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The RmInt1 group II intron is an efficient self-splicing mobile retroelement that catalyzes its own excision as lariat, linear and circular molecules. In vivo , the RmInt1 lariat and the reverse transcriptase (IEP) it encodes form a ribonucleoprotein particle (RNP) that recognizes the DNA target for site-specific full intron insertion via a two-step reverse splicing reaction. RNPs containing linear group II intron RNA are generally thought to be unable to complete the reverse splicing reaction. Here, we show that reconstituted in vitro RNPs containing linear RmInt1 ΔORF RNA can mediate the cleavage of single-stranded DNA substrates in a very precise manner with the attachment of the intron RNA to the 3´exon as the first step of a reverse splicing reaction. Notably, we also observe molecules in which the 5´exon is linked to the RmInt1 RNA, suggesting the completion of the reverse splicing reaction, albeit rather low and inefficiently. That process depends on DNA target recognition and can be successful completed by RmInt1 RNPs with linear RNA displaying 5´ modifications.

Group II introns are large catalytic RNAs and mobile retroelements that encode a reverse transcriptase. Here, we provide methods for their identification in bacterial genomes and further analysis of their splicing and mobility capacities.

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

Group II introns are self-splicing catalytic RNAs that probably originated in bacteria and act as mobile retroelements. The dispersal and dynamics of group II intron spread within a bacterial genome are thought to follow a selection-driven extinction model. Likewise, various studies on the evolution of group II introns have suggested that they are evolving toward an inactive form by fragmentation, with the loss of the intron 3'-terminus, but with some intron fragments remaining and continuing to evolve in the genome. RmInt1 is a mobile group II intron that is widespread in natural populations of Sinorhizobium meliloti, but some strains of this species have no RmInt1 introns. We studied the splicing ability and mobility of the three full-length RmInt1 copies harbored by S. meliloti 1021, and obtained evidence suggesting that specific mutations may lead to the impairment of intron splicing and retrohoming. Our data suggest that the RmInt1 copies in this strain are undergoing a process of inactivation.

Group II introns are catalytic RNAs that are excised from their precursors in a protein-dependent manner in vivo. Certain group II introns can also react in a protein-independent manner under nonphysiological conditions in vitro. The efficiency and fidelity of the splicing reaction is crucial, to guarantee the correct formation and expression of the protein-coding mRNA. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. The RmInt1 intron self-splices in vitro, but this reaction generates side products due to a predicted cryptic IBS1* sequence within the 3' exon. We engineered an RmInt1 intron lacking the cryptic IBS1* sequence, which improved the fidelity of the splicing reaction. However, atypical circular forms of similar electrophoretic mobility to the lariat intron were nevertheless observed. We analyzed a run of four cytidine residues at the 3' splice site potentially responsible for a lack of fidelity at this site leading to the formation of circular intron forms. We showed that mutations of residues base-pairing in the tertiary EBS3-IBS3 interaction increased the efficiency and fidelity of the splicing reaction. Our results indicate that RmInt1 has developed strategies for decreasing its splicing efficiency and fidelity. RmInt1 makes use of unproductive splicing reactions to limit the transposition of the insertion sequence into which it inserts itself in its natural context, thereby preventing potentially harmful dispersion of ISRm2011-2 throughout the genome of its host., (© 2014 Chillón et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Group II introns are self-splicing RNAs and site-specific mobile retroelements found in bacterial and organellar genomes. The group II intron RmInt1 is present at high copy number in Sinorhizobium meliloti species, and has a multifunctional intron-encoded protein (IEP) with reverse transcriptase/maturase activities, but lacking the DNA-binding and endonuclease domains. We characterized two RmInt1-related group II introns RmInt2 from S. meliloti strain GR4 and Sr.md.I1 from S. medicae strain WSM419 in terms of splicing and mobility activities. We used both wild-type and engineered intron-donor constructs based on ribozyme ΔORF-coding sequence derivatives, and we determined the DNA target requirements for RmInt2, the element most distantly related to RmInt1. The excision and mobility patterns of intron-donor constructs expressing different combinations of IEP and intron RNA provided experimental evidence for the co-operation of IEPs and intron RNAs from related elements in intron splicing and, in some cases, in intron homing. We were also able to identify the DNA target regions recognized by these IEPs lacking the DNA endonuclease domain. Our results provide new insight into the versatility of related group II introns and the possible co-operation between these elements to facilitate the colonization of bacterial genomes.

