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.     

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.     

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.     

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.     

Conjugation mediates transfer of the Ll.LtrB group II intron between different bacterial species

Some self-splicing group II introns (ribozymes) are mobile retroelements. These retroelements, which can insert themselves into cognate intronless alleles or ectopic sites by reverse splicing, are thought to be the evolutionary progenitors of the widely distributed eukaryotic spliceosomal introns. Lateral or horizontal transmission of introns (i.e. between species), although never experimentally demonstrated, is a well-accepted model for intron dispersal and evolution. Horizontal transfer of the ancestral bacterial group II introns may have contributed to the dispersal and wide distribution of spliceosomal introns present in modern eukaryotic genomes. Here, the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was used as a model system to address the dissemination of introns in the bacterial kingdom. We report the first experimental demonstration of horizontal transfer of a group II intron. We show that the Ll.LtrB group II intron, originally discovered on an L. lactis conjugative plasmid (pRS01) and within a chromosomally located sex factor in L. lactis 712, invades new sites using both retrohoming and retrotransposition pathways after its transfer by conjugation. Ll.LtrB lateral transfer is shown among different L. lactis strains (intraspecies) (retrohoming and retrotransposition) and between L. lactis and Enterococcus faecalis (interspecies) (retrohoming). These results shed light on long-standing questions about intron evolution and propagation, and demonstrate that conjugation is one of the mechanisms by which group II introns are, and probably were, broadly disseminated between widely diverged organisms.     

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.     

Group II Introns Generate Functional Chimeric Relaxase Enzymes with Modified Specificities through Exon Shuffling at Both the RNA and DNA Level

Group II introns are large self-splicing RNA enzymes with a broad but somewhat irregular phylogenetic distribution. These ancient retromobile elements are the proposed ancestors of approximately half the human genome, including the abundant spliceosomal introns and non-long terminal repeat retrotransposons. In contrast to their eukaryotic derivatives, bacterial group II introns have largely been considered as harmful selfish mobile retroelements that parasitize the genome of their host. As a challenge to this view, we recently uncovered a new intergenic trans-splicing pathway that generates an assortment of mRNA chimeras. The ability of group II introns to combine disparate mRNA fragments was proposed to increase the genetic diversity of the bacterial host by shuffling coding sequences. Here, we show that the Ll.LtrB and Ef.PcfG group II introns from Lactococcus lactis and Enterococcus faecalis respectively can both use the intergenic trans-splicing pathway to catalyze the formation of chimeric relaxase mRNAs and functional proteins. We demonstrated that some of these compound relaxase enzymes yield gain-of-function phenotypes, being significantly more efficient than their precursor wild-type enzymes at supporting bacterial conjugation. We also found that relaxase enzymes with shuffled functional domains are produced in biologically relevant settings under natural expression levels. Finally, we uncovered examples of lactococcal chimeric relaxase genes with junctions exactly at the intron insertion site. Overall, our work demonstrates that the genetic diversity generated by group II introns, at the RNA level by intergenic trans-splicing and at the DNA level by recombination, can yield new functional enzymes with shuffled exons, which can lead to gain-of-function phenotypes.     

Multiple physical forms of excised group II intron RNAs in wheat mitochondria

Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3′terminaladenosines, as well as heterogeneous circular introns, which lack 3′ nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo.     

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.     

Group II introns in wheat mitochondria have degenerate structural features and varied splicing pathways

Mitochondrial introns in flowering plant genes are virtually all classified as members of the group II ribozyme family although certain structural features have degenerated to varying degrees over evolutionary time. We are interested in the impact that unconventional intron architecture might have on splicing biochemistry in vivo and we have focused in particular on intronic domains V and VI, which for self-splicing introns provide a key component of the catalytic core and the bulged branchpoint adenosine, respectively. Notably, the two transesterification steps in classical group II splicing are the same as for nuclear spliceosomal introns and release the intron as a lariat. Using RT-PCR and circularized RT-PCR, we had previously demonstrated that several wheat mitochondrial introns which lack a branchpoint adenosine have atypical splicing pathways, and we have now extended this analysis to the full set of wheat introns, namely six trans-splicing and sixteen cis-splicing ones. A number of introns are excised using non-lariat pathways and interestingly, we find that several introns which do have a conventional domain VI also use pathways that appear to exploit other internal or external nucleophiles, with the lariat form being relatively minor. Somewhat surprisingly, several introns with weakly-structured domain V/VI helices still exhibit classical lariat splicing, suggesting that accessory factors aid in restoring a splicing-competent conformation. Our observations illustrate that the loss of conventional group II features during evolution is correlated with altered splicing biochemistry in an intron-distinctive manner.     

Group II intron in Bacillus cereus has an unusual 3' extension and splices 56 nucleotides downstream of the predicted site

All group II introns known to date fold into six functional domains. However, we recently identified an intron in Bacillus cereus ATCC 10987, B.c.I4, that splices 56nt downstream of the expected 3′ splice site in vivo (Tourasse et al. 2005, J. Bacteriol., 187, 5437–5451). In this study, we confirmed by ribonuclease protection assay that the 56-bp segment is part of the intron RNA molecule, and computational prediction suggests that it might form a stable stem-loop structure downstream of domain VI. The splicing of B.c.I4 was further investigated both in vivo and in vitro. Lariat formation proceeded primarily by branching at the ordinary bulged adenosine in domain VI without affecting the fidelity of splicing. In addition, the splicing efficiency of the wild-type intron was better than that of a mutant construct deleted of the 56-bp 3′ extension. These results indicate that the intron has apparently adapted to the extra segment, possibly through conformational adjustments. The extraordinary group II intron B.c.I4 harboring an unprecedented extra 3′ segment constitutes a dramatic example of the flexibility and adaptability of group II introns.