Translational Recoding of UGA as Selenocysteine in Selenoprotein Synthesis

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。对硒的营养需求可能是由于它在硒蛋白中的功能，硒蛋白中含有以硒半胱氨酸(SEC)的形式存在的元素，即第21种氨基酸。哺乳动物硒蛋白在甲状腺激素代谢、抗氧化性防御性炎症和发育过程中发挥重要作用。这项提议的目的是了解硒蛋白合成的机制，并确定控制这一过程效率的决定因素。将SEC整合到不断增长的多肽链中需要翻译重新编码事件，在该事件中UGA终止密码子被读作SEC。在真核生物中，UGA重新编码为SEC依赖于硒蛋白mRNA 3‘非翻译区的SEC插入序列(SECIS)。SECIS与SECIS结合蛋白2(SBP2)和核糖体蛋白L30相互作用，它们在重新编码机制中起着关键作用。我们在SBP2中定义了一个新的两部分RNA结合域，并表明该结构域中的一个自然发生的点突变与人类甲状腺功能低下有关，改变了蛋白质的SECIS结合活性，并选择性地影响硒蛋白的子集的表达，包括那些参与甲状腺激素代谢的蛋白质。多条证据支持L30参与UGA重新编码的假设，但这种核糖体蛋白如何在SEC掺入中发挥作用尚不清楚。我们的研究表明，SECIS上存在SBP2和L30的动态交换，并且这两种蛋白在UGA重新编码过程中依次发挥作用。这种循序渐进的组装机制可以通过防止非生产性相互作用和协调一系列特定的事件来提高硒蛋白合成的效率。在这个项目中，我们建议：1)了解SBP2：SECIS相互作用的分子基础；2)检验SBP2在体外和体内决定硒蛋白质组表达的假设；3)阐明L30在哺乳动物细胞硒蛋白合成中的功能。该项目中产生的信息和分子工具将为未来将人类硒蛋白活性缺陷与编码SEC掺入途径组成部分的基因突变或多态联系起来的研究提供坚实的基础。 与公众健康相关：硒是饮食中的一种基本矿物质，对人类健康至关重要。世界上许多地区都存在膳食中硒摄入量低的情况，这种情况与疾病风险增加有关，包括甲状腺问题、心脏病、炎症、关节炎、病毒感染和癌症。这个项目的目标是了解硒是如何被结合到一小群重要的蛋白质中的，这些蛋白质在细胞中发挥关键作用，并可能对这种基本微量元素的有益影响负责。)

DESCRIPTION (provided by applicant): Selenium is an essential micronutrient that exerts many important health benefits. The nutritional requirement for selenium is likely due to its function in selenoproteins, which contain the element in the form of selenocysteine (Sec), the 21st amino acid. Mammalian selenoproteins perform critical functions in thyroid hormone metabolism, anti-oxidant defense inflammation, and development. The goal of this proposal is to understand the mechanism of selenoprotein synthesis and identify the determinants that control the efficiency of this process. The incorporation of Sec into the growing polypeptide chain requires a translational recoding event in which the UGA stop codon is read as Sec. In eukaryotes, the recoding of UGA as Sec depends on the Sec Insertion Sequence (SECIS) in the 3' untranslated region of the selenoprotein mRNA. The SECIS interacts with SECIS Binding Protein 2 (SBP2) and ribosomal protein L30, which play critical roles in the recoding mechanism. We defined a novel bipartite RNA- binding domain in SBP2 and showed that a naturally occurring point mutation in this domain, which is associated with hypothyroidism in humans, alters the SECIS-binding activity of the protein and selectively affects the expression of a subset of selenoproteins, including those involved in thyroid hormone metabolism. Multiple lines of evidence support the hypothesis that L30 is involved in UGA recoding but how this ribosomal protein functions in Sec incorporation is not known. Our studies suggest that there is a dynamic exchange of SBP2 and L30 on the SECIS and that the two proteins act sequentially during UGA recoding. Such a stepwise assembly mechanism may enhance the efficiency of selenoprotein synthesis by preventing nonproductive interactions and orchestrating a specific series of events. In this project, we propose to: 1) understand the molecular basis for the SBP2:SECIS interaction; 2) test the hypothesis that SBP2 dictates the expression of the selenoproteome in vitro and in vivo; and 3) elucidate the function of L30 in selenoprotein synthesis in mammalian cells. The information and molecular tools generated in this project will provide a strong foundation for future studies linking defects in selenoprotein activity in humans to genetic mutations or polymorphisms in genes that encode components of the Sec incorporation pathway. PUBLIC HEALTH RELEVANCE: Selenium, an essential mineral in the diet, is critical for human health. Low dietary intake of selenium, which occurs in many regions of the world, is associated with an increased risk of disease, including thyroid problems, heart disease, inflammation, arthritis, viral infection, and cancer. The goal of this project is to understand how selenium is incorporated into a small but important group of proteins, which play critical roles in the cell and are likely responsible for the beneficial effects of this essential trace element. )