Proteome-wide identification of RNA-binding proteins

描述(由申请人提供)：项目概述RNA在许多细胞过程中都是重要的，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。这方面的研究将检验这样一个假设，即蛋白质可以使用蛋白质组芯片以及生化和基于细胞的分析来区分RNA基序结构中的微小变化。这一假设将通过一个定义良好的简单RNA结构、定义良好的GNRA四环以及丙型肝炎病毒基因组RNA的3‘非翻译区(UTR)中的高度保守部分来验证。这两个目标将：1)确定发夹GNRA Tetraloop的结构变化如何影响它们与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能够特异性识别四环蛋白的蛋白质，确定大肠杆菌蛋白质组的同源物是否具有类似的特异性，测量RNA与蛋白质相互作用的亲和力和特异性，并在体内验证这些相互作用。2)筛选能识别丙型肝炎病毒基因组RNA3‘端非编码区的细胞蛋白。我们将筛选一个蛋白质芯片，该芯片携带3000个人类DNA/RNA结合蛋白和11个带有丙型肝炎病毒3‘非编码区X区的蛋白质。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分析中的丙型肝炎病毒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。这些信息应为今后发展蛋白质-RNA相互作用数据库提供起点。AIM 2的预期结果将确定与丙型肝炎病毒RNA具有高亲和力的细胞蛋白，这些蛋白可能会影响病毒感染。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鉴定在细胞中具有新活性的核酸结合蛋白。我们现在寻求确定将结合一个简单的RNA基序，GNRA四环，以及来自医学上重要的丙型肝炎病毒的调节RNA基序的蛋白质。这项为期两年的研究将提供原则证明，并制定出进一步研究蛋白质-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局的宿主因素。

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.