Downregulation of eNOS mRNA expression by TNFα:: identification and functional characterization of RNA-protein interactions in the 3′UTR
Downregulation of eNOS mRNA expression by TNFα:: identification and functional characterization of RNA-protein interactions in the 3′UTR
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DOI：
10.1016/s0008-6363(03)00296-7
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发表时间：
2003-07-01
影响因子：
10.8
通讯作者：
Stewart, DJ
中科院分区：
文献类型：
--
作者：
Lai, PFH;Mohamed, F;Stewart, DJ
目的：我们之前已经表明，肿瘤坏死因子- α (TNFalpha)对内皮型一氧化氮合酶（eNOS）表达的下调完全是由于内皮型一氧化氮合酶mRNA的显著不稳定。由于许多真核mRNA中的3'-非翻译区（3' utr）已被充分证明可以结合调控转录物稳定性的反式因子，因此我们研究了蛋白质与eNOS mRNA该区域的结合。人类和牛3'UTR的高度同源性也表明，通过进化保守的重要功能特征存在于该区域。方法：将编码人eNOS 3'UTR或选定序列的放射性标记RNA与培养的人脐静脉内皮细胞（HUVECs）的细胞质提取物孵育，通过交联实验研究RNA与蛋白质的相互作用。产生eNOS YUTR序列的5‘和3’截断缺失突变，以确定特定的结合序列。RT-PCR检测HUVECs中eNOS rnRNA的表达。结果：利用放射性标记的RNA编码整个418个核苷酸的3'UTR，我们在内皮提取物中鉴定了分子量约为53,56和66 kDa的核糖核蛋白复合物（RNPs）。53- kda和56-kDa RNPs的形成被TNFalpha上调。而66-kDa RNP的形成被下调。含有3'UTR近端和远端序列的RNA片段有利于53-kDa RNP的形成，而富含au的远端RNA片段有利于66-kDa RNP的形成。来自eNOS YUTR中间部分的含有富含cu的158个核苷酸序列的RNA片段（命名为M158）有利于56 kda RNP的形成。腺病毒基因转移和m158 RNA的过表达，作为阻止内源性转录物上56-kDa RNP形成的蛋白结合诱饵，减弱了培养内皮细胞中tnfalpha诱导的eNOS mRNA下调。结论：我们的研究结果表明，eNOS表达的调控涉及细胞质蛋白与沿YUTR高度保守元件的特异性结合，56-kDa RNP代表了eNOS转录物不稳定的一种新的调控反式因子。(C) 2003年欧洲心脏病学会。Elsevier Science B.V.版权所有。
Objective: We have previously shown that downregulation of endothelial nitric oxide synthase (eNOS) expression by tumour necrosis factor-alpha (TNFalpha) resulted entirely from the marked destabilization of the cNOS mRNA. As the 3'-untranslated region (3'UTR) in many eukaryotic mRNA has been well documented to bind regulatory trans-factors in the control of transcript stability, we have examined protein binding to this region of the eNOS mRNA. A high degree of homology amongst human and bovine 3'UTR also suggests that important functional features that are conserved through evolution are present within this region. Methods: RNA-protein interactions were studied in cross-linking assays, in which radiolabelled RNA encoding the human eNOS 3'UTR or selected sequences was incubated with cytoplasmic extracts of cultured human umbilical vein endothelial cells (HUVECs). Serial 5'- and 3'-truncated deletional mutations of the eNOS YUTR were generated to identify the specific binding sequences. eNOS rnRNA expression in HUVECs was assessed by RT-PCR analysis. Results: Using radiolabelled RNA encoding the entire 418-nucleotide 3'UTR, we have identified ribonucleoprotein complexes (RNPs) of approximate molecular weights of 53, 56 and 66 kDa in the endothelial extracts. The formation of the 53- and 56-kDa RNPs was upregulated by TNFalpha,. while the formation of the 66-kDa RNP was downregulated. Formation of the 53-kDa RNP was favoured by RNA fragments that contained sequences from the proximal and distal portions of the 3'UTR, whereas the formation of the 66-kDa RNP was favoured by RNA fragments with the AU-rich distal end. RNA fragments containing a CU-rich 158-nucleotide sequence from the medial portion of the eNOS YUTR (designated M158) favoured the formation of the 56-kDa RNP. Adenoviral gene transfer and overexpression of M 158 RNA, as a protein-binding decoy to prevent the formation of the 56-kDa RNP on the endogenous transcripts, attenuated the TNFalpha-induced downregulation of eNOS mRNA in Cultured endothelial cells. Conclusion: Our results demonstrate that the regulation of eNOS expression involves the specific binding of cytoplasmic proteins to highly conserved elements along the YUTR, and the 56-kDa RNP represents a novel regulatory trans-factor in the destabilization of eNOS transcripts. (C) 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.