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，只有少数指导转录后调控的调控元件被实验表征。RNA结合蛋白（RNA-binding proteins，RBP）是真核生物中转录后调控基因表达的关键蛋白，在转录、剪接、输出、稳定和翻译等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（称为核糖体组学）鉴定相关的RNA靶标。这一进展使得大规模识别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，类似于转录调控，功能相关的RNA组的协调调节，在一个组合的方式由不同类别的RBP通过靶向相关的顺式调控元件位于转录本。作为早期ENCODE技术资助的一部分，我们通过开发一种基于RIP芯片平铺阵列的检测方法来改进我们的技术，该方法包括消化步骤，以便于识别靶向顺式调节元件/RBP结合位点。使用这种方法，本项目的目的是全面编目的顺式调控元件/RBP结合位点CREBS表达ENCODE mRNA使用五个ENCODE细胞系和几个细胞扰动。这将通过（1）使用核糖组分析和全基因组表达阵列表征表达的mRNA与一组代表性RBP组的全基因组关联;（2）使用基于平铺阵列的RIP-Chip鉴定ENCODE表达的mRNA子集的CREBS;（3）验证并进一步提高预测的ENCODE顺式调节元件/RBP的分辨率，结合位点，以及（4）使用报告基因测定生物学验证所鉴定的CREBS的功能和RBP结合活性。总之，我们将使用RNA结合蛋白来鉴定人类基因组的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.