title: The organization of RNA contacts by PTB for regulation of FAS splicing.
abstract: Post-transcriptional steps of gene expression are regulated by RNA binding proteins. Major progress has been made in characterizing RNA-protein interactions, from high resolution structures to transcriptome-wide profiling. Due to the inherent technical challenges, less attention has been paid to the way in which proteins with multiple RNA binding domains engage with target RNAs. We have investigated how the four RNA recognition motif (RRM) domains of Polypyrimidine tract binding (PTB) protein, a major splicing regulator, interact with FAS pre-mRNA under conditions in which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl radical probing, targeted inactivation of individual RRMs and single molecule analyses revealed an unequal division of labour between the four RRMs of PTB. RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA. The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker. Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.
authors: Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK.
authors: Ian
authors: I
authors: Mickleburgh
authors: Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK kafasla@fleming.gr.
authors: Panagiota
authors: P
authors: Kafasla
authors: Department of Biochemistry, Henry Wellcome Building, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors: Dmitry
authors: D
authors: Cherny
authors: Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK.
authors: Miriam
authors: M
authors: Llorian
authors: Division of Cell and Molecular Biology, Imperial College, Exhibition Road, London SW7 2AZ, UK.
authors: Stephen
authors: S
authors: Curry
authors: Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK.
authors: Richard J
authors: RJ
authors: Jackson
authors: Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK cwjs1@cam.ac.uk.
authors: Christopher W J
authors: CW
authors: Smith
mesh_terms: D017398:Alternative Splicing; D001665:Binding Sites; D009154:Mutation; D038941:Polypyrimidine Tract-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism
chemical_list: D012322:RNA Precursors; D012333:RNA, Messenger; D019014:fas Receptor; D038941:Polypyrimidine Tract-Binding Protein