Function of Sequence-Specific Regulators of RNA Splicing

描述(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信使核糖核酸和蛋白质亚型，这些剪接可能具有不同的甚至是拮抗的生物学功能。选择性剪接由一组RNA结合蛋白(RBPs)调控，这些蛋白在发育和许多疾病中发挥重要作用。我们的长期目标是了解限制性商业惯例的功能及其在选择性剪接中的作用，我们的方法是 围绕以下具体目标组织：SA1。建立一种在体外以前所未有的深度定量测定蛋白质的RNA结合特异性的方法，并测定RBFox家族蛋白质的RNA亲和力。SA2.了解MBNL和CELF家族剪接因子与RNA结合的序列和RNA结构基础，协同和竞争的程度，以及在发育和致病背景下不同靶子集的调节。SA3.了解识别内含子3‘端基序的因子的结合亲和力图景及其在调控中的作用 可供选择的3‘剪接位点(3’SS)。我们最近开发了一种名为HITS-Flip的方法，它提供了对体外DNA结合亲和力图景的定量描述 蛋白质达到了前所未有的深度。我们建议开发这种方法的变体“HITS-Flip-R”，以同样高的分辨率评估RNA结合亲和力。其基本思想是在Illumina Genome Analyzer 2(Illumina Genome Analyzer 2)Flow细胞上对数千万个DNA簇进行测序，通过对锚定的DNA接头进行引物延伸来产生相应的RNA序列，在Flow细胞中添加不同浓度的荧光标记的RNA结合蛋白，并使用GA2的S光学器件成像与RNA簇的结合。这一方法将被应用于参与发育和疾病的关键因子，包括RBFOX2和强直性肌营养不良(DM)相关因子MBNL1和CELF1，以及识别3‘剪接位点并促进结构性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，以预测特定限制性商业惯例水平上明确的扰动对结合和调节的影响，以及这些因素在发育和糖尿病中的作用。

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.