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

Published online by Cambridge University Press:  01 February 2000

PETER M. GORDON
Affiliation:
Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, USA
ERIK J. SONTHEIMER
Affiliation:
Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, USA Present address: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 4-120 Hogan Hall, 2153 North Campus Drive, Evanston, Illinois 60208-3500, USA.
JOSEPH A. PICCIRILLI
Affiliation:
Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, USA Department of Chemistry, Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, USA
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Abstract

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.

Type
Research Article
Copyright
2000 RNA Society

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