Gene-targeting vectors derived from mobile group II introns capable of forming a ribonucleoprotein (RNP) complex containing excised intron lariat RNA and an intron-encoded protein (IEP) with reverse transcriptase (RT), maturase, and endonuclease (En) activities have been described. RmInt1 is an efficient mobile group II intron with an IEP lacking the En domain. We performed a comprehensive study of the rules governing RmInt1 target site recognition based on selection experiments with donor and recipient plasmid libraries, with randomization of the elements of the intron RNA involved in target recognition and the wild-type target site. The data obtained were used to develop a computer algorithm for identifying potential RmInt1 targets in any DNA sequence. Using this algorithm, we modified RmInt1 for the efficient recognition of DNA target sites at different locations in the Sinorhizobium meliloti chromosome. The retargeted RmInt1 integrated efficiently into the chromosome, regardless of the location of the target gene. Our results suggest that RmInt1 could be efficiently adapted for gene targeting.

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

Group II introns are self-splicing RNAs that act as mobile retroelements in the organelles of plants, fungi and protists. They are also widely distributed in bacteria, and are generally assumed to be the ancestors of nuclear spliceosomal introns. Most bacterial group II introns have a multifunctional intron-encoded protein (IEP) ORF within the ribozyme domain IV (DIV). This ORF encodes an N-terminal reverse transcriptase (RT) domain, followed by a putative RNA-binding domain with RNA splicing or maturase activity and, in some cases, a C-terminal DNA-binding (D) region followed by a DNA endonuclease (En) domain. In this study, we focused on bacterial group II intron ORF phylogenetic classes containing only reverse transcriptase/maturase open reading frames, with no recognizable D/En region (classes A, C, D, E, F and unclassified introns). On the basis of phylogenetic analyses of the maturase domain and its C-terminal extension, which appears to be a signature characteristic of ORF phylogenetic class, with support from the phylogeny inferred from the RT domain, we have revised the proposed new class F, defining new intron ORF varieties. Our results increase knowledge of the lineage of group II introns encoding proteins lacking the En-domain.

Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products. We also found that a single substitution at the EBS3 position (G329C), preventing EBS3-IBS3 pairing, resulted in the production of 50 to 100 times more RNA molecules in which the 5' and 3' extremities were joined. We provide evidence that these intron molecules may correspond to both, intron circles linked by a 2'-5' phosphodiester bond, and tandem, head-to-tail intron copies.

Group II introns act as both large catalytic RNAs and mobile retroelements. They are found in organelle and bacterial genomes and are spliced via a lariat intermediate, in a mechanism similar to that of spliceosomal introns. However, their distribution and insertion patterns, particularly for bacterial group II introns, suggest that they function and behave more like retroelements than organelle introns. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. This group II intron is excised, in vivo and in vitro, as intron lariats. However, the complete splicing reaction in vivo remains to be elucidated. A lacZ reporter gene system, northern blotting and real-time reverse transcription were carried out to investigate RmInt1 splicing activity. Splicing efficiency of 0.07 ± 0.02% was recorded. These findings suggest that bacterial group II introns function more like retroelements than spliceosomal introns. Their location is consistent with a role for these introns in preventing the spread of other potentially harmful mobile elements in bacteria.

The group IIA intron Ll.LtrB from Lactococcus lactis and the group IIB intron EcI5 from Escherichia coli have intron-encoded proteins (IEP) with a DNA-binding domain (D) and an endonuclease domain (En). Both have been successfully retargeted to invade target DNAs other than their wild-type target sites. RmInt1, a subclass IIB3/D intron with an IEP lacking D and En domains, is highly active in retrohoming in its host, Sinorhizobium meliloti. We found that RmInt1 was also mobile in E. coli and that retrohoming in this heterologous host depended on temperature, being more efficient at 28°C than at 37°C. Furthermore, we programmed RmInt1 to recognize target sites other than its wild-type site. These retargeted introns efficiently and specifically retrohome into a recipient plasmid target site or a target site present as a single copy in the chromosome, generating a mutation in the targeted gene. Our results extend the range of group II introns available for gene targeting.

