The focus of this proposed project is to develop the novel technologies necessary for exploiting RNA-binding proteins (RBPs) to comprehensively identify regulatory elements present in expressed human RNA. Eukaryotic organisms depend on the actions of RBPs for successful gene expression, providing not only a link between transcriptional and translational regulation, but also playing essential roles in established regulatory processes including transcription, splicing, export, stability and translation. Understanding posttranscriptional gene regulation and identifying the RNA regulatory elements responsible for this regulation is an important aspect of fulfilling the ENCODE project objectives. We previously developed methods for purifying endogenous mRNA-protein complexes (mRNPs) and identifying the associated mRNA targets using genomic array technologies, a method we termed ribonomics. Our studies have demonstrated that, analogous to transcriptional regulation, groups of mRNAs are coordinately regulated in a combinatorial manner by distinct classes of RBPs at the post-transcriptional level and that RBPs can coordinate the regulation of groups of mRNAs by targeting related regulatory elements located in the transcripts. Presently, ribonomic technology only identifies mRNA targets of RBPs. New high-throughput technologies will need to be developed for the exhaustive identification and mapping of RBP binding sites that function as regulatory elements. Newly developed tiling array technology represents a tremendous breakthrough for the potential identification of regulatory elements in expressed RNA. This new array platform represents a significant advance in microarray technology that utilizes contiguous probe sets to interrogate entire genomic sequences in exhaustive detail.
The aim of this project is to develop novel tiling array based ribonomic profiling methods including high-throughput RBP foot-printing, which will in turn, facilitate the comprehensive mapping of RNA regulatory elements. ? ?
