Importance of base pairing during trans-splicing of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis.

CECILIA QUIROGA; LISA KRONSTAD; CHRISTINE RITLOP; BENOIT COUSINEAU

Congreso; Riboclub Annual Metting; 2009

Group II (GII) introns are large ribozymes that possess a highly conserved RNA secondary structure. This structure is formed of six domains (D1 to D6) radiating from a central wheel. D4 usually harbors an open reading frame (ORF) that codes for the intron encoded protein (IEP). GII introns self-splice by two consecutive transesterification reactions, resulting in the release of the intron and ligation of the flanking exons. In nature, splicing can also be achieved by organellar GII introns fragmented into two or three pieces. In order to trans-splice, these introns need to reassemble either by RNA base pairing, tertiary interactions or IEP stabilization. Previous results from our laboratory showed that the Lactococcus lactis Ll.LtrB GII intron can trans-splice, which means that the fragmented transcripts of this intron are able to reassemble and splice correctly. The aim of this work was to address the role of the RNA base pairing during the trans-splicing mechanism by an in vivo splicing/conjugation assay. The Ll.LtrB intron was fragmented in bipartite structures at different positions (S1 to S9) by standard molecular techniques. Positions S1, S2, S3, S5, S6 and S7 are located in domain D4, while positions S8 and S9 are present in domain D1. Fragmentation sites located at the bottom of a stem (S1, S6, S9) did not lead to potential base pairing between the intron fragments. The remaining fragmentations led to potential base pairings between the intron fragments. We also observed that disruption of the base pairing reduces trans-splicing efficiency; subsequent restoration of a stem by complementation led to complete recovery of the Ll.LtrB trans-splicing efficiency. This work shows that RNA base pairing significantly contributes to the reassembly and trans-splicing efficiency of fragmented GII introns.