CONTROL OF CELL FUNCTION VIA SELECTIVE MRNA TRANSLATION

对疾病的分子机制有全面的了解 将需要阐明定义细胞的控制机制 结构和功能。该项目的目标是定义 选择性信使RNA翻译和TO的分子基础 表征mRNA序列和/或结构的特征 特定信使RNA蛋白复合体的信号形成。 应激或病毒感染的细胞选择性地翻译 总信使核糖核酸池，以及5‘非翻译前导序列中的核苷酸 信使RNA的序列有助于差异表达。 一种植物病毒信使RNA，苜蓿花叶病毒RNA4，将被 被用于这些研究，因为它被高效地表达和 从感染了病毒的人细胞中制备的提取物中选择性地 脊髓灰质炎病毒，其中其他信使RNA易位是 废止了。序列和/或结构的鉴定 紫花苜蓿花叶病毒RNA4(AMV RNA4)的持续特性 病变细胞的翻译活性将会增加 了解信使RNA通常是如何被选择用于 翻译，并可能建议对抗病毒的方案 感染或在应激期间防止细胞关闭 条件。初步研究表明，允许 选择性翻译。核苷酸缺失的组合 实验和多肽互补分析将用于 表征选择性AMV RNA4的分子基础 翻译。尽管有暗示说特定的信使RNA- 蛋白质相互作用构成重要调控的基础 机制，很少有特定的信使核糖核酸-蛋白质复合体 对其进行了详细的描述和研究。初步研究 证明两个不相关的蛋白质与3‘- AMV RNA末端片段4.特异性结合分析 AMV外壳蛋白(CP)和哺乳动物双链的 RNA依赖蛋白激酶(DSI)到一个180个核苷酸的片段 RNA4为检测信使核糖核酸蛋白提供了一个难得的机会 互动。AMV RNA4-CP复合体Will的特性 有助于了解病毒交叉感染的分子基础 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 激活剂。迁移率带移电泳法与硝化棉 将使用过滤器保留研究来表征特定的 苜蓿花叶病毒RNA4与CP/DSI的相互作用 核苷酸缺失分析和定点突变将是 用于探测RNA和蛋白质结合位点。这个 大量CP的供应允许延长 分析包括物理研究。直接临床相关性 可能会在最近的报告中找到，这些报告详细描述了 与肿瘤特征相关的肿瘤表型的发展 癌基因mRNAs的5‘未翻译前导序列；此外， 蛋白质(S)与人5‘末端的相互作用 免疫缺陷病毒mRNAs对病毒基因表达的影响 产品。

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.