Distinctive properties of the 5′-untranslated region of human Hsp70 mRNA
Distinctive properties of the 5′-untranslated region of human Hsp70 mRNA
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DOI：
10.1074/jbc.m303213200
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发表时间：
2003-06-20
影响因子：
4.8
通讯作者：
Shatsky, IN
中科院分区：
文献类型：
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作者：
Rubtsova, MP;Sizova, DV;Shatsky, IN
哺乳动物热休克蛋白Hsp 70的5 '端非翻译区（UTR）具有内部核糖体进入位点（IRES）。在这项研究中，这种可能性已经在转染细胞中使用表达双顺反子mRNA的质粒进行了测试。使用报告基因构建体，海肾荧光素酶/Photinus pyralis荧光素酶，我们表明，热休克蛋白70 mRNA的216-nt长的5 '-非翻译区作为一个IRES，指导核糖体的下游起始密码子通过帽独立的机制。该IRES的相对活性（空载体的100倍）与经典的小核糖核酸病毒IRES相似。另外的对照表明，下游报告基因的这种高表达不是由于上游顺反子的通读，也不是由于隐蔽的单顺反子转录物的翻译。Hsp 70 mRNA的5 '-UTR内的小缺失对IRES活性的影响取决于它们在5'-UTR序列内的位置。除了nt 33-50的缺失之外，它对于5 '-UTR的5'-末端一半是小的，而对于3 '-末端部分是相当强的。然而，这些小的缺失都不能完全消除IRES活性。通过从5 '端截短5'-UTR或通过内部缺失切除较大部分（> 50 nt）导致IRES功能的显著损害。总之，这些数据表明Hsp 70 mRNA的5 '-UTR的IRES活性需要5'-UTR的几乎整个序列的完整性。数据进行了讨论的模型，允许一个三维的，而不是线性模式的选择的起始区域周围的Hsp 70 mRNA的起始密码子。
A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (> 50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.