %K Base Pairing %K Base Sequence %K Binding Sites %K Deoxyribonuclease I %K Deoxyuridine %K DNA %K DNA Footprinting %K DNA Methylation %K DNA-Binding Proteins %K Endonucleases %K Exons %K Introns %K Kinetics %K Lactococcus lactis %K Models, Molecular %K Molecular Sequence Data %K Mutation %K Nucleic Acid Denaturation %K Potassium Permanganate %K Protein Structure, Tertiary %K Ribonucleoproteins %K RNA Splicing %K Substrate Specificity %X Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position -25 to +19 from the intron-insertion site on the top strand and from -28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5'-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions -12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3' exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3' exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site. %B J Mol Biol %V 309 %P 361-86 %8 2001 Jun 1 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/11371159?dopt=Abstract %R 10.1006/jmbi.2001.4658 %0 Journal Article %J J Mol Biol %D 2001 %T Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) with the group I intron P4-P6 domain. Thermodynamic analysis and the role of metal ions. %A Caprara, M G %A Myers, C A %A Lambowitz, A M %K Base Sequence %K Cations %K Diethyl Pyrocarbonate %K Entropy %K Ethylnitrosourea %K Introns %K Iodine %K Magnesium %K Metals %K Mitochondria %K Molecular Sequence Data %K Neurospora crassa %K Nucleic Acid Conformation %K Pliability %K Potassium Chloride %K Protein Binding %K RNA, Fungal %K RNA, Transfer, Tyr %K RNA-Binding Proteins %K Sulfuric Acid Esters %K Temperature %K Tyrosine-tRNA Ligase %X The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron's catalytic core. Previous studies suggested a model in which the protein binds first to the intron's P4-P6 domain, and then makes additional contacts with the P3-P9 domain to stabilize the two domains in the correct relative orientation to form the intron's active site. Here, we analyzed the interaction of CYT-18 with a small RNA (P4-P6 RNA) corresponding to the isolated P4-P6 domain of the N. crassa mitochondrial large subunit ribosomal RNA intron. RNA footprinting and modification-interference experiments showed that CYT-18 binds to this small RNA around the junction of the P4-P6 stacked helices on the side opposite the active-site cleft, as it does to the P4-P6 domain in the intact intron. The binding is inhibited by chemical modifications that disrupt base-pairing in P4, P6, and P6a, indicating that a partially folded structure of the P4-P6 domain is required. The temperature-dependence of binding indicates that the interaction is driven by a favorable enthalpy change, but is accompanied by an unfavorable entropy change. The latter may reflect entropically unfavorable conformational changes or decreased conformational flexibility in the complex. CYT-18 binding is inhibited at > or =125 mM KCl, indicating a strong dependence on phosphodiester-backbone interactions. On the other hand, Mg(2+) is absolutely required for CYT-18 binding, with titration experiments showing approximately 1.5 magnesium ions bound per complex. Metal ion-cleavage experiments identified a divalent cation-binding site near the boundary of P6 and J6/6a, and chemical modification showed that Mg(2+) binding induces RNA conformational changes in this region, as well as elsewhere, particularly in J4/5. Together, these findings suggest a model in which the binding of Mg(2+) near J6/6a and possibly at one additional location in the P4-P6 RNA induces formation of a specific phosphodiester-backbone geometry that is required for CYT-18 binding. The binding of CYT-18 may then establish the correct structure at the junction of the P4/P6 stacked helices for assembly of the P3-P9 domain. The interaction of CYT-18 with the P4-P6 domain appears similar to the TyrRS interaction with the D-/anticodon arm stacked helices of tRNA(Tyr). %B J Mol Biol %V 308 %P 165-90 %8 2001 Apr 27 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/11327760?dopt=Abstract %R 10.1006/jmbi.2001.4581 %0 Journal Article %J EMBO J %D 2001 %T Mechanism of maturase-promoted group II intron splicing. %A Matsuura, M %A Noah, J W %A Lambowitz, A M %K Aldehydes %K Base Sequence %K Introns %K Iodine %K Molecular Sequence Data %K Nucleic Acid Conformation %K RNA %K RNA Splicing %K RNA-Directed DNA Polymerase %K Saccharomyces cerevisiae Proteins %X Mobile group II introns encode reverse transcriptases that also function as intron-specific splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high affinity binding site at the beginning of its own coding region in an idiosyncratic structure, DIVa. Here, we identify potential secondary binding sites in conserved regions of the catalytic core and show via chemical modification experiments that binding of the maturase