Group II intron-like reverse transcriptases function in double-strand break repair.

Bacteria encode reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. 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 expressed in Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase Î¸ and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ). We also found that a group II intron RT can function similarly in DNA repair, with reciprocal active-site substitutions showing isoleucine favors MMEJ and alanine favors primer extension in both enzymes. These DNA repair functions 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 inherent ability to function in DSBR in a wide range of organisms.

A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.

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.

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein.

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.

Mechanisms used for genomic proliferation by thermophilic group II introns.

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.

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein.

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 Marinomonas mediterranea RT-Cas1/Cas2 adds short 3'-DNA (dN) tails to RNA protospacers enabling their direct integration into CRISPR arrays as 3'-dN-RNA/cDNA duplexes or 3'-dN-RNAs at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers occurs 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 cDNAs from diverse RNAs without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded (ss) DNAs 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 novel mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

Mne1 is a novel component of the mitochondrial splicing apparatus responsible for processing of a COX1 group I intron in yeast.

Saccharomyces cerevisiae cells lacking Mne1 are deficient in intron splicing in the gene encoding the Cox1 subunit of cytochrome oxidase but contain wild-type levels of the bc(1) complex. Thus, Mne1 has no role in splicing of COB introns or expression of the COB gene. Northern experiments suggest that splicing of the COX1 aI5ß intron is dependent on Mne1 in addition to the previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing of the aI5ß intron is similarly impaired in mne1Δ and mrs1Δ cells and overexpression of Mrs1 partially restores the respiratory function of mne1Δ cells. Mrs1 is known to function in the initial transesterification reaction of splicing. Mne1 is a mitochondrial matrix protein loosely associated with the inner membrane and is found in a high mass ribonucleoprotein complex specifically associated with the COX1 mRNA even within an intronless strain. Mne1 does not appear to have a secondary function in COX1 processing or translation, because disruption of MNE1 in cells containing intronless mtDNA does not lead to a respiratory growth defect. Thus, the primary defect in mne1Δ cells is splicing of the aI5ß intron in COX1.

A Highly Proliferative Group IIC Intron from Geobacillus stearothermophilus Reveals New Features of Group II Intron Mobility and Splicing.

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.

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

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 CRISPR-associated 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.IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis, a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic.

Putative proteins related to group II intron reverse transcriptase/maturases are encoded by nuclear genes in higher plants.

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.

Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein.

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.

Recent mobility of plastid encoded group II introns and twintrons in five strains of the unicellular red alga Porphyridium.

Group II introns are closely linked to eukaryote evolution because nuclear spliceosomal introns and the small RNAs associated with the spliceosome are thought to trace their ancient origins to these mobile elements. Therefore, elucidating how group II introns move, and how they lose mobility can potentially shed light on fundamental aspects of eukaryote biology. To this end, we studied five strains of the unicellular red alga Porphyridium purpureum that surprisingly contain 42 group II introns in their plastid genomes. We focused on a subset of these introns that encode mobility-conferring intron-encoded proteins (IEPs) and found them to be distributed among the strains in a lineage-specific manner. The reverse transcriptase and maturase domains were present in all lineages but the DNA endonuclease domain was deleted in vertically inherited introns, demonstrating a key step in the loss of mobility. P. purpureum plastid intron RNAs had a classic group IIB secondary structure despite variability in the DIII and DVI domains. We report for the first time the presence of twintrons (introns-within-introns, derived from the same mobile element) in Rhodophyta. The P. purpureum IEPs and their mobile introns provide a valuable model for the study of mobile retroelements in eukaryotes and offer promise for biotechnological applications.

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

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.

The contribution of cellulosomal scaffoldins to cellulose hydrolysis by Clostridium thermocellum analyzed by using thermotargetrons.

BACKGROUND: 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. RESULTS: We constructed C. thermocellum mutants with the primary scaffoldin CipA (cellulosome-integrating protein A) truncated at different positions or lacking four different secondary scaffoldins by using a newly developed thermotargetron system, and we analyzed cellulose hydrolysis, cellulosome formation, and cellulose binding of the mutants. A CipA truncation that deletes six type I cohesin modules, which bind cellulolytic enzymes, decreased cellulose hydrolysis rates by 46%, and slightly longer truncations that also delete the carbohydrate binding module decreased rates by 89 to 92%, indicating strong cellulosome-substrate synergy. By contrast, a small CipA truncation that deletes only the C-terminal type II dockerin (XDocII) module detached cellulosomes from the cells, but decreased cellulose hydrolysis rates by only 9%, suggesting a relatively small contribution of cellulosome-cell synergy. Disruptants lacking any of four different secondary scaffoldins (OlpB, 7CohII, Orf2p, or SdbA) showed moderately decreased cellulose hydrolysis rates, suggesting additive contributions. Surprisingly, the CipA-ΔXDocII mutant, which lacks cell-associated polycellulosomes, adheres to cellulose almost as strongly as wild-type cells, revealing an alternate, previously unknown cellulose-binding mechanism. CONCLUSIONS: Our results emphasize the important role of cellulosome-substrate synergy in cellulose degradation, demonstrate a contribution of secondary scaffoldins, and suggest a previously unknown, non-cellulosomal system for binding insoluble cellulose. Our findings provide new insights into cellulosome function and impact genetic engineering of microorganisms to enhance bioconversions of cellulose substrates.

A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum.

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.

High-throughput genetic identification of functionally important regions of the yeast DEAD-box protein Mss116p.

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.

Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro.

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.

Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase.

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.

The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns.

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.