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

描述（由申请人提供）：基因的协调表达和蛋白质的产生在多个步骤中受到控制。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（如microRNA（miRNA）和小干扰RNA（siRNA））介导。这些调控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。了解指导这些组合mRNP复合物组装的顺式作用调控密码是理解这一复杂过程的第一步，并且才开始使用基因组水平的工具进行系统研究。我们已经开发出了高效鉴定人细胞中RBP可能靶点的方法。我们已经在主要实验证明中表明，我们可以使用RNA免疫沉淀（IP），然后进行微阵列分析（RIP-Chip），以高置信度鉴定组蛋白茎环结合蛋白（SLBP）的靶标。该方法已通过使用重组RBP来检查纯化的总RNA中的潜在结合位点而得到扩展。我们称这种方法为重组，或rRIP芯片。rRIP-Chip方法允许对总RNA池中所有可能的RBP结合位点进行采样，并提供了一种简单的方法来区分直接的RNA-蛋白质相互作用与通过蛋白质桥发生的相互作用。在本申请中，我们描述了进一步开发这些方法的实验。具体而言，将通过使用有限的核酸酶消化和平铺的mRNA阵列来确定由特定RBP结合的mRNA中的顺式作用调控序列。我们还概述了将这种方法扩展到Argonaute（AGO）蛋白质家族的实验，这些蛋白质在介导miRNA和siRNA的作用中发挥关键作用。RIP-Chip分析与AGO蛋白相关的RNA将鉴定哪些miRNA与AGO蛋白相关以及哪些mRNA被这些miRNA靶向。有限的核酸酶双链反应将用于鉴定受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。了解在所有水平（转录，前mRNA加工，翻译控制和RNA稳定性）指导基因表达的调控密码将提供对这一基本过程的更好理解。

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.