Group II introns are both catalytic RNAs (ribozymes) and mobile retroelements that were discovered almost 14 years ago. It has been suggested that eukaryotic mRNA introns might have originated from the group II introns present in the alphaproteobacterial progenitor of the mitochondria. Bacterial group II introns are of considerable interest not only because of their evolutionary significance, but also because they could potentially be used as tools for genetic manipulation in biotechnology and for gene therapy. This review summarizes what is known about the splicing mechanisms and mobility of bacterial group II introns, and describes the recent development of group II intron-based gene-targetting methods. Bacterial group II intron diversity, evolutionary relationships, and behaviour in bacteria are also discussed.

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase-maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (DeltaORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic DeltaORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature.

Excision of group II introns as circles has been described only for a few eukaryotic introns and little is known about the mechanisms involved, the relevance or consequences of the process. We report that splicing of the bacterial group II intron RmInt1 in vivo leads to the formation of both intron lariat and intron RNA circles. We determined that besides being required for the intron splicing reaction, the maturase domain of the intron-encoded protein also controls the balance between lariat and RNA intron circle production. Furthermore, comparison with in vitro self-splicing products indicates that in vivo, the intron-encoded protein appears to promote the use of a correct EBS1/IBS1 intron-exon interaction as well as cleavage at, or next to, the expected 3' splice site. These findings provide new insights on the mechanism of excision of group II introns as circles.

RmInt1 is a mobile group II intron which interrupts ISRm2011-2, another mobile element from the bacterium Sinorhizobium meliloti. Ribozyme constructs derived from intron RmInt1 self-splice in vitro when incubated under permissive conditions, but the excised intron and ligated exons are largely replaced by unconventional products. These include a slightly shorter, 5'-end truncated 3' exon, truncated variants of the linear and lariat forms of the intron-3' exon reaction intermediate, as well as presumably circular molecules derived from the latter. Two factors explain the abundance of these products: (i) nucleotides 5-11 of the 3' exon (IBS1*) provide a better match to the EBS1 5'-exon-binding site than the authentic IBS1 sequence in the 5' exon; (ii) exon ligation is unusually inefficient, and especially so when the 5' exon is truncated close to the second (IBS2) intron-binding site. We propose that reactions at the IBS1* site play a part in the regulation of the intron ISRm2011-2 host in vivo.

Sinorhizobium meliloti RmInt1 is an efficient mobile group II intron that uses an unknown reverse transcriptase priming mechanism as the intron ribonucleoprotein complex can reverse splice into DNA target substrates but cannot carry out site-specific second strand cleavage due to the lack of a C-terminal DNA endonuclease domain. We show here that, like other mobile group II introns, RmInt1 moves around by an efficient RNA-based retrohoming mechanism. We found evidence of two distinct RmInt1 retrohoming pathways for mobility depending on the orientation of the target site relative to the direction of DNA replication. The preferred retrohoming pathway is consistent with reverse splicing of the intron RNA into single-stranded DNA at a replication fork, using a nascent lagging DNA strand as the primer for reverse transcription. This strand bias is the opposite of that reported for mobility of the lactococcal Ll.ltrB intron in the absence of second strand cleavage. The mobility mechanism found here for RmInt1 may be used for dissemination by many bacterial group II introns encoding proteins lacking the DNA endonuclease domain.

The mobile group II introns characterized to date encode ribonucleoprotein complexes that promote mobility by a major retrohoming mechanism in which the intron RNA reverse splices directly into the sense strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase/maturase cleaves the antisense strand and uses it as primer for reverse transcription of the inserted intron RNA. Here, we show that the Sinorhizobium meliloti group II intron RmInt1, which encodes a protein lacking a DNA endonuclease domain, similarly uses both the intron RNA and an intron-encoded protein with reverse transcriptase and maturase activities for mobility. However, while RmInt1 reverse splices into both single-stranded and double-stranded DNA target sites, it is unable to carry out site-specific antisense-strand cleavage due to the lack of a DNA endonuclease domain. Our results suggest that RmInt1 mobility involves reverse splicing into double-stranded or single-stranded DNA target sites, but due to the lack of DNA endonuclease function, it requires an alternate means of procuring a primer for target DNA-primed reverse transcription.

