The NMD pathway in RNA surveillance and gene expression

RNA 监测和基因表达中的 NMD 通路

• 批准号: 6622911
• 负责人: MICHAEL R CULBERTSON
• 金额: $ 30.2万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6622911
• 关键词: Saccharomyces cerevisiae; antisense nucleic acid; binding sites; density gradient ultracentrifugation; eukaryote; fluorescent in situ hybridization; fungal genetics; gene expression; genetic translation; immunofluorescence technique; messenger RNA; microarray technology; molecular cloning; northern blottings; nuclear transfer; posttranscriptional RNA processing; protein protein interaction; protein structure function; receptor binding; suppressor mutations; western blottings; yeast two hybrid system

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.

大多数无义mRNA是高度不稳定的，因为它们通过称为无义介导的mRNA衰变（NMD）的衰变途径降解。监测mRNA以消除那些编码潜在有害蛋白质片段的mRNA的过程称为RNA监视。监测发生在真菌，植物，线虫和脊椎动物，包括人类。在NMD中催化步骤的蛋白质，称为Upf蛋白，起两个作用，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，作为基因表达正常库的一部分。对NMD所需蛋白质的研究有很大的希望告诉我们基因表达中的家庭错误是监测的，以及基因表达是如何由诺埃尔转录后机制控制的。NMD通路对人类群体中的数百种遗传疾病具有直接影响，其中导致遗传性遗传疾病和各种形式的癌症的所有突变的四分之一到三分之一是可以靶向NMD的相应mRNA的类型。该计划的主要目的是了解NMD的机制，NMD和翻译之间的联系，以及NMD在RNA监视和基因表达中的作用。酵母酿酒酵母将被开发作为一个模型系统适用于复杂的真核生物。我们将确定Upf2和Upf3在无义mRNA募集到NMD通路中的作用。我们提出的假设是，募集需要Upf3p和可能的Upf2p在细胞核和细胞质之间穿梭，以标记细胞核中的无义mRNA，以便随后在细胞质中衰变。我们将在两个新的基因中研究Upf样抑制因子。其中一个抑制突变残基在EGS1基因，这是感兴趣的，因为它在翻译中的潜在作用，因为它的表达是由NMD途径控制。我们对NMD机制的假设是Upf3p首先通过结合细胞核中的mRNP颗粒起作用，然后作为mRNP结合蛋白输出到细胞质。我们将通过使用系链分析、mRNP输出的遗传研究和mRNP的生化分析来解决该模型的各个方面。最后，我们将研究Upf蛋白在全球基因表达中的作用。使用DNA阵列，我们已经确定了超过500个基因，其表达受到Upf蛋白的影响。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。