Mutation of the conserved first nucleotide of a group II intron from yeast mitochondrial DNA reduces the rate but allows accurate splicing

Abstract

Group II introns have a phylogenetically conserved 5'-terminal pentanucleotide, -G1U2G3C4G5-, that resembles the conserved 5' end sequence of nuclear pre-mRNA introns. No functional interaction or catalytic role for the conserved G1 position has been proposed, although a tertiary structure involving -G3C4- has been implicated in splicing in vitro. We have analyzed splicing phenotypes both in vitro and in vivo for all three point mutants affecting guanosine at position 1 (G1) of intron 5 gamma from the COXI gene of yeast mitochondrial DNA. While all of these G1N substitutions slow splicing in vitro, G1C is clearly the most defective. All three mutant transcripts splice as accurately as the wild-type transcript, although the yield of lariat intron is reduced. The branched trinucleotide core includes the mutated position 1 nucleotide linked to the canonical branchpoint adenosine. The mutant lariats vary significantly in their susceptibility to the debranching activity from human cells. After wild-type, G1A was most sensitive, G1U was somewhat resistant, while G1C was highly resistant to debranching. These mutant lariats had normal ribozyme activity for promoting spliced exon reopening. The three mutant introns were transformed into otherwise normal yeast mitochondrial DNA. These mutants grow on nonfermentable carbon sources and splce aI5 gamma to yield excised intron lariat and mRNA. Nonetheless, each mutant splices with reduced efficiency, roughly parallel to their in vitro activity. In vivo, all three mutants accumulate both the pre-mRNA retaining intron 5 gamma and the lariat splicing intermediate containing intron and 3' exon. Clearly, this primary sequence element, shared with nuclear pre-mRNA introns, has a very different functional significance in group II splicing.