COMPLEX INVOLVED IN MRNA DECAY IN YEAST

参与酵母 mRNA 衰变的复合物

• 批准号: 6384329
• 负责人: STUART W PELTZ
• 金额: $ 27.27万
• 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/6384329
• 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.

我们研究的目标是了解调控真核生物细胞质mRNA衰退的分子机制。改变对mRNA稳定性的控制可能会导致基因调控异常，从而导致疾病。我们的研究主要集中在酿酒酵母中涉及mRNA周转的顺式作用元件和反式作用因子的特征。通过许多实验室的努力，现在清楚的是，在哺乳动物和酵母细胞中，许多转录本的聚(A)缩短和/或5‘帽结构的水解是控制给定转录本稳定性的重要速率决定事件。基于这些结果，这项拨款计划中实验的目标是识别和表征调控酵母去壳活性的基因及其产物。为了做到这一点，开发了一种生物化学筛查来监测细胞提取物中的脱壳活性。从一组对温度敏感的菌株中提取的提取物进行了筛选，以确定是否降低了脱壳活性。这项分析成功地确定了调节去顶活动的因素。结果表明，VPS16、MRT1和热休克蛋白SSA1/SSA2编码的蛋白可能是脱壳活性的调节蛋白，而Ssa1p/2p是脱壳酶的直接影响因子。Ssa1p/2p调节脱壳活性的能力取决于反应中是否存在ADP。此外，有证据表明，Dcp1p蛋白可能是一个降解体复合体的一部分，该复合体至少由Dcp1p、Xrn1p、Vps16p、mRt1p和热休克蛋白Ssa1p/2p组成。我们还鉴定了一个dcp2等位基因，它可能编码一种可诱导的剥离活动。基于这些结果，资助提案的目的是：1)表征胞质Hsp70p及其调节因子在控制mRNA周转中的作用；2)。“降解体复合体”的生物化学特征；c)与降解体复合体有关的个别因素的分子、遗传和生物化学特征。