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3′ splice site recognition in nematode trans-splicing involves enhancer- dependent recruitment of U2 snRNP

Published online by Cambridge University Press:  29 June 2001

CHARLES M. ROMFO
Affiliation:
Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA
PATRICIA A. MARONEY
Affiliation:
Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA
SHAOPING WU
Affiliation:
Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605, USA
TIMOTHY W. NILSEN
Affiliation:
Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA
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Abstract

Trans-splicing requires that 5′ and 3′ splice sites be independently recognized. Here, we have used mutational analyses and a sensitive nuclease protection assay to determine the mechanism of trans-3′ splice site recognition in vitro. Efficient recognition of the 3′ splice site is dependent upon both the sequence of the 3′ splice site itself and enhancer elements located in the 3′ exon. We show that the presence of three distinct classes of enhancers results in increased binding of U2 snRNP to the branchpoint region. Several lines of evidence strongly suggest that the increased binding of U2 snRNP is mediated by U2AF. These results expand the roles of enhancers in constitutive splicing and provide direct support for the recruitment model of enhancer function.

Type
REPORT
Copyright
2001 RNA Society

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3′ splice site recognition in nematode trans-splicing involves enhancer- dependent recruitment of U2 snRNP
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