The Role of Phosphorylation in the NMD RNA Surveillance Mechanism

摘要 三分之一的人类遗传性疾病是由携带提前终止密码子的mRNA引起的 （PTC）作为无义或移码突变的结果。这些类型的突变引起异常的 被无义介导的mRNA衰变（NMD）途径识别并快速降解的转录物。 这种RNA监视途径是重要的，因为它大大减少了截短蛋白质的合成，其中一些截短蛋白质的合成是通过RNA监视途径进行的。 其具有有害的功能获得或显性负效应。此外，最近它还成为 很明显，NMD调节约5%的正常基因的转录。这表明，NMD不仅是一个 RNA监视途径，而且还在基因表达中起调节作用。参与的核心基因 NMD（UPF1、UPF2和LJPF3）首先在酿酒酵母中鉴定。这些基因的直系同源物 在秀丽隐杆线虫、黑腹果蝇和人类中也被发现，这表明， NMD是真核生物中高度保守的RNA监视机制。虽然越来越多的证据表明， NMD的生理相关性，这种RNA监视的潜在机制和调节 对这一途径仍然知之甚少。一个刚刚开始处理的特别重要的问题是 磷酸化在NMD中的作用。许多研究提供了证据表明，磷酸化和 RNA解旋酶UPF 1的去磷酸化在许多生物体中的NMD中起作用。然而，功能 UPF1中磷酸化的残基和UPF1磷酸化的生物化学相关性， 去磷酸化在任何生物体中都没有被清楚地阐明。同样，UPF2已被证明是 磷酸化，但不知道为什么。本提案通过确定和功能测试 UPF1和UPF2磷酸化的保守位点，使用分子、生物化学和 酿酒酵母和哺乳动物细胞系的遗传方法。更好地理解 NMD通路可能允许发现调节异常蛋白的稳定性和翻译的方法。 mRNA作为对抗癌症和其他由无义和移码引起的人类遗传疾病的手段 突变。

Abstract One third of inherited human genetic diseases are caused by mRNAs harboring premature termination codons (PTCs) as a result of nonsense or frameshift mutations. These types of mutations give rise to aberrant transcripts that are recognized and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. This RNA surveillance pathway is important, as it greatly reduces the synthesis of truncated proteins, some of which possess deleterious gain-of-function or dominant-negative effects. In addition, recently it has become clear that NMD regulates transcripts from about 5% of normal genes. This suggests that NMD is not only an RNA surveillance pathway but also performs a regulatory role in gene expression. The core genes involved in NMD (UPF1, UPF2, and LJPF3) were first identified in Saccharomyces cerevisiae. Orthologues of these genes have also been identified in Caenorhabditis elegans, Drosophila melanogaster, and humans, suggesting that NMD is a highly conserved RNA surveillance mechanism in eukaryotes. While there is increasing evidence for the physiological relevance of NMD, the underlying mechanism and regulation of this RNA surveillance pathway remains poorly understood. A particularly important issue that has only begun to be addressed is the role of phosphorylation in NMD. Numerous studies have provided evidence that both the phosphorylation and dephosphorylation of the RNA helicase UPF1 have a role in NMD in many organisms. However, the functional residues phosphorylated in UPF1 and the biochemical relevance of UPF1 phosphorylation and dephosphorylation has not been clearly elucidated in any organism. Likewise, UPF2 has been shown to be phosphorylated but it is not known why. This proposal fills this gap by identifying and functionally testing the conserved sites of UPF1 and UPF2 phosphorylation using a combination of molecular, biochemical, and genetic approaches in Saccharomyces cerevisiae and mammalian cell lines. A better understanding of the NMD pathway may permit the discovery of approaches to modulate the stability and translation of aberrant mRNAs as a means to combat cancer and other human genetic disorders caused by nonsense and frameshift mutations.