Regulation of Selenoprotein Synthesis by SECIS-binding Proteins

描述（由申请人提供）：硒是一种必需的微量营养素，具有许多重要的健康益处。该元素以硒代半胱氨酸（Sec）的形式并入硒蛋白中，即第21位氨基酸。哺乳动物硒蛋白在抗氧化防御、甲状腺激素代谢、雄性生殖和发育中发挥重要作用。Sec由UGA密码子编码，其通常被读作终止密码子。UGA作为Sec的后退需要在硒蛋白mRNA的3'非翻译区中的Sec插入序列（SECIS）元件。我们以前确定了两个SECIS结合蛋白，SECIS结合蛋白2（SBP 2）和核糖体蛋白L30，这在后退机制中发挥关键作用。虽然已经取得了很大的进展，在了解SEC的纳入机制，少有人知道这个途径的监管。在缺硒期间，该元素优先用于大脑和内分泌器官。也有个别硒蛋白的表达层次。即使在硒充足的条件下，某些硒蛋白对生命和健康是必需的，而另一些则不是。我们的建议的中心假设是，在SECIS顺式作用序列招募多个反式作用因子，优先利用硒是一个组织特异性和硒蛋白依赖的方式。我们最近发现了两个额外的SECIS结合蛋白：一个110 kDa的蛋白质，通过RNA亲和层析纯化，并通过质谱分析鉴定为核仁素，和一个未知的蛋白质的~ 45 kDa，这里称为SBP 45。与SBP 2和L30不同，核仁素和SBP 45可以区分硒蛋白mRNA。这两种蛋白质具有不同的结合特异性，核仁素优先结合硒蛋白的SECIS元素，这些元素对正常健康和发育至关重要。我们还表明，核仁素在体外特异性调节UGA消退。在本项目中，我们建议使用各种生物化学，细胞生物学和分子生物学的方法：1）确定功能上重要的核仁素和SECIS元件之间的相互作用; 2）阐明核仁素在调节硒蛋白mRNA翻译的功能; 3）确定SBP 45并确定其在调节硒蛋白表达中的功能。成功实现这些目标可以确定限制因素和调节途径，可用于治疗性调节体内硒蛋白表达。

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.