GENETIC & MOLECULAR STUDIES OF NEMATODE MRNA DEGRADATION

基因

• 批准号: 2189145
• 负责人: Philip Anderson
• 金额: $ 25.33万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2189145
• 关键词: Caenorhabditis elegans; RNA biosynthesis; antibody; gene complementation; messenger RNA; molecular cloning; molecular genetics; mutant; nucleic acid metabolism; nucleic acid sequence; polymerase chain reaction; pulsed field gel electrophoresis; regulatory gene; site directed mutagenesis; transcription factor

The steady-state level of a messenger RNA is established by its relative rates of synthesis and degradation. It is increasingly apparent that mRNA degradation is an important aspect of gene expression and its regulation. Although numerous cis-acting sequences that regulate mRNA stability have been defined, little is known about trans-acting factors that selectively degrade a message. Most mRNA turnover appears to be intimately coupled to translation. This is particular clear in the case of nonsense mutant mRNAs. mRNAs that contain nonsense mutations (premature stop codons) are unstable in all organisms, including humans. We will study this phenomenon, termed nonsense-mediated mRNA decay, in the nematode Caenorhabditis elegans. Loss-of-function mutations affecting any of six different genes (smg-1 through smg-6) eliminate the C. elegans system of nonsense-mediated mRNA decay. The six smg genes define proteins that unambiguously act in trans to accomplish specific mRNA turnover. We will study genetic and molecular properties of the smg genes and answer the following questions: What are the smg gene products? Where are smg proteins located in the cell? With what do they associate? And, how are nonsense mutant mRNAs targeted for degradation? Our methods combine genetic, molecular, and biochemical approaches. Specific smg genes will be cloned and sequenced. smg genes essential for viability of the animal will be identified using a novel genetic screen. Polyclonal anti-Smg antibodies will be used to locate smg proteins in situ and in fractionated cell extracts. Site-directed mutations that test specific models for the specificity of nonsense-mediated decay will be constructed. Our results should identify the proteins involved in nonsense-mediated decay and provide an outline understanding of how the system selects mRNAs to be destroyed. Our long range goals are to understand the molecular mechanisms of mRNA turnover. Nonsense-mediated decay in C. elegans may prove to be similar to many cases of constitutive or regulated mRNA turnover. Nonsense-mediated mRNA decay is a universal phenomenon and may contribute to human genetic disease. Patients having nonsense alleles of an affected gene usually express low steady-state levels of the mutant mRNA. We believe that these and other mRNAs are degraded by the human system of nonsense-mediated mRNA decay. Understanding the smg genes of C. elegans will contribute to understanding the etiology of these diseases.

信使RNA的稳态水平是由其相对分子质量来确定的。 合成和降解的速率。越来越明显的是， 降解是基因表达及其调节的一个重要方面。 尽管许多调节mRNA稳定性的顺式作用序列具有 虽然已经定义，但对选择性地 降低消息的质量。 大多数mRNA周转似乎与以下因素密切相关： 翻译.这在无义突变体的情况下特别清楚 mRNA。 含有无义突变（过早终止密码子）的mRNA是 在所有生物体中都不稳定，包括人类。我们会研究这个 这种现象，称为无义介导的mRNA衰变，在线虫 秀丽隐杆线虫功能缺失突变影响六种基因中的任何一种 不同的基因（smg-1至smg-6）消除C.优雅系统 无义介导的mRNA衰变。六个smg基因定义了 明确地反式作用以完成特定的mRNA周转。我们将 研究smg基因的遗传和分子特性， 以下问题：什么是smg基因产物？smg在哪里 细胞内的蛋白质？与他们有什么关系呢？你怎么样 针对降解的无义突变mRNA？我们的方法联合收割机 基因、分子和生物化学方法。 特定的smg基因将 进行克隆和测序动物生存所必需的smg基因 将通过一种新的基因筛选进行鉴定。多克隆抗Smg 抗体将被用来定位sMG蛋白在原位和在分级的 细胞提取物定点突变，测试特定的模型， 将构建无义介导的衰变的特异性。 我们的结果 应该确定参与无义介导的衰变的蛋白质， 提供了一个系统如何选择mRNA的轮廓理解， 摧毁. 我们的长期目标是了解mRNA的分子机制， 周转无义介导的C.秀丽线虫可能被证明与 许多情况下的组成或调节mRNA周转。 无义介导 mRNA衰变是一种普遍现象，可能有助于人类遗传 疾病具有受影响基因的无义等位基因的患者通常 表达低稳态水平的突变mRNA。 我们相信这些 而其他mRNA则被人类无义介导的mRNA系统降解， 腐烂 了解C. elegans将有助于 了解这些疾病的病因。