Metal ion catalysis during the exon-ligation step of nuclear pre-mRNA splicing: Extending the parallels between the spliceosome and group II introns

Mechanistic analyses of nuclear pre-mRNA splicing by the spliceosome and gr oup II intron self-splicing provide insight into both the catalytic strateg ies of splicing and the evolutionary relationships between the different sp licing systems. We previously showed that 3'-sulfur substitution at the 3' splice site of a nuclear pre-mRNA has no effect on splicing, We now report that 3'-sulfur substitution at the 3' splice site of a nuclear pre-mRNA cau ses a switch in metal specificity when the second step of splicing is monit ored using a bimolecular exon-ligation assay. This suggests that the splice osome uses a catalytic metal ion to stabilize the 3'-oxyanion leaving group during the second step of splicing, as shown previously for the first step . The lack of a metal-specificity switch under cis splicing conditions indi cates that a rate-limiting conformational change between the two steps of s plicing may mask the subsequent chemical step and the metal-specificity swi tch. As the group II intron, a true ribozyme, uses identical catalytic stra tegies for splicing, our results strengthen the argument that the spliceoso me is an RNA catalyst that shares a common molecular ancestor with group II introns.