A Discovery Tool for Variations that Affect Splicing

描述（由申请人提供）：Pre-mRNA剪接是一个关键和受调节的加工事件，内含子被精确地从新生RNA转录物中切除。多达三分之一的遗传性疾病突变导致剪接缺陷。该基金旨在通过剪接中断的机制，鉴定具有自身免疫/炎症性疾病遗传风险的单核苷酸多态性（snp）。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。该筛选适用于LD中与自身免疫性或炎症性疾病相关的遗传位点的所有snp。PTB是交替剪接的主要调控因子之一，主要在免疫系统细胞中高表达。该建议的重点是筛选基础剪接机制（包括PTB）与与自身免疫性疾病（即GWAs的输出）相关的pre- mrna多态性区域之间的相互作用。配体的结合是用一种新的大规模平行高通量结合试验来测试的，这是一种megasshift协议的改编。然后，我们通过CLIP和功能分析在体内的内源性位点验证这些预测。

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.