Mechanisms of 3'UTR Control: Yeast PUF Proteins

描述(由申请人提供)： PUF蛋白是转录后抑制因子，控制着发育和分化、线粒体功能、细胞生长和寿命等不同的过程。这些序列特异的RNA结合蛋白与靶mRNAs的3‘非翻译区(3’UTRs)的调控序列结合，以抑制基因表达。PUF蛋白的抑制机制尚不清楚。我们的工作假设是，PUF蛋白结合到一个mRNA的3‘非编码区，并招募基本的mRNA周转机制来降解该mRNA。我们选择了酵母PUF5蛋白作为一个模型系统来确定PUF蛋白的功能。Puf5p结合并抑制参与交配类型转换的HO内切酶mRNA的表达。我们建议使用双杂交筛选来鉴定Puf5p和酵母蛋白之间的蛋白质-蛋白质相互作用。此外，Puf5p蛋白复合体的生化纯化和蛋白质显微测序将用于鉴定Puf5p相关的蛋白质复合体。我们将通过三杂交蛋白质-RNA相互作用筛选来鉴定与Puf5p一起组装在HO mRNA的3‘UTR上的调节蛋白复合体的成分。体内功能分析将被用来确定Puf5p介导的抑制所需的基因，并确定由Puf5p控制的mRNA周转途径。最后，将使用体外mRNA转换实验进一步剖析Puf5p抑制的机制。

DESCRIPTION (provided by applicant): PUF proteins are post-transcriptional repressors that control diverse processes such as development and differentiation, mitochondrial function, cellular growth, and lifespan. These sequence specific RNA-binding proteins bind to regulatory sequences in the 3' untranslated regions (3'UTRs) of target mRNAs to repress gene expression. The mechanism of repression by PUF proteins is not known. Our working hypothesis is that PUF proteins bind to the 3'UTR of an mRNA and recruit the basal mRNA turnover machinery to degrade that mRNA. We have chosen the yeast PUF5 protein as a model system for determining how PUF proteins function. Puf5p binds to and represses expression of the HO endonuclease mRNA, which is involved in mating type switching. We propose to identify protein-protein interactions between the Puf5p and yeast proteins using a two-hybrid screen. Also, biochemical purification of Puf5p protein complexes and protein microsequencing will be used to identify Puf5p-associated protein complexes. We will identify the components of the regulatory protein complex that assembles on the 3'UTR of the HO mRNA with Puf5p by using a three-hybrid protein-RNA interaction screen. An in vivo functional assay will be used to identify genes that are required for Puf5p-mediated repression and to identify the pathway of mRNA turnover that is controlled by Puf5p. Finally, an in vitro mRNA turnover assay will used to further dissect the mechanism of Puf5p repression.