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Proteome-wide identification of RNA-binding proteins

RNA 结合蛋白的蛋白质组范围鉴定

基本信息

批准号：
7843516
负责人：
Cheng C Kao
金额：
$ 19.64万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
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

项目摘要

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 在许多细胞过程中都很重要，这些过程从基因表达的调节到充当先天防御机制中的特异性决定因素。这方面的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细胞分析来区分 RNA 基序结构中的微小变化。该假设将通过明确的简单 RNA 结构、明确的 GNRA 四环以及丙型肝炎病毒 (HCV) 基因组 RNA 3' 非翻译区 (UTR) 中的高度保守部分进行检验。这两个目标将： 1) 确定发夹 GNRA 四环结构的变化将如何影响其对目标蛋白的特异性和亲和力。我们将鉴定来自酵母蛋白质组芯片的能够特异性识别四环的蛋白质，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) 鉴定能够识别HCV基因组RNA 3' UTR的细胞蛋白。我们将筛选一种蛋白质芯片，该芯片携带人类中的 3000 个 DNA/RNA 结合蛋白和 HCV 中的 11 种蛋白质，并具有 HCV 3' UTR 的 X 区域。我们将通过体内 Pull down 测定进一步确认候选靶标，并确定它们是否会在细胞测定中影响 HCV 亚基因组复制子复制。目标 1 的预期结果将确定蛋白质-RNA 相互作用的基本规则，但很可能揭示以前未知的蛋白质功能。这些信息应该为蛋白质-RNA 相互作用数据库的未来开发提供起点。 Aim 2 的预期结果将鉴定与 HCV RNA 具有高亲和力的细胞蛋白，从而可能影响病毒感染。公共健康相关性：定义蛋白质-RNA 相互作用的规则将有益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在细胞中具有新活性的核酸结合蛋白。我们现在寻求鉴定能够结合简单 RNA 基序（GNRA 四环）以及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理证明，并制定出对蛋白质-RNA 相互作用组进行更深入研究所需的技术，并确定可能影响丙型肝炎病毒感染结果的宿主因素。