YEAST GENES IN RNA PROCESSING & NUCLEUS/CYTOSOL EXCHANGE

RNA 加工中的酵母基因

• 批准号: 2389488
• 负责人: Anita K Hopper
• 金额: $ 26.8万
• 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/2389488
• 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 Rnalp, a regulatory protein of this cycle, raises questions as to whether it 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 an 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. Aim5. 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 affects 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， 生物化学专业化 细胞核/细胞质交换是必要的 因为RNA在细胞核中合成 并被转运到细胞质中，在那里它们以蛋白质的形式发挥作用， 合成和许多蛋白质合成的细胞质中的功能， 细胞核在有丝分裂、DNA复制和 RNA合成。 强大的酵母遗传系统将 用于研究控制核/胞质溶胶的机制 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。 对于核能，需要Ran-GT循环 进出口和RNA加工，但细胞分布 Rnalp，这个周期的调节蛋白，提出了一些问题， 它是否对出口有直接作用。 目标2.为了识别基因 在RAN非依赖性途径中起作用的核蛋白产物 输出编码应激蛋白（Hsp）的mRNA。 以前的研究 表明Hsp mRNA通过Ran非依赖性 路径 原位杂交将用于鉴定突变体 Hsp mRNA的核输出缺陷。目标3. 测试 酵母中tRNA核输出不依赖于Ran假说 并鉴定在tRNA核中起作用的基因产物， 导出. 其他研究表明，在哺乳动物细胞中， 核通过RAN独立途径。 原位杂交和 3-杂交技术将用于研究细胞核/胞质溶胶 酵母前体和成熟tRNA的分布，并鉴定 变异的分布。目标4。区分 Reg 1 p在调节Ran-GT循环组分中的作用， 核/胞质交换的RAN非依赖性途径。 Reg 1是 RNA 1等位基因的抑制子。为了确定Reg 1 p是否 调节Ran-GT循环组分的活性， 亚细胞位置，或者相反，调节RAN独立 途径，遗传学，免疫荧光，原位 将采用杂交和生物化学测定。 目标5。到 测试Ran-GT3通路在以下假设中起作用： 除了细胞核/胞质溶胶交换之外的过程。 Ran途径 与多种细胞过程有关， 细胞核/胞质交换。 私家侦探的实验室制造了一个 编码Ran GAP的基因似乎不会影响 细胞核/胞质溶胶交换，而是影响细胞周期进程。 测试此GAP参与其他蜂窝的模型 该突变体的第二位点和多拷贝抑制子 将研究等位基因，并将研究与Rna 1 p相互作用的蛋白质 通过使用双杂交技术来鉴定。