Proteome-wide identification of RNA-binding proteins

rna在许多细胞过程中都很重要，从基因表达的调控到作为先天防御机制的特异性决定因素。这方面的研究将检验这样一种假设，即蛋白质可以通过蛋白质组芯片、生化和基于细胞的分析来区分RNA基序结构的微小变化。这一假设将通过明确定义的简单RNA结构、明确定义的GNRA四环和丙型肝炎病毒（HCV）基因组RNA 3'非翻译区（UTR）的高度保守部分来检验。这两个目标将是：1)确定发夹型GNRA四足蛋白结构的变化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能够特异性识别四足虫的蛋白质，确定大肠杆菌蛋白质组的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)鉴定能识别HCV基因组RNA 3′UTR的细胞蛋白。我们将筛选一种蛋白质芯片，该芯片携带人类3000种DNA/RNA结合蛋白和HCV 3' UTR X区域的11种HCV蛋白。我们将通过体内拉下试验进一步确认候选靶点，并在基于细胞的试验中确定它们是否会影响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。这些信息应该为未来蛋白质- rna相互作用数据库的开发提供起点。Aim 2的预期结果将鉴定出可能影响病毒感染的与HCV RNA高亲和力的细胞蛋白。公共卫生相关性：确定蛋白质- rna相互作用的规则将有利于生物科学的许多领域。我们已经成功地使用蛋白质组芯片来鉴定细胞中具有新活性的核酸结合蛋白。我们现在正在寻找能够结合简单RNA基序（即GNRA四环）和医学上重要的丙型肝炎病毒的调节性RNA基序的蛋白质。这项为期两年的研究将为更深入地研究蛋白质- rna相互作用组和确定影响丙型肝炎病毒感染结果的宿主因素提供原理证明和所需的技术。

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.