Mechanisms of Alternative Splicing Regulation by Rbfox Proteins

项目摘要 选择性剪接是调节由不同前体mRNA指导的遗传输出的关键机制 结合蛋白虽然最近的基因组分析已经深入了解了调控基因的广度， 虽然这些蛋白质控制着神经网络，但我们对这一过程的机械理解还很初步。之甚少 已知调节蛋白影响剪接体组装的分子相互作用， 这些信息对于理解许多形式的人类疾病是必不可少的， 拼接本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 谱系障碍我们最近发现，核Rbfox亚型与一种新的 一种大分子复合物，含有八种其他RNA结合蛋白，称为剪接大集合体 调节器，LASR。几乎所有与未剪接RNA结合的Rbfox蛋白都与LASR复合物结合， 并且数据表明Rbfox与LASR一起起作用以控制剪接。我们现在建议描述 Rbfox/LASR互动和活动的详细信息。使用体内和体外试验，我们将鉴定蛋白质-蛋白质 LASR从其亚基组装、Rbfox缔合及其多聚化所必需的相互作用 变成更高级的复合物。我们将表征与LASR共纯化的受保护的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 针对特定的RNA特征，我们将测试Rbfox/LASR和纯化的LASR亚基与 单个基序和体外基序的组合。使用剪接报告基因和全基因组 通过RNAseq测定，我们将定义LASR和Rbfox剪接调控的共同靶标。最后我们将 研究Rbfox和单个LASR亚基如何在调节特定靶外显子中合作， CRISPR敲除细胞系和神经元中的RNAi。这些研究将产生对复杂的 介导剪接调节的RNA元件和结合蛋白的组合，及其 人类疾病中的失调。

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.