induces the formation of key tertiary interactions required for RNA splicing. The interaction with conserved as well as idiosyncratic regions explains how maturases in some organisms could evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns. %B EMBO J %V 20 %P 7259-70 %8 2001 Dec 17 %G eng %N 24 %1 http://www.ncbi.nlm.nih.gov/pubmed/11743002?dopt=Abstract %R 10.1093/emboj/20.24.7259 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2001 %T Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites. %A Dickson, L %A Huang, H R %A Liu, L %A Matsuura, M %A Lambowitz, A M %A Perlman, P S %K Base Sequence %K DNA Primers %K DNA, Fungal %K Introns %K Mutation %K Polymerase Chain Reaction %K Retroelements %K RNA, Fungal %K Saccharomyces cerevisiae %X Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes. %B Proc Natl Acad Sci U S A %V 98 %P 13207-12 %8 2001 Nov 6 %G eng %N 23 %1 http://www.ncbi.nlm.nih.gov/pubmed/11687644?dopt=Abstract %R 10.1073/pnas.231494498 %0 Journal Article %J Nucleic Acids Res %D 2000 %T Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid. %A Mohr, S %A Wanner, L A %A Bertrand, H %A Lambowitz, A M %K Base Sequence %K Blotting, Southern %K Cloning, Molecular %K Deoxyribonuclease EcoRI %K DNA Primers %K DNA, Fungal %K DNA, Mitochondrial %K Genetic Variation %K Introns %K Mitochondria %K Molecular Sequence Data %K Mutagenesis, Insertional %K Neurospora %K Neurospora crassa %K Nucleic Acid Conformation %K Physical Chromosome Mapping %K Plasmids %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Fungal %K RNA, Transfer, Trp %K Transcription, Genetic %X We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64) and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74 A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich palindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like mtRNA. Our results suggest that mt tRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells. %B Nucleic Acids Res %V 28 %P 1514-24 %8 2000 Apr 1 %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/10710417?dopt=Abstract %0 Journal Article %J Nucleic Acids Res %D 2000 %T Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid. %A Mohr, S %A Wanner, L A %A Bertrand, H %A Lambowitz, A M %K Base Sequence %K Blotting, Southern %K Cloning, Molecular %K Deoxyribonuclease EcoRI %K DNA, Fungal %K DNA, Mitochondrial %K Genetic Variation %K Introns %K Mitochondria %K Molecular Sequence Data %K Mutagenesis, Insertional %K Neurospora %K Neurospora crassa %K Nucleic Acid Conformation %K Physical Chromosome Mapping %K Plasmids %K Reverse Transcriptase Polymerase Chain Reaction %K RNA Processing, Post-Transcriptional %K RNA, Fungal %K RNA, Transfer, Trp %K Transcription, Genetic %X We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64)and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich pallindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like tmRNA. Our results suggest that mtRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells. %B Nucleic Acids Res %V 28 %P 1514-24 %8 2000 Jul 1 %G eng %N 13 %1 http://www.ncbi.nlm.nih.gov/pubmed/11001704?dopt=Abstract %0 Journal Article %J Genes Dev %D 2000 %T Rules for DNA target-site recognition by a lactococcal group II intron enable retargeting of the intron to specific DNA sequences. %A Mohr, G %A Smith, D %A Belfort, M %A Lambowitz, A M %K Base Pairing %K Base Sequence %K DNA, Antisense %K DNA, Bacterial %K Escherichia coli %K Exons %K Introns %K Lactococcus lactis %K Models, Genetic %K Molecular Sequence Data %K Nucleic Acid Conformation %K Polymerase Chain Reaction %K Saccharomyces cerevisiae %K Sequence Analysis, DNA %K Templates, Genetic %X Group II intron homing occurs primarily by a mechanism in which the intron RNA reverse splices into a DNA target site and is then reverse transcribed by the intron-encoded protein. The DNA target site is recognized by an RNP complex containing the intron-encoded protein and the excised intron RNA. Here, we analyzed DNA target-site requirements for the Lactococcus lactis Ll.LtrB group II intron in vitro and in vivo. Our results suggest a model similar to yeast mtDNA introns, in which the intron-encoded protein first recognizes a small number of nucleotide residues in double-stranded DNA and causes DNA unwinding, enabling the intron RNA to base-pair with the DNA for reverse splicing. Antisense-strand cleavage requires additional interactions between the protein and 3' exon. Key nucleotide residues are recognized directly by the intron-encoded protein independent of sequence context, and there is a stringent requirement for fixed spacing between target site elements recognized by the protein and RNA components of the endonuclease. Experiments with DNA substrates containing GC-clamps or "bubbles" indicate a requirement for DNA unwinding in the 3' exon but not the distal 5' exon