Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein.
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.
Liu
Haiying
H
Howard Hughes Medical Institute and Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
Zhang
Wenqing
W
Reed
Robyn B
RB
Liu
Weiqun
W
Grabowski
Paula J
PJ
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D007438:Introns; D008836:Micrococcal Nuclease; D008969:Molecular Sequence Data; D009154:Mutation; D038941:Polypyrimidine Tract-Binding Protein; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D038941:Polypyrimidine Tract-Binding Protein; D008836:Micrococcal Nuclease