Nuclease sensitive element binding protein 1 associates with the selenocysteine insertion sequence and functions in mammalian selenoprotein translation.
Nuclease sensitive element binding protein 1 associates with the selenocysteine insertion sequence and functions in mammalian selenoprotein translation.
复制标题
核酸酶敏感元件结合蛋白 1 与硒代半胱氨酸插入序列相关，并在哺乳动物硒蛋白翻译中发挥作用。
DOI：
10.1002/jcp.20619
复制
发表时间：
2006
影响因子：
5.6
通讯作者：
Newburger,PeterE
中科院分区：
文献类型：
--
作者：
Shen,Qichang;Fan,Lin;Newburger,PeterE
含硒蛋白的生物合成需要通过UGA密码子的交替翻译插入不寻常的氨基酸硒代半胱氨酸，UGA密码子通常用作终止密码子。在真核生物中，硒蛋白的翻译依赖于位于mRNA 3′非翻译区的一个或多个硒代半胱氨酸插入序列（SECIS）元件，以及几个SECIS结合蛋白。我们的实验室以前已经确定了核酸酶敏感元件结合蛋白1（NSEP 1）作为另一种SECIS结合蛋白，但已经提出了支持和反对其在体内SECIS结合和硒蛋白翻译中的作用的证据。我们目前的研究试图解决这一争议，首先通过调查NSEP 1是否与完整细胞内的SECIS元件密切相互作用。在可逆的体内交联和核糖核蛋白免疫沉淀后，编码两种谷胱甘肽过氧化物酶家族成员的mRNA与人和大鼠细胞系中的NSEP 1共沉淀。表位标记GPX 1构建体的免疫共沉淀依赖于其3′-非翻译区的完整SECIS元件。为了测试这种相互作用对硒蛋白翻译的功能重要性，我们使用小的抑制性RNA来减少组织培养细胞的NSEP 1含量，然后检查这种减少对SECIS依赖性荧光素酶报告基因活性的影响，该报告基因的表达取决于UGA密码子的通读。针对NSEP 1的小抑制性RNA的共转染使其表达降低约50%，并显著降低荧光素酶活性。这些研究表明，NSEP 1是一个真正的SECIS结合蛋白，在结构上与硒蛋白翻译复合物和功能上参与哺乳动物细胞中硒蛋白的翻译。J.细胞。Physiol. © 2006 Wiley利斯公司
Biosynthesis of selenium‐containing proteins requires insertion of the unusual amino acid selenocysteine by alternative translation of a UGA codon, which ordinarily serves as a stop codon. In eukaryotes, selenoprotein translation depends upon one or more selenocysteine insertion sequence (SECIS) elements located in the 3′‐untranslated region of the mRNA, as well as several SECIS‐binding proteins. Our laboratory has previously identified nuclease sensitive element binding protein 1 (NSEP1) as another SECIS‐binding protein, but evidence has been presented both for and against its role in SECIS binding in vivo and in selenoprotein translation. Our current studies sought to resolve this controversy, first by investigating whether NSEP1 interacts closely with SECIS elements within intact cells. After reversible in vivo cross‐linking and ribonucleoprotein immunoprecipitation, mRNAs encoding two glutathione peroxidase family members co‐precipitated with NSEP1 in both human and rat cell lines. Co‐immunoprecipitation of an epitope‐taggedGPX1construct depended upon an intact SECIS element in its 3′‐untranslated region. To test the functional importance of this interaction on selenoprotein translation, we used small inhibitory RNAs to reduce the NSEP1 content of tissue culture cells and then examined the effect of that reduction on the activity of a SECIS‐dependent luciferase reporter gene for which expression depends upon readthrough of a UGA codon. Co‐transfection of small inhibitory RNAs directed against NSEP1 decreased its expression by approximately 50% and significantly reduced luciferase activity. These studies demonstrate that NSEP1 is an authentic SECIS binding protein that is structurally associated with the selenoprotein translation complex and functionally involved in the translation of selenoproteins in mammalian cells. J. Cell. Physiol. © 2006 Wiley‐Liss, Inc.