REGULATED SPLICING OF THE NEURON SPECIFIC HUMAN TAU GENE

对神经细胞的调节通路的理解 系统将是预防和治疗神经元疾病的关键。 细胞骨架元件的组织是神经元迁移的关键 和轴突的形成。Tau蛋白，它结合并组织 微管是建立神经元形态的重要工具。这个 神经元特异性tau转录本通过以下途径产生多种亚型 发育阶段和组织特定的选择性剪接。 Tau拼接障碍会导致神经元的破坏 细胞骨架与病理性tau结构的形成 (神经原纤维缠结)在痴呆症患者的大脑中发现。这个 这项建议的目的是调查其背后的机制 人类tau基因区域的选择性剪接以及 由这些区域编码的结构域的功能。具体地说，这 本研究旨在探索tau蛋白的剪接调控和功能 通过下列方法替换外显子：1)tau的测序 包含选择性剪接的外显子和 这些外显子在胎儿和成人中利用的建立 在大脑和人类神经母细胞瘤细胞中。2)微型燃气轮机的建设 包含tau选择性剪接外显子和 观察它们在神经母细胞瘤和非神经细胞中的行为。 在tau基因选择性加工中起作用的顺式元件 将由内含子和/或外显子的缺失决定 原始微型基因及其体内和体内转录本的研究 体外培养。3)确定参与剪接的反式因子 在体外通过对普通外显子的互补获得tau可选外显子 与神经母细胞瘤细胞提取物或已有的 表征了剪接因素。4)鉴定和表征 与编码的区域相互作用的细胞因子 利用酵母双杂交系统替代tau外显子。C DNA脑 将检查文库是否与携带tau的靶质粒结合 另一种外显子。卵巢癌的组织特异性和发育特征 通过这种方法鉴定的新蛋白质将作为一种 确定其编码基因的初步步骤。这个 这种相互作用的生理相关性将在体内得到验证 或体外结合或共沉淀试验。这项工作的成果 将有助于理解神经元的可塑性和 发展中的专业化。从长远来看，这项研究将授予 对神经源性级联的洞察，从而对异常过程的洞察 唐氏综合症和阿尔茨海默病的常见症状。

Understanding of the regulatory pathways which operate on the nervous system will be crucial to prevention and cure of neuronal disorders. Organization of cytoskeletal elements is critical for neuron migration and axon formation. Tau protein, which binds to and organizes microtubules, is instrumental in establishing neuron morphology. The neuron-specific tau transcript gives rise to multiple isoforms via developmental stage- and tissue-specific alternative splicing. Disturbances in tau splicing result in disruption of the neuronal cytoskeleton and formation of pathological tau structures (neurofibrillary tangles) found in brains of dementia sufferers. The objective of this proposal is to investigate the mechanisms that underlie the alternative splicing of regions of the human tau gene as well as the function of the domains encoded by these regions. Specifically, this study aims to explore the splicing regulation and function of tau alternative exons by the following methods: 1) Sequencing of the tau gene cloned segments that contain the alternatively spliced exons and establishment of utilization of these exons in fetal and adult human brain and in human neuroblastoma cells. 2) Construction of minigene constructs that contain the tau alternatively spliced exons and examination of their behavior in neuroblastoma and non-neuronal cells. Cis elements that play a role in alternative processing of the tau gene will be determined by deletions of introns and/or exons included in the original minigenes and investigation of their transcripts in vivo and in vitro. 3) Identification of trans factors that are involved in splicing of the tau optional exons by complementation of the generic in vitro splicing extract with either extracts from neuroblastoma cells or already characterized splicing factors. 4) Identification and characterization of cellular factors which interact with the domains encoded by the alternative tau exons by use of the yeast two-hybrid system. cDNA brain libraries will be examined for binding to target plasmids bearing the tau alternative exons. The tissue specificity and developmental profile of novel proteins identified by this method will be investigated as a preliminary step to characterizing their encoding genes. The physiological relevance of the interaction will be validated by in vivo or in vitro binding or co-precipitation assays. Results from this work will contribute to the understanding of neuronal plasticity and specialization during development. Long term, this research will grant insights into the neurogenic cascade and hence into abnormal processes common to Down syndrome and Alzheimer's disease.