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. ? ? ? ?
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