Importance of base pairing during trans-splicing of the Ll.LtrB group II.

CECILIA QUIROGA; LISA KRONSTAD; CHRISTINE RITLOP; BENOIT COUSINEAU

Congreso; 59th Annual CSM Meeting of the Canadian Society of Microbiologist.; 2009

Group II introns are large ribozymes able to self-splice from their pre-mRNAs. These retroelements possess 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 Domain 4 (D4) called intron encoded protein (IEP). These IEPs code for a multifunctional protein with reverse transcriptase and maturase, with occasional endonuclease, activities. Group II introns self-splice by two consecutive transesterification reactions that result in the release of the intron and ligation of the flanking exons. The maturase activity of the IEP is essential for splicing in vivo since it stabilizes the catalytically active tertiary conformation of the ribozyme. In nature, splicing can also be achieved by organellar group II introns fragmented into two or three pieces. In order to trans-splice, these introns need to reassemble. Reassembly of the intron fragments may occur by RNA base pairing, tertiary interactions and IEP stabilization. The Ll.LtrB intron from the gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II intron. Previous results from our laboratory showed that Ll.LtrB 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. S1, S6 and S9 do not lead to potential base pairing between the two intron fragments since they are located at the bottom of a stem. On the contrary, the remaining fragmentations led to potential base pairings between the two intron fragments. Our results show that disruption of base pairing reduces significantly the trans-splicing efficiency of Ll.LtrB. We also observed that subsequent restoration of the disrupted base pairing in the D4a stem by complementation leads to complete recovery of the Ll.LtrB trans-splicing efficiency. This work thus show that RNA base pairing significantly contributes to the reassembly and trans-splicing efficiency of fragmented group II introns.