NUCLEOCYTOPLASMIC TRANSPORT OF mRNA IN YEAST

酵母中 mRNA 的核质转运

• 批准号: 7097925
• 负责人: CHARLES N. COLE
• 金额: $ 46.37万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7097925
• 关键词: RNA binding protein; Saccharomyces cerevisiae; active transport; adenosine triphosphate; affinity chromatography; cell nucleus; cytoplasm; ethanol; fluorescence recovery after photobleaching; gel mobility shift assay; heat shock proteins; heat stimulus; intracellular transport; messenger RNA; microorganism culture; microorganism metabolism; nuclear membrane; nuclear proteins; physiologic stressor; polyadenylate; protein localization; protein protein interaction; protein structure function; small nuclear RNA; transport proteins

DESCRIPTION (provided by applicant): The long-term objectives of this research program remain directed at defining the mechanisms by which mRNA molecules are exported from the nucleus and determining under what circumstances and by what mechanisms nucleocytoplasmic transport is regulated. Our understanding of nucleocytoplasmic transport of proteins and small RNAs (e.g. tRNAs) has grown substantially through the identification of transport signals and karyopherin receptors that recognize them. We know much less about mRNA export, which is considerably more complex, in part because it is tightly coupled to mRNA biogenesis and checkpoints exist that permit only completely- and accurately-processed mRNAs to be exported. mRNAs are exported as RNA/protein complexes (mRNPs). Many factors required uniquely for mRNA export have been identified but how they work together is not known. A major focus is to understand the role in mRNA export of the DEADbox protein (DBP) Dbp5/Rat8. DBFs play essential roles in virtually all aspects of RNA metabolism. Dbp5 shuttles and interacts directly with components of the nuclear pore complex (NPC). To address two centrals questions about RNA export, the following specific aims are proposed: Aim 1) To test the hypothesis that Dbp5 acts when bound to the cvtoplasmic filaments of NPCs to couple ATP hydrolysis with mRNP translocation through the pore and removal of mRNP proteins. The interactions between Dbp5, proteins of the cytoplasmic filaments of the NPC, and accessory proteins will be mapped. Genetic and physical approaches will be used to determine whether Dbp5 must bind to NPC filaments to mediate mRNA export. The dynamics of interaction between Dbp5 and NPCs will be analyzed using fluorescence recovery after photobleaching (FRAP) and with mating assays. Whether Dbp5 can disassemble model RNA/protein complexes in vitro will be determined. Aim 2) To test the hypothesis that they key to export lies in the composition and organization of mRNP complexes. The studies will compare export-competent mRNPs from wild-type cells and mRNPs that accumulate in export mutant strains. Other studies will determine how exported heat shock mRNPs differ from mRNPs that accumulate in nuclei following heat shock. Aim 3) To determine whether Dbp5 functions not only at NPCs but also in the nucleus and what it does in the nucleus.

描述（由申请人提供）：该研究计划的长期目标仍然旨在定义从核中导出mRNA分子的机制，并在哪种情况下确定了哪种机制核细胞质转运受调节。我们对蛋白质和小RNA（例如TRNA）的核细胞质转运的理解已通过鉴定识别它们的转运信号和核粘蛋白受体的大大生长。我们对mRNA输出的了解少得多，这是相当复杂的，部分原因是它与mRNA生物发生紧密耦合，并且存在检查点仅允许完全和准确地处理的mRNA出口。 mRNA作为RNA/蛋白质复合物（MRNP）出口。已经确定了许多用于mRNA导出所需的因素，但是尚不清楚它们如何一起工作。主要重点是了解死盒蛋白（DBP）DBP5/Rat8中的mRNA输出中的作用。 DBF在RNA代谢的几乎所有方面都起着重要作用。 DBP5穿梭和直接与核孔复合物（NPC）的成分相互作用。为了解决有关RNA出口的两个中心问题，提出了以下具体目标： 目的1）检验以下假设：DBP5与NPC的CVTOPLASMIC细丝结合到将ATP水解与MRNP易位通过孔隙和去除MRNP蛋白质结合起来。 DBP5，NPC细胞质细丝的蛋白质与辅助蛋白之间的相互作用将进行映射。遗传和物理方法将用于确定DBP5是否必须与NPC丝结合以介导mRNA输出。 DBP5和NPC之间相互作用的动力学将使用光漂白后的荧光回收（FRAP）和交配分析分析。 DBP5是否可以在体外拆卸RNA/蛋白质复合物模型。 目的2）检验它们出口关键的假设在于MRNP复合物的组成和组织。这些研究将比较从出口突变菌株中积累的野生型细胞和MRNP的出口功能mRNP。其他研究将确定输出热休克mRNP与热休克后在核中积累的MRNP有何不同。 目标3）确定DBP5是否不仅在NPC中功能，而且在核中及其在细胞核中的作用。