Evidence of a tertiary interaction functional in group I 3'-splicing

It has been recently shown that the schedule of 3'-splicing events for yeast mitochondrial group I introns requires that conserved helix P10 materializes only after 5'-cleavage has taken place. A scenario compatible with experimental findings has been proposed [(1992) FEBS Lett. 297, 201-204; (1990) Proc. Natl. Acad. Sci. USA 87, 8192-8196] where the formation of P10 is postponed by competition for standard base-pairing with an extended P1 interaction engaging the same portion of the internal guide sequence (IGS) which is subsequently involved in P10. Although the formation of P10 in the fifth intron of yeast apocytochrome b gene (YCOB5) has been confirmed by site-directed mutagenesis, we cannot be confident that this interaction is merely stabilized by Watson-Crick base-pairing, involving the 3'-exon and the IGS, especially given that its formation requires closure of an intron loop of 159 unpaired bases. Thus, our aim is to establish the participation of the 5'-extremity of the intron in the formation of P10. By deoxyribose substitution at positions 1 and 2 of the 5'-extremity of the intron, we are able to confirm the existence of tertiary interactions stabilizing the 3'-splicing site. We show that selective deoxyribose substitution renders the intron inefficient for 3'-splicing when compared with wild type levels.