COMPLEX INVOLVED IN MRNA DECAY IN YEAST

参与酵母 mRNA 衰变的复合物

• 批准号: 2849128
• 负责人: STUART W PELTZ
• 金额: $ 27.02万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2849128
• 关键词: RNA binding protein; Saccharomyces cerevisiae; adenosine diphosphate; fungal genetics; fungal proteins; genetic strain; immunofluorescence technique; messenger RNA; molecular cloning; nucleic acid metabolism; phosphorylation; polysomes; posttranscriptional RNA processing; protein protein interaction; protein purification; site directed mutagenesis; stress proteins; western blottings; yeast two hybrid system

The goal of our investigations is the understanding of the molecular mechanisms that regulate cytoplasmic mRNA decay in eucaryotes. Altered control of mRNA stability can lead to aberrant gene regulation and, consequently to disease. Our studies have focused on the characterization of the cis-acting elements and trans-acting factors involved in mRNA turnover in the yeast Saccharomyces cerevisiae. Through the efforts of a number of laboratories it is now clear that poly(A) shortening and/or hydrolysis of the 5' cap structures for many transcripts are important rate determining events in controlling the stability of a given transcript in both mammalian and yeast cells. Based on these results, the goal of the experiments in this grant proposal is the identification and characterization of the genes and their products that regulate decapping activity in yeast. To accomplish this, a biochemical screen was developed to monitor decapping activity in cell extracts. Extracts prepared from a collection of temperature-sensitive strains were screened for reduced decapping activity. This analysis successfully identified factors that modulate decapping activity. The results demonstrated that proteins encoded by the VPS16, MRT1 and the heat shock protein SSA1/SSA2 appear to be regulators of the decapping activity, with Ssa1p/2p being a direct effector of the decapping enzyme. The ability of Ssa1p/2p to modulate decapping activity depends on whether ADP is present in the reaction. Furthermore, evidence is presented suggesting that the Dcp1p protein may be part of a degradosome complex consisting of, at a minimum, Dcp1p, Xrn1p, Vps16p, Mrt1p and the heat shock protein Ssa1p/2p. We have also identified a dcp2 allele that may encode an inducible decapping activity. Based on these results, the aims of the grant proposal are; 1) Characterization of the role cytosolic Hsp70p and its regulators in controlling mRNA turnover; 2). Biochemical characterization of the "degradosome complex"; C) Molecular, genetic and biochemical characterization of the individual factors that are associated with the degradosome complex.

我们研究的目的是了解调节桉树中细胞质mRNA衰变的分子机制。 对mRNA稳定性的控制改变会导致基因调节异常，从而导致疾病。 我们的研究集中于酿酒酵母中的顺式作用元件和涉及mRNA转换的跨作用因子的表征。 通过许多实验室的努力，现在很明显，多（a）对许多转录本的5'帽结构的缩短和/或水解是确定在控制哺乳动物和酵母细胞中给定转录物稳定性的事件的重要速率。 基于这些结果，该赠款提案中实验的目标是对基因及其产物的识别和表征，这些基因及其产物调节酵母中的dec依活动。 为此，开发了一个生化筛选，以监视细胞提取物中的替代活动。 筛选从温度敏感性菌株的集合中制备的提取物，以减少脱皮活性。 该分析成功地识别了调节decap灭活动的因素。 结果表明，由VPS16，MRT1和热休克蛋白SSA1/SSA2编码的蛋白质似乎是decapping活性的调节剂，而SSA1P/2P是Decapping酶的直接效应。 SSA1P/2P调节脱皮活性的能力取决于反应中是否存在ADP。此外，提供了证据表明DCP1P蛋白可能是降解体复合物的一部分，该复合物至少包括DCP1P，XRN1P，VPS16P，MRT1P和热震蛋白SSA1P/2P。 我们还确定了一个可能编码诱导式替代活动的DCP2等位基因。 基于这些结果，赠款提案的目的是： 1）表征胞质HSP70P的角色及其在控制mRNA周转方面的调节剂； 2）。 “降解体复合物”的生化表征； c）与降解体复合物相关的各个因素的分子，遗传和生化表征。