Characterization of Small Molecule Splicing Modulators of MAPT and FGFR1

描述（由申请人提供）： 我的项目的目标是进一步表征和治疗目标，选择性剪接的两个转录本，异常调节神经系统疾病状态。tau蛋白病是一组以微管相关蛋白tau（tau）的改变剪接为特征的神经退行性疾病，而胶质细胞肿瘤与成纤维细胞生长因子受体1（FGFR1）的不正确剪接相关，两者都是神经疾病方面令人难以置信的社会负担。本申请中提出的方法需要使用高通量筛选测定来鉴定这两种神经系统前mRNA转录物剪接的小分子调节剂。随后对这些化合物的分析，如洋地黄毒苷作为MAPT外显子10的调节剂，将集中在转录因子组范围的剪接效应上。对药物处理细胞的剪接敏感性微阵列数据的生物信息学分析将提供一组序列基序，这些序列基序是药物能够改变剪接的潜在调控元件。对这些基序的研究将需要分析已知与基序结合的RNA结合蛋白，以及表征以前未知的调控元件。分析将包括小分子对候选靶蛋白表达、翻译后修饰和细胞定位的影响。 高通量筛选的应用并不限于鉴定可以改变重要剪接事件的药物。通过筛选siRNA构建体的文库，我打算鉴定在MAPT和FGFR1剪接中发挥关键调节作用的蛋白质。此类筛选的数据补充了小分子数据，因为关键调节蛋白也可能是药物靶点候选者。总之，这种表征MAPT和FGFR1剪接的策略不仅将进一步了解这两种转录本的基本选择性剪接调控，而且还将朝着开发针对导致神经变性和恶性胶质细胞转化的剪接异常的疗法迈出第一步。 从公共卫生的角度来看，该项目建议在分子水平上进一步表征两种多产的神经系统疾病过程，神经变性和神经胶质细胞肿瘤发生。这种可以特异性靶向这些分子畸变的药物的表征和鉴定，例如在选择性剪接中，是将神经系统疾病识别为需要集中诊断和治疗的个体和特定过程的关键步骤。

DESCRIPTION (provided by applicant): The goals of my project are to further characterize, and therapeutically target, the alternative splicing of two transcripts that are aberrantly regulated in nervous system disease states. Tauopathies, which are a set of neurodegenerative diseases characterized by altered splicing of microtubule associated protein tau (MAPI), and glial cell tumors, which are associated with incorrect splicing of fibroblast growthfactor receptor 1 (FGFR1), are both incredible societal burdens in terms of neurological disease. The methods proposed in this application entail using a high-throughput screening assay to identify small molecule modulators of the splicing of these two nervous system pre-mRNA transcripts. Subsequent analysis of such compounds, such as digitoxin as a modulator of MAPT exon 10, will focus on transciptome-wide splicing effects. Bioinformatics analysis of splicing-sensitive microarraydata from drug-treated cells will provide a set of sequence motifs which are potential regulatory elements by which a drug is able to alter splicing. Investigation of such motifs will entail analysis of RNA binding proteins which are known to bind to the motifs, as well as characterization of previously unknown regulatory elements. Analyses will consist of the effects of small molecules on a candidate target protein's expression, post-translational modifications, and cellular localization. The application of high-throughput screening is not limited to identification of drugs that can alter important splicing events. By screening libraries of siRNA constructs, I intend to identify proteins that play a key regulatory role in MAPT and FGFR1 splicing. Data from such screens complements the small molecule data as key regulatory proteins are also likely drug target candidates. In total, this strategy for characterizing MAPT and FGFR1 splicing will not only further the understanding of basic alternative splicing regulation of the two transcripts, but will also make the first steps toward developing therapies aimed at the splicing abnormalities that contribute to neurodegeneration and malignant glial cell transformation. From a public health standpoint, this project proposes to further characterize two prolific nervous system disease processes, neurodegeneration and glial cell tumorigenesis, at the molecular level. This characterization and identification of drugs that can specifically target these molecular aberrations, such as in alternative splicing, are key steps in the transition to recognition of neurological diseases as individual and specific processes that require focused diagnosis and treatment.