Protein factor requirements of the Apaf-1 internal ribosome entry segment:Roles of polypyrimidine tract binding protein and upstream of N-ras

It has been reported previously that the 5' untranslated region of the mRNA encoding; Apaf-1 (apoptotic protease-activating , factor 1) has an interna l 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 retic ulocyte lysates, provided that the system is supplemented with polyprimidin e 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 as says with individual recombinant proteins, the Apaf-1 IRES binds unr hilt n ot PTB; however PTB binding occurs if unr is present. Over, a range of diff erent cell types there is a broad correlation between the activity of the A paf-1 IRES and their content of PTB and unr, In cell lines deficient in the se 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 tiro, on specific cellul ar RNA binding proteins. Given the critical role of Apaf-1 in apoptosis? th ese results have important implications for the control of the apoptotic ca scade.