The Ll.LtrB group II intron from the gram-positive bacterium Lactococcus lactis excisses as circles and generates double-stranded head to tail DNA junctions in vivo

CAROLINE MONAT; CECILIA QUIROGA; BENOIT COUSINEAU

Congreso; International Union of Microbiological Societies Congresses; 2014

Group II introns are large ribozymes that require the assistance of intron-encoded or free-standing maturases to splice from pre-mRNAs in vivo. The branching pathway, releasing group II introns as lariats, is the major and most studied splicing pathway. However, group II introns can also splice through secondary pathways like hydrolysis and circularization that are not as well characterized as branching. The Ll.LtrB intron from the gram-positive bacterium Lactococcus lactis is the best model to study group II intron splicing and mobility. Here, we assessed splicing of the Ll.LtrB group II intron in L. lactis by amplifying the splice junction of its ligated exons and released introns. Ligated exons revealed that different mutants of Ll.LtrB undergo alternative splicing using the same remote 5? and 3? alternative splice sites. We also found that the maturase activity of LtrA controls 5? and 3? splice site selection and the balance between accurate and alternative splicing. The study of excised introns revealed the presence of lariats, circles and alternatively spliced products for all Ll.LtrB variants studied albeit at different ratios. Removal of the branch point residue prevented Ll.LtrB excision through the branching pathway but did not hinder circle formation and alternative splicing. A number of circular and alternatively spliced introns were found harboring extra non- encoded nucleotides at the splice junction. The presence of identifiable mRNA fragments at the junction of some intron RNA circles provides insights into the circularization mechanism of group II introns. Complete intron RNA circles were found associated with LtrA but forming inactive RNPs. Traces of double-stranded head to tail intron DNA junctions were also detected from L. lactis total RNA and nucleic acid extracts. This work unveils that Ll.LtrB excises not only as lariats but also as circles in vivo and shed light on the circularization pathway of group II introns.