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Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein

Published online by Cambridge University Press:  13 February 2002

HAIYING LIU
Affiliation:
Howard Hughes Medical Institute and Department of Biological Sciences, A507LH, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA
WENQING ZHANG
Affiliation:
Howard Hughes Medical Institute and Department of Biological Sciences, A507LH, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA
ROBYN B. REED
Affiliation:
Howard Hughes Medical Institute and Department of Biological Sciences, A507LH, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA
WEIQUN LIU
Affiliation:
Howard Hughes Medical Institute and Department of Biological Sciences, A507LH, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA
PAULA J. GRABOWSKI
Affiliation:
Howard Hughes Medical Institute and Department of Biological Sciences, A507LH, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA
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Abstract

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.

Type
Research Article
Copyright
© 2002 RNA Society

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