Molecular mechanism of mRNA 3'-end formation in yeast

描述(由申请人提供)：项目摘要：真核细胞中信使核糖核酸的合成及其在细胞质中的应用，需要在核糖核酸的3‘端添加聚(A)尾进行修饰。这个过程也是一个点，在这个点上，细胞可以调节来自特定基因的mRNA的类型和数量。尽管mRNA3‘末端的形成发生在一个出人意料的大复合体中，但这一机制的大部分，如果不是全部的亚基，已经在酵母中被鉴定出来。然而，关于这21种蛋白质如何相互协作以确保加工准确以及与mRNA合成和包装中的其他事件及时耦合，人们知之甚少。随着3‘端加工组件的问世，以及对其中几个因子的活性的新定义，现在存在着一个独特的机会来严格解决基因表达中这一基本和普遍步骤发生和调控的机制。这一提议的中心假设是，当评估加工位点的真实性时，蛋白质伙伴在复合体内发生可定义的重排，承诺在聚(A)位点切割，重组定位聚(A)聚合酶用于尾部合成，并释放最终的RNA产物。目的是了解这种重组是如何驱动mRNA 3‘末端加工的循环的。这项研究的重点是四个事件，它们可能是本轮周期中的关键转折点，但其潜在机制尚不清楚。这些包括切割的启动和Ssuy2蛋白在这一步骤中的作用，从切割到聚(A)加成的转变及其通过PtaI支架蛋白的磷酸化来调节，通过其氨基末端的相互作用控制聚(A)聚合酶的活性，以及在尾部合成后加工因子的释放。这些研究将使用酵母作为模式生物，因为它易于将标签和突变引入基因组，大量3‘端加工突变体的可获得性，用于生化研究的加工因子易于纯化，以及真核生物中3’端加工机制的高度保守。相关性：如果没有Poly(A)尾巴，mRNA的目标是在细胞核中降解，它不能很好地离开细胞核，它不能有效地翻译或在细胞质中以适当的速度翻转。M RNA 3‘端的成熟在功能上与转录、m RNA输出、染色体分离、DNA修复和组织特异性蛋白表达等其他基本过程有关。聚腺苷酸化过程中的错误可能会影响所有这些过程。这项拟议的研究将极大地促进我们对mRNA合成这一关键步骤的动态的洞察，并确定构成过程可能受到调控的点。

DESCRIPTION (provided by applicant): Project summary: Synthesis of mRNA in eukaryotes, and its utilization in the cytoplasm, requires modification at the RNA's 3' end by addition of a poly(A) tail. This process also serves as a point at which the cell can regulate the type and amount of mRNA derived from a particular gene. Even though mRNA 3' end formation occurs in an unexpectedly large complex, most, if not all, of the subunits of this machinery have been identified in the yeast S. cerevisiae. However, little is known about how these 21 proteins cooperate with each other to insure processing that is accurate as well as coupled in a timely fashion to other events in mRNA synthesis and packaging. With the 3'-end processing components in hand, and activities for several of these factors newly defined, a unique opportunity now exists to rigorously address the mechanism by which this essential and universal step in gene expression occurs and is regulated. The central hypothesis of this proposal is that definable rearrangements of protein partners occur within the complex as it evaluates the authenticity of the processing site, commits to cleavage at the poly(A) site, reorganizes to position the poly(A) polymerase for tail synthesis, and releases the final RNA product. The objective is to understand how such reorganizations drive the cycle of mRNA 3' end processing. The research focuses on four events that are likely to be critical transition points in this cycle yet whose underlying mechanisms are not understood. These include the initiation of cleavage and the role of the Ssuy2 protein in this step, the transition from cleavage to poly(A) addition and its regulation by phosphorylation of the Ptai scaffold protein, the control of poly(A) polymerase activity by interactions at its amino-terminus, and the release of processing factors following tail synthesis. The studies will use yeast as the model organism because of the ease of introducing tags and mutations into the genome, the availability of numerous 3' end processing mutants, the ease of purifying processing factors for biochemical studies, and the high degree of conservation of the 3' end processing machinery across eukaryotes. Relevance: Without poly(A) tails, mRNA is targeted for degradation in the nucleus, it does not get out of the nucleus very well, and it is not translated efficiently or turned over at the appropriate rate in the cytoplasm. Maturation of mRNA 3' ends is functionally linked to other essential processes such as transcription, mRNA export, chromosome segregation, DNA repair, and tissue-specific protein expression. Mistakes in polyadenylation can impact on all of these processes. The proposed research should significantly advance our insight into the dynamics of this essential step in mRNA synthesis and identify points at which the constitutive process is likely to be regulated.