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。真核生物依赖于RNA结合蛋白(RNAbindingProteins，RBPs)在转录后调控基因的成功表达，它们在转录、剪接、输出、稳定和翻译等许多调控过程中发挥着重要的作用。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，我们开发了纯化内源性RBP-RNA复合体并使用全基因组表达阵列技术(称为核基因组学)识别相关RNA靶标的方法。这一进展使大规模鉴定限制性商业惯例的许多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，不同类别的限制性商业惯例通过靶向位于转录本中的相关顺式调控元件，以组合方式调节一组功能相关的RNA。作为早期ENCODE技术资助的一部分，我们改进了我们的技术，开发了一种基于RIP-Chip平铺阵列的分析方法，该方法结合了一个消化步骤，以便于识别目标顺式调节元件/RBP结合位点。使用这种方法，该项目的目标是使用五个ENCODE细胞系和几个细胞扰动，全面地对表达的ENCODE mRNA中存在的顺式调控元件/RBP结合位点Creb进行分类。这将通过以下方式实现：(1)使用核糖体分析和全基因组表达阵列来表征表达的mRNA与一组具有代表性的限制性商业惯例的全基因组相关性；(2)使用基于平铺阵列的RIP-Chip来鉴定ENCODE表达的mRNA子集的CREB；(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；以及(4)使用报告分析从生物学上验证所鉴定的CREB的功能和RBP结合活性。综上所述，我们将使用RNA结合蛋白来确定人类基因组ENCODE序列中基于RNA的顺式调节元件/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.