Self-splicing group II introns are present in the organelles of lower eukaryotes, plants and Bacteria and have been found recently in Archaea. It is generally accepted that group II introns originated in bacteria before spreading to mitochondria and chloroplasts. These introns are thought to be related to the progenitors of spliceosomal introns. Group II introns are also mobile genetic elements. In bacteria, they appear to spread using either other mobile genetic elements or low-expression regions as target sites. Bacteria and Archaea genome sequence annotations have revealed the diversity of group II intron classes and that they are involved in vertical and horizontal inheritance.

Group II intron-encoded proteins (IEPs), which have maturase and reverse transcriptase activities, form a ribonucleoprotein (RNP) complex with the intron RNA. Some IEPs also have a C-terminal DNA-binding region and conserved DNA endonuclease domain involved in the recognition and cleavage of specific DNA target sites used for intron homing. RmInt1 is a mobile group II intron of Sinorhizobium meliloti, the IEP of which lacks the endonuclease domain, as do over half of their bacterial counterparts. Here, we analyzed the DNA target sequence requirements for homing in vivo of intron RmInt1 and compared these requirements to those established for the Lactococcus lactis Ll.LtrB intron, a representative of mobile subgroup IIA introns encoding proteins with functional C-terminal DNA endonuclease domains. As for Ll.LtrB, RmInt1 homing requires modifiable base-pairing interactions between the intron RNA and the DNA target, involving 13 nucleotides. However, instead of the delta-delta' interaction, typical of subgroup IIA introns, we demonstrate that RmInt1 recognizes the first nucleotide within the 3' exon of the target site by a new EBS3/IBS3 pairing predicted for subgroup IIB self-splicing introns. Unlike Ll.LtrB, there are less stringent requirements for RmInt1 recognition of distal 5' and 3' exon regions, where only single nucleotide positions are fixed constraints for intron homing. Our results predict differences in the DNA target-site requirements among group II introns, which may have mechanistic and evolutionary implications.

Group II introns are catalytic RNAs and mobile genetic elements. Phylogenetic characterization of group II intron-encoded reverse transcriptases (RTs) established seven classes: the mitochondrial class, chloroplast-like classes 1 and 2, and bacterial classes A, B, C, and D. In this study, we identified and characterized a new bacterial class of group II introns, bacterial class E, on the basis of phylogenetic analysis of the intron-encoded protein (IEP) RT and determination of a consensus intron RNA structure.

Reverse transcriptases (RTs) closely related to those encoded by group II introns but lacking the intron RNA structure have been found associated with type III clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, a prokaryotic immune system against invading viruses and foreign genetic elements. Two models have been proposed to explain the origin and evolutionary relationships of these RTs: (i) the "single point of origin" model, according to which these RTs originated from a single acquisition event in bacterial, with the various protein domains (RT, RT-Cas1, and Cas6-RT-Cas1 fusions) corresponding to single points in evolution; and (ii) the "various origins" model, according to which, independent acquisition events in different evolutionary episodes led to these fusions. We tested these alternative hypotheses, by analyzing and integrating published datasets of RT sequences associated with CRISPRCas systems and inferring phylogenetic trees by maximum likelihood (ML) methods. The RTs studied could be grouped into 13 clades, mostly in bacteria, in which they probably evolved. The various clades appear to form three independent lineages in bacteria and a recent lineage in archaea. Our data show that the Cas6 domain was acquired twice, independently, through RT-Cas1 fusion, in the bacterial lineages. Taken together, there more evidence to support the "various origins" hypothesis. [ABSTRACT FROM AUTHOR]

Bacterial group II introns are self-splicing catalytic RNAs and mobile retroelements that have an open reading frame encoding an intron-encoded protein (IEP) with reverse transcriptase (RT) and RNA splicing or maturase activity. Some IEPs carry a DNA endonuclease (En) domain, which is required to cleave the bottom strand downstream from the intron-insertion site for target DNA-primed reverse transcription (TPRT) of the inserted intron RNA. Host factors complete the insertion of the intron. By contrast, the major retrohoming pathway of introns with IEPs naturally lacking endonuclease activity, like the Sinorhizobium meliloti intron RmInt1, is thought to involve insertion of the intron RNA into the template for lagging strand DNA synthesis ahead of the replication fork, with possible use of the nascent strand to prime reverse transcription of the intron RNA. The host factors influencing the retrohoming pathway of such introns have not yet been described. Here, we identify key candidates likely to be involved in early and late steps of RmInt1 retrohoming. Some of these host factors are common to En+ group II intron retrohoming, but some have different functions. Our results also suggest that the retrohoming process of RmInt1 may be less dependent on the intracellular free Mg2+ concentration than those of other group II introns. [ABSTRACT FROM AUTHOR]

