Molecular mechanism of mRNA 3'-end formation in yeast

项目概述：真核生物mRNA的合成及其在细胞质中的利用，需要在RNA的3'端添加聚(a)尾进行修饰。这个过程也可以作为一个点，细胞可以调节来自特定基因的mRNA的类型和数量。尽管mRNA 3'端形成发生在一个意想不到的大复合体中，但大多数（如果不是全部的话）这种机制的亚基已经在酿酒酵母中被鉴定出来。然而，对于这21种蛋白质如何相互合作以确保加工的准确性以及及时耦合到mRNA合成和包装中的其他事件，人们知之甚少。随着3'端加工成分的掌握，以及其中几个因素的活动新定义，现在存在一个独特的机会来严格解决基因表达中这一重要和普遍步骤发生和调节的机制。该建议的中心假设是，当复合体评估加工位点的真实性，在poly(A)位点进行切割，重组以定位poly(A)聚合酶的尾部合成，并释放最终的RNA产物时，可定义的蛋白质伴侣重排发生在复合体内。目的是了解这种重组如何驱动mRNA 3'端加工周期。研究集中在四个事件上，这些事件可能是这个周期的关键转折点，但其潜在机制尚不清楚。这些包括裂解的起始和Ssuy2蛋白在这一步骤中的作用，从裂解到聚(A)添加的转变及其通过Ptai支架蛋白磷酸化的调控，通过其氨基端相互作用控制聚(A)聚合酶活性，以及尾部合成后加工因子的释放。研究将使用酵母作为模式生物，因为酵母很容易将标签和突变引入基因组，有大量的3‘端加工突变体，易于纯化生化研究的加工因子，以及真核生物中3’端加工机制的高度保守性。相关性：没有多聚(A)尾，mRNA在细胞核中是降解的目标，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。mRNA 3'端的成熟与转录、mRNA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在功能上相关。聚腺苷化过程中的错误会影响所有这些过程。拟议的研究将显著推进我们对mRNA合成中这一重要步骤的动力学的了解，并确定构成过程可能受到调节的点。

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.