A splicing repressor domain in polypyrimidine tract-binding protein.
Polypyrimidine tract-binding protein (PTB) is an hnRNP with four RRM type domains. It plays roles as a repressive alternative splicing regulator of multilple target genes, as well as being involved in pre-mRNA 3' end processing, mRNA localization, stability, and internal ribosome entry site-mediated translation. Here we have used a tethered function assay, in which a fusion protein of PTB and the bacteriophage MS2 coat protein is recruited to a splicing regulatory site by binding to an artificially inserted MS2 binding site. Deletion mutations of PTB in this system allowed us to identify RRM2 and the following inter-RRM linker region as the minimal region of PTB that can act as splicing repressor domain when recruited to RNA. Splicing repression by the minimal repressor domain remained cell type-specific and dependent upon other defined regulatory elements in the alpha-tropomyosin test minigene. Our results highlight the fact that splicing repression by PTB can be uncoupled from the mode by which it binds to RNA.
Robinson
Fiona
F
Department of Biochemistry, University of Cambridge, UK.
Smith
Christopher W J
CW
D017398:Alternative Splicing; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D017353:Gene Deletion; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D038941:Polypyrimidine Tract-Binding Protein / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000737:chemistry*; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000737:chemistry; D013379:Substrate Specificity; D014162:Transfection
D011743:Pyrimidines; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D038941:Polypyrimidine Tract-Binding Protein; D012313:RNA; C030986:pyrimidine