Development of a zebrafish model for selenoprotein synthesis and function

 描述（由申请人提供）：硒是一种必需的微量元素，作为氨基酸硒代半胱氨酸（Sec）掺入25种人类蛋白质中。有趣的是，硒代半胱氨酸的共翻译掺入发生在被称为硒代半胱氨酸插入序列（SECIS）元件的RNA结构的上游发现的框内终止密码子（UGA）处，其位于所有硒蛋白mRNA的3' UTR中。我们最近已经证明，SEC成立所必需的已知因素实际上是足够的。 这些因子包括特化延伸因子（eEFSec）、SECIS结合蛋白（SBP 2）和特化转移RNA（Sec-tRNASec）。关于哪些蛋白质结构域对于Sec掺入活性是重要的，已经取得了重大进展，但是这些因子允许Sec掺入的机制尚未进行体内研究，特别是在胚胎发育的背景下。此外，存在一种SBP 2蛋白，称为SECISBP 2L，但它不参与体外Sec掺入，其功能仍然未知。因此，硒蛋白的合成和功能的研究仍然是一个新兴的领域，需要开发新的和负担得起的模型系统。在这里，我们建议开发一个斑马鱼模型系统，在其中研究的SEC纳入机制以及硒蛋白的功能。使用斑马鱼是有理由的，主要有四个原因：1）Sec掺入所需的因子在所有脊椎动物中是保守的; 2）鱼类中硒蛋白的补体与人类非常相似; 3）开发鱼类模型的成本大大低于小鼠，以及4）作为脊椎动物发育的易处理模型，斑马鱼的使用将允许前所未有地进入硒蛋白在早期发育期间发挥作用的机制。在第一个目标中，我们将通过研究Sec掺入因子的表达动力学和发育概况来奠定基础，之后我们将确定SBP 2 parastrophin SECISBP 2L的功能。虽然SECISBP 2L与SBP 2同时被鉴定，但其功能仍然难以捉摸。我们从注射抗SECISBP 2L吗啉代寡核苷酸的胚胎中获得的初步数据表明，它可能在造血和/或血管发育中发挥作用。在第二个目标中，我们建议通过在内源性SBP 2中引入特定突变并确定对硒蛋白产生的影响来模拟鱼类中的人类疾病。此外，虽然我们预计，SBP 2缺失将导致胚胎致死，我们建议精确定义的发育过程中，改变了硒蛋白的情况下。重要的是，这种方法将允许解决长期寻求的SBP 2的N-末端一半的功能，这是脊椎动物特异性的，没有已知的功能。最后，作为研究硒蛋白在斑马鱼中的基本功能的初步尝试，我们建议研究巨噬细胞的功能，以确定硒蛋白在巨噬细胞迁移中所起作用的基础。

 DESCRIPTION (provided by applicant): Selenium is an essential trace element that is incorporated into 25 human proteins as the amino acid selenocysteine (Sec). Interestingly, the co-translational incorporation of selenocysteine occurs at in-frame stop codons (UGA) that are found upstream of RNA structures known as selenocysteine insertion sequence (SECIS) elements, which reside in the 3' UTR of all selenoprotein mRNAs. We have recently demonstrated that the factors known to be essential for Sec incorporation are, in fact, sufficient. These factors include a specialized elongation factor (eEFSec), a SECIS binding protein (SBP2), and a specialized transfer RNA (Sec-tRNASec). Significant progress has been made regarding which protein domains are important for Sec incorporation activity, but the mechanism by which these factors allow Sec incorporation has not been subjected to study in vivo, particularly in the context of embryonic development. In addition, an SBP2 paralogue exists, termed SECISBP2L, but it does not participate in Sec incorporation in vitro and its function remains unknown. As such, the study of selenoprotein synthesis and function is still an emerging field and requires the development of novel and affordable model systems. Here we propose to develop a zebrafish model system in which to study both the mechanism of Sec incorporation as well as selenoprotein function. The use of zebrafish is warranted for four major reasons: 1) the factors required for Sec incorporation are conserved in all vertebrates; 2) the complement of selenoproteins in fish is very similar to that in humans; 3) the cost of developing fish models is vastly lower than that in mice and 4) as a tractable model for vertebrate development, the use of zebrafish will allow unprecedented access to the mechanisms by which selenoprotein function during early development. In the first Aim we will lay the groundwork by investigating expression dynamics and developmental profile for Sec incorporation factors, after which we will determine the function of the SBP2 paralogue, SECISBP2L. Although SECISBP2L was identified at the same time as SBP2, its function has remained elusive. Our preliminary data derived from embryos injected with anti SECISBP2L morpholino oligonucleotides suggests that it may play a role in hematopoeisis and/or vascular development. In the second aim, we propose to model human disease in fish by introducing specific mutations in endogenous SBP2 and determining the effects on selenoprotein production. In addition, although we expect that SBP2 deletion will result in embryonic lethality, we propose to precisely define the developmental process that is altered in the absence of selenoproteins. Importantly, this methodology will allow the resolution of the long-sought function for the N-terminal half of SBP2, which is vertebrate-specific and has no known function. Finally, as an initial foray into studying the basic functions of selenoproteins in zebrafish, we propose to study macrophage function in order to determine the underlying basis for the role that selenoproteins are known to play in macrophage migration.