Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found that Marinomonas mediterranea RT-Cas1/Cas2 adds short 3'-DNA (dN) tails to RNA protospacers, enabling their direct integration into CRISPR arrays as 3'-dN-RNAs or 3'-dN-RNA/cDNA duplexes at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers is initiated at 3' proximal sites by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of near full-length cDNAs of diverse RNAs without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded DNAs (ssDNAs) is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage defense nucleases. Our findings reveal mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

Background Understanding the mechanisms of resistance to CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is pivotal in exploring new therapeutic strategies for hormone receptor-positive (HR+), HER2-negative metastatic breast cancer. To decipher these resistance mechanisms, we analyzed the alterations in comprehensive RNA-seq (coding and non-coding RNAs) of HR+ HER2-negative BC cell lines via thermostable group II intron reverse transcriptase sequencing (TGIRT-seq). Methods We established Tamoxifen-resistant (TMR), Abemaciclib-resistant (ACR), Palbociclib-resistant (PCR), Tamoxifen/Abemaciclib double-resistant (TMR-ACR), and Tamoxifen/Palbociclib double-resistant (TMR-PCR) BC cell lines from MCF7 and T47D HR+/HER2- BC cell lines through stepwise dose-escalation continuous drug exposure. We performed a TGIRT-seq transcriptomic analysis using a protocol that allows the …

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here we identify C12orf29 as an atypical ATP-grasp enzyme that ligates RNA. Human C12orf29 and its homologs auto-adenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5’-phosphate and a 3’-hydroxyl. C12orf29 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Genetic depletion of c12orf29 in female mice alters global tRNA levels in brain. Furthermore, crystal structures of a C12orf29 homolog from Yasminevirus bound to nucleotides reveal a minimal and atypical RNA ligase fold with a unique active site architecture that participates in catalysis. Collectively, our results identify C12orf29 as an RNA ligase and suggest its involvement in tRNA biology.

Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 tRNA^His 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 tRNA^His. Sequence analysis revealed that miR-4454 is identical to the 3’ end of tRNA^His. Moreover, we were able to generate this ‘miRNA’ in vitro through incubation of mature tRNA^His with Dicer. As found previously for several pre-miRNAs, a 5’P-tRNA^His appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNA^His. Moreover, tRNA^His 3’-fragment/‘miR-4454’ levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNA^His 3’-fragment processing without negatively affecting tRNA^His’s canonical function of aminoacylation.

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.

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.