Biochemical Characterization of the splicing regulation of nPTB

描述(由申请人提供)：替代前信使RNA剪接是真核基因调控的关键手段，允许单个基因产生各种mRNAs和蛋白质。许多对神经元发育和活动至关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却知之甚少。我建议研究神经元外显子剪接，重点是调节蛋白神经元多嘧啶结合蛋白(NPTB)。NPTB及其同源基因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仅限于非神经元谱系，而nPTB仅在有丝分裂后神经元中发现。在神经元分化过程中，pTb被nptb取代。这种开关对神经元中一大组可选外显子的剪接重新编程。C-src的神经元特异性N1外显子的调控已在体外构建。PTB抑制N1外显子的剪接。然而，神经元PTB并不抑制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有何不同。我将使用N1模型系统来分析PTB和nPTB在预剪接体复合体组装中的不同作用。已经去除了PTB并且只含有nPTB的细胞的提取物将被用来分析与被抑制的PTB复合体相比在成分和构象上的差异。PTB抑制的决定因素将通过嵌合的PTB/nPTB构建来检验，该构建将在体内和体外检测抑制、结合和复合体的形成。高阶PtB-RNA和nPTB-RNA络合物的结构将通过电子显微镜和X射线结晶学进行探索。通过这些实验，我希望从分子上详细了解这两种高度相似的蛋白质如何产生不同的剪接结果，从而影响神经细胞生物学。对选择性剪接的理解对于我们理解多种形式的遗传病是必不可少的。脊髓性肌萎缩症、强直性肌营养不良和前额叶痴呆是剪接调节的神经性疾病。许多人类疾病的突变改变了剪接调控元件以产生异常蛋白质。为了将这些疾病用于治疗，还需要更多关于剪接调节机制及其在神经功能中的作用的信息。

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.