Biochemical Characterization of the splicing regulation of nPTB

描述（由申请人提供）：选择性前信使RNA剪接是真核生物基因调控的关键手段，允许单个基因产生多种mrna和蛋白质。许多对神经元发育和活动重要的蛋白质通过不同的外显子的不同包含而在功能上多样化。尽管它对神经元功能和疾病很重要，但控制选择性剪接的机制尚不清楚。我建议对神经元外显子剪接进行研究，重点关注调控蛋白神经元多嘧啶束结合蛋白（nPTB）。nPTB及其同源PTB是多个外显子的剪接抑制因子。PTB和nPTB的表达是相互排斥的，PTB局限于非神经元谱系，而nPTB仅在有丝分裂后的神经元中发现。在神经元分化过程中，PTB被nPTB取代。这个开关重新编程了神经元中大量可选外显子的剪接。体外构建了c-src神经元特异性N1外显子的调控。PTB抑制N1外显子的剪接。然而，神经元PTB不抑制N1和其他神经元外显子的剪接。实验将检验这种高度同源的蛋白质在活性上的差异。我将使用N1模型系统来分析PTB和nPTB对剪接前复合物组装的影响如何不同。已耗尽PTB且仅含有nPTB的细胞提取物将用于分析与被抑制的PTB复合物相比在成分和构象方面的差异。PTB抑制的决定因素将通过嵌合PTB / nPTB构建体进行检查，该构建体将在体内和体外进行抑制，结合和复合物形成的分析。高阶PTB-RNA和nPTB -RNA复合物的结构将通过电子显微镜和x射线晶体学进行研究。通过这些实验，我希望从分子细节上了解这两种高度相似的蛋白质如何产生不同的剪接结果，从而影响神经元细胞生物学。了解选择性剪接对我们了解多种形式的遗传疾病至关重要。脊髓性肌萎缩症、肌强直性营养不良症和前额叶痴呆是剪接调节的神经系统疾病。许多人类疾病突变改变剪接调控元件产生异常蛋白。为了治疗这些疾病，需要更多的信息来了解剪接调节的机制及其在神经元功能中的作用。

DESCRIPTION (provided by applicant): Alternative pre-messenger RNA splicing is a critical means of eukaryotic gene regulation that allows a single gene to produce a variety of mRNAs and proteins. Many proteins important for neuronal development and activity are functionally diversified through the differential inclusion of alternative exons. In spite of its importance to neuronal function and disease, the mechanisms controlling alternative splicing are poorly understood. I propose to study neuronal exon splicing with a focus on the regulatory protein neuronal Polypyrimidine Tract Binding Protein (nPTB). nPTB and its homolog PTB are splicing repressors for multiple exons. The expression of PTB and nPTB are mutually exclusive with PTB restricted to non-neuronal lineages and nPTB found only in post mitotic neurons. During neuronal differentiation PTB is replaced by nPTB. This switch reprograms the splicing of a large set of alternative exons in neurons. The regulation of the neuron specific N1 exon of the c-src has been constructed in vitro. PTB represses the splicing of N1 exon. However neuronal PTB does not repress the splicing of N1 and other neuronal exons. Experiments will examine how this highly homologous protein differs in activity. I will use the N1 model system to analyze how PTB and nPTB differ in their effect on pre-spliceosomal complex assembly. Extracts of cells that have been depleted of PTB and only contain nPTB will be used to analyze for differences in components and conformation compared to the repressed PTB complexes. The determinants of PTB repression will be examined through chimeric PTB / nPTB constructs that will be assayed in vivo and in vitro for repression, binding and complex formation. The Structure of the higher order PTB-RNA and nPTB -RNA complexes will be explored by electron microscopy and X-ray crystallography. Through these experiments I hope to understand in molecular detail how these two highly similar proteins generate different splicing outcomes and thus affect neuronal cell biology. The understanding of alternative splicing is essential to our understanding of multiple forms of genetic disease. Spinal muscular Atrophy, Myotonic Dystrophy, and Prefrontal Dementia are neurologic disorders of splicing regulation. Many human disease mutations alter splicing regulatory elements to produce aberrant proteins. For these diseases to be approached therapeutically, much more information is needed on the mechanisms of splicing regulation and its role in neuronal function.