CONTROL OF CELL FUNCTION VIA SELECTIVE MRNA TRANSLATION

通过选择性 mRNA 翻译控制细胞功能

• 批准号: 3301115
• 负责人: Lee Gehrke
• 金额: $ 28.81万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3301115
• 关键词: HeLa cells; RNA binding protein; X ray crystallography; capsid; cell growth regulation; gel electrophoresis; gene deletion mutation; gene expression; genetic manipulation; genetic regulation; genetic translation; messenger RNA; nucleic acid sequence; nucleic acid structure; plant virus; poliovirus; protein biosynthesis; protein kinase; protein sequence; site directed mutagenesis; tobacco mosaic virus; transcription factor; virus RNA; virus infection mechanism; virus protein

A complete understanding of the molecular mechanisms of disease will require elucidation of control mechanisms which define cell structure and function. The goals of this project are to define the molecular basis of selective messenger RNA translation and to characterize features of mRNA sequence and/or structure which signal formation of a specific messenger RNA protein complex. Stressed or virus-infected cells selectively translate a subset of the total mRNA pool, and nucleotides in the 5' untranslated leader sequence of the messenger RNAs facilitate differential expression. A plant viral messenger RNA, alfalfa mosaic virus RNA 4, will be exploited for these studies because it is expressed efficiently and selectively in extracts prepared from human cells infected with poliovirus wherein translocation of other messenger RNAs is abolished. Identification of the sequence and/or structural features of alfalfa mosaic virus RNA 4(AMV RNA 4) which sustain translational activity in diseased cells will increase understanding of how messenger RNAs are normally selected for translation and may suggest protocols for combating viral infection or for prevention of cell shut-down during stress conditions. Preliminary studies show that signals which permit selective translation. A combination of nucleotide deletion experiments and peptide complementation analyses will be used to characterize the molecular basis of selective AMV RNA 4 translation. Despite the implication that specific messenger RNA- protein interactions form the basis of important regulatory mechanisms, very few examples of specific mRNA-protein complexes have been described an studied in detail. Preliminary studies demonstrate that two unrelated proteins bind specifically to a 3'- terminal fragment of AMV RNA 4. Analysis of the specific binding of the AMV coat protein (CP) and of the mammalian double strand RNA-dependent protein kinase (dsI) to a 180 nucleotide fragment of RNA 4 offers an uncommon opportunity to examine mRNA protein interactions. Characterization of the AMV RNA 4-CP complex will be useful for understanding the molecular basis of viral cross- protection in plants; moreover, preliminary studies show that the plant viral RNA fragment is a potent activator of the mammalian dsI kinase. Mobility band shift electrophoresis and nitrocellulose filter retention studies will be used to characterize the specific interaction between alfalfa mosaic virus RNA 4 and CP/dsI. Nucleotide deletion analysis and site-directed mutagenesis will be used to probe both the RNA and the protein binding sites. The availability of large quantities of CP permits extension of the analysis to include physical studies. Direct clinical relevance may be found in recent reports detailing significant changes in the development of a neoplastic phenotypic associated with features of the 5' untranslated leader sequence of oncogene mRNAs; moreover, interactions of protein(s) with the 5' leader of the human immunodeficiency virus mRNAs influence expression of viral gene products.

对疾病分子机制的完整理解 需要阐明定义细胞的控制机制 结构和功能。 本项目的目标是定义 选择性信使RNA翻译的分子基础， 表征mRNA序列和/或结构的特征， 形成特异性信使RNA蛋白复合物的信号。 应激或病毒感染的细胞选择性地翻译一个亚群， 总mRNA库和5'非翻译前导序列中的核苷酸 信使RNA的序列促进差异表达。 一种植物病毒信使RNA，苜蓿花叶病毒RNA 4， 因为它被有效地表达， 选择性地在从感染了 脊髓灰质炎病毒，其中其他信使RNA的易位是 废除 序列和/或结构的鉴定 苜蓿花叶病毒RNA 4（AMV RNA 4）的特征， 病变细胞中的翻译活性将增加 了解信使RNA通常是如何被选择用于 翻译，并可能建议协议，以打击病毒 感染或用于防止细胞在应激期间关闭 条件 初步研究表明， 选择性翻译 核苷酸缺失的组合 实验和肽互补分析将用于 描述选择性AMV RNA 4的分子基础 翻译. 尽管暗示特定的信使RNA- 蛋白质的相互作用形成了重要的调控基础， 机制，很少有特定的mRNA-蛋白质复合物的例子 已经被详细描述和研究。 初步研究 证明两种不相关的蛋白质特异性结合3 '- AMV RNA末端片段4. 特异性结合分析 AMV外壳蛋白（CP）和哺乳动物双链 RNA依赖性蛋白激酶（dsI）的180个核苷酸的片段， RNA 4提供了一个不寻常的机会来检查mRNA蛋白质 交互. AMV RNA 4-CP复合物的表征将 有助于理解病毒交叉的分子基础， 保护植物;此外，初步研究表明， 植物病毒RNA片段是哺乳动物dsI的有效激活剂 激酶。 迁移率谱带位移电泳与硝酸纤维素 过滤器保留研究将用于表征特定的 苜蓿花叶病毒RNA 4与CP/dsI相互作用 核苷酸缺失分析和定点突变将在 用于探测RNA和蛋白质结合位点。 的 大量CP的可用性允许扩展 分析包括物理研究。 直接临床相关性 在最近的报告中可以发现， 与以下特征相关的肿瘤表型的发展 癌基因mRNA的5'非翻译前导序列;此外， 蛋白质与人的5'前导序列的相互作用 免疫缺陷病毒mRNAs影响病毒基因表达 产品.