Development of a zebrafish model for selenoprotein synthesis and function

 描述(申请人提供)：硒是一种必需的微量元素，以氨基酸硒半胱氨酸(SEC)的形式被结合到25种人类蛋白质中。有趣的是，硒半胱氨酸的共翻译掺入发生在框内终止密码子(UGA)，该密码子位于RNA结构的上游，称为硒半胱氨酸插入序列(SECIS)元件，位于所有硒蛋白mRNAs的3‘UTR中。我们最近已经证明，已知的对证券交易委员会成立至关重要的因素实际上是充分的。 这些因子包括特化延伸因子(EEFSec)、SECIS结合蛋白(SBP2)和特化转移RNA(SEC-tRNASec)。关于哪些蛋白质结构域对SEC掺入活性很重要，已经取得了重大进展，但这些因素允许SEC掺入的机制还没有在体内进行研究，特别是在胚胎发育的背景下。此外，存在一个SBP2类似物，称为SECISBP2L，但它不参与SEC的体外掺入，其功能尚不清楚。因此，对硒蛋白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，我们建议建立一个斑马鱼模型系统，在其中既研究SEC的掺入机制，又研究硒蛋白的功能。有理由使用斑马鱼有四个主要原因：1)所有脊椎动物中SEC掺入所需的因子都是保守的；2)鱼中硒蛋白的组成与人类非常相似；3)建立鱼类模型的成本远低于小鼠；4)作为脊椎动物发育的易驯化模型，使用斑马鱼将使人们能够前所未有地了解硒蛋白在早期发育中的作用机制。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，然后我们将确定SBP2类似物SECISBP2L的功能。尽管SECISBP2L与SBP2同时被鉴定，但其功能仍不清楚。我们在胚胎中注射抗SECISBP2L吗啉寡核苷酸的初步数据表明，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SBP2中引入特定突变并确定其对硒蛋白生产的影响来在鱼类中模拟人类疾病。此外，尽管我们预计SBP2缺失会导致胚胎死亡，但我们建议精确定义在没有硒蛋白的情况下发生变化的发育过程。重要的是，这一方法将能够解决SBP2的N-末端一半的长期寻找的功能，它是脊椎动物特有的，没有已知的功能。最后，作为研究斑马鱼中硒蛋白基本功能的初步尝试，我们建议研究巨噬细胞的功能，以确定已知的硒蛋白在巨噬细胞迁移中所起作用的潜在基础。

 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.