Identifying functional regulatory elements in RNA

描述(由申请人提供)：这项拟议项目的重点是开发利用RNA结合蛋白(RBPs)来全面识别存在于表达的人RNA中的调控元件所需的新技术。真核生物依赖限制性商业惯例的作用来成功表达基因，不仅在转录和翻译调控之间提供联系，而且在转录、剪接、输出、稳定和翻译等已建立的调控过程中发挥重要作用。了解转录后基因调控并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。我们以前开发了利用基因组阵列技术纯化内源性mRNA-蛋白质复合体(MRNPs)并识别相关mRNA靶标的方法，我们称之为核基因组学方法。我们的研究表明，与转录调控类似，mRNAs组在转录后水平上由不同类别的限制性商业惯例以组合方式协调调节，限制性商业惯例可以通过靶向位于转录本中的相关调控元件来协调对mRNAs组的调控。目前，核糖体技术仅识别限制性商业惯例的信使核糖核酸。将需要开发新的高通量技术，以详尽地识别和绘制起调控元件作用的RBP结合位点。新发展的平铺阵列技术代表着对表达RNA中调控元件的潜在鉴定的巨大突破。这一新的阵列平台代表了微阵列技术的重大进步，该技术利用连续的探针组来详尽地询问整个基因组序列。该项目的目的是开发新的基于平铺阵列的核基因组图谱方法，包括高通量RBP足迹，这反过来将促进RNA调控元件的全面图谱。

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.