Proteome-wide identification of RNA-binding proteins

描述（由申请人提供）：项目概述RNA在许多细胞过程中是重要的，从基因表达的调节到作为先天防御机制中的特异性决定因素。这方面的研究将测试蛋白质可以使用蛋白质组芯片和生物化学和基于细胞的测定来区分RNA基序结构中的小变化的假设。这一假设将用明确定义的简单RNA结构、明确定义的GNRA四环和丙型肝炎病毒（HCV）基因组RNA的3 '非翻译区（UTR）中的高度保守部分来检验。这两个目标将：1）确定发夹GNRA四环结构的变化将如何影响它们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筛选出能特异性识别四环的蛋白质，确定是否存在来自大肠杆菌的直系同源物。大肠杆菌蛋白质组将具有相似的特异性，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)鉴定能够识别HCV基因组RNA的3 'UTR的细胞蛋白。我们将用HCV 3 'UTR的X区筛选一个携带3000个人类DNA/RNA结合蛋白和11个HCV蛋白的蛋白芯片。我们将通过体内下拉试验进一步确认候选靶点，并确定它们是否会影响基于细胞的试验中的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，但可能会揭示蛋白质以前未知的功能。这些信息应该为蛋白质-RNA相互作用数据库的未来发展提供起点。目标2的预期结果将鉴定出对HCV RNA具有高亲和力的细胞蛋白，这些蛋白可能影响病毒感染。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。我们已经成功地使用蛋白质组芯片来识别在细胞中具有新活性的核酸结合蛋白。我们现在试图确定蛋白质，将结合一个简单的RNA基序，GNRA四环，也从医学上重要的丙型肝炎病毒的调控RNA基序。这项为期两年的研究将提供原理证明，并制定出更深入研究蛋白质-RNA相互作用组所需的技术，并确定可能影响丙型肝炎病毒感染结果的宿主因素。

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.