Mechanisms of 3'UTR Control: Yeast PUF Proteins

描述（由申请人提供）： PUF蛋白是转录后阻遏物，其控制多种过程，例如发育和分化、线粒体功能、细胞生长和寿命。这些序列特异性RNA结合蛋白与靶mRNA的3'非翻译区（3' UTR）中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mRNA的3 'UTR，并招募基础mRNA周转机制来降解该mRNA。我们选择酵母PUF 5蛋白作为模型系统，用于确定PUF蛋白如何发挥功能。Puf 5 p结合并抑制HO内切核酸酶mRNA的表达，其参与交配型转换。我们建议使用双杂交筛选鉴定Puf 5 p和酵母蛋白之间的蛋白质-蛋白质相互作用。此外，Puf 5 p蛋白复合物的生化纯化和蛋白质微测序将用于鉴定Puf 5 p相关蛋白复合物。我们将通过使用三杂交蛋白-RNA相互作用筛选来鉴定与Puf 5 p组装在HO mRNA的3 'UTR上的调节蛋白复合物的组分。体内功能测定将用于鉴定Puf 5 p介导的抑制所需的基因，并鉴定由Puf 5 p控制的mRNA周转途径。最后，将使用体外mRNA周转测定来进一步剖析Puf 5 p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.