Functional RNA elements in the human genome

描述（由申请人提供）： 该提案寻求更新多PI项目（Fu和Yeo），以使用全球方法阐明哺乳动物基因组中选择性剪接调控的调控原则。基于我们在过去的资助周期中取得的成就，包括广泛的初步结果，我们建议进行几项系统的功能丧失和获得研究，以确定三个特定目标中参与选择性剪接调控的基因、基因网络和途径。第一个目标是进行大规模的网络分析的调节可变剪接。使用我们实验室开发的二维mRNA异构体分析平台，我们建议在HEK 293细胞中进行全基因组RNAi和过表达筛选，并在每种处理条件下对一组常见的调节剪接事件（约400）进行评分。我们还将通过全面的RNA-seq对约300种精心选择的RNA结合蛋白（RBP）的shRNA介导的耗尽（已经完成）和异位表达进行转录组学分析，以补充这些全基因组干扰研究。这些数据将有助于识别新的剪接因子，将RBP整合到转录，表观遗传和信号转导途径中，并破译单个RBP的独特和主导功能。在第二个目标中，我们提出分析RBP为中心的蛋白质-蛋白质相互作用网络。特别是，我们建议充分利用我们的验证库的开放阅读框架编码RBPs的定量蛋白质组学分析的300 RBPs与RNA酶处理和不确定RNA依赖和独立的相互作用的框架内的RBP复合物。使用功能性RNA靶标（Aim 1）和RBP中心蛋白质-蛋白质相互作用（Aim 2）的大数据集，我们将在Aim 3中对RNA基因组学数据进行综合分析，以建立RBP调节选择性剪接的预测模型。我们将使用或开发一套计算工具来预测新的RBP靶点，完善单个RBP中心基因网络，最重要的是，将全面的RBP顺式和反式相互作用组与系统范围的扰动相结合，以建立细胞特异性调节选择性剪接的预测模型。我们相信，这种综合分析将对我们理解调控剪接和相关疾病机制产生重大影响。

DESCRIPTION (provided by applicant): This proposal seeks renewal of a multi-PI project (Fu and Yeo) to use global approaches to elucidate regulatory principles in the regulation of alternative splicing in mammalian genomes. Built upon our accomplishments in the past funding cycle, including extensive preliminary results, we propose to conduct several systematic loss- and gain-of-function studies to identify genes, gene networks, and pathways involved in the regulation of alternative splicing in three specific aims. The first aim is to perform large-scale network analysis of regulated alternative splicing. Using the two-dimensional mRNA isoform profiling platform developed in our labs, we propose to conduct both genome-wide RNAi and overexpression screening in HEK293 cells and score a set of commonly regulated splicing events (~400) in each treatment condition. We will also complement these genome-wide perturbation studies with transcriptomic analyses by comprehensive RNA-seq against shRNA-mediated depletion (already completed) and ectopic expression of ~300 carefully selected RNA binding proteins (RBPs). These data will help identify new splicing factors, integrate RBPs into transcription, epigenetic and signaling pathways, and decipher both unique and dominant functions of individual RBPs. In the second aim, we propose to analyze RBP-centric protein-protein interaction networks. In particular, we propose to take full advantage of our validated library of open reading frames encoding RBPs for quantitative proteomic analysis of 300 RBPs with and without RNase treatment to identify both RNA-dependent and independent interactions within the framework of RBP complexes. Using the large datasets of functional RNA targets (Aim 1) and RBP-centric protein-protein interactions (Aim 2), we will in Aim 3 perform integrated analysis of RNA genomics data to build predictive models of the regulation of alternative splicing by RBPs. We will use or develop a set of computational tools to predict novel RBP targets, refine individual RBP-central gene networks, and most importantly, integrate comprehensive RBP cis and trans interactomes with system-wide perturbation to build predictive models for cell-specific regulation of alternative splicing. We believe that such integrated analysis will have major impacts on our understanding of regulated splicing and associated disease mechanisms.