Regulation of Selenoprotein Synthesis by SECIS-binding Proteins

说明(申请人提供)：硒是一种基本的微量营养素，对健康有许多重要的益处。这种元素以硒半胱氨酸(SEC)的形式被结合到硒蛋白中，硒半胱氨酸是第21种氨基酸。哺乳动物硒蛋白在抗氧化防御、甲状腺激素代谢、雄性生殖发育等方面发挥着重要作用。SEC由UGA密码子编码，通常读作终止密码子。UGA作为SEC的消退需要在硒蛋白mRNA的3‘非翻译区有SEC插入序列(SECIS)元件。我们以前发现了两个SECIS结合蛋白，SECIS结合蛋白2(SBP2)和核糖体蛋白L30，它们在消退机制中发挥关键作用。尽管在理解SEC的合并机制方面已经取得了很大进展，但对这一途径的监管知之甚少。在缺硒期间，这种元素优先用于大脑和内分泌器官。单个硒蛋白的表达也有层级结构。即使在硒充足的条件下，某些硒蛋白对生命和健康是必不可少的，而另一些则不是。我们建议的中心假设是，SECIS中的顺式作用序列招募了多种反式作用因子，这些反式作用因子优先利用硒是一种组织特异性和硒蛋白依赖的方式。我们最近发现了另外两种SECIS结合蛋白：一种是110 kDa的蛋白，经RNA亲和层析纯化后经质谱仪鉴定为核仁蛋白；另一种是-45 kDa的未知蛋白，这里称为SBP45。与SBP2和L30不同，核仁素和SBP45可以区分硒蛋白mRNAs。这两种蛋白质具有不同的结合特异性，核仁优先与硒蛋白中对正常健康和发育至关重要的SECIS元件结合。我们还表明，在体外，核仁特异性地调节UGA的消退。在本项目中，我们建议使用多种生化、细胞生物学和分子生物学方法：1)确定核仁与SECIS元件之间重要的功能相互作用；2)阐明核仁在调节硒蛋白mRNA翻译中的功能；3)鉴定SBP45并确定其在调控硒蛋白表达中的功能。对这些目标的成功追求可能确定限制因素和调控途径，这些限制因素和调控途径可以用于治疗，以调节体内硒蛋白的表达。

DESCRIPTION (provided by applicant): Selenium is an essential micronutrient that exerts many important health benefits. The element is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. The mammalian selenoproteins perform important functions in anti-oxidant defense, thyroid hormone metabolism, male reproduction, and development. Sec is encoded by a UGA codon, which is normally read as a stop codon. The receding of UGA as Sec requires a Sec Insertion Sequence (SECIS) element in the 3' untranslated region of the selenoprotein mRNA. We previously identified two SECIS-binding proteins, SECIS Binding Protein 2 (SBP2) and ribosomal protein L30, which play critical roles in the receding mechanism. Although much progress has been made in understanding the Sec incorporation machinery, less is known about the regulation of this pathway. During selenium deficiency, the element is preferentially utilized in the brain and endocrine organs. There is also a hierarchy of expression of individual selenoproteins. Even under selenium adequate conditions, certain selenoproteins are essential for life and health whereas others are not. The central hypothesis of our proposal is that cis-acting sequences in the SECIS recruit multiple trans-acting factors, which prioritize the utilization of selenium is a tissue-specific and selenoprotein-dependent manner. We recently discovered two additional SECIS-binding proteins: a 110 kDa protein which was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis, and an unknown protein of - 45 kDa, referred to here as SBP45. Unlike SBP2 and L30, nucleolin and SBP45 can distinguish between selenoprotein mRNAs. The two proteins have distinct binding specificities, with nucleolin preferentially binding to SECIS elements from selenoproteins that are essential for normal health and development. We also show that nucleolin specifically regulates UGA receding in vitro. In this project, we propose to use a variety of biochemical, cell biology, and molecular biology approaches to: 1) identify functionally important interactions between nucleolin and the SECIS element; 2) elucidate the function of nucleolin in regulating selenoprotein mRNA translation, and 3) identify SBP45 and determine its function in regulating selenoprotein expression. The successful pursuit of these aims may identify limiting factors and regulatory pathways that could be used therapeutically to modulate selenoprotein expression in vivo.