Use of RmInt1, a group IIB intron lacking the intron-encoded protein endonuclease domain, in gene targeting

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.     

Dispersal and evolution of the Sinorhizobium meliloti group II RmInt1 intron in bacteria that interact with plants

Group II introns are both self-splicing RNAs and mobile retroelements found in bacterial and archaeal genomes and in organelles of eukaryotes. They are thought to be the ancestors of eukaryote spliceosomal introns and non-long terminal repeat retrotransposons. We show here that RmInt1, a bacterial group II intron first described in the nitrogen-fixing symbiont of alfalfa (Medicago sativa) Sinorhizobium meliloti, is also present in other Sinorhizobium and Rhizobium species. The intron-homing sites in these species are IS elements of the ISRm2011-2 group as in S. meliloti, but ectopic insertion is also observed. We present evidence that these related bacteria have acquired RmInt1 by vertical inheritance from a common ancestor and by independent horizontal transfer events. We also show that RmInt1 is mobile in related taxa of bacteria that interact with plants and tends to evolve toward an inactive form by fragmentation, with loss of the 3' terminus including the intron-encoded protein. Our results provide an overview of the evolution and dispersion of a bacterial group II intron.     

Diversity of group II introns in the genome of <Emphasis Type="Italic"> Sinorhizobium meliloti </Emphasis>strain 1021: splicing and mobility of RmInt1

The number and diversity of known group II introns in eubacteria are continually increasing with the addition of new data from sequencing projects, but the significance of these introns in the evolution of bacterial genomes is unknown. We analyzed the main features of the group II introns present in the genome of the soil microorganism Sinorhizobium meliloti (strain 1021), the nitrogen-fixing symbiont of alfalfa, the DNA sequence of which was recently determined. Strain 1021 harbors three different classes of group II introns: RmInt1, of bacterial class D; SMb2147/SMb21167, which cluster within bacterial class C; and SMa1875, the phylogenetic class of which is uncertain. The group II introns SMb2147/SMb21167 and SMa1875 are widely distributed in S. meliloti, but are present in lower copy numbers than RmInt1. Strain 1021 harbors three copies of RmInt1, which is pSym-specific. Although RmInt1 is spliced in strain 1021, mobility assays suggested that, in contrast to other S. meliloti strains, the genetic background of strain 1021 does not support intron homing events.     

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

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.     

Ectopic transposition of a group II intron in natural bacterial populations

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.     

Insights into the history of a bacterial group II intron remnant from the genomes of the nitrogen-fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae

Group II introns are self-splicing catalytic RNAs that act as mobile retroelements. In bacteria, they are thought to be tolerated to some extent because they self-splice and home preferentially to sites outside of functional genes, generally within intergenic regions or in other mobile genetic elements, by mechanisms including the divergence of DNA target specificity to prevent target site saturation. RmInt1 is a mobile group II intron that is widespread in natural populations of Sinorhizobium meliloti and was first described in the GR4 strain. Like other bacterial group II introns, RmInt1 tends to evolve toward an inactive form by fragmentation, with loss of the 3′ terminus. We identified genomic evidence of a fragmented intron closely related to RmInt1 buried in the genome of the extant S. meliloti/S. medicae species. By studying this intron, we obtained evidence for the occurrence of intron insertion before the divergence of ancient rhizobial species. This fragmented group II intron has thus existed for a long time and has provided sequence variation, on which selection can act, contributing to diverse genetic rearrangements, and to generate pan-genome divergence after strain differentiation. The data presented here suggest that fragmented group II introns within intergenic regions closed to functionally important neighboring genes may have been microevolutionary forces driving adaptive evolution of these rhizobial species.     

RecA-independent ectopic transposition in vivo of a bacterial group II intron

RmInt1 is a group II intron of Sinorhizobium meliloti which was initially found within the insertion sequence ISRm2011-2. Although the RmInt1 intron-encoded protein lacks a recognizable endonuclease domain, it is able to mediate insertion of RmInt1 at an intron-specific location in intronless ISRm2011-2 recipient DNA, a phenomenon termed homing. Here we have characterized three additional insertion sites of RmInt1 in the genome of S.meliloti. Two of these sites are within IS elements closely related to ISRm2011-2, which appear to form a characteristic group within the IS630-Tc1 family. The third site is in the oxi1 gene, which encodes a putative oxide reductase. The newly identified integration sites contain conserved intron-binding site (IBS1 and IBS2) and δ′ sequences (14 bp). The RNA of the intron-containing oxi1 gene is able to splice and the oxi1 site is a DNA target for RmInt1 transposition in vivo. Ectopic transposition of RmInt1 into the oxi1 gene occurs at 20-fold lower efficiency than into the homing site (ISRm2011-2) and is independent of the major RecA recombination pathway. The possibility that transposition of RmInt1 to the oxi1 site occurs by reverse splicing into DNA is discussed.     

Potential for alternative intron-exon pairings in group II intron RmInt1 from Sinorhizobium meliloti and its relatives

Ribozyme constructs derived from group II intron RmInt1 of Sinorhizobium meliloti self-splice in vitro when incubated under permissive conditions, but exon ligation is unusually inefficient when the 5' exon is truncated close to the IBS2 intron-binding site. One plausible explanation for this observation is the presence of an alternative intron-exon pairing between an intron segment that overlaps with the EBS2 exon-binding site and a 5' exon site located just distal of IBS2 relative to the splice junction. Strikingly, the existence of this pairing is supported by comparative sequence analysis of introns related to RmInt1.     

An alternative intron-exon pairing scheme implied by unexpected in vitro activities of group II intron RmInt1 from Sinorhizobium meliloti

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.     

In vitro characterization of the splicing efficiency and fidelity of the RmInt1 group II intron as a means of controlling the dispersion of its host mobile element

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.     

Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

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.     

Characterization of IS666, a newly described insertion element of Mycobacterium avium

The insertion sequence IS666 was isolated from Mycobacterium avium strain 101. IS666 is a 1474 bp insertion sequence belonging to the IS256 family, that includes IS6120 from Mycobacterium smegmatis, IS1166 and IS1295 from Rhodococcus sp. IGTS8, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus, and ISRm3 from Rhizobium meliloti. IS666 has 24 bp imperfect inverted repeats that fit the consensus described for the family, and generates 9 bp duplications upon insertion into the host DNA with no apparent specificity in the target sequence. In contrast with its two closest homologues, IS1166 and IS6120, IS666 contains a single ORF that would codify a transposase of 434 aa. IS666 is restricted to M. avium, where it is present in 21% of the isolates in a number ranging between 1 to 7 copies.     

Relevance of the branch point adenosine, coordination loop, and 3' exon binding site for in vivo excision of the Sinorhizobium meliloti group II intron RmInt1

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.