A Discovery Tool for Variations that Affect Splicing

描述(申请人提供)：前mRNA剪接是一个关键的和受调控的加工事件，内含子被精确地从新生的RNA转录本中删除。在所有可遗传疾病突变中，多达三分之一会导致剪接缺陷。这项拨款建议通过剪接中断的机制识别单核苷酸多态(SNPs)，这些SNPs通过剪接中断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识别蛋白质/RNA结合和剪接中的等位基因差异。这一筛查适用于LD中具有与自身免疫性或炎症性疾病相关的遗传位点的所有SNPs。PTB是交替剪接的主要调节因子之一，主要在免疫系统的细胞中高表达。这一建议侧重于筛选基本剪接机制(包括PTB)和与自身免疫疾病相关的前mRNAs的多态区域(即GWA的输出)之间的相互作用。配体的结合是用一种新的大规模并行的高通量结合试验来测试的，这是MEGAShift协议的改编。然后，我们通过CLIP和功能分析在体内验证这些预测的内源性位点。

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.