title: Protein factor requirements of the Apaf-1 internal ribosome entry segment: roles of polypyrimidine tract binding protein and upstream of N-ras.
abstract: It has been reported previously that the 5' untranslated region of the mRNA encoding Apaf-1 (apoptotic protease-activating factor 1) has an internal ribosome entry site (IRES), whose activity varies widely among different cell types. Here it is shown that the Apaf-1 IRES is active in rabbit reticulocyte lysates, provided that the system is supplemented with polypyrimidine tract binding protein (PTB) and upstream of N-ras (unr), two cellular RNA binding proteins previously identified to be required for rhinovirus IRES activity. In UV cross-linking assays and electrophoretic mobility shift assays with individual recombinant proteins, the Apaf-1 IRES binds unr but not PTB; however, PTB binding occurs if unr is present. Over a range of different cell types there is a broad correlation between the activity of the Apaf-1 IRES and their content of PTB and unr. In cell lines deficient in these proteins, overexpression of PTB and unr stimulated Apaf-1 IRES function. This is the first example where an IRES in a cellular mRNA has been shown to be functionally dependent, both in vitro and in vivo, on specific cellular RNA binding proteins. Given the critical role of Apaf-1 in apoptosis, these results have important implications for the control of the apoptotic cascade.
authors: Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
authors: S A
authors: SA
authors: Mitchell
authors: E C
authors: EC
authors: Brown
authors: M J
authors: MJ
authors: Coldwell
authors: R J
authors: RJ
authors: Jackson
authors: A E
authors: AE
authors: Willis
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D053477:Apoptotic Protease-Activating Factor 1; D016923:Cell Death / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D005786:Gene Expression Regulation; D011905:Genes, ras; D006801:Humans; D038941:Polypyrimidine Tract-Binding Protein; D011506:Proteins / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics*; D014162:Transfection
chemical_list: C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D011506:Proteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D038941:Polypyrimidine Tract-Binding Protein