The effects of dietary selenium on translational control of protein synthesis

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，该机制涉及一个专门的SEC-tRNA，它解码通常指定终止的UGA密码子。这种对UGA的翻译“重新定义”是一个高度调控的步骤，这不仅对我们理解控制硒蛋白合成的机制感兴趣，而且因为它也提供了一个机会来更广泛地研究如何改变翻译延伸和解码的过程来调节基因表达。在这里，我们建议使用新的深度测序方法和已建立的生化方法相结合的方法来研究UGA重新定义的分子机制及其受生理条件的调节。在目标1中，我们将测试新的假设，当细胞经历内质网(ER)应激时，UGA-近端核糖体暂停及其释放的作用。我们认为这种现象是一种快速诱导硒蛋白表达的新机制，以响应细胞压力。此外，我们将讨论这些硒蛋白在保护内质网应激和细胞凋亡过程中所起的生物学作用。在目标2中，我们研究了RNA结构在UGA-近端核糖体暂停、对内质网应激的反应以及对硒的感知中的作用。最后，在目标3中，我们将利用高通量测序技术、质谱仪和直接从小鼠组织中分离的硒蛋白核糖核蛋白复合体的RNA结构分析来研究硒对UGA重新定义和硒蛋白表达的影响。这些研究将为如何改变翻译延伸的控制和解码过程以调节基因表达提供新的见解。

Selenocysteine is incorporated into selenoproteins during translation by an unconventional mechanism involving a dedicated Sec-tRNA that decodes UGA codons, which normally specify termination. This translational “redefinition” of UGA is a highly regulated step that is of interest not only for our understanding of the mechanisms controlling selenoprotein synthesis but because it also provides an opportunity to examine more generally how the processes of translation elongation and decoding can be altered to regulate gene expression. Here we propose to examine the molecular mechanisms of UGA redefinition and its regulation by physiological conditions using a combination of new deep-sequencing approaches and established biochemical methods. In Aim 1, we will test new hypotheses regarding the role of UGA-proximal ribosome pausing and its release when cells undergo endoplasmic reticulum (ER) stress. We propose this phenomenon to be a novel mechanism to rapidly induce selenoprotein expression in response to cellular stress. Further, we will address the biological role that these selenoproteins play in protection from ER stress and the progression to apoptosis. In Aim 2, we examine the role of RNA structures in UGA-proximal ribosome pausing, response to ER stress, and sensing selenium. Finally, in Aim 3, we will utilize high throughput sequencing techniques, mass spectrometry, and RNA structural analysis of selenoprotein ribonucleoprotein complexes isolated directly from tissues of mice to examine the how selenium effects UGA redefinition and selenoprotein expression. These studies will provide new insights into how the control of translational elongation and the very process of decoding can be altered to regulate gene expression.