Martineau Yvan, Le Bec Christine, Monbrun Laurent, Allo Valérie, Chiu Ing-Ming, Danos Olivier, Moine Hervé, Prats Hervé, Prats Anne-Catherine
Institut National de la Santé et de la Recherche Médicale U589, Hormones, Facteurs de Croissance et Physiopathologie Vasculaire, Institut Louis Bugnard, IFR31, CHU Rangueil, 31059 Toulouse Cedex 09, France.
Mol Cell Biol. 2004 Sep;24(17):7622-35. doi: 10.1128/MCB.24.17.7622-7635.2004.
Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases.
成纤维细胞生长因子1（FGF - 1）是一种强大的血管生成因子，其基因结构包含四个启动子，导致可变剪接过程，产生四种具有可变5'非翻译区（5'UTR）的mRNA。在这里，我们通过使用双荧光素酶双顺反子载体，确定了人类FGF - 1 5'UTR中存在内部核糖体进入位点（IRES），特别是在A和C前导序列中，它们在哺乳动物细胞中具有不同的活性。小鼠肌肉中的DNA电转染显示，FGF - 1前导序列A中存在的IRES在体内具有高活性。我们开发了一种新的可调控的TET OFF双顺反子系统，这使我们能够排除FGF - 1前导序列中任何隐蔽启动子的可能性。分别定位在118和103个核苷酸片段中的FGF - 1 IRES A和C，在起始密码子位置方面具有灵活性，从生物技术角度来看很有趣。此外，我们表明小鼠和人类来源的FGF - 1 IRES A显示出相似的IRES活性谱。对FGF - 1 IRES A RNA的酶促和化学探测揭示了在哺乳动物中核苷酸序列和RNA结构水平上都保守的一个结构域。通过点突变，包括补偿性突变，已经证明了这个结构基序的功能作用。这些数据支持IRES在FGF - 1表达控制中的重要作用，并提供了一个新的IRES结构基序，这有助于在5'UTR数据库中进行IRES预测。
