Function of Sequence-Specific Regulators of RNA Splicing

RNA 剪接的序列特异性调节因子的功能

• 批准号: 8371260
• 负责人: CHRISTOPHER B BURGE
• 金额: $ 31.85万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8371260
• 关键词: 3' Splice Site; Address; Affect; Affinity; Alternative Splicing; Binding; Binding Proteins; Biological Process; Cells; Complex; Computer software; DNA; DNA Binding; Data; Data Analyses; Development; Disease; Exons; Family; Genes; Genome; Goals; Human; Image; In Vitro; Introns; Ligands; Location; Malignant Neoplasms; Messenger RNA; Methods; Modeling; Mus; Muscle Development; Myotonic Dystrophy; Optics; Pathogenesis; Play; Primer Extension; Protein Analysis; Protein Dynamics; Protein Family; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Sequences; RNA Splicing; RNA-Binding Proteins; Recruitment Activity; Regulation; Resolution; Resources; Role; SF1; Site; Specificity; Spinal Muscular Atrophy; Staging; Structure; Tissues; U2 Small Nuclear Ribonucleoprotein; Variant; base; cardiogenesis; cellular imaging; exon skipping; high throughput analysis; human disease; in vivo; mRNA Precursor; method development; predictive modeling; protein function; research study; tool

DESCRIPTION (provided by applicant): Most human genes produce multiple distinct mRNA and protein isoforms through alternative splicing that may have distinct or even antagonistic biological functions. Alternative splicing is regulated by a set of RNA binding proteins (RBPs) whose functions are important in development and in a number of diseases. Our approach to the long-term goal of understanding the function of RBPs and their roles in alternative splicing is organized around the following specific aims: SA1. To develop a method for determination of the quantitative in vitro RNA binding specificity of a protein at unprecedented depth and to determine the RNA affinity landscape of RBFOX family proteins. SA2. To understand the sequence and RNA structural basis of RNA binding by MBNL and CELF family splicing factors, the extent of cooperativity and competition, and to understand the regulation of distinct subsets of targets in developmental and pathogenic contexts. SA3. To understand the binding affinity landscapes of factors that recognize motifs at the 3' ends of introns and their roles in regulation of alternative 3' splice site (3'SS) choice. We have recently developed a method called HiTS-FLIP, which provides a quantitative description of the in vitro DNA binding affinity landscape of a protein at unprecedented depth. We propose to develop a variation of this method, "HiTS-FLIP-R", to assess RNA binding affinity at a similarly high resolution. The essential idea is to sequence tens of millions of DNA clusters on an Illumina Genome Analyzer 2 (GA2) flow cell, to generate the corresponding RNA sequences by primer extension of anchored DNA adapters, to add a fluorescently tagged RNA binding protein to the flow cell at various concentrations and to image binding to RNA clusters using the GA2's optics. This approach will be applied to key factors involved in development and disease, including RBFOX2 and the myotonic dystrophy (DM) related factors MBNL1 and CELF1, as well as major factors that recognize the 3' splice site and contribute to constitutive and alternative splicing. These data will be used to develop quantitative models that predict the effects on binding and regulation of defined perturbations in the levels of specific RBPs, and the roles of these factors in development and in DM. PUBLIC HEALTH RELEVANCE: This project will provide comprehensive resources, tools and concepts for understanding the binding of proteins to RNA. It will also generate comprehensive binding affinity data for several important RNA binding proteins that function in constitutive splicing, which is required for expression of most human genes, or alternative splicing, including analysis of proteins that play central roles in development and in diseases such as myotonic dystrophy and cancer.

描述（由申请人提供）：大多数人类基因通过选择性剪接产生多种不同的mRNA和蛋白质同种型，这些基因可能具有不同甚至拮抗的生物学功能。选择性剪接受一组RNA结合蛋白（RBP）的调节，其功能在发育和许多疾病中很重要。我们对理解RBP的功能及其在选择性剪接中的作用的长期目标的方法是 围绕以下具体目标组织：SA 1。开发一种用于在前所未有的深度上定量测定蛋白质的体外RNA结合特异性的方法，并确定RBFOX家族蛋白质的RNA亲和力景观。SA 2.了解MBNL和CELF家族剪接因子结合RNA的序列和RNA结构基础，协同和竞争的程度，并了解发育和致病背景下不同靶点子集的调节。SA 3.了解识别内含子3'端基序的因子的结合亲和力景观及其在调控中的作用 选择性3'剪接位点（3' SS）。我们最近开发了一种称为HiTS-FLIP的方法，该方法提供了一种体外DNA结合亲和力的定量描述， 蛋白质在前所未有的深度。我们建议开发这种方法的变体，“HiTS-FLIP-R”，以类似的高分辨率评估RNA结合亲和力。其基本思想是在Illumina Genome Analyzer 2（GA 2）流动池上对数千万个DNA簇进行测序，通过锚定DNA接头的引物延伸产生相应的RNA序列，以各种浓度将荧光标记的RNA结合蛋白添加到流动池中，并使用GA 2的光学器件对与RNA簇的结合进行成像。这种方法将应用于参与发育和疾病的关键因子，包括RBFOX 2和强直性肌营养不良（DM）相关因子MBNL 1和CELF 1，以及识别3'剪接位点并有助于组成性剪接和选择性剪接的主要因子。这些数据将被用来开发定量模型，预测特定的限制性商业惯例的水平，以及这些因素在发展中的作用和DM的约束力和规定的扰动的影响。 公共卫生相关性：该项目将为理解蛋白质与RNA的结合提供全面的资源，工具和概念。它还将生成几个重要的RNA结合蛋白的综合结合亲和力数据，这些蛋白在组成性剪接中发挥作用，这是大多数人类基因表达或选择性剪接所必需的，包括分析在发育和强直性肌营养不良和癌症等疾病中发挥核心作用的蛋白质。