Comprehensive Identification of ENCODE RNA based Cis-Regulatory Elements

描述（由申请人提供）：我们对转录后调控的理解相对较差，只有少数直接转录后调控的调控元件被实验表征。真核生物依赖rna结合蛋白（rna binding protein, rbp）的作用来成功控制转录后基因的表达，它们提供了转录和翻译调控之间的联系，在转录、剪接、输出、稳定性和翻译等许多调控过程中发挥着重要作用。对表达RNA中顺式调控元件的全面鉴定是NIH/NHGRI ENCODE项目的基础，但目前还非常有限。以前，我们开发了纯化内源性RBP-RNA复合物的方法，并使用全基因组表达阵列技术（称为核糖组学）鉴定相关的RNA靶点。这一进展使rbp的许多mRNA靶点得以大规模鉴定，并为转录后基因调控的原理提供了新的见解。我们的研究表明，与转录调控类似，不同种类的rbp通过靶向转录本中相关的顺式调控元件，以组合方式协调调节功能相关的rna群。作为早期ENCODE技术授权的一部分，我们改进了我们的技术，开发了一种基于rip芯片的切片阵列检测，该检测包含一个消化步骤，以促进鉴定靶向顺式调控元件/RBPbinding位点。使用这种方法，本项目的目的是利用5种ENCODE细胞系和几种细胞扰动，全面编目在表达的ENCODE mRNA中存在的顺式调控元件/ rbp结合位点CREBS。这将通过(1)利用核糖组分析和全基因组表达阵列，用一组具有代表性的rbp来表征表达mRNA的全基因组关联；(2)利用基于平铺阵列的RIP-Chip技术鉴定ENCODE表达mrna子集的CREBS；(3)利用生物信息学和定量Real-Time PCR验证并进一步提高预测的ENCODE顺式调控元件/ rbp结合位点的分辨率；(4)利用报告基因试验对鉴定的CREBS的功能和rbp结合活性进行生物学验证。总之，我们将使用RNA结合蛋白来鉴定人类基因组ENCODE序列中基于RNA的顺式调控元件/ rbp结合位点。

DESCRIPTION (provided by applicant): Our understanding of post-transcriptional regulation is comparatively poor, with only a handful of regulatory elements that direct post-transcriptional control being experimentally characterized. Eukaryotic organisms depend on the actions of RNA-binding proteins (RBPs) for successful post-transcriptional control gene expression and they provide the link between transcriptional and translational regulation, playing essential roles in many regulatory processes including transcription, splicing, export, stability and translation. The comprehensive identification of cis-regulatory elements residing in expressed RNA is fundamental to the NIH/NHGRI ENCODE project but is extremely limited at the present time. Previously we developed methods for purifying endogenous RBP-RNA complexes and identifying the associated RNA targets using whole genome expression array technologies (termed ribonomics). This advance enabled the large-scale identification of many mRNA targets of RBPs and provided new insight into the principles governing posttranscriptional gene regulation. Our studies demonstrated that, analogous to transcriptional regulation, groups of functionally related RNAs are coordinately regulated in a combinatorial manner by distinct classes of RBPs by targeting related cis-regulatory elements located in the transcripts. As part of an earlier ENCODE technology grant, we improved our technology by developing a RIP-Chip tiling-array based assay that incorporates a digestion step to facilitate the identification of targeted cis-regulatory elements/RBPbinding sites. Using this method, the objective of this project is to comprehensively catalog the cis regulatory elements/RBP-binding sites CREBS present in expressed ENCODE mRNA using the five ENCODE cell lines and several cellular perturbations. This will be accomplished by (1) characterizing the genome-wide associations of expressed mRNA with a set of representative set of RBPs using ribonomic profiling and whole-genome expression arrays; (2) identifying the CREBS of the subset of ENCODE expressed mRNAs using tiling-array based RIP-Chip; (3) verifying and further increasing the resolution of predicted ENCODE cis-regulatory elements/RBP-binding sites using bioinformatics followed by quantitative Real-Time PCR and (4) biologically validating the function and RBP-binding activity of identified CREBS using a reporter assay. ln summary, we will use RNA-binding proteins to identify RNA based cis-regulatory elements / RBP-binding sites in the ENCODE sequence of the human genome.