HOME /

Publications

2013
Truong DM, Sidote DJ, Russell R, Lambowitz AM. Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core. Proc Natl Acad Sci U S A [Internet]. 110 (40) :E3800-9. Publisher's VersionAbstract

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.

Enyeart PJ, Chirieleison SM, Dao MN, Perutka J, Quandt EM, Yao J, Whitt JT, Keatinge-Clay AT, Lambowitz AM, Ellington AD. Generalized bacterial genome editing using mobile group II introns and Cre-lox. Mol Syst Biol [Internet]. 9 :685. Publisher's VersionAbstract

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%.

Yao J, Truong DM, Lambowitz AM. Genetic and biochemical assays reveal a key role for replication restart proteins in group II intron retrohoming. PLoS Genet [Internet]. 9 (4) :e1003469. Publisher's VersionAbstract

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.

Saldanha RJ, Pemberton A, Shiflett P, Perutka J, Whitt JT, Ellington A, Lambowitz AM, Kramer R, Taylor D, Lamkin TJ. Rapid targeted gene disruption in Bacillus anthracis. BMC Biotechnol [Internet]. 13 :72. Publisher's VersionAbstract

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.

Mohr G, Hong W, Zhang J, Cui G-Z, Yang Y, Cui Q, Liu Y-J, Lambowitz AM. A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum. PLoS One [Internet]. 8 (7) :e69032. Publisher's VersionAbstract

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.

Mohr S, Ghanem E, Smith W, Sheeter D, Qin Y, King O, Polioudakis D, Iyer VR, Hunicke-Smith S, Swamy S, et al. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA [Internet]. 19 (7) :958-70. Publisher's VersionAbstract

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.

Russell R, Jarmoskaite I, Lambowitz AM. Toward a molecular understanding of RNA remodeling by DEAD-box proteins. RNA Biol [Internet]. 10 (1) :44-55. Publisher's VersionAbstract

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.

2012
White TB, Lambowitz AM. The retrohoming of linear group II intron RNAs in Drosophila melanogaster occurs by both DNA ligase 4-dependent and -independent mechanisms. PLoS Genet. 8 (2) :e1002534.Abstract
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.
Mallam AL, Del Campo M, Gilman B, Sidote DJ, Lambowitz AM. Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p. Nature. 490 (7418) :121-5.Abstract
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.
2011
Potratz JP, Del Campo M, Wolf RZ, Lambowitz AM, Russell R. ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo. J Mol Biol. 411 (3) :661-79.Abstract
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.
Lambowitz AM, Zimmerly S. Group II introns: mobile ribozymes that invade DNA. Cold Spring Harb Perspect Biol. 3 (8) :a003616.Abstract
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.
Mohr G, Del Campo M, Turner KG, Gilman B, Wolf RZ, Lambowitz AM. High-throughput genetic identification of functionally important regions of the yeast DEAD-box protein Mss116p. J Mol Biol. 413 (5) :952-72.Abstract
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.
Paukstelis PJ, Chari N, Lambowitz AM, Hoffman D. NMR Structure of the C-terminal domain of a tyrosyl-tRNA synthetase that functions in group I intron splicing. Biochemistry. 50 (18) :3816-26.Abstract
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.
Mallam AL, Jarmoskaite I, Tijerina P, Del Campo M, Seifert S, Guo L, Russell R, Lambowitz AM. Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail. Proc Natl Acad Sci U S A. 108 (30) :12254-9.Abstract
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.
2010
Gu S-Q, Cui X, Mou S, Mohr S, Yao J, Lambowitz AM. Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase. RNA. 16 (4) :732-47.Abstract
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.
Mohr G, Ghanem E, Lambowitz AM. Mechanisms used for genomic proliferation by thermophilic group II introns. PLoS Biol. 8 (6) :e1000391.Abstract
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.
2009
Del Campo M, Lambowitz AM. Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP. Acta Crystallogr Sect F Struct Biol Cryst Commun. 65 (Pt 8) :832-5.Abstract
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.
Zhuang F, Karberg M, Perutka J, Lambowitz AM. EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting. RNA. 15 (3) :432-49.Abstract
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.
Markov DA, Savkina M, Anikin M, Del Campo M, Ecker K, Lambowitz AM, De Gnore JP, McAllister WT. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification. Yeast. 26 (8) :423-40.Abstract
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.
Zhuang F, Mastroianni M, White TB, Lambowitz AM. Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation. Proc Natl Acad Sci U S A. 106 (43) :18189-94.Abstract
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.

Pages