Biosynthesis of RNAs

描述（申请人提供）：我实验室的工作主要集中在基因表达的两个主要步骤，mRNA剪接和mRNA输出。 我们的长期目标是在分子水平上了解这些途径中负责特异性和保真度的机制。我们未来的工作将解决三个基本问题：1）剪接体NTPases的活动是如何具体调控的？一个长期存在的问题是剪接体死亡盒ATP酶如何在剪接周期的精确时间被激活。我们最近确定了一个区域的U 5 snRNP蛋白Prp 8，特异性地刺激Brr 2 ATP酶解开U4从U6 snRNA，在剪接体的催化激活的关键事件。使用基于活性和FRET的测定，我们现在将确定控制这一步骤的全套分子相互作用。我们将专注于积极的激活剂Snu 114，EF 2样GTdR的作用，并提出下调Brr 2的Prp 8相互作用因子的泛素化。2)转录如何与mRNA剪接和输出耦合？虽然很明显，在基因表达的核步骤的时间耦合，很少了解这种耦合的潜在机制基础。 我们正在采用一种创新的高通量遗传平台，以促进定量遗传相互作用的识别;这种上位性微阵列配置文件已被证明是新的功能关系的强大预测因子。我们将测试我们正在进行的分析中的具体预测，这些预测表明蛋白酶体和核孔之间以及剪接体和染色质重塑机制之间存在意想不到的联系。3)剪接是如何响应环境调节的？使用全球微阵列为基础的分析，我们最近证明，氨基酸饥饿选择性地抑制核糖体蛋白基因转录的剪接。我们现在将确定介导这种反应的新信号转导途径的分子基础。我们还将扩大我们的压力源电池，以确定其他剪接调控模块。更广泛地说，我们将通过定量分析约270株含有精确内含子缺失的工程菌株来询问酵母内含子的生物学影响。

DESCRIPTION (provided by applicant): The work in my laboratory focuses on two major steps in gene expression, mRNA splicing and mRNA export. Our long-term goal is to understand at a molecular level the mechanisms responsible for specificity and fidelity in these pathways. Our future work will address three fundamental questions: 1) How are the activities of the spliceosomal NTPases specifically regulated? A long-standing question is how the spliceosomal DEAD-box ATPases are activated at precise times in the splicing cycle. We recently identified a region of the U5 snRNP protein Prp8 that specifically stimulates the Brr2 ATPase to unwind U4 from U6 snRNA, the key event in catalytic activation of the spliceosome. Using activity- and FRET-based assays, we will now identify the full set of molecular interactions that control this step. We will focus on the roles of the positive activator Snu114, an EF2-like GTPase, and the proposed down-regulation of Brr2 by ubiquitylation of a Prp8-interacting factor. 2) How is transcription coupled to mRNA splicing and export? While it is apparent that the nuclear steps in gene expression are temporally coupled, little is understood about the underlying mechanistic bases of this coupling. We are employing an innovative high-throughput genetic platform to facilitate identification of quantitative genetic interactions; such Epistasis Mini-Array Profiles have proven powerful predictors of novel functional relationships. We will test specific predictions from our ongoing analysis that suggest unexpected connections between the proteasome and the nuclear pore, and between the spliceosome and the chromatin remodeling machinery. 3) How is splicing regulated in response to the environment? Using a global microarray-based assay, we recently demonstrated that amino acid starvation selectively inhibits the splicing of ribosomal protein gene transcripts. We will now determine the molecular basis of the novel signal transduction pathway mediating this response. We will also expand our battery of stressors to identify other splicing regulatory modules. More broadly, we will interrogate the biological impact of yeast introns by the quantitative analysis of each of ~270 strains engineered to contain a precise intron deletion.