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）：基因的协调表达和蛋白质的产生是在多个步骤中控制的。除了转录之外，mRNA 在前 mRNA 加工、mRNA 输出、翻译和稳定性水平上受到调节。基因表达的转录后调控可以由RNA结合蛋白（RBP）和非编码RNA（例如微小RNA（miRNA）和小干扰RNA（siRNA））介导。这些调节步骤被认为是通过影响信使核糖核蛋白颗粒（mRNP）的组成或状态来发挥作用的。了解指导这些组合 mRNP 复合物组装的顺式作用调控代码是了解这一复杂过程的第一步，并且刚刚开始使用基因组水平工具进行系统研究。我们已经开发出高效识别人类细胞中 RBP 可能靶点的方法。我们在原理验证实验中证明，我们可以使用 RNA 免疫沉淀 (IP) 和微阵列分析 (RIP-Chip) 来高度可信地识别组蛋白茎环结合蛋白 (SLBP) 的靶标。该方法已通过使用重组 RBP 来检查纯化总 RNA 中的潜在结合位点进行了扩展。我们将这种方法称为重组法或 rRIP 芯片。 rRIP-Chip 方法允许对总 RNA 池中 RBP 的所有可能结合位点进行采样，并提供一种简单的方法来区分直接的 RNA-蛋白质相互作用和通过蛋白质桥发生的相互作用。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，将通过使用有限核酸酶消化和平铺的mRNA阵列来确定与特定RBP结合的mRNA中的顺式作用调节序列。我们还概述了将该方法扩展到 argonaute (AGO) 蛋白质家族的实验，该蛋白质家族在介导 miRNA 和 siRNA 的作用中发挥着关键作用。对与 AGO 蛋白相关的 RNA 进行 RIP 芯片分析将确定哪些 miRNA 与 AGO 蛋白相关以及哪些 mRNA 是这些 miRNA 的目标。有限的核酸酶消化将用于鉴定受 miRNA/AGO 蛋白保护的 mRNA 序列，生物信息学分析将用于确定特定的 miRNA 靶序列。基因表达的失调是大多数（如果不是全部）疾病状态的基础。了解指导基因在各个水平（转录、前 mRNA 加工、翻译控制和 RNA 稳定性）表达的调控代码将有助于更好地理解这一基本过程。