FUNCTIONAL-CHARACTERIZATION OF THE INTERNAL RIBOSOME ENTRY SITE OF EIF4G MESSENGER-RNA

The eIF4 group initiation factors are required for cap-dependent trans lation initiation. Infection of mammalian cells by picornaviruses resu lts in proteolytic cleavage of one of these factors, eIF4G, which seve rely restricts cap-dependent initiation but permits cap independent in itiation to proceed from an internal ribosome entry site (IRES) in pic ornaviral RNAs. The first 357 nucleotides (nt) of the 5'-untranslated region of eIF4G mRNA also contains an IRES, Using bicistronic construc ts for expression in K562 cells, we have now shown that progressive de letions of the 5'-untranslated region can have either stimulatory or i nhibitory effects. Furthermore, a 101-nt segment exhibits full IRES ac tivity, and an 81-nt segment exhibits detectable IRES activity. A poly pyrimidine tract (PPT) at the 3' terminus is essential for internal in itiation, a property which is characteristic of picornaviral IRESs but not the other host cellular IRESs studied to date. IRES activity does not require sequences beyond 357 nt. Out-of-frame AUGs have no effect on IRES-driven luciferase expression when introduced upstream of the PPT but markedly decrease expression when introduced at sites between the PPT and the authentic initiation codon at nt 369. These results su ggest that the ribosomal subunit enters at or near the PPT and then sc ans downstream for the initiation codon.