title: Defining the roles and interactions of PTB.
abstract: PTB (polypyrimidine tract-binding protein) is an abundant and widely expressed RNA-binding protein with four RRM (RNA recognition motif) domains. PTB is involved in numerous post-transcriptional steps in gene expression in both the nucleus and cytoplasm, but has been best characterized as a regulatory repressor of some ASEs (alternative splicing events), and as an activator of translation driven by IRESs (internal ribosome entry segments). We have used a variety of approaches to characterize the activities of PTB and its molecular interactions with RNA substrates and protein partners. Using splice-sensitive microarrays we found that PTB acts not only as a splicing repressor but also as an activator, and that these two activities are determined by the location at which PTB binds relative to target exons. We have identified minimal splicing repressor and activator domains, and have determined high resolution structures of the second RRM domain of PTB binding to peptide motifs from the co-repressor protein Raver1. Using single-molecule techniques we have determined the stoichiometry of PTB binding to a regulated splicing substrate in whole nuclear extracts. Finally, we have used tethered hydroxyl radical probing to determine the locations on viral IRESs at which each of the four RRM domains bind. We are now combining tethered probing with single molecule analyses to gain a detailed understanding of how PTB interacts with pre-mRNA substrates to effect either repression or activation of splicing.
authors: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors: Panagiota
authors: P
authors: Kafasla
authors: Ian
authors: I
authors: Mickleburgh
authors: Miriam
authors: M
authors: Llorian
authors: Miguel
authors: M
authors: Coelho
authors: Clare
authors: C
authors: Gooding
authors: Dmitry
authors: D
authors: Cherny
authors: Amar
authors: A
authors: Joshi
authors: Olga
authors: O
authors: Kotik-Kogan
authors: Stephen
authors: S
authors: Curry
authors: Ian C
authors: IC
authors: Eperon
authors: Richard J
authors: RJ
authors: Jackson
authors: Christopher W J
authors: CW
authors: Smith
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D005091:Exons / Q000235:genetics; D006801:Humans; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D016601:RNA-Binding Proteins; D038941:Polypyrimidine Tract-Binding Protein; D012313:RNA