YEAST GENES IN RNA PROCESSING & NUCLEUS/CYTOSOL EXCHANGE

RNA 加工中的酵母基因

• 批准号: 6018518
• 负责人: Anita K Hopper
• 金额: $ 25.31万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-09-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018518
• 关键词: RNA biosynthesis; RNA splicing; Saccharomyces cerevisiae; biological signal transduction; cell nucleus; cytoplasm; enzyme activity; fluorescence microscopy; fungal genetics; gene expression; gene mutation; guanosinetriphosphatase activating protein; immunofluorescence technique; in situ hybridization; intracellular transport; messenger RNA; posttranscriptional RNA processing; protein structure function; ribosomal RNA; stress proteins; transfer RNA; yeast two hybrid system

Eukaryotic cells are characterized by organelles, sites of biochemical specialization, Nucleus/cytosol exchange is essential for all eukaryotic organisms as RNAs are synthesized in the nucleus and are transported to the cytosol where they function in protein synthesis and many proteins synthesized in the cytosol function in the nucleus in processes such as mitosis, DNA replication, and RNA synthesis. The powerful yeast genetic system will be employed to study the mechanism(s) controlling nucleus/cytosol exchange and how exchange is coupled to RNA processing. Aim 1. To test the hypothesis that Rna1p participates directly in mRNA nuclear export. The Ran-GTPase cycle is required for nuclear import and export and RNA processing, but the cellular distribution of Rna1p, a regulatory protein of this cycle, raises questions as to whether is has a direct role in export. Aim 2. To identify gene products that function in the Ran-independent pathway for nuclear export of mRNAs encoding stress proteins (Hsp). Previous studies showed that Hsp mRNAs exit the nucleus via a Ran-independent path. In situ hybridization will be used to identify mutants defective in nuclear export of Hsp mRNAs. Aim 3. To test the hypothesis that tRNA nuclear export is Ran-independent in yeast and to identify the gene products that function in tRNA nuclear export. Others have shown that in mammalian cells tRNAs exit the nucleus via a Ran- independent pathway. In situ hybridization and the 3-hybrid technique will be used to study the nucleus/cytosol distribution of yeast precursor and mature tRNAs and to identify mutants with altered distribution. Aim 4. To distinguish between roles of Reg1p in regulating the Ran-GTPase cycle components or a Ran-independent path of nucleus/cytosol exchange. reg1 is a suppressor of RNA1 allele. To determine whether Reg1p modulates the activities of the Ran-GTPase cycle components their subcellular locations or, instead, regulates Ran-independent pathways, a combination of genetics, immunofluorescence, in situ hybridization and biochemical assays will be employed. Aim 5. To test the hypothesis that the Ran-GTPase pathway functions in processes other than nucleus/cytosol exchange. The Ran pathway has been implicated in a variety of cellular processes in addition to nucleus/cytosol exchange. The PI's lab generated an allele of the gene encoding the Ran GAP that appears not to affect nucleus/cytosol exchange, but rather effects cell cycle progression. To test the model that this GAP participates in other cellular processes, second-site and multicopy suppressors of this mutant allele will be studied and the proteins that interact with Rna1p will be identified by use of the 2- hybrid technique.

真核细胞的特征是细胞器，生化位点， 核/胞质交换对于所有真核生物都是必不可少的。 RNA在细胞核中合成，并被运输到 它们在蛋白质合成和许多蛋白质中起作用胞质溶胶 在细胞质中合成，在细胞核中发挥作用， 有丝分裂、DNA复制和RNA合成。强大的酵母基因 系统将被用来研究机制（S）控制 核/胞质溶胶交换以及交换如何与RNA加工耦合。 目标1.为了验证Rna 1 p直接参与mRNA表达的假设， 核出口。核进口需要Ran-GT循环， 出口和RNA加工，但Rna 1 p的细胞分布， 这个周期的调节蛋白，提出了一个问题，即是否有一个 直接出口。目标二。为了鉴定基因产物， Ran非依赖性应激基因核输出途径 蛋白（Hsp）。以往的研究表明，热休克蛋白的mRNA出核， 通过Ran独立的途径。原位杂交将用于鉴定 Hsp mRNA核输出缺陷突变体。目标3。测试 假设tRNA核输出在酵母中是Ran非依赖性， 鉴定在tRNA核输出中起作用的基因产物。别人 已经表明，在哺乳动物细胞中，tRNA通过Ran- 独立的道路。原位杂交和三杂交技术将 用于研究酵母前体的细胞核/胞质溶胶分布， 成熟的tRNA，并确定突变体与改变分布。目标4。到 区分Reg 1 p在调节Ran-GT循环中的作用 组分或核/胞质溶胶交换的Ran独立路径。reg 1是 RNA 1等位基因的抑制基因。为了确定Reg 1 p是否调节了 Ran-GT循环组分的活性及其亚细胞位置 或者，相反，调节Ran非依赖性途径， 遗传学、免疫荧光、原位杂交和生化分析 将被雇用。目标5。为了验证Ran-GTdirt 在细胞核/胞质溶胶交换以外的过程中起作用。的 Ran通路参与多种细胞过程， 除了细胞核/胞质溶胶交换。私家侦探的实验室产生了一个 编码Ran GAP的基因似乎不影响细胞核/细胞质 交换，而是影响细胞周期进程。检验模型 该GAP参与其他细胞过程，第二位点和 将研究该突变等位基因的多拷贝抑制子， 与Rna 1 p相互作用的蛋白质将通过使用2- 混合技术