Biochemical Characterization of the splicing regulation of nPTB

nPTB 剪接调控的生化表征

• 批准号: 8112644
• 负责人: Niroshika a M Keppetipola
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8112644
• 关键词: Affect; Alternative Splicing; Binding; Biochemical; Biological Assay; Biological Models; Brain; Cell Extracts; Cell physiology; Cellular biology; Complex; Dementia; Development; Disease; Electron Microscopy; Embryo; Exons; Foxes; Gene Expression Regulation; Genes; Goals; Hereditary Disease; Higher Order Chromatin Structure; Homologous Gene; Homologous Protein; Human; In Vitro; Laboratory Study; Lead; Length; Mammalian Cell; Mediating; Messenger RNA; Mitotic; Molecular; Molecular Conformation; Mutation; Myotonic Dystrophy; Nervous system structure; Neuroglia; Neurologic; Neuronal Differentiation; Neurons; Outcome; Pattern; Polypyrimidine Tract-Binding Protein; Protein Splicing; Proteins; RNA; RNA Recognition Motif; RNA Splicing; RNA, Messenger, Splicing; Regulation; Regulatory Element; Repression; Role; SRC gene; Site; Spinal Muscular Atrophy; Spliceosome Assembly Pathway; Structure; X-Ray Crystallography; cell type; genetic regulatory protein; human disease; in vivo; mRNA Precursor; nerve stem cell; nervous system disorder; neuron development; protein complex; research study

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.

描述（由申请人提供）：替代性前信使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选择性外显子的差异包含而功能多样化。尽管它对神经元功能和疾病的重要性，控制选择性剪接的机制知之甚少。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道结合蛋白（nPTB）。nPTB及其同源物PTB是多个外显子的剪接阻遏物。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。这个开关重新编程神经元中大量选择性外显子的剪接。在体外构建了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。实验将研究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在前剪接体复合体组装中的作用如何不同。已耗尽PTB且仅含有nPTB的细胞的提取物将用于分析与阻遏的PTB复合物相比组分和构象的差异。将通过嵌合PTB / nPTB构建体检查PTB抑制的决定簇，所述嵌合PTB / nPTB构建体将在体内和体外测定抑制、结合和复合物形成。高级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，从而影响神经元细胞生物学。对选择性剪接的理解对于我们理解多种形式的遗传疾病至关重要。脊髓性肌肉萎缩、强直性肌营养不良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节元件以产生异常蛋白质。为了治疗这些疾病，需要更多关于剪接调节机制及其在神经元功能中的作用的信息。