Translational control of selenoprotein synthesis

描述（由申请人提供）： 翻译后摘要：环境元素硒（Se）是人类健康所必需的少量，但有毒的高水平。有强有力的证据表明，即使是硒状态的微小变化也会对人类健康产生多方面的影响，包括免疫功能的改变和对病毒感染、生化应激、癌症甚至糖尿病的易感性。硒的许多有益作用可归因于硒蛋白;以硒蛋白形式含有硒的蛋白质， 第21位氨基酸硒代半胱氨酸（Sec）。硒蛋白作用的底物和生化途径的多样性可能解释了与硒摄入相关的多种健康效应。Se通过重新定义UGA密码子编码Sec而共同参与硒蛋白的合成。在哺乳动物中，UGA-Sec密码子由Sec-tRNA[Ser]Sec的两种同种型解码，不同之处仅在于反密码子位置Um 34处是否存在甲基。该残基在体内的甲基化取决于Se状态。未甲基化的亚型占主导地位时，硒是缺乏的，而相反的情况下，硒充足。以前的研究表明，管家硒蛋白可以使用任一异构体掺入Sec，而胁迫相关硒蛋白更喜欢甲基化形式的Sec-tRNA[Ser]Sec。我们推测，硒依赖性的变化，在未甲基化的甲基化Sec-tRNA[Ser]Sec的比例提供了硒的可用性和基因特异性调节硒蛋白的生产之间的直接联系。为了验证这一假设，并进一步探讨在体内硒蛋白表达的翻译控制（目标1），我们将开发和验证核糖体分析作为一种方法来量化核糖体的位置和密度硒蛋白mRNA的组织中的小鼠提出的硒缺乏，足够或补充的饮食。此外，我们将研究核糖体分布在组织中的转基因小鼠过表达的非甲基化亚型的Sec-tRNA[Ser]Sec。在目标2中，我们将建立一个在体外tRNA定义的兔网织红细胞翻译系统编程与甲基化或未甲基化的异构体的Sec-tRNA[Ser]Sec直接测试Sec掺入效率在翻译的一个子集的管家和应力相关的硒蛋白mRNA，并确定涉及的顺式作用元件。实验结果表明，硒蛋白的合成与硒的健康效应密切相关。

DESCRIPTION (provided by applicant): Abstract: The environmental element selenium (Se) is essential for human health in small amounts, but toxic at high levels. There is strong evidence that even small changes in Se status have multi-faceted effects on human health which include altered immune function and susceptibility to viral infections, biochemical stresses, cancer, and even diabetes. Many of the beneficial effects of Se can be attributed to selenoproteins; proteins containing Se in the form of the 21st amino acid selenocysteine (Sec). The diversity of substrates and biochemical pathways that selenoproteins act upon likely explains the multiple health effects associated with Se intake. Se is incorporated into selenoproteins co-translationally by redefinition of a UGA codon to encode Sec. In mammals UGA-Sec codons are decoded by two isoforms of Sec-tRNA[Ser]Sec differing only by the presence or absence of a methyl group at the anticodon position Um34. Methylation of this residue in vivo is dependent upon Se status. The unmethylated isoform predominates when Se is deficient, whereas the opposite is true under Se sufficiency. Previous studies suggest that the house-keeping selenoproteins can incorporate Sec using either isoform, whereas the stress-related selenoproteins prefer the methylated form of Sec-tRNA[Ser]Sec. We hypothesize that Se-dependent changes in the ratio of unmethylated to methylated Sec-tRNA[Ser]Sec provides the direct link between Se availability and gene-specific regulation of selenoprotein production. To test this hypothesis and further explore translational control of selenoprotein expression in vivo (Aim 1), we will develop and validate ribosome profiling as a methodology to quantify ribosome position and density on selenoprotein mRNAs in tissues from mice raised on Se deficient, adequate or supplemented diets. In addition, we will examine ribosome distribution in tissues from transgenic mice overexpressing the unmethylated isoform of Sec-tRNA[Ser]Sec. In Aim 2, we will establish an in vitro tRNA-defined rabbit reticulocyte translation system programmed with the methylated or unmethylated isoforms of Sec-tRNA[Ser]Sec to directly test Sec incorporation efficiency during translation of a subset of the house-keeping and stress-related selenoprotein mRNAs and identify the cis-acting elements involved. The results from experiments proposed here promise to establish a new methodology to examine selenoprotein synthesis and to further elucidate the mechanism of Se-dependent regulation of selenoprotein expression, a finding of direct relevance to the health effects of Se.