Deletion of nonconserved helices near the 3' end of the rRNA intron of Tetrahymena thermophila alters self-splicing but not core catalytic activity

Abstract

The self-splicing rRNA intron of Tetrahymena thermophila contains two stem-loop structures (P9.1 and P9.2) near its 3' end that are not conserved among group I introns. As a step toward deriving the smallest active self-splicing RNA, 78 nucleotides encompassing P9.1 and P9.2 have been deleted. This deletion has no effect on the core catalytic activity of the intron, as judged by its ability to catalyze poly(C) polymerization and other related reactions. In contrast, reactions at the 3' splice site of the rRNA precursor--exon ligation and intermolecular exon ligation--take place with reduced efficiency, and exon ligation becomes rate-limiting for self-splicing. Moreover, intermolecular exon ligation with pentaribocytidylic acid is inaccurate, occurring primarily at a cryptic site in the 3' exon. A deletion of 79 nucleotides that disrupts P9, as well as removing P9.1 and P9.2, has more severe effects on both the first and second steps of splicing. P9, a conserved helix at the 5' edge of the deletion point, can form stable alternative structures in the deletion mutants. This aberrant folding may be responsible for the reduced activity and accuracy of reactions with mutant precursors. Analysis of the cryptic site suggests that choice of the 3' splice site may not only depend on sequence but also on proximity to P9. In the course of these studies, evidence has been obtained for an alternative 5' exon-binding site distinct from the normal site in the internal guide sequence.