Proteome-wide identification of RNA-binding proteins

RNA 结合蛋白的蛋白质组范围鉴定

• 批准号: 7470332
• 负责人: Cheng C Kao
• 金额: $ 24.75万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7470332
• 关键词: 3' Untranslated Regions; Affect; Affinity; Antiviral Agents; Area; Binding; Biochemical; Biological; Biological Assay; Biological Sciences; Catalytic RNA; Cell physiology; Cells; Databases; Defense Mechanisms; Development; Engineering; Escherichia coli; Future; Gene Expression Regulation; Genomics; Hepatitis C; Hepatitis C virus; Human; Integration Host Factors; Introns; Life; Measures; Orthologous Gene; Outcome; Process; Protein Binding; Protein Engineering; Protein Microchips; Proteins; Proteome; RNA; RNA-Binding Proteins; RNA-Protein Interaction; Replicon; Research; Ribosomal RNA; Role; Specificity; Structure; Technology; Testing; Untranslated Regions; Variant; Virus Diseases; Work; base; design; improved; in vivo; novel; nucleic acid binding protein; protein function; public health relevance; viral RNA; yeast protein

DESCRIPTION (provided by applicant): Project Summary RNAs are important in numerous cellular processes that range from the regulation of gene expression to serving as the specificity determinants in innate defense mechanisms. Research in this will test the hypothesis that proteins can distinguish small variations in the structures of an RNA motif using proteome chips and biochemical and cell-based assays. This hypothesis will be examined with a well-defined simple RNA structure, the well-defined GNRA tetraloop and with the highly conserved portion in the 3' untranslated region (UTR) of the hepatitis C virus (HCV) genomic RNA. The two aims will: 1) Determine how changes in the structures of hairpin GNRA tetraloops will affect their specificity and affinity for target proteins. We will identify proteins from the yeast proteome chips that can specifically recognize the tetraloops, determine whether orthologs from E. coli proteome will have the similar specificities, measure the affinities and specificities of the RNA-protein interactions, and validate the interactions in vivo. 2) Identify the cellular proteins that could recognize the 3' UTR of the HCV genomic RNA. We will screen a protein chip that carries 3000 DNA/RNA binding proteins in humans and eleven proteins in HCV with the X region of the HCV 3' UTR. We will further confirm the candidate targets by in vivo pull down assays and determine whether they will affect HCV subgenomic replicon replication in cell-based assays. Anticipated results from Aim 1 will identify basic rules for protein-RNA interaction, but will likely reveal previously unknown functions of proteins. This information should provide the starting point for the future development of a protein-RNA interaction database. Anticipated results from Aim 2 will identify cellular proteins with high affinity to the HCV RNA that could influence viral infection. PUBLIC HEALTH RELEVANCE: Defining the rules for protein-RNA interaction will benefit many areas of biological science. We have successfully used proteome chips to identify nucleic acid-binding proteins that have novel activities in cells. We now seek to identify proteins that will bind a simple RNA motif, the GNRA tetraloop, and also the regulatory RNA motifs from the medically important hepatitis C virus. This two-year study will provide proof of principle and work out the technology needed for a more through study of the protein-RNA interactome and identify host factors that can influence the outcome of hepatitis C virus infection.

描述（由申请人提供）：项目摘要RNA在众多细胞过程中很重要，范围从基因表达的调节到先天防御机制的特异性决定因素。在此研究将检验以下假设：蛋白质可以使用蛋白质组芯片以及生化和基于细胞的测定方法来区分RNA基序的结构的微小变化。该假设将通过定义明确的简单RNA结构，定义明确的GNRA四臂以及高度保守的部分在乙型肝炎病毒（HCV）基因组RNA的3'未翻译区域（UTR）中进行检查。这两个目的将：1）确定发夹gnra四环的结构的变化将如何影响其对靶蛋白的特异性和亲和力。我们将从酵母蛋白质组芯片中鉴定出蛋白质，这些蛋白质可以特异性地识别四边形，确定来自大肠杆菌蛋白质组的直系同源物是否具有相似的特异性，测量RNA - 蛋白质相互作用的亲和力和特异性，并在体内验证相互作用。 2）确定可以识别HCV基因组RNA的3'UTR的细胞蛋白。我们将筛选一个蛋白芯片，该蛋白芯片在HCV 3'UTR的X区域中携带3000个DNA/RNA结合蛋白，在HCV中含有11种蛋白。我们将通过体内下拉分析来进一步确认候选靶标，并确定它们是否会影响基于细胞的测定中的HCV亚基因组复制子复制。 AIM 1的预期结果将确定蛋白质RNA相互作用的基本规则，但可能会揭示以前未知的蛋白质功能。该信息应为蛋白质RNA相互作用数据库的未来开发提供起点。 AIM 2的预期结果将确定可能影响病毒感染的HCV RNA高亲和力的细胞蛋白。公共卫生相关性：定义蛋白质RNA相互作用的规则将使生物科学的许多领域受益。我们已经成功使用蛋白质组芯片来鉴定在细胞中具有新活性的核酸结合蛋白。现在，我们试图鉴定将结合简单的RNA基序的蛋白质，GNRA四边形，以及来自医学上重要的丙型肝炎病毒的调节RNA基序。这项为期两年的研究将提供原理证明，并通过研究蛋白RNA Itsportome的更多技术来确定可能影响丙型肝炎病毒感染结果的宿主因素。