Complex Involved In mRNA Decay in Yeast

参与酵母 mRNA 衰变的复合物

• 批准号: 6941767
• 负责人: STUART W PELTZ
• 金额: $ 30.81万
• 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 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6941767
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; adenosine diphosphate; binding sites; fungal genetics; genetic transcription; immunoprecipitation; messenger RNA; microarray technology; nucleic acid metabolism; nucleic acid sequence; phosphorylation; posttranscriptional RNA processing; posttranslational modifications; protein protein interaction; stimulus /response; stress proteins; translation factor

DESCRIPTION (provided by applicant): The decay of mRNA is central to the post-transcriptional regulation of gene expression. The expression of many clinically relevant genes, including cytokines, proto-oncogenes and growth factors, is regulated at the level of mRNA turnover through AU-rich elements (AREs) in their 3' untranslated regions. Therefore, studies of this process provide valuable insight into the deregulation of cellular mechanisms in some cancers and immune disorders. This proposal aims to use the yeast, Saccharomyces cerevisiae, as a model system to study the phenomenon of ARE-mediated mRNA decay. The enzymes and pathways of mRNA turnover are well characterized in this organism. Regulation of AREmediated mRNA decay in yeast occurs in response to at least two cellular stimuli and involves three identified factors; Publp, Cthlp and Cth2p. The goals of this proposal are to determine how (i) the ARE-binding complex regulates mRNA decay rates (ii) the ARE-binding complex is modulated in response to cellular stimuli and (iii) the complex of factors that assembles on the ARE is specified its sequence context. The first part of the proposal concentrates on characterization of the three ARE-binding proteins and the interactions among them and between them and the ARE. One important aspect focuses on dissecting the interaction between the Cth proteins and Pablp. In addition, experiments in this Aim will determine how these factors are post-translationally modified in response to cellular conditions. Aim II of the proposal is to isolate ARE-binding complexes formed in vivo using both RNA tags and protein tags. Together the results of this part of the study will identify novel components of the ARE-binding complex and give us insight into how these complexes are altered in response to cellular conditions. The final Aim of the proposal is to identify novel AU-rich elements in yeast through microarray analysis of mRNA decay rates in strains lacking the known ARE-binding factors and by analysis of candidate ARE-containing transcripts discovered by searching the Transterm database. The goal is to amass a set of AU-rich elements large enough to facilitate computer analysis of the sequences to identify commonalities between them that might specify the proteins they interact with and their regulation.

描述(申请人提供)：信使核糖核酸的衰变是基因表达转录后调控的中心。许多临床相关基因的表达，包括细胞因子、原癌基因和生长因子，都是通过其3‘非翻译区富含AU的元件(Ares)在mRNA周转水平上进行调节的。因此，对这一过程的研究为一些癌症和免疫疾病的细胞机制的放松提供了有价值的见解。这项建议旨在利用酿酒酵母作为模型系统来研究ARE介导的mRNA衰退现象。在这种生物体中，mRNA周转的酶和途径被很好地描述。酵母中ARE介导的mRNA衰退的调节发生在至少两个细胞刺激的反应中，涉及三个已确定的因子：Publp、Cthlp和Cth2p。本提案的目的是确定(I)ARE结合复合体如何调节mRNA衰减率(Ii)ARE结合复合体响应细胞刺激而被调制，以及(Iii)聚集在ARE上的因子复合体是其特定的序列背景。该提案的第一部分集中于三种ARE结合蛋白的特征以及它们之间以及它们与ARE之间的相互作用。一个重要的方面是解剖Cth蛋白和Pablp之间的相互作用。此外，这个目标的实验将确定这些因子是如何在翻译后修改以响应细胞条件的。该提案的目的II是使用RNA标签和蛋白质标签分离在体内形成的ARE结合复合体。总之，这部分研究的结果将确定ARE结合复合体的新成分，并使我们深入了解这些复合体是如何随着细胞条件的变化而改变的。该提案的最终目的是通过对缺乏已知ARE结合因子的菌株的mRNA衰减率的微阵列分析，以及通过搜索Transterm数据库发现的含有ARE的候选转录本的分析，在酵母中鉴定新的富含AU的元素。目标是积累一组足够大的富含AU的元素，以便于对序列进行计算机分析，以确定它们之间的共同点，这些共同点可能指定它们相互作用的蛋白质及其调控。