Background: Population genetic analyses based on genome-wide sequencing data have been carried out for Sinorhizobium medicae and S. meliloti, two closely related bacterial species forming nitrogen-fixing symbioses with plants of the genus Medicago. However, genome coverage was low or the isolates had a broad geographic distribution, making it difficult to interpret the estimated diversity and to unravel the early events underlying population genetic variations and ecological differentiation. Results: Here, to gain insight into the early genome level variation and diversification within S. meliloti populations, we first used Illumina paired-end reads technology to sequence a new clone of S. meliloti strain GR4, a highly competitive strain for alfalfa nodulation. The Illumina data and the GR4 genome sequence previously obtained with 454 technology were used to generate a high-quality reference genome sequence. We then used Illumina technology to sequence the genomes of 13 S. meliloti isolates representative of the genomic variation within the GR4-type population, obtained from a single field site with a high degree of coverage. The genome sequences obtained were analyzed to determine nucleotide diversity, divergence times, polymorphism and genomic variation. Similar low levels of nucleotide diversity were observed for the chromosome, pSymB and pSymA replicons. The isolates displayed other types of variation, such as indels, recombination events, genomic island excision and the transposition of mobile elements. Conclusions: Our results suggest that the GR4-type population has experienced a process of demographic expansion and behaves as a stable genotypic cluster of genome-wide similarity, with most of the genome following a clonal pattern of evolution. Although some of genetic variation detected within the GR4-type population is probably due to genetic drift, others might be important in diversification and environmental adaptation. [ABSTRACT FROM AUTHOR]

Background: Group II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP) assists the splicing of the intron in vivo and remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP) that promotes intron mobility. Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA)] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC))] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ') in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron. Results: In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo. Conclusions: The excision in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting. [ABSTRACT FROM AUTHOR]

Group II introns are both catalytic RNAs and mobile retroelements that move through a process catalyzed by a RNP complex consisting of an intron-encoded protein and the spliced intron lariat RNA. Group II intron-encoded proteins are multifunctional and contain an N-terminal reverse transcriptase domain, followed by a putative RNA-binding domain (domain X) associated with RNA splicing or maturase activity and a C-terminal DNA binding/DNA endonuclease region. The intron-encoded protein encoded by the mobile group II intron RmInt1, which lacks the DNA binding/DNA endonuclease region, has only a short C-terminal extension (C-tail) after a typical domain X, apparently unrelated to the C-terminal regions of other group II intron-encoded proteins. Multiple sequence alignments identified features of the C-terminal portion of the RmInt1 intron-encoded protein that are conserved throughout evolution in the bacterial ORF class D, suggesting a group-specific functionally important protein region. The functional importance of these features was demonstrated by analyses of deletions and mutations affecting conserved amino acid residues. We found that the C-tail of the RmInt1 intron-encoded protein contributes to the maturase function of this reverse transcriptase protein. Furthermore, within the C-terminal region, we identified, in a predicted α-helical region and downstream, conserved residues that are specifically required for the insertion of the intron into DNA targets in the orientation that would make it possible to use the nascent leading strand as a primer. These findings suggest that these group II intron intron-encoded proteins may have adapted to function in mobility by different mechanisms to make use of either leading or lagging-oriented targets in the absence of an endonuclease domain. [ABSTRACT FROM AUTHOR]

Self-splicing group II introns are thought to be the evolutionary progenitors of eukaryotic spliceosomal introns. The invasion of novel (ectopic) sites by group II introns is considered to be a key mechanism by which spliceosomal introns may have become widely dispersed. However, the dynamics of these events in populations are unknown. In bacteria, only two group II introns have been shown to splice and to be mobile in vivo. One of these introns, RmInt1 from Sinorhizobium meliloti, which encodes a protein with no endonuclease domain, has been shown to invade the ectopic oxi1 site independently of recombinase. In this study, we analysed ectopic transposition of the RmInt1 intron in a natural population of S. meliloti. We characterized S. meliloti isolates by polymerase chain reaction amplification of a gene, dapB, which is found only on the pRmeGR4b plasmid diagnostic of GR4-type strains. The diversity within this specific field population of bacteria was analysed by restriction fragment length polymorphism using ISRm2011-2 (homing site of RmInt1) and RmInt1 as probes. We found that ectopic transposition of RmInt1 to the oxi1 site occurred in this natural bacterial population. This ectopic transposition was also the most frequent genetic event observed. This work provides further evidence that the ectopic transposition of group II introns is an important mechanism for their spread in natural bacterial populations. [ABSTRACT FROM AUTHOR]

