MUTATION OF THE CONSERVED 1ST-NUCLEOTIDE OF A GROUP-II INTRON FROM YEAST MITOCHONDRIAL-DNA REDUCES THE RATE BUT ALLOWS ACCURATE SPLICING

Group II introns have a phylogenetically conserved, 5'-terminal pentan ucleotide, -G1U2G3C4G5-, that resembles the conserved 5' end sequence of nuclear pre-mRNA introns. No functional interaction or catalytic ro le for the conserved G1 position has been proposed, although a tertiar y structure involving -G3C4- has been implicated in splicing in vitro. We have analyzed splicing phenotypes both in vitro and in vivo for al l three point mutants affecting guanosine at position 1 (G1) of intron 5gamma from the COXI gene of yeast mitochondrial DNA. While all of th ese G1N substitutions slow splicing in vitro, G1C is clearly the most defective. All three mutant transcripts splice as accurately as the wi ld-type transcript, although the yield of lariat intron is reduced. Th e branched trinucleotide core includes the mutated position 1 nucleoti de linked to the canonical branchpoint adenosine. The mutant lariats v ary significantly in their susceptibility to the debranching activity from human cells. After wild-type, G1A was most sensitive, G1U was som ewhat 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 n ormal yeast mitochondrial DNA. These mutants grow on nonfermentable ca rbon sources and splice aI5gamma to yield excised intron lariat and mR NA.. Nonetheless, each mutant splices with reduced efficiency, roughly parallel to their in vitro activity. In vivo, all three mutants accum ulate both the pre-mRNA retaining intron 5gamma and the lariat splicin g intermediate containing intron and 3' exon. Clearly, this primary se quence element, shared with nuclear pre-mRNA introns, has a very diffe rent functional significance in group II splicing.