Two independent internal ribosome entry sites are involved in translation initiation of vascular endothelial growth factor mRNA
Two independent internal ribosome entry sites are involved in translation initiation of vascular endothelial growth factor mRNA
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DOI：
10.1128/mcb.18.11.6178
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发表时间：
1998-11-01
影响因子：
5.3
通讯作者：
Prats, H
中科院分区：
文献类型：
--
作者：
Huez, I;Créancier, L;Prats, H
血管内皮生长因子（VEGF）是主要的血管生成生长因子，其mRNA包含一个异常长（1，038个核苷酸）且结构化的5'非翻译区（UTR）。根据核糖体扫描的经典翻译起始模型，预期这样的5' UTR是强翻译抑制剂。在体外和双顺反子的策略被用来表明，VEGF mRNA的翻译是帽独立的，并通过内部核糖体进入过程发生。我们首次证明了在这个5' UTR中存在两个独立的内部核糖体进入位点（IRES）。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允许鉴定IRES A活性所必需的49个核苷酸的结构域（D4）。UV交联实验表明，IRES A活性与100 kDa的蛋白质与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和483之间的元件。免疫沉淀实验表明，IRES B的主要结合蛋白是多聚嘧啶束结合蛋白（PT B），它是一种众所周知的小核糖核酸病毒IRES的调节因子。然而，我们发现PT B与IRES B的结合似乎与其活性无关。证据提供了一个原始的累积效应的两个IRES，可能由不同的因素控制，以促进有效的启动翻译在同一AUG密码子。
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.