Bacterial group II introns: splicing, function and evolution

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The over-arching and long-term goal of my Research Program is to address fundamental questions related to the splicing, function and evolution of group II introns. Our model system is the Ll.LtrB group II intron from the industrially important Gram-positive bacterium Lactococcus lactis.******Group II introns are large ribozymes that self-splice from pre-mRNAs. Eukaryotic nuclear introns and fragmented group II introns use the exact same splicing pathway strongly suggesting that group II and nuclear introns have a common evolutionary origin. Another fascinating characteristic about group II introns is their potential, following their autocatalytic excision, to invade new identical DNA sites by retrohoming and non-homologous DNA sites by retrotransposition.******Aim 1: We demonstrated that the Ll.LtrB group II intron can be fragmented in two or three pieces at multiple locations and still assemble and splice in trans. This work strongly supports the evolutionary theories claiming that group II introns are the progenitors of eukaryotic nuclear introns and that the five small nuclear RNAs, that are part of the spliceosome machinery, were derived from group II intron fragments. We propose to study how the pieces of fragmented introns fold and assemble in vivo and the contribution of the intron-encoded protein LtrA in their folding and assembly process (1.1), to identify and study Ll.LtrB trans-splicing factors in L. lactis (1.2) and assess if Ll.LtrB can be further fragmented in four and five pieces and still assemble and trans-splice (1.3).******Aim 2: We recently showed that a significant proportion of Ll.LtrB is released as perfect RNA circles demonstrating that this intron is not only released as lariats in L. lactis. Overall, our work shows that circularization is a conserved group II intron splicing pathway generating spliced products with potentially new interesting biological functions. We propose to complete the characterization of the primary group II intron circularization pathway (2.1), to study in further details the new Ll.LtrB circularization pathway (2.2) and the function of its splicing products: the intron RNA circles (2.3) and the chimeric mRNAs (2.4).******Aim 3: We provided the first experimental demonstrations of the well-accepted and long-standing theory stating that group II introns can be laterally transferred between diverged species. Our work strongly suggests that the unexpectedly high representation of group II introns associated with other mobile elements reflects the evolutionary advantage held by these introns shuttling between bacterial cells. We propose to study the host range of Ll.LtrB lateral transfer by conjugation in the bacterial kingdom (3.1), to evaluate the dissemination potential of Ll.LtrB within bacterial biofilms (3.2), to investigate the lateral transfer of Ll.LtrB by bacteriophage transduction (3.3) and through bacterial membrane vesicles (3.4).