Identifying functional regulatory elements in RNA

描述（由申请人提供）：本项目的重点是开发开发RNA结合蛋白（rbp）所需的新技术，以全面鉴定表达的人类RNA中存在的调控元件。真核生物依赖rbp的作用来成功表达基因，rbp不仅是转录和翻译调控之间的纽带，而且在转录、剪接、输出、稳定性和翻译等已建立的调控过程中发挥着重要作用。了解转录后基因调控和确定负责这种调控的RNA调控元件是实现ENCODE项目目标的一个重要方面。我们之前开发了纯化内源性mRNA-蛋白复合物（mRNPs）和使用基因组阵列技术鉴定相关mRNA靶点的方法，我们将这种方法称为核糖组学。我们的研究表明，与转录调控类似，mrna组在转录后水平上由不同类别的rbp以组合方式协调调控，rbp可以通过靶向转录本中的相关调控元件来协调mrna组的调控。目前，核糖组学技术只能识别rbp的mRNA靶标。需要开发新的高通量技术，以详尽地识别和绘制作为调控元件的RBP结合位点。新开发的平铺阵列技术代表了潜在的RNA调控元件鉴定的巨大突破。这种新的阵列平台代表了微阵列技术的重大进步，该技术利用连续探针集来查询整个基因组序列的详尽细节。该项目的目的是开发新的基于平铺阵列的核糖组分析方法，包括高通量RBP足迹，这将反过来促进RNA调控元件的全面定位。

DESCRIPTION (provided by applicant): 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.