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理解这些通路中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的活性是如何被特异性调控的？一个长期存在的问题是剪接体DEAD-box atp酶是如何在剪接周期的精确时间被激活的。我们最近发现了U5 snRNP蛋白Prp8的一个区域，该区域特异性地刺激Brr2 atp酶将U4从U6 snRNA中解开，这是催化剪接体激活的关键事件。使用基于活性和fret的分析，我们现在将确定控制这一步骤的全套分子相互作用。我们将重点关注阳性激活剂Snu114（一种类似ef2的GTPase）的作用，以及prp8相互作用因子的泛素化对Brr2的下调。2)转录是如何耦合到mRNA剪接和输出的？虽然很明显，基因表达中的核步骤是暂时耦合的，但对这种耦合的潜在机制基础知之甚少。我们正在采用创新的高通量遗传平台来促进定量遗传相互作用的鉴定；这种上位迷你阵列谱已被证明是新型功能关系的有力预测因子。我们将测试我们正在进行的分析的具体预测，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)剪接是如何受环境调控的？利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抑制核糖体蛋白基因转录物的剪接。我们现在将确定介导这种反应的新型信号转导途径的分子基础。我们还将扩展我们的应激源电池，以确定其他剪接调节模块。更广泛地说，我们将通过对约270株含有精确内含子缺失的菌株进行定量分析，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.