Complex Involved In mRNA Decay in Yeast

描述（由申请人提供）：mRNA的衰变是基因表达转录后调控的核心。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，在mRNA转换水平上通过富au元件（AREs）在其3'非翻译区进行调节。因此，对这一过程的研究为一些癌症和免疫疾病中细胞机制的失调提供了有价值的见解。本课题旨在以酵母（Saccharomyces cerevisiae）为模型系统，研究are介导的mRNA衰变现象。mRNA转换的酶和途径在这种生物体中被很好地表征。酵母中arem介导的mRNA衰变的调节发生在对至少两种细胞刺激的响应中，涉及三个已确定的因素；public， Cthlp和Cth2p。本提案的目标是确定(i) are结合复合体如何调节mRNA衰减率（ii） are结合复合体在响应细胞刺激时被调节，以及（iii） are上组装的因子复合体被指定其序列上下文。该提案的第一部分集中在三个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。一个重要的方面集中在剖析Cth蛋白和Pablp之间的相互作用。此外，本目的实验将确定这些因子如何在响应细胞条件下进行翻译后修饰。该提案的目的二是分离在体内使用RNA标签和蛋白质标签形成的ARE-binding复合物。总之，这部分研究的结果将确定are结合复合物的新成分，并使我们深入了解这些复合物是如何响应细胞条件而改变的。该提案的最终目的是通过微阵列分析缺乏已知的are结合因子的菌株的mRNA衰变率，以及通过搜索Transterm数据库发现的候选ARE-containing转录本来鉴定酵母中新的富含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性，从而可能指定与它们相互作用的蛋白质及其调控。

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.