region. Finally, by applying the target-site recognition rules, we show that the L1.LtrB intron can be modified to insert at new sites in a plasmid-borne thyA gene in Escherichia coli. This strategy should be generally applicable to retargeting group II introns and to delivering foreign sequences to specific sites in heterologous genomes. %B Genes Dev %V 14 %P 559-73 %8 2000 Mar 1 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/10716944?dopt=Abstract %0 Journal Article %J J Mol Biol %D 2000 %T Function of tyrosyl-tRNA synthetase in splicing group I introns: an induced-fit model for binding to the P4-P6 domain based on analysis of mutations at the junction of the P4-P6 stacked helices. %A Chen, X %A Gutell, R R %A Lambowitz, A M %K Bacteriophage T4 %K Catalytic Domain %K Edetic Acid %K Escherichia coli %K Introns %K Models, Biological %K Mutation %K Neurospora crassa %K Nucleic Acid Conformation %K Protein Denaturation %K Protein Structure, Secondary %K Protein Structure, Tertiary %K RNA Splicing %K Suppression, Genetic %K Tyrosine-tRNA Ligase %X We used an Escherichia coli genetic assay based on the phage T4 td intron to test the ability of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) to suppress mutations that cause structural defects around its binding site in the P4-P6 domain of the group I intron catalytic core. We analyzed all possible combinations of nucleotides at either P4 bp-1 or P6 bp-1, which together form the junction of the P4-P6 stacked helices, and looked for synergistic effects in double mutants. Most mutations at either position inhibit self-splicing, but can be suppressed by CYT-18. CYT-18 can compensate efficiently for mutations that disrupt base-pairing at either P4 bp-1 or P6 bp-1, for mutations at P6 bp-1 that disrupt the base-triple interaction with J3/4-3, and for nucleotide substitutions at either position that are predicted to be suboptimal for base stacking, based on the analysis of DNA four-way junctions. However, CYT-18 has difficulty suppressing combinations of mutations at P4 bp-1 and P6 bp-1 that simultaneously disrupt base-pairing and base stacking. Thermal denaturation and Fe(II)-EDTA analysis showed that mutations at the junction of the P4-P6 stacked helices lead to grossly impaired tertiary-structure formation centered in the P4-P6 domain. CYT-18-suppressible mutants bind the protein with K(d) values up to 79-fold higher than that for the wild-type intron, but in all cases tested, the k(off) value for the complex remains within twofold of the wild-type value, suggesting that the binding site can be formed properly and that the increased K(d) value reflects primarily an increased k(on) value for the binding of CYT-18 to the misfolded intron. Our results indicate that the P4/P6 junction is a linchpin region, where even small nucleotide substitutions grossly disrupt the catalytically-active group I intron tertiary structure, and that CYT-18 binding induces the formation of the correct structure in this region, leading to folding of the group I intron catalytic core. %B J Mol Biol %V 301 %P 265-83 %8 2000 Aug 11 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/10926509?dopt=Abstract %R 10.1006/jmbi.2000.3963 %0 Journal Article %J Science %D 2000 %T Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. %A Guo, H %A Karberg, M %A Long, M %A Jones, J P %A Sullenger, B %A Lambowitz, A M %K Base Pairing %K Base Sequence %K Cell Line %K DNA %K DNA, Viral %K Escherichia coli %K Gene Targeting %K Genes, pol %K Genetic Therapy %K HIV-1 %K Humans %K Introns %K Lactococcus lactis %K Molecular Sequence Data %K Proviruses %K Receptors, CCR5 %K Recombination, Genetic %K RNA, Catalytic %K Transfection %X Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy. %B Science %V 289 %P 452-7 %8 2000 Jul 21 %G eng %N 5478 %1 http://www.ncbi.nlm.nih.gov/pubmed/10903206?dopt=Abstract %0 Journal Article %J Mol Cell Biol %D 2000 %T Multiple homing pathways used by yeast mitochondrial group II introns. %A Eskes, R %A Liu, L %A Ma, H %A Chao, M Y %A Dickson, L %A Lambowitz, A M %A Perlman, P S %K Base Sequence %K Crosses, Genetic %K DNA Repair %K DNA, Complementary %K Endonucleases %K Exons %K Introns %K Mitochondria %K Molecular Sequence Data %K Mutation %K Recombination, Genetic %K Retroelements %K RNA-Directed DNA Polymerase %K Yeasts %X The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells. %B Mol Cell Biol %V 20 %P 8432-46 %8 2000 Nov %G eng %N 22 %1 http://www.ncbi.nlm.nih.gov/pubmed/11046140?dopt=Abstract %0 Journal Article %J J Mol Biol %D 1999 %T Group II intron reverse transcriptase in yeast mitochondria. Stabilization and regulation of reverse transcriptase activity by the intron RNA. %A Zimmerly, S %A Moran, J V %A Perlman, P S %A Lambowitz, A M %K Amino Acid Sequence %K Conserved Sequence %K DNA Primers %K DNA, Complementary %K