The effects of dietary selenium on translational control of protein synthesis

 说明(申请人提供)：环境元素硒(Se)对人体健康是必不可少的，少量是必需的，但高浓度是有毒的。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以及对病毒感染、生化压力、癌症甚至糖尿病的易感性。硒的许多有益作用可以归因于硒蛋白，即以第21个氨基酸硒半胱氨酸(SEC)的形式含有硒的蛋白质。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影响。硒半胱氨酸在翻译过程中通过一种非传统的机制被结合到硒蛋白中，该机制涉及UGA密码子的SEC-tRNA解码。这种UGA密码子的翻译“重新定义”提供了一种独特的机制，可以通过这种机制来控制硒蛋白的表达。因此，硒蛋白合成的调控是相当复杂的，除了调控转录和翻译启动外，可能还包括控制硒半胱氨酸的掺入效率和mRNA的稳定性(由于无义介导的衰退)。在这里，我们建议使用新的深度测序方法和成熟的生物化学方法相结合的方法，系统地研究和阐明硒半胱氨酸掺入和控制硒蛋白合成的分子机制。核糖体分析(核糖体保护的mRNA足迹的深度测序)将被用来检查体内(小鼠组织)中主动翻译硒蛋白mRNAs的核糖体的位置和密度。根据这些信息，将有可能量化在饮食或遗传背景受控变化的背景下的翻译启动和SEC掺入效率。结合信使核糖核酸丰度的测量，这种方法提供了一种理想的工具，可以准确地确定不断变化的生物条件如何影响硒蛋白的合成。提供的初步数据挑战了有关硒半胱氨酸掺入的机制以及顺式和反式作用因子如何调节硒蛋白表达的传统观点。在目标1中，我们建议继续研究改变膳食硒摄入量影响硒蛋白表达的机制。在目标2中，我们建议使用含有SEC掺入所需的关键顺式和反式作用因子突变的转基因小鼠来检验关于SEC掺入机制的假说。最后，在目标3中，我们建议研究硒蛋白表达的改变对整个基因组中基因表达的转录和翻译控制的影响。

 DESCRIPTION (provided by applicant): 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. Selenocysteine is incorporated into selenoproteins during translation by an unconventional mechanism involving Sec-tRNA decoding of UGA codons. This translational "redefinition" of UGA codons provides a unique mechanism by which selenoprotein expression may be controlled. Consequently, the regulation of selenoprotein synthesis is quite complex and likely involves control of selenocysteine incorporation efficiency and mRNA stability (due to nonsense mediated decay) in addition to regulation of transcription and translation initiation. Here we propose to systematically examine and clarify the molecular mechanisms of selenocysteine incorporation and control of selenoprotein synthesis using a combination of new deep-sequencing approaches and well established biochemical methods. Ribosome profiling (deep-sequencing of ribosome protected mRNA footprints) will be used to examine the location and density of ribosomes actively translating selenoprotein mRNAs in vivo (mouse tissues). From this information it will be possible to quantify both translation initiation and Sec incorporation efficiency in the context of controlled changes in diet or genetic backgrounds. Combined with measurements of mRNA abundance, this methodology provides an ideal tool to determine precisely how changing biological conditions affect selenoprotein synthesis. Preliminary data is presented which challenges conventional thinking about the mechanism of selenocysteine incorporation and how cis- and trans-acting factors regulate selenoprotein expression. In Aim 1, we propose to continue our studies of the mechanisms by which altered dietary selenium intake affects selenoprotein expression. In Aim 2, we propose to test hypothesis regarding the mechanism of Sec incorporation using genetically modified mice containing mutations in key cis- and trans-acting factors required for Sec incorporation. Finally, in Aim 3 we propose to examine the impact of altered selenoprotein expression on transcriptional and translational control of gene expression across the genome.