Development of RIP-Chip methods and tiled arrays to identify functional elements

开发 RIP-Chip 方法和平铺阵列来识别功能元素

• 批准号: 7347325
• 负责人: MICHAEL L WHITFIELD
• 金额: $ 36万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7347325
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Applications Grants; Binding; Binding Proteins; Binding Sites; Biogenesis; Bioinformatics; Cells; Chromatin Structure; Code; Complex; Computer software; Coupled; Cytoplasm; Development; Digestion; Disease; Electrophoretic Mobility Shift Assay; Elements; Functional RNA; Gene Expression; Genetic Transcription; Genome; Hela Cells; Heterogeneous Nuclear RNA; Histones; Human; Human Genome; Immunoprecipitation; Indium; Link; Mediating; Messenger RNA; Methods; MicroRNAs; Microarray Analysis; Molecular Conformation; Play; Post-Transcriptional Regulation; Process; Production; Protein Family; Proteins; Purpose; RNA; RNA Processing; RNA Stability; RNA analysis; RNA-Binding Proteins; RNA-Protein Interaction; Recombinants; Regulation; Regulatory Element; Role; Sampling; Site; Small Interfering RNA; Testing; Thinking; Translations; Untranslated RNA; cis acting element; combinatorial; mRNA Export; mRNA Precursor; mRNA Stability; mRNA tagging; messenger ribonucleoprotein; nuclease; particle; research study; stem; technology development; tool

DESCRIPTION (provided by applicant): The coordinate expression of genes and production of protein is controlled at multiple steps. In addition to transcription, mRNAs are regulated at the level of pre- mRNA processing, mRNA export, translation, and stability. Post-transcriptional regulation of gene expression can be mediated by RNA-binding proteins (RBPs) and by non-coding RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). These regulatory steps are thought to function by impacting the composition or state of the messenger ribonucleoprotein particle (mRNP). Understanding the cis-acting regulatory code that directs the assembly of these combinatorial mRNP complexes is the first step towards understanding this complex process and is only beginning to be investigated systematically using genome-level tools. We have developed methods to identify with high efficiency the possible targets of RBPs in human cells. We have shown in proof-of-principal experiments that we can use RNA Immunoprecipitations (IPs) followed by microarray analysis (RIP-Chip) to identify the targets of the histone stem-loop binding protein (SLBP) with high confidence. This method has been extended by using recombinant RBPs to examine potential binding sites in purified total RNA. We have termed this method recombinant, or rRIP-chip. The rRIP-Chip method allows sampling of all possible binding sites for an RBP in a pool of total RNA, and provides a simple method to distinguish direct RNA-protein interactions from interactions that occur via a protein bridge. In this application we describe experiments to develop these methods further. Specifically, the cis-acting regulatory sequences in mRNAs bound by specific RBPs will be determined by using limited nuclease digestion and tiled mRNA arrays. We also outline experiments to extend this method to the argonaute (AGO) family of proteins that play key roles in mediating the effects of miRNAs and siRNAs. RIP-Chip analysis of RNAs associated with AGO proteins will identify which miRNAs associate with AGO proteins and which mRNAs are targeted by these miRNAs. Limited nuclease digestions will be used to identify the mRNA sequences protected by miRNAs/AGO proteins and bioinformatic analyses will be used to determine specific miRNA target sequences.The misregulation of gene expression underlies most, if not all, disease states. Understanding the regulatory code that directs the expression of genes at all levels (transcription, pre-mRNA processing, translational control and RNA stability) will provide a better understanding of this basic process.

描述（由申请人提供）：基因的坐标表达和蛋白质的产生由多个步骤控制。除了转录外，在mRNA处理，mRNA输出，翻译和稳定性的水平上还调节mRNA。基因表达的转录后调节可以通过RNA结合蛋白（RBP）和非编码RNA（例如microRNA（miRNA）（miRNA）和小的干扰RNA（siRNA）介导。这些调节步骤被认为通过影响信使核糖核蛋白颗粒（MRNP）的组成或状态来起作用。了解指导这些组合MRNP复合物的顺式作用监管代码是理解这一复杂过程的第一步，并且刚刚开始使用基因组级工具系统地研究。我们已经开发了高效识别RBP在人类细胞中的可能目标的方法。我们已经在主要验证实验中表明，我们可以使用RNA免疫沉淀（IPS），然后进行微阵列分析（RIP-CHIP），以高度置信度鉴定组蛋白Stem-Roop结合蛋白（SLBP）的靶标。通过使用重组RBP检查纯化的总RNA中的潜在结合位点，该方法已扩展。我们称这种方法重组或RRIP-CHIP。 RRIP-CHIP方法允许在总RNA池中为RBP取样所有可能的结合位点，并提供了一种简单的方法，可以将直接RNA - 蛋白质相互作用与通过蛋白桥发生的相互作用区分出来。在此应用中，我们描述了进一步开发这些方法的实验。具体而言，通过使用有限的核酸酶消化和瓷砖mRNA阵列来确定由特定RBP结合的mRNA中的顺式作用调节序列。我们还概述了将此方法扩展到蛋白质的Argonaute（Ago）蛋白家族，这些蛋白质在介导miRNA和siRNA的作用中起着关键作用。与AGO蛋白相关的RNA的RIP-CHIP分析将确定哪种miRNA与AGO蛋白相关，以及这些mRNA是由这些miRNA靶向的。有限的核酸酶消化将用于识别受miRNA/AGO蛋白保护的mRNA序列，并且生物信息学分析将用于确定特定的miRNA靶靶序列。基因表达的不正直是疾病状态的大多数（即使不是全部）。了解指导各级基因表达的监管代码（转录，前MRNA处理，翻译控制和RNA稳定性）将为这一基本过程提供更好的理解。