Group II introns are large ribozymes that require the assistance of intron-encoded or free-standing maturases to splice from their premRNAs in vivo. They mainly splice through the classical branching pathway, being released as RNA lariats. However, group II introns can also splice through secondary pathways like hydrolysis and circularization leading to the release of linear and circular introns, respectively. Here, we assessed in vivo splicing of various constructs of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. The study of excised intron junctions revealed, in addition to branched intron lariats, the presence of perfect end-to-end intron circles and alternatively circularized introns. Removal of the branch point A residue prevented Ll.LtrB excision through the branching pathway but did not hinder intron circle formation. Complete intron RNA circles were found associated with the intron-encoded protein LtrA forming nevertheless inactive RNPs. Traces of double-stranded head-to-tail intron DNA junctions were also detected in L. lactis RNA and nucleic acid extracts. Some intron circles and alternatively circularized introns harbored variable number of non-encoded nucleotides at their splice junction. The presence of mRNA fragments at the splice junction of some intron RNA circles provides insights into the group II intron circularization pathway in bacteria.

Group II introns are ancient retroelements that significantly shaped the origin and evolution of contemporary eukaryotic genomes. These self-splicing ribozymes share a common ancestor with the telomerase enzyme, the spliceosome machinery as well as the highly abundant spliceosomal introns and non-LTR retroelements. More than half of the human genome thus consists of various elements that evolved from ancient group II introns, which altogether significantly contribute to key functions and genetic diversity in eukaryotes. Similarly, group II intron-related elements in bacteria such as abortive phage infection (Abi) retroelements, diversity generating retroelements (DGRs) and some CRISPR-Cas systems have evolved to confer important functions to their hosts. In sharp contrast, since bacterial group II introns are scarce, irregularly distributed and frequently spread by lateral transfer, they have mainly been considered as selfish retromobile elements with no beneficial function to their host. Here we unveil a new group II intron function that generates genetic diversity at the RNA level in bacterial cells. We demonstrate that Ll.LtrB, the model group II intron from Lactococcus lactis, recognizes specific sequence motifs within cellular mRNAs by base pairing, and invades them by reverse splicing. Subsequent splicing of ectopically inserted Ll.LtrB, through circularization, induces a novel trans-splicing pathway that generates exon 1-mRNA and mRNA-mRNA intergenic chimeras. Our data also show that recognition of upstream alternative circularization sites on intron-interrupted mRNAs release Ll.LtrB circles harboring mRNA fragments of various lengths at their splice junction. Intergenic trans-splicing and alternative circularization both produce novel group II intron splicing products with potential new functions. Overall, this work describes new splicing pathways in bacteria that generate, similarly to the spliceosome in eukaryotes, genetic diversity at the RNA level while providing additional functional and evolutionary links between group II introns, spliceosomal introns and the spliceosome.

BackgroundGroup II introns are catalytically active RNA and mobile retroelements present in certain eukaryotic organelles, bacteria and archaea. These ribozymes self-splice from the pre-mRNA of interrupted genes and reinsert within target DNA sequences by retrohoming and retrotransposition. Evolutionary hypotheses place these retromobile elements at the origin of over half the human genome. Nevertheless, the evolution and dissemination of group II introns was found to be quite difficult to infer.ResultsWe characterized the functional and evolutionary relationship between the model group II intron from Lactococcus lactis, Ll.LtrB, and Ef.PcfG, a newly discovered intron from a clinical strain of Enterococcus faecalis. Ef.PcfG was found to be homologous to Ll.LtrB and to splice and mobilize in its native environment as well as in L. lactis. Interestingly, Ef.PcfG was shown to splice at the same level as Ll.LtrB but to be significantly less efficient to invade the Ll.LtrB recognition site. We also demonstrated that specific point mutations between the IEPs of both introns correspond to functional adaptations which developed in L. lactis as a response to selective pressure on mobility efficiency independently of splicing. The sequence of all the homologous full-length variants of Ll.LtrB were compared and shown to share a conserved pattern of mutation acquisition.ConclusionsThis work shows that Ll.LtrB and Ef.PcfG are homologous and have a common origin resulting from a recent lateral transfer event followed by further adaptation to the new target site and/or host environment. We hypothesize that Ef.PcfG is the ancestor of Ll.LtrB and was initially acquired by L. lactis, most probably by conjugation, via a single event of horizontal transfer. Strong selective pressure on homing site invasion efficiency then led to the emergence of beneficial point mutations in the IEP, enabling the successful establishment and survival of the group II intron in its novel lactococcal environment. The current colonization state of Ll.LtrB in L. lactis was probably later achieved through recurring episodes of conjugation-based horizontal transfer as well as independent intron mobility events. Overall, our data provide the first evidence of functional adaptation of a group II intron upon invading a new host, offering strong experimental support to the theory that bacterial group II introns, in sharp contrast to their organellar counterparts, behave mostly as mobile elements.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0789-7) contains supplementary material, which is available to authorized users.

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-LTR 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.

Group II introns can self-splice from RNA transcripts through branching, hydrolysis and circularization, being released as lariats, linear introns and circles, respectively. In contrast to branching, the circularization pathway is mostly based on assumptions and has been largely overlooked. Here, we address the molecular details of both transesterification reactions of the group II intron circularization pathway in vivo. We show that free E1 is recruited by the intron through base pairing interactions and that released intron circles can generate free E1 by the spliced exon reopening reaction. The first transesterification reaction was found to be induced inaccurately by the 3′OH of the terminal residue of free E1 at the 3′ splice site, producing circularization intermediates with heterogeneous 3′ ends. Nevertheless, specific terminal 3′OH, selected by a molecular ruler, was shown to precisely attack the 5′ splice site and release intron circles with 3′–5′ rather than 2′–5′ bonds at their circularization junction. Our work supports a circularization model where the recruitment of free E1 and/or displacement of cis-E1 induce a conformational change of the intron active site from the pre-5′ to the pre-3′ splice site processing conformation, suggesting how circularization might initiate at the 3′ instead of the 5′ splice site.