Translational Recoding of UGA as Selenocysteine in Selenoprotein Synthesis

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。对硒的营养需求可能是由于它在硒蛋白中的功能，硒蛋白以硒半胱氨酸（Sec）的形式含有硒，这是第21种氨基酸。哺乳动物硒蛋白在甲状腺激素代谢、抗氧化防御炎症和发育中起着关键作用。本提案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整合到生长的多肽链中需要一个翻译重编码事件，其中UGA停止密码子被读取为Sec。在真核生物中，UGA重编码为Sec取决于硒蛋白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，在编码机制中起关键作用。我们在SBP2中定义了一个新的二部RNA结合结构域，并表明该结构域自然发生的点突变与人类甲状腺功能减退有关，改变了该蛋白的secis结合活性，并选择性地影响了硒蛋白亚群的表达，包括那些参与甲状腺激素代谢的硒蛋白。多种证据支持L30参与UGA重新编码的假设，但这种核糖体蛋白如何在Sec整合中起作用尚不清楚。我们的研究表明，SBP2和L30在SECIS上存在动态交换，并且这两种蛋白在UGA重新编码过程中是顺序作用的。这种逐步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事件来提高硒蛋白合成的效率。在本项目中，我们提出：1)了解SBP2:SECIS相互作用的分子基础；2)在体内和体外验证SBP2调控硒蛋白组表达的假说；3)阐明L30在哺乳动物细胞硒蛋白合成中的作用。该项目产生的信息和分子工具将为未来研究将人类硒蛋白活性缺陷与编码Sec结合途径组分的基因突变或多态性联系起来提供坚实的基础。

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. )