Huez I, Créancier L, Audigier S, Gensac M C, Prats A C, Prats H
INSERM U397, Endocrinologie et Communication Cellulaire, Institut Fédératif de Recherche Louis Bugnard, CHU Rangueil, 31403 Toulouse cedex 04, France.
Mol Cell Biol. 1998 Nov;18(11):6178-90. doi: 10.1128/MCB.18.11.6178.
The mRNA of vascular endothelial growth factor (VEGF), the major angiogenic growth factor, contains an unusually long (1,038 nucleotides) and structured 5' untranslated region (UTR). According to the classical translation initiation model of ribosome scanning, such a 5' UTR is expected to be a strong translation inhibitor. In vitro and bicistronic strategies were used to show that the VEGF mRNA translation was cap independent and occurred by an internal ribosome entry process. For the first time, we demonstrate that two independent internal ribosome entry sites (IRESs) are present in this 5' UTR. IRES A is located within the 300 nucleotides upstream from the AUG start codon. RNA secondary structure prediction and site-directed mutagenesis allowed the identification of a 49-nucleotide structural domain (D4) essential to IRES A activity. 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 nucleotides 379 and 483 is required for its activity. Immunoprecipitation experiments demonstrated that a main IRES B-bound protein was the polypyrimidine tract binding protein (PTB), a well-known regulator of picornavirus IRESs. However, we showed that binding of the PTB on IRES B does not seem to be correlated with its activity. Evidence is provided of an original cumulative effect of two IRESs, probably controlled by different factors, to promote an efficient initiation of translation at the same AUG codon.
血管内皮生长因子（VEGF）是主要的血管生成生长因子，其信使核糖核酸（mRNA）含有一个异常长（1038个核苷酸）且具有结构的5'非翻译区（UTR）。根据核糖体扫描的经典翻译起始模型，这样的5'UTR预计是一种强大的翻译抑制剂。采用体外和双顺反子策略表明，VEGF mRNA的翻译不依赖于帽子结构，而是通过内部核糖体进入过程发生。我们首次证明，在这个5'UTR中存在两个独立的内部核糖体进入位点（IRESs）。IRES A位于AUG起始密码子上游300个核苷酸内。RNA二级结构预测和定点诱变使得能够鉴定出对IRES A活性至关重要的一个49个核苷酸的结构域（D4）。紫外线交联实验表明，IRES A的活性与一种100 kDa蛋白质与D4结构域的结合相关。IRES B位于5'UTR的前半部分。其活性需要核苷酸379至483之间的一个元件。免疫沉淀实验表明，一种主要与IRES B结合的蛋白质是多嘧啶序列结合蛋白（PTB），它是微小核糖核酸病毒IRESs的一种著名调节因子。然而，我们表明PTB与IRES B的结合似乎与其活性无关。有证据表明，两个IRESs存在一种原始的累积效应，可能受不同因素控制，以促进在同一个AUG密码子处高效起始翻译。
