Functional RNA elements in the human genome

人类基因组中的功能性RNA元件

基本信息

批准号：
8773860
负责人：
XIANG-DONG FU
金额：
$ 69.75万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-06-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8773860
关键词：
Alternative Splicing Binding Cells Code Collection Complement Complex Computing Methodologies Coupling Custom Data Data Set Development Disease Dissection Double-Stranded RNA Ectopic Expression Epigenetic Process Event Functional RNA Funding Gene Expression Gene Expression Regulation Gene Mutation Generations Genes Genetic Transcription Genomics Grant Health Human Human Genome Indium Individual Laboratories Length Libraries Link Malignant Neoplasms Mammalian Cell Mediating Messenger RNA Molecular Mutation National Human Genome Research Institute Nerve Degeneration Neurodegenerative Disorders Open Reading Frames Pathway Analysis Pathway interactions Positioning Attribute Process Productivity Protein Binding Protein Isoforms Proteins Proteomics Publications Publishing RNA RNA Interference RNA Processing RNA Splicing RNA analysis RNA-Binding Proteins Reagent Regulation Regulator Genes Research Resources Ribonucleases Series Signal Pathway Signaling Molecule Structure System Technology Translations base cell type cis acting element computerized tools effective therapy gain of function genome-wide human disease innovation insight leukemia mammalian genome novel overexpression predictive modeling protein TDP-43 protein complex protein protein interaction response screening small hairpin RNA tool transcriptome sequencing transcriptomics treatment strategy two-dimensional

项目摘要

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.

描述（由申请人提供）：该提案寻求更新多 PI 项目（Fu 和 Yeo），以使用全球方法来阐明哺乳动物基因组中选择性剪接的监管原则。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性的功能丧失和功能获得研究，以识别基因、基因网络和参与选择性剪接调节的途径，以实现三个具体目标。第一个目标是对受调控的选择性剪接进行大规模网络分析。使用我们实验室开发的二维 mRNA 同工型分析平台，我们建议在 HEK293 细胞中进行全基因组 RNAi 和过度表达筛选，并在每种治疗条件下对一组常见调节的剪接事件（~400）进行评分。我们还将通过针对 shRNA 介导的耗竭（已完成）和约 300 个精心挑选的 RNA 结合蛋白 (RBP) 的异位表达的综合 RNA-seq 进行转录组分析来补充这些全基因组扰动研究。这些数据将有助于识别新的剪接因子，将 RBP 整合到转录、表观遗传和信号通路中，并破译单个 RBP 的独特和主导功能。在第二个目标中，我们建议分析以 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经过验证的编码 RBP 的开放阅读框架文库，对经过或不经过 RNase 处理的 300 个 RBP 进行定量蛋白质组分析，以识别 RBP 复合物框架内依赖于 RNA 的和独立的相互作用。利用功能性 RNA 靶点（目标 1）和以 RBP 为中心的蛋白质-蛋白质相互作用（目标 2）的大型数据集，我们将在目标 3 中对 RNA 基因组数据进行综合分析，以建立 RBP 选择性剪接调节的预测模型。我们将使用或开发一套计算工具来预测新的 RBP 靶标，完善单个 RBP 中央基因网络，最重要的是，将全面的 RBP 顺式和反式相互作用组与全系统扰动相结合，以建立细胞特异性调节的预测模型选择性拼接。我们相信，这种综合分析将对我们对调控剪接和相关疾病机制的理解产生重大影响。