Exons %K Introns %K Mitochondria %K Mutation %K Ribonucleoproteins %K RNA Precursors %K RNA Splicing %K RNA, Fungal %K RNA-Directed DNA Polymerase %K Templates, Genetic %K Transcription, Genetic %K Yeasts %X Group II introns encode reverse transcriptases that function in both intron mobility and RNA splicing. The proteins bind specifically to unspliced precursor RNA to promote splicing, and then remain associated with the excised intron to form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription. Here, immunoblotting and UV cross-linking experiments show that the reverse transcriptase activity encoded by the yeast mtDNA group II intron aI2 is associated with an intron-encoded protein of 62 kDa (p62). p62 is bound tightly to endogenous RNAs in mitochondrial ribonucleoprotein particles, and the reverse transcriptase activity is rapidly and irreversibly lost when the protein is released from the endogenous RNAs by RNase digestion. Non-denaturing gel electrophoresis and activity assays show that the aI2 reverse transcriptase is associated predominantly with the excised intron RNA, while a smaller amount is associated with unspliced precursor RNA, as expected from the role of the protein in RNA splicing. Although the reverse transcriptase in wild-type yeast strains is bound tightly to endogenous RNAs, it is regulated so that it does not copy these RNAs unless a suitable DNA oligonucleotide primer or DNA target site is provided. Certain mutations in the intron-encoded protein or RNA circumvent this regulation and activate reverse transcription of endogenous RNAs in the absence of added primer. Although p62 is bound to unspliced precursor RNA in position to initiate cDNA synthesis in the 3' exon, the major template for target DNA-primed reverse transcription in vitro is the reverse-spliced intron RNA, as found previously for aI1. Together, our results show that binding to intron-containing RNAs stabilizes and regulates the activity of p62. %B J Mol Biol %V 289 %P 473-90 %8 1999 Jun 11 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/10356323?dopt=Abstract %R 10.1006/jmbi.1999.2778 %0 Journal Article %J Manag Care Q %D 1999 %T How to implement a case-mix system in less than a year. %A Lambowitz, S %K Certification %K Diagnosis-Related Groups %K Health Plan Implementation %K Humans %K Long-Term Care %K Managed Care Programs %K Medicaid %K Nursing Homes %K Ohio %K Prospective Payment System %K State Health Plans %K United States %B Manag Care Q %V 7 %P 64-9 %8 1999 Winter %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/10350800?dopt=Abstract %0 Journal Article %J Mol Cell %D 1999 %T A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA. %A Wank, H %A SanFilippo, J %A Singh, R N %A Matsuura, M %A Lambowitz, A M %K Bacterial Proteins %K Base Sequence %K Binding Sites %K Cloning, Molecular %K Codon, Initiator %K DNA Transposable Elements %K Escherichia coli %K Exons %K Introns %K Kinetics %K Lactococcus lactis %K Models, Molecular %K Molecular Sequence Data %K Nucleic Acid Conformation %K Open Reading Frames %K Recombinant Proteins %K RNA Splicing %K RNA-Directed DNA Polymerase %K Saccharomyces cerevisiae Proteins %K Transcription, Genetic %X Group II introns encode reverse transcriptases that promote RNA splicing (maturase activity) and then with the excised intron form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription (TPRT). Here, we show that the primary binding site for the maturase (LtrA) encoded by the Lactococcus lactis Ll.LtrB intron is within a region of intron domain IV that includes the start codon of the LtrA ORF. This binding is enhanced by other elements, particularly domain I and the EBS/IBS interactions, and helps position LtrA to initiate cDNA synthesis in the 3' exon as occurs during TPRT. Our results suggest how the maturase functions in RNA splicing and support the hypothesis that the reverse transcriptase coding region was derived from an independent genetic element that was inserted into a preexisting group II intron. %B Mol Cell %V 4 %P 239-50 %8 1999 Aug %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/10488339?dopt=Abstract %0 Journal Article %J Biochemistry %D 1999 %T RNA and protein catalysis in group II intron splicing and mobility reactions using purified components. %A Saldanha, R %A Chen, B %A Wank, H %A Matsuura, M %A Edwards, J %A Lambowitz, A M %K Bacterial Proteins %K Catalysis %K Deoxyribonucleases, Type II Site-Specific %K DNA Transposable Elements %K Enzyme Stability %K Escherichia coli %K Hydrogen-Ion Concentration %K Introns %K Lactococcus lactis %K Nucleic Acid Conformation %K Recombinant Proteins %K Ribonucleoproteins %K RNA Splicing %K RNA, Bacterial %K RNA-Binding Proteins %K RNA-Directed DNA Polymerase %K Sodium Chloride %K Substrate Specificity %X Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of