Biosynthesis of RNAs

RNA的生物合成

基本信息

批准号：
7904471
负责人：
CHRISTINE GUTHRIE
金额：
$ 39.4万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-18 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7904471
关键词：
ATP phosphohydrolase Address Affect Alternative Splicing Amino Acids Amitrole Binding Biochemical Biological Biological Assay Boxing C-terminal Chimera organism Complex Coupled Couples Coupling DNA Sequence Rearrangement Down-Regulation Engineering Environment Event Family Future Gene Expression Gene Expression Regulation Genes Genetic Genetic Epistasis Genetic Transcription Goals Growth Guanosine Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Histidine Hydrolysis In Vitro Introns Laboratories Libraries Mediating Messenger RNA Modification Molecular Mutation Nuclear Nuclear Pore Nucleotides Pathway interactions Proteins Quantitative Genetics RNA RNA Processing RNA Splicing RNA biosynthesis RNA-dependent ATPase Reaction Regulation Ribosomal Proteins Role SAGA Signal Transduction Pathway Small Nuclear Ribonucleoproteins Specificity Spliced Genes Spliceosomes Starvation System Testing Time Transcript U5 Small Nuclear Ribonucleoprotein U6 small nuclear RNA Ubiquitin Work Yeasts base chromatin remodeling density design environmental change environmental stressor expectation in vivo innovation mRNA Export molecular phenotype multicatalytic endopeptidase complex mutant novel reconstitution response stressor ubiquitin-protein ligase

项目摘要

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.

描述（由申请人提供）：我的实验室中的工作重点介绍了基因表达，mRNA剪接和mRNA导出的两个主要步骤。我们的长期目标是在分子级别了解这些途径中特异性和忠诚度的机制。我们未来的工作将解决三个基本问题：1）剪接体NTPases的活动如何受到调节？一个长期以来的问题是，如何在剪接周期中的精确时间激活剪接体死盒ATPases。我们最近确定了U5 SNRNP蛋白PRP8的一个区域，该区域专门刺激BRR2 ATPase从u6 snRNA放松u4，u6 snRNA是酶子剪接体催化激活的关键事件。使用基于活动和FRET的测定，我们现在将确定控制此步骤的完整分子相互作用。我们将重点关注阳性激活剂SNU114，类似于EF2的GTPase的作用，以及通过PRP8相互作用因子的泛素化对BRR2提出的下调。 2）如何将转录耦合到mRNA剪接和导出？虽然很明显，基因表达的核步骤是时间耦合的，但几乎没有什么理解的关于该耦合的基本机械基础。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识别；这种上学迷你阵列概况已证明了新型功能关系的有力预测指标。我们将从我们正在进行的分析中测试特定的预测，这些预测表明蛋白酶体与核孔之间以及剪接体和染色质重塑机械之间的意外连接。 3）如何对环境响应剪接？使用基于全球微阵列的测定法，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。现在，我们将确定介导此响应的新型信号转导途径的分子基础。我们还将扩大电池压力源，以识别其他剪接调节模块。更广泛地说，我们将通过对〜270种菌株的定量分析来询问酵母内含子的生物学影响，以包含精确的内含子缺失。