Regulation of mRNA splicing by intronic genetic variants

 描述(申请人提供)：内含子的遗传变异通常通过引起信使核糖核酸剪接的改变来影响细胞功能。外显子的异常包含性和排斥性常常改变蛋白质的功能和细胞表型。虽然许多内含子变体具有已知的功能，但随着下一代测序的采用，更多的内含子变体被发现，其功能影响尚不清楚。因此，重要的是能够预测变种的影响，而不需要在昂贵而费力的测试中对它们进行全部测试。虽然已经有信息学算法来预测遗传变异对前mRNA剪接的影响，但它们缺乏预测对蛋白质功能的影响，并最终预测疾病和治疗表型的能力。此外，还需要高通量的细胞分析来测试这些对细胞功能的预测结果。这里提出的研究将通过开发算法来满足这些需求，根据内含子变体对剪接和基因功能的潜在影响来确定它们的优先顺序，并开发一种高通量测试来从功能上测试数千种预测。这些新技术将应用于内含子变体对两种临床重要肿瘤药物氯法拉滨和紫杉醇的药物基因组学的影响。我们的长期目标是能够预测基因组变异对人类疾病和治疗反应的功能影响。我们的中心假设是，内含子遗传变异改变了mRNA的剪接，从而改变了蛋白质的功能，最终影响了细胞对药物治疗的反应。我们的第一个目标将是开发计算算法，根据内含子变体对前mRNA剪接和蛋白质功能的影响来确定它们的优先顺序。利用各种基因组和结构特征以及大量的基因组数据，我们将开发一种专门设计的生物信息学算法，根据内含子变体对前mRNA剪接和蛋白质功能的潜在影响来确定它们的优先顺序。我们的第二个目标将是确定与药物诱导的细胞毒性相关的功能性内含子变体。利用现有的基因组学和来自人类细胞系群体的细胞毒性反应数据，我们将识别与个体对氯法拉滨和紫杉醇细胞毒性反应有关的功能性内含子变异。我们的第三个目标 将从功能上测试优先考虑的内含子变体对前mRNA剪接和药物细胞毒性的影响。使用我们的新型高通量功能剪接测试，我们将测试 来自Aim 2的预测功能变异对前-mRNA剪接的影响。此外，我们将使用外显子特异的siRNA和CRISPR/Cas技术来操纵靶基因的剪接，以验证内含子变体对细胞毒性的影响。在这些研究完成后，我们希望开发出生物信息学算法，能够根据内含子变体对前mRNA剪接和蛋白质功能的功能影响准确地区分它们的优先顺序。此外，我们将在细胞前mRNA剪接试验中测试数千个变体，并验证其中几个功能变体对紫杉醇和氯法拉滨细胞毒性的影响。

 DESCRIPTION (provided by applicant): Genetic variations in introns commonly impact cellular functions by causing alterations in mRNA splicing. The abnormal inclusion and exclusion of exons often change protein functions and cellular phenotypes. Although many intronic variations have known functions, with the adoption of next generation sequencing, many more intronic variants have been discovered for which the functional impact is unknown. Thus, it is important to be able to predict the impact of the variants without needing to test them all in expensive and laborious assays. Although there are informatics algorithms that predict the impact of genetic variants on pre-mRNA splicing, their ability to predict the effect on protein function and ultimately disease and therapeutic phenotypes is lacking. In addition, there is a need for high-throughput cellular assays to test the results of these predictions on cellular functions. The studies proposed here will fulfill these needs by developing algorithms that prioritize the intronic variants by their potential impact on splicing and gene function, and developing a high-throughput assay to functionally test thousands of these predictions. These novel technologies will be applied to the effect of intronic variants on the pharmacogenomics of two clinically important oncology drugs, clofarabine and paclitaxel. Our long-term goals are to be able to predict the functional impact of genomic variants on human disease and therapeutic response. Our central hypothesis is that intronic genetic variants alter mRNA splicing and consequently protein function that ultimately affects the cellular response to drug therapy. Our first aim will be to develop computational algorithms that prioritize intronic variants based on their impacts on pre-mRNA splicing and protein function. Using a variety of genomic and structural features and large sets of genomic data, we will develop a bioinformatics algorithm specifically designed to prioritize intronic variants based on their potential impacts on pre-mRNA splicing and protein function. Our second aim will be to identify functional intronic variants associated with drug-induced cytotoxicity. Using existing genomics and cellular cytotoxic response data from populations of human cell lines, we will identify functional intronic variants that contribute to individuals' responses to clofarabine and paclitaxel cytotoxicity. Our third aim will be to functionally test the impact of the prioritized intronic variants on pre-mRNA splicing and drug cytotoxicity. Using our novel high- throughput functional splicing assay, we will test the effects of predicted functional variants from Aim 2 on pre- mRNA splicing. In addition, we will validate the effect of the intronic variants on cytotoxicity using exon specific siRNA and CRISPR/Cas technology to manipulate the target gene splicing. Upon completion of these studies, we expect to have developed bioinformatics algorithms that can accurately prioritize the intronic variants based on their functional impact on pre-mRNA splicing and protein function. Also, we will have tested thousands of variants in a cellular pre-mRNA splicing assay and validated the impact of several of these functional variants on paclitaxel and clofarabine cytotoxicity.