Group II introns are large catalytic RNA molecules that act as mobile genetic elements. They were initially identified in the organelle genomes of lower eukaryotes and plants, and it has been suggested that they are the progenitors of nuclear spliceosomal introns. Group II self-splicing introns were shown to be present in bacteria in 1993, since when the various bacterial genome sequencing projects have led to a significant increase in the number of group II intron sequences present in databases. However, few of these introns have been characterized, and most were identified on the basis of their intron-encoded protein (IEP), with little data available concerning their ribozyme/RNA structure. Their frequency in prokaryotes is also unknown. We attempt here to provide a first comprehensive review of bacterial group II introns based on recent genome sequencing data and mechanistic studies. [ABSTRACT FROM AUTHOR]

RmInt1 is a functional group II intron found in Sinorhizobium meliloti where it interrupts a group of IS elements of the IS630-Tc1 family. In contrast to many other group II introns, the intron-encoded protein (IEP) of RmInt1 lacks the characteristic conserved part of the Zn domain associated with the IEP endonuclease activity. Nevertheless, in this study, we show that RmInt1 is capable of inserting into a vector containing the DNA spanning the RmInt1 target site from the genome of S. meliloti. Efficient homing was also observed in the absence of homologous recombination (RecA- strains). In addition, it is shown that RmInt1 is able to move to its target in a heterologous host (S. medicae). Homing of RmInt1 occurs very efficiently upon DNA target uptake (conjugation/electroporation) by the host cell resulting in a proportion of invaded target of 11–30%. Afterwards, the remaining intronless target DNA is protected from intron invasion. [ABSTRACT FROM AUTHOR]

By sequence analysis of Sinorhizobium meliloti strain GR4 plasmid pRmeGR4b, we have identified a group II intron named RmInt1 inserted within the insertion sequence ISRm2011-2 of the IS630 -Tc1/IS3 retroposon superfamily. Like some other group II introns, RmInt1 possesses, in addition to the structurally conserved ribozyme core, an open reading frame (ORF) with homology to reverse transcriptases. Using a T7 expression system in Escherichia coli , we show that the intron is active in splicing in vivo and that splicing efficiency requires the intron-encoded ORF, which suggests that the putative intron encoded protein has a maturase function. DNA hybridization studies indicate that intron RmInt1 is widespread within S. meliloti native populations and appears to be mostly located within this IS element. Nevertheless, some S. meliloti strains harbour one copy of RmInt1 at a different location. DNA sequence analysis of the 5′ exon of one of these heterologous intron insertion sites revealed the presence of a putative IS element closely related to insertion sequence ISRm2011-2 . The intron-binding sites (IBS1 and IBS2 motifs) are conserved, although a transition of a G→A in the IBS1 has occurred. Our results demonstrate an association of intron RmInt1 with particular insertion sequences of the IS630 -Tc1/IS3 retroposon superfamily that may have ensured the spread and maintenance of this group II intron in S. meliloti . [ABSTRACT FROM AUTHOR]

Group II introns are self-splicing catalytic RNAs that probably originated in bacteria and act as mobile retroelements. The dispersal and dynamics of group II intron spread within a bacterial genome are thought to follow a selection-driven extinction model. Likewise, various studies on the evolution of group II introns have suggested that they are evolving toward an inactive form by fragmentation, with the loss of the intron 3′-terminus, but with some intron fragments remaining and continuing to evolve in the genome. RmInt1 is a mobile group II intron that is widespread in natural populations of Sinorhizobium meliloti, but some strains of this species have no RmInt1 introns. We studied the splicing ability and mobility of the three full-length RmInt1 copies harbored by S. meliloti 1021, and obtained evidence suggesting that specific mutations may lead to the impairment of intron splicing and retrohoming. Our data suggest that the RmInt1 copies in this strain are undergoing a process of inactivation. [ABSTRACT FROM AUTHOR]