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 group II intron self-splicing provide insight into both the catalytic strategies of splicing and the evolutionary relationships between the different splicing 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 causes a switch in metal specificity when the second step of splicing is monitored using a bimolecular exon-ligation assay. This suggests that the spliceosome 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 indicates that a rate-limiting conformational change between the two steps of splicing may mask the subsequent chemical step and the metal-specificity switch. As the group II intron, a true ribozyme, uses identical catalytic strategies for splicing, our results strengthen the argument that the spliceosome is an RNA catalyst that shares a common molecular ancestor with group II introns.
Gordon
P M
PM
Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Illinois 60637, USA.
Sontheimer
E J
EJ
Piccirilli
J A
JA
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D019143:Evolution, Molecular; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008670:Metals / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism*; D013455:Sulfur / Q000737:chemistry
D017931:DNA Primers; D008670:Metals; D012322:RNA Precursors; D012367:RNA, Viral; D013455:Sulfur