2 INDEPENDENT INTERNAL RIBOSOME ENTRY SITES ARE INVOLVED IN TRANSLATION INITIATION OF VASCULAR ENDOTHELIAL GROWTH-FACTOR MESSENGER-RNA

The mRNA of vascular endothelial growth factor (VEGF), the major angio genic growth factor, contains an unusually long (1,038 nucleotides) an d structured 5' untranslated region (UTR). According to the classical translation initiation model of ribosome scanning, such a 5' UTR is ex pected to be a strong translation inhibitor. In vitro and bicistronic strategies were used to show that the VEGF mRNA translation was cap in dependent and occurred by an internal ribosome entry process. For the first time, we demonstrate that two independent internal ribosome entr y sites (IRESs) are present in this 5' UTR. IRES A is located within t he 300 nucleotides upstream from the AUG start codon. RNA secondary st ructure prediction and site-directed mutagenesis allowed the identific ation of a 49-nucleotide structural domain (D4) essential to IRES A ac tivity. UV cross-linking experiments revealed that IRES A activity was correlated with binding of a 100-kDa protein to the D4 domain. IRES B is located in the first half of the 5' UTR, An element between nucleo tides 379 and 483 is required for its activity. Immunoprecipitation ex periments demonstrated that a main IRES B-bound protein was the polypy rimidine tract binding protein (PTB), a well-known regulator of picorn avirus IRESs, However, we showed that binding of the PTB on IRES B doe s not seem to be correlated with its activity. Evidence is provided of an original cumulative effect of two IRESs, probably controlled by di fferent factors, to promote an efficient initiation of translation at the same AUG codon.