Assembly of group II intron fragments by base pairing during trans-splicing.

CECILIA QUIROGA; LISA KRONSTAD; CHRISTINE RITLOP; BENOIT COUSINEAU

Congreso; Mobile DNA II; 2010

Group II introns are large ribozymes that self-splice from their pre-mRNAs. They fold into a highly conserved RNA secondary structure formed of six domains (D1 to D6) radiating from a central wheel. Most group II introns harbor an open reading frame (ORF) within D4, which encodes for a multifunctional protein with reverse transcriptase, maturase, and occasionally endonuclease, activities. Group II introns self-splice by two consecutive trans-esterification reactions that result in the release of the intron and ligation of the flanking exons. The maturase activity of the ORF is essential for splicing in vivo since it stabilizes the catalytically active tertiary conformation of the ribozyme by binding to the high affinity binding site D4a. Some organellar group II introns that are fragmented into two or three pieces are also able to splice in trans. In order to trans-splice, these fragmented introns need to assemble, an event that may depend on RNA base pairing interactions, tertiary interactions and RNA stabilization by the maturase. We recently showed that the Ll.LtrB group II intron from the gram-positive bacterium Lactococcus lactis trans-splices efficiently in vivo. Numerous bipartite variants of Ll.LtrB are able to assemble and splice in trans accurately leading to the precise ligation of their flanking exons. The aim of this work was to address the role of RNA base pairing between intron fragments during Ll.LtrB trans-splicing. Bipartite Ll.LtrB introns fragmented at different positions in D1, D3 and D4 were analyzed using a trans-splicing/conjugation assay. Ll.LtrB trans-splicing efficiency was compared between close fragmentation sites allowing or not potential base pairs between the intron fragments. We noticed that all Ll.LtrB trans-splicers with potential base pairing trans-spliced more efficiently than their counterparts with no base pairing. Disruption of the base pairing at the different engineered fragmentation sites and sequential restoration by sequence complementation led to a reduced trans-splicing efficiency followed by a step-wise recovery. This work shows that RNA base pairing between intron fragments significantly contributes to the assembly and trans-splicing of fragmented group II introns in vivo.