YEAST GENES IN RNA PROCESSING & NUCLEUS/CYTOSOL EXCHANGE

真核细胞的特征是细胞器，部位 生化专门化。核/胞浆交换是必不可少的 对于所有真核生物来说，因为RNA是在细胞核中合成的 并被运输到胞浆中，在那里它们在蛋白质中发挥作用 合成与胞浆中合成的多种蛋白质的功能 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是 用来研究(S)控制核/胞浆的机制 交换以及交换是如何耦合到RNA处理的。目标1。 检验RNA1p直接参与信使核糖核酸的假设 核出口。Ran-GTP酶循环是核所必需的 进出口和RNA加工，但细胞分布 Rnalp是这个周期的一种调节蛋白，这引发了关于 它是否对出口有直接的作用。目的2.鉴定基因 在核糖核酸非依赖性途径中发挥作用的产物 输出编码应激蛋白(HSP)的mRNAs。以前的研究 显示热休克蛋白mRNAs通过Ran非依赖性的方式离开细胞核。 路径。将使用原位杂交来鉴定突变株 热休克蛋白mRNAs核出口存在缺陷。目标3.测试 酵母中tRNA核输出不依赖Run的假说 并鉴定在tRNA核中起作用的基因产物 出口。其他研究表明，在哺乳动物细胞中，tRNAs退出 细胞核通过一条Ran非依赖性途径。原位杂交和 3杂交技术将被用于研究核/胞浆 酵母前体和成熟tRNAs的分布及鉴定 分布发生改变的突变体。目标4.区分 Reg1p在调节Ran-GTP酶循环成分或a中的作用 核/胞浆交换的Ran非依赖性路径。REG1是一种 RNA1等位基因的抑制子。要确定Reg1p是否 调节Ran-GTP酶循环组件的活性，其 亚细胞位置，或者，相反，调节RAN无关 途径，遗传学，免疫荧光，原位 将采用杂交和生化分析。Aim5.至 验证RAN-GTP酶途径在体内发挥作用的假设 除核/胞浆交换外的其他过程。RAN途径 与多种细胞过程有关，除了 核/胞浆交换。PI的实验室产生了一个等位基因 编码RAN缺口的基因似乎不影响 细胞核/胞浆交换，但影响细胞周期进程。 为了测试这个缺口参与其他细胞的模型 该突变体的过程、第二位点和多拷贝抑制子 将研究等位基因，与Rna 1p相互作用的蛋白质将 通过使用双杂交技术进行鉴定。

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.