The Role of Phosphorylation in the NMD RNA Surveillance Mechanism

摘要 三分之一的遗传性人类遗传病是由含有提前终止密码子的mRNAs引起的。 (PTCS)是由于无意义或移码突变所致。这些类型的突变会导致异常 被无义介导的信使核糖核酸衰变(NMD)途径识别并迅速降解的转录产物。 这种RNA监测途径很重要，因为它极大地减少了截短蛋白质的合成，其中一些 具有有害的功能增益或显性负效应。此外，最近它已经变成了 显然，NMD调节大约5%正常基因的转录本。这表明，NMD不仅是一种 RNA监视途径还在基因表达中起着调节作用。涉及到的核心基因 NMD(UPF1、UPF2和LJPF3)是在酿酒酵母中首次发现的。这些基因的同源基因 在秀丽线虫、黑腹果蝇和人类中也被发现，这表明 NMD是真核生物中一种高度保守的RNA监测机制。虽然有越来越多的证据表明 NMD的生理相关性、这种RNA监测的潜在机制和调节 这条途径仍然知之甚少。一个特别重要的问题才刚刚开始解决，那就是 磷酸化在NMD中的作用。许多研究都提供了证据，证明磷酸化和 RNA解旋酶UPF1的去磷酸化在许多生物体的NMD中起作用。然而，功能性的 UPF1的磷酸化残基和UPF1磷酸化与生化相关性 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，但原因尚不清楚。该提案通过确定并在功能上测试 UPF1和UPF2磷酸化的保守位点结合分子、生化和 酿酒酵母和哺乳动物细胞系的遗传途径。更好地理解 NMD途径可能允许发现调节异常的稳定性和翻译的方法 作为对抗癌症和其他由废话和移码引起的人类遗传疾病的手段的mRNAs 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.