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，位置 生化专业。 细胞核/细胞质交换是必不可少的 对于所有真核生物作为RNA，均在细胞核中合成 并将其运输到蛋白质中作用的细胞质 合成和许多在细胞质功能中合成的蛋白质 诸如有丝分裂，DNA复制和 RNA合成。 强大的酵母菌系统将是 用于研究控制核/细胞质的机制 交换以及如何与RNA处理耦合。目标1。 测试RNA1p直接参与mRNA的假设 核出口。 核需要RAN GTPase周期 导入，导出和RNA处理，但细胞分布 RNALP是该周期的调节蛋白，提出了有关的问题 它是否在出口中起着直接作用。 目标2。识别基因 在核能独立途径中起作用的产品 编码应力蛋白（HSP）的mRNA导出。 先前的研究 表明HSP mRNA通过RAN独立地退出核 小路。 原位杂交将用于识别突变体 HSP mRNA的核出口缺陷。目标3。测试 假设tRNA核输出在酵母中独立于 并确定在tRNA核中起作用的基因产物 出口。 其他人则表明，在哺乳动物细胞中，trnas退出 核通过独立途径。 原位杂交和 3杂交技术将用于研究核/细胞质 酵母前体和成熟的trnas的分布并确定 分布变化的突变体。目标4。区分 REG1P在调节RAN-GTPase周期组件或A中的作用 核/胞质交换的不依赖性路径。 reg1是 RNA1等位基因的抑制剂。确定reg1p是否 调节RAN-GTPase周期组件的活动，它们 亚细胞位置或改为调节独立的 途径，遗传学，免疫荧光的结合，原位 将采用杂交和生化测定。 AIM5。到 测试RAN-GTPase途径在功能中起作用的假设 除核/胞质交换以外的过程。 跑路 除了 细胞核/细胞质交换。 Pi的实验室产生了一个等位基因 编码似乎不影响的RAN间隙的基因 细胞核/细胞质交换，而是影响细胞周期进程。 测试该差距参与其他细胞的模型 该突变体的过程，第二站点和多拷贝抑制剂 将研究等位基因，与RNA1P相互作用的蛋白质将 通过使用2杂交技术来识别。