Mechanisms of Alternative Splicing Regulation by Rbfox Proteins

Rbfox 蛋白的选择性剪接调控机制

基本信息

批准号：
9753010
负责人：
Douglas L Black
金额：
$ 41.66万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-16 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9753010
关键词：
Affect Alternative Splicing Amyotrophic Lateral Sclerosis Binding Binding Proteins Binding Sites Biological Assay Cell Line Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Data Development Disease Elements Epilepsy Event Exons Frontotemporal Dementia Genetic Genomics Goals Heterogeneous-Nuclear Ribonucleoprotein Group M In Vitro Individual Knock-out Macromolecular Complexes Maps Mass Spectrum Analysis Mediating Multiprotein Complexes Mutation Myotonic Dystrophy Nervous system structure Neurons Nuclear Output Process Protein Family Protein Isoforms Proteins RNA RNA Interference RNA Sequences RNA Splicing RNA-Binding Proteins Reaction Recombinants Regulation Reporter Reporter Genes Role Site Spinal Muscular Atrophy Spliceosomes Synapses Tertiary Protein Structure Testing Transcript autism spectrum disorder crosslink genetic regulatory protein genome-wide human disease in vitro Assay in vivo insight mRNA Precursor nervous system disorder novel polypeptide protein protein interaction stoichiometry targeted treatment transcriptome sequencing virtual

项目摘要

PROJECT SUMMARY Alternative splicing is a key mechanism for regulating genetic output that is directed by diverse pre-mRNA binding proteins. Although recent genomic analyses have lent insight into the breadth of the regulatory networks controlled by these proteins, our mechanistic understanding of the process is rudimentary. Little is known of the molecular interactions by which regulatory proteins affect the assembling spliceosome, and such information is essential to understanding the many forms of human disease attributed to misregulated splicing. This project will study the Rbfox RNA binding proteins that control the splicing of many transcripts important for neuronal function and synaptic activity, and which are implicated in epileptic and autism spectrum disorders. We recently showed that the nuclear Rbfox isoforms are bound with a novel macromolecular complex containing eight other RNA binding proteins and called a large assembly of splicing regulators, LASR. Virtually all the Rbfox protein bound to unspliced RNA is associated with a LASR complex, and data indicate that Rbfox functions with LASR to control splicing. We now propose to characterize Rbfox/LASR interactions and activity in detail. Using in vivo and in vitro assays, we will identify protein-protein interactions necessary for LASR assembly from its subunits, for Rbfox association, and for its multimerization into higher order complexes. We will characterize the protected RNA sequences that copurify with LASR and will define which fragments associate with particular subunits. Genomewide iCLIP analysis will map the binding of LASR subunits relative to the known Rbfox binding sites. With the goal of understanding how it is targeted to particular RNA features, we will test the binding of Rbfox/LASR and purified LASR subunits to individual motifs and to combinations of motifs in vitro. Using splicing reporter genes and genomewide RNAseq assays, we will define the common targets of splicing regulation by LASR and Rbfox. Finally, we will examine how Rbfox and individual LASR subunits cooperate in regulating particular target exons using CRISPR knockout cell lines and RNAi in neurons. These studies will yield new understanding of the intricate combinations of RNA elements and binding proteins that mediate the regulation of splicing, and its misregulation in human disease.

项目概要选择性剪接是调节遗传输出的关键机制，由不同的前 mRNA 指导结合蛋白。尽管最近的基因组分析让我们深入了解了监管的广度由于这些蛋白质控制的网络，我们对该过程的机械理解还很初级。小的是已知调节蛋白影响剪接体组装的分子相互作用，以及这些信息对于了解由于监管不当而导致的多种人类疾病至关重要拼接。该项目将研究控制许多转录本剪接的 Rbfox RNA 结合蛋白对神经元功能和突触活动很重要，与癫痫和自闭症有关频谱障碍。我们最近证明核 Rbfox 亚型与一种新的结合含有其他八种RNA结合蛋白的大分子复合物，称为剪接大组装体监管机构，LASR。事实上，所有与未剪接 RNA 结合的 Rbfox 蛋白都与 LASR 复合物相关，数据表明，Rbfox 与 LASR 一起发挥作用来控制剪接。我们现在建议表征 Rbfox/LASR 相互作用和活动的详细信息。使用体内和体外测定，我们将鉴定蛋白质-蛋白质 LASR 从其亚基组装、Rbfox 关联及其多聚化所需的相互作用成更高阶的复合物。我们将表征与 LASR 共纯化的受保护 RNA 序列，将定义哪些片段与特定的子单元相关联。全基因组 iCLIP 分析将绘制 LASR 亚基相对于已知 Rbfox 结合位点的结合。目的是了解它是如何发生的针对特定的 RNA 特征，我们将测试 Rbfox/LASR 和纯化的 LASR 亚基的结合单个基序和体外基序组合。使用剪接报告基因和全基因组 RNAseq 分析中，我们将定义 LASR 和 Rbfox 剪接调节的共同目标。最后，我们将使用以下方法检查 Rbfox 和各个 LASR 亚基如何合作调节特定目标外显子 CRISPR 敲除细胞系和神经元中的 RNAi。这些研究将对复杂的问题产生新的认识介导剪接调节的RNA元件和结合蛋白的组合及其人类疾病的失调。