Investigation into the role of Secisbp2 in re-coding of UGA codons in selenoproteins by global ribosomal footprinting

硒蛋白对哺乳动物来说是必不可少的。它们含有稀有的氨基酸硒半胱氨酸(SEC)。SEC是已知的第一个遗传密码的自然扩展。在蛋白质中加入SEC需要对UGA(终止)密码子进行重新编码。对于重新编码，硒蛋白mRNAs在3(主)非翻译区包含硒半胱氨酸插入序列(SECIS)元件。人类SECIS结合蛋白2(SECISBP2)的突变导致硒蛋白生物合成的先天性缺陷，导致神经、内分泌和免疫症状。在第一个资助期，我们已经建立并研究了带有Secisbp2突变的小鼠模型。我们发现Secisbp2不仅刺激UGA重新编码，而且通过稳定硒蛋白mRNAs而具有意想不到的功能。在下一个资助期，我们将利用核糖体图谱，在全球范围内以不同寻常的细节分析硒蛋白的生物合成。我们将遵循四个目标：(1)确定在不存在Secisbp2的情况下UGA密码子的重编码效率。利用我们的新小鼠模型，我们将确定Secisbp2对UGA翻译为SEC的影响。(2)Secisbp2(针对SID结构域或L7Ae结构域)的点突变是否会改变UGA的重新编码效率？这种方法将使我们能够理解病原点突变是如何影响Secisbp2功能的。(3)停止并不等于停止：分别缺乏Secisbp2或tRNA(SEC)的停滞核糖体在UGA重新编码和无意义介导的衰变和No-Go衰变等mRNA监控途径方面有什么不同？(4)如果缺少Secisbp2或tRNA(SEC)，那么mRNA降解的确切机制是什么？这个问题将通过在肝细胞模型中进行遗传操作来解决。我们期望获得对当前非规范氨基酸翻译问题的基本见解，以及哺乳动物中不去腐烂的正常作用。

Selenoproteins are essential for mammals. They contain the rare amino acid selenocysteine (Sec). Sec is the first known natural expansion of the genetic code. Incorporation of Sec in proteins requires the re-coding of a UGA (stop) codon. For re-coding, selenoprotein mRNAs contain the selenocysteine insertion sequence (SECIS) element in the 3(prime)-untranslated region. Mutations in SECIS-binding protein 2 (SECISBP2) in human lead to congenital defects of selenoprotein biosynthesis entailing neurological, endocrinological, and immunological symptoms. In the first funding period, we have generated and studied mouse models with mutations in Secisbp2. We have found that Secisbp2 does not only stimulate UGA re-coding, but carries an unexpected function by stabilizing selenoprotein mRNAs. In the next funding period, we will analyze selenoprotein biosynthesis on a global scale in unusual detail by using Ribosomal Profiling. We will follow four aims: (1) Determine the re-coding efficiency at UGA codons in the presence of not of Secisbp2. Using our novel mouse models, we will determine the influence of Secisbp2 on translation of UGA as Sec. (2) Do point mutations in Secisbp2 (targeting the SID-domain or L7Ae domain) alter the re-coding efficiency at UGA? This approach will allow us to understand how pathogenic point mutations affect Secisbp2 functions. (3) STOP does not equal STOP: What is the difference between stalling ribosomes lacking Secisbp2 or tRNA(Sec), respectively, with respect to UGA re-coding and mRNA surveillance pathways like Nonsense-Mediated Decay and No-Go Decay? (4) What is the exact mechanism of mRNA degradation, if Secisbp2 or tRNA(Sec) is lacking? This question will be approached by genetic manipulation in a hepatocyte cell model. We expect to gain fundamental insights into current questions of translation of non-canonical amino acids as well as into the normal role of No-Go Decay in mammals.