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Genetic interactions between the 5′ and 3′ splice site consensus sequences and U6 snRNA during the second catalytic step of pre-mRNA splicing

Published online by Cambridge University Press:  11 January 2002

CATHERINE A. COLLINS
Affiliation:
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143, USA Present address: Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
CHRISTINE GUTHRIE
Affiliation:
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143, USA
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Abstract

The YAG/ consensus sequence at the 3′ end of introns (the slash indicates the location of the 3′ splice site) is essential for catalysis of the second step of pre-mRNA splicing. Little is known about the interactions formed by these three nucleotides in the spliceosome. Although previous observations have suggested that the G of the YAG/ interacts with the first nucleotide of the /GUA consensus sequence at the 5′ end of the intron, additional interactions have not been identified. Here we report several striking genetic interactions between A+3 of the 5′ /GUA with Y−3 of the 3′ YAG/ and G50 of the highly conserved ACAGAG motif in U6 snRNA. Two mutations in U6 G50 of the ACAGAG can weakly suppress two mutations in A+3 of the 5′ /GUA. This suppression is significantly enhanced upon the inclusion of a specific mutation Y−3 in the 3′ YAG/. RNA analysis confirmed that the severe splicing defect observed in A+3 and Y−3 double mutants can be rescued to near wild-type levels by the mutations in U6 G50. The contributions of each mutation to the genetic interaction and the strong position specificity of suppression, combined with previous findings, support a model in which the 5′ /GUA and the GAG of U6 function in binding the 3′ YAG/ during the second catalytic step.

Type
Research Article
Information
RNA , Volume 7 , Issue 12 , December 2001 , pp. 1845 - 1854
Copyright
2001 RNA Society

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