The NMD pathway in RNA surveillance and gene expression

大多数非义mRNA是高度不稳定的，因为它们是通过一种称为非义介导的mRNA衰变（NMD）的衰变途径降解的。监测mrna以消除那些编码潜在有害蛋白质片段的过程称为RNA监测。监测发生在真菌、植物、线虫和包括人类在内的脊椎动物中。催化NMD步骤的蛋白质被称为Upf蛋白，有两个作用，一个是监测基因表达中的错误，另一个是控制内源性野生型mrna的丰度，作为正常基因表达库的一部分。对NMD所需蛋白质的研究很有希望告诉我们基因表达中的home错误是如何被监测的，以及基因表达是如何被新的转录后机制控制的。NMD途径对人类中数百种遗传疾病有直接影响，其中四分之一到三分之一的导致遗传性遗传疾病和各种形式癌症的突变是可以针对NMD的相应mRNA的类型。本研究的主要目的是了解NMD的机制，NMD与翻译之间的联系，以及NMD在RNA监测和基因表达中的作用。酿酒酵母将被开发为适用于复杂真核生物的模型系统。我们将确定Upf2和Upf3在募集无义mrna进入NMD通路中的作用。我们提出的假设是，招募需要在细胞核和细胞质之间穿梭Upf3p和可能的Upf2p来标记细胞核中的无义mrna，以便随后在细胞质中衰变。我们将研究两个新发现的基因中的upf样抑制因子。EGS1基因中的这些抑制突变残基之一，由于其在翻译中的潜在作用和其表达受NMD途径控制而引起了人们的兴趣。我们对NMD机制的假设是，Upf3p首先与细胞核中的mRNP颗粒结合，然后作为mRNP结合蛋白出口到细胞质。我们将通过使用栓系分析、mRNP输出的遗传研究和mRNP的生化分析来解决这个模型的各个方面。最后，我们将研究Upf蛋白在全局基因表达中的作用。利用DNA阵列，我们已经确定了500多个表达受Upf蛋白影响的基因。我们将确定目标，区分UPF基因的直接和间接目标，建立靶向机制，并表征UPF菌株的全球基因表达谱。

Most non-sense mRNAs are highly unstable because they are degraded by a decay pathway called nonsense-mediated mRNA decay (NMD). The process whereby mRNAs are monitored to eliminate those that code for potentially deleterious protein fragments is called RNA surveillance. Surveillance occurs in fungi, plants, nematodes, and vertebrates including humans. The proteins that catalyze steps in NMD, called Upf proteins, serve two roles, one to monitor errors in gene expression and the other to control the abundance of endogenous wild-type mRNAs as part of the normal repertoire of gene expression. Studies of the proteins required for NMD hold great promise for telling us home errors in gene expression are monitored and how gene expression is controlled by noel post-transcriptional mechanisms. The NMD pathway has a direct impact on hundreds of genetic disorders in the human population, where one fourth to one third of all mutations causing inherited genetic disorders and various forms of cancer are of the type that could target the corresponding mRNA for NMD. The primary thrust of this proposal is to understand the mechanism of NMD, the connection between NMD and translation, and the role of NMD in RNA surveillance and gene expression. The yeast Saccharomyces cerevisiae will be exploited as a model system applicable to complex eukaryotes. We will establish the role of Upf2 and Upf3 in the recruitment of nonsense mRNAs into the NMD pathway. The hypothesis we have put forward is that recruitment requir5es the shuttling Upf3p and possibly Upf2p between the nucleus and cytoplasm to mark nonsense mRNAs in the nucleus for subsequent decay in the cytoplasm. We will examine Upf-like suppressors in two new genes that have been identified. One of these suppressor mutations residues in the EGS1 gene, which is of interest because of its potential role in translation and because its expression is controlled by the NMD pathway. Our hypothesis for the mechanism of NMD is that Upf3p acts first by binding to mRNP particles in the nucleus after which it exports to the cytoplasm as an mRNP-bound protein. We will address various aspects of this model by using tethering assays, genetic studies of mRNP export, and biochemical analysis of mRNP's. Finally, we will examine the role of Upf proteins in global gene expression. Using DNA arrays, we have identified over 500 genes whose expression is affected by the Upf proteins. We will identify targets and distinguish direct from indirect targets of the UPF genes, establish mechanisms of targeting, and characterize the global gene expression profile for uPF-strains.