Mechanisms of Alternative Splicing Regulation by Rbfox Proteins

项目总结 选择性剪接是调控基因输出的一种关键机制，该机制由不同的Pre-mRNA控制 结合蛋白。尽管最近的基因组分析让人们对监管的广度有了深入的了解 由这些蛋白质控制的网络，我们对这一过程的机械理解是初步的。小才是 已知调控蛋白影响拼接剪接体的分子相互作用，以及 这些信息对于理解多种形式的人类疾病是必不可少的，这些疾病被归因于调控不当 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，这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 含有其他八种RNA结合蛋白的大分子复合体，称为大剪接组装 监管机构，LASR。实际上，所有与未剪接RNA结合的RBFox蛋白都与LASR复合体有关， 数据表明，RBFox与LASR一起控制剪接。我们现在建议将 详细介绍RBFox/LASR的相互作用和活动。利用体内和体外实验，我们将鉴定蛋白质-蛋白质 LASR从其亚基组装、RBFox结合及其多聚体所必需的相互作用 转化为更高阶的复合体。我们将对与LASR和LASR共同验证的受保护RNA序列进行表征 将定义哪些片段与特定亚基相关联。全基因组的iCLIP分析将绘制出 LASR亚基与已知的RBFox结合位点的结合。其目的是了解它是如何 针对特定的RNA功能，我们将测试RBFox/LASR和纯化的LASR亚单位与 单个模体和体外组合的模体。使用剪接报告基因和全基因组 RNAseq分析，我们将定义LASR和RBFox剪接调控的共同靶点。最后，我们会 研究RBFox和单个LASR亚基如何合作调节特定的靶基因外显子 CRISPR基因敲除细胞系和神经元中的RNAi。这些研究将对错综复杂的 介导剪接调控的RNA元件和结合蛋白的组合及其 人类疾病中的错误调控。

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.