Bacterial group II introns generate genetic diversity by circularization and trans-splicing from a population of intron-invaded mRNAs

Abstract

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.

The circle to lariat ratio of the Ll.LtrB group II intron from Lactococcus lactis is greatly influenced by a variety of biological determinants in vivo

“…This is concurring with the fact that intron circles are dead end spliced products that can only accumulate since they cannot reverse splice through the circularization pathway (Fig 1B). In contrast, released intron lariats are active ribozymes that can invade various RNA [6] and/or DNA [17,28] substrates by reverse splicing through the branching pathway ( Fig 1A).…”

“…Altogether, these observations have led to a general consensus that bacterial group II introns behave solely as selfish retromobile elements, conferring no beneficial function to their hosts [13]. However, a potentially beneficial function for group II introns has recently been observed, which combines aspects of both branching and circularization [6]. Ll.LtrB, the model group II intron from the gram-positive bacterium Lactococcus lactis, was shown to generate genetic diversity at the RNA level by shuffling mRNA fragments.…”

“…First, released intron lariats reverse the branching pathway to generate a population of intron-invaded mRNA transcripts in vivo. Next, they self-splice through circularization, joining non-contiguous mRNA fragments by trans-splicing [6]. This delicate balance between branching and circularization has the effect of shuffling fragments of interrupted transcripts together and increasing the genetic diversity of the bacterial transcriptome by producing chimeric mRNA transcripts [6].…”

“…After binding to their IEP and adopting an active tridimensional structure, these ribozymes excise autocatalytically from their primary RNA transcripts by two consecutive transesterification reactions while concurrently ligating their flanking exons ( Fig 1 , steps 1 and 2) [ 3 ]. Group II introns can self-splice by three different splicing pathways, branching ( Fig 1A ), hydrolysis ( Fig 1B ) and circularization ( Fig 1C ) and be released respectively as branched structures called lariats, linear introns and perfect end-to-end closed circles [ 3 – 6 ].…”

“…Although branching is the primary and most studied group II intron splicing pathway, circularization was always considered as a secondary pathway, principally studied in vitro and not as properly characterized [ 3 , 16 ]. We recently conducted the first molecular analyses of the group II intron circularization pathway [ 4 – 6 ]. Our work demonstrated that a significant proportion of Ll.LtrB, excises through the circularization pathway while no trace of perfect linear intron could be detected [ 4 , 5 ].…”