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，仅对少数指导转录后控制的调控元件进行了实验表征。真核生物依赖于 RNA 结合蛋白 (RBP) 的作用来成功实现转录后控制基因表达，它们提供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重要作用。全面鉴定表达 RNA 中的顺式调控元件是 NIH/NHGRI ENCODE 项目的基础，但目前极其有限。 此前，我们开发了使用全基因组表达阵列技术（称为核糖组学）纯化内源性 RBP-RNA 复合物并鉴定相关 RNA 靶标的方法。这一进展使得大规模鉴定 RBP 的许多 mRNA 靶点成为可能，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，功能相关的 RNA 组通过靶向位于转录本中的相关顺式调控元件，由不同类别的 RBP 以组合方式协调调控。作为早期 ENCODE 技术资助的一部分，我们通过开发基于 RIP-Chip 平铺阵列的检测改进了我们的技术，该检测结合了消化步骤，以促进目标顺式调控元件/RBP 结合位点的识别。使用这种方法，该项目的目标是使用五种 ENCODE 细胞系和几种细胞扰动对表达的 ENCODE mRNA 中存在的顺式调控元件/RBP 结合位点 CREBS 进行全面编目。这将通过 (1) 使用核糖组分析和全基因组表达阵列来表征表达的 mRNA 与一组代表性 RBP 的全基因组关联； (2)使用基于平铺阵列的RIP芯片识别ENCODE表达的mRNA子集的CREBS； (3) 使用生物信息学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，然后进行定量实时 PCR，以及 (4) 使用报告基因测定对已识别的 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 结合蛋白来识别人类基因组 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.