A Discovery Tool for Variations that Affect Splicing

描述（由申请人提供）：前体mRNA剪接是一个关键的和受调控的加工事件，其中内含子从新生RNA转录物中精确切除。多达三分之一的遗传性疾病突变导致剪接缺陷。这项资助旨在鉴定单核苷酸多态性（SNPs），这些多态性通过破坏剪接的机制赋予自身免疫性/炎症性疾病的遗传风险。我们使用一种新的高通量方法来鉴定蛋白质/RNA结合和剪接中的等位基因差异。该筛选应用于具有与自身免疫性或炎性疾病相关的遗传基因座的LD中的所有SNP。PTB是可变剪接的主要调节因子之一，并且主要在免疫系统的细胞中高度表达。该建议的重点是筛选基础剪接机制（包括PTB）和与自身免疫性疾病相关的前体mRNA的多态性区域（即GWA的输出）之间的相互作用。用一种新的大规模平行高通量结合测定法测试配体的结合，该测定法是MEGAshift方案的改编。然后，我们在体内验证这些预测在其内源性位点的CLIP和功能测定。

DESCRIPTION (provided by applicant): Pre-mRNA splicing is a critical and regulated processing event where introns are precisely excised from nascent RNA transcripts. As many as one third of all heritable disease mutations result in splicing defects. This grant proposes to identify single nucleotide polymorphisms (SNPs) that confer genetic risk for autoimmune/inflammatory diseases through a mechanism of disrupted splicing. We use a novel high throughput method for identifying allelic differences in protein/RNA binding and splicing. This screen is applied to all SNPs in LD with genetic loci associated with autoimmune or inflammatory diseases. PTB is one of the major regulators of alternate splicing and is mostly highly expressed in the cells of the immune system. This proposal focuses on screening interactions between the basal splicing machinery (including PTB) and polymorphic regions of pre-mRNAs that are associated with autoimmune disorders (i.e. the output of GWAs). The binding of ligand is tested with a novel massively parallel high- throughput binding assay that is an adaptation of the MEGAshift protocol. We then validate these predictions in vivo at their endogenous loci by CLIP and functional assays.