Mechanisms of Alternative Splicing Regulation by Rbfox Proteins

Rbfox 蛋白的选择性剪接调控机制

• 批准号: 9175889
• 负责人: Douglas L Black
• 金额: $ 41.62万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-16 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9175889
• 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; Neurologic; 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; Tertiary Protein Structure; Testing; Transcript; autism spectrum disorder; crosslink; genetic regulatory protein; genome-wide; human disease; in vitro Assay; in vivo; insight; mRNA Precursor; novel; polypeptide; protein protein interaction; stoichiometry; synaptic function; targeted treatment; transcriptome sequencing

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.

项目摘要 替代剪接是调节遗传输出的关键机制，该机制由不同的前MRNA指导 结合蛋白。尽管最近的基因组分析对调节的广度有所了解 由这些蛋白质控制的网络，我们对过程的机械理解是基本的。几乎没有 已知调节蛋白会影响组装剪接体的分子相互作用，并且 这样的信息对于理解归因于未经调节的多种形式的人类疾病至关重要 剪接。该项目将研究控制许多转录本的剪接的RBFOX RNA结合蛋白 对神经元功能和突触活动很重要，并且与癫痫和自闭症有关 频谱障碍。我们最近表明，核RBFOX同工型与一种新颖 大分子复合物，含有其他八种RNA结合蛋白，称为大型剪接组件 监管机构，拉斯尔。几乎所有与未填充RNA结合的RBFOX蛋白都与LASR复合物有关， 数据表明，RBFOX用LASR函数来控制剪接。我们现在建议描述 RBFOX/LASR相互作用和活动详细。使用体内和体外测定，我们将识别蛋白质蛋白 LASR组装从其亚基，RBFOX关联及其多聚化所需的相互作用 进入高阶复合物。我们将表征与LASR相关的受保护的RNA序列 将定义哪些片段与特定亚基相关联。全基因组ICLIP分析将绘制 LASR亚基相对于已知的RBFOX结合位点的结合。以了解它的目标 针对特定RNA特征，我们将测试RBFOX/LASR和纯化的LASR亚基的结合 单个主题和体外基序的组合。使用剪接报告基因和全基因组 RNASEQ分析，我们将定义LASR和RBFOX的剪接调节的共同靶标。最后，我们会的 检查RBFOX和单个LASR亚基如何使用 CRISPR敲除细胞系和神经元中的RNAi。这些研究将对复杂的新了解 RNA元素和结合蛋白的组合，介导剪接调节及其的结合蛋白 人类疾病的正调。