REGULATED SPLICING OF THE NEURON SPECIFIC HUMAN TAU GENE

了解对神经系统起作用的调节途径 系统将是预防和治疗神经元疾病关键。 细胞骨架元素的组织对于神经元迁移至关重要 和轴突形成。 Tau蛋白，结合并组织 微管是建立神经元形态的工具。 的 神经元特异性tau转录物通过以下途径产生多种同种型： 发育阶段和组织特异性选择性剪接。 tau蛋白剪接的干扰导致神经元的破坏， 细胞骨架和病理性tau结构的形成 在痴呆症患者的大脑中发现的（神经系统缠结）。 的 本提案的目的是调查 人tau基因区域的选择性剪接以及 这些区域编码的结构域的功能。 具体来说， 本研究旨在探讨tau蛋白的剪接调控和功能 1）tau蛋白的测序 含有可变剪接外显子的基因克隆片段， 在胎儿和成人中建立这些外显子利用 脑和人类神经母细胞瘤细胞中。 2)minigene的构建 含有tau可变剪接外显子的构建体， 检查它们在神经母细胞瘤和非神经元细胞中的行为。 在tau基因的替代加工中起作用的顺式元件 将通过包括在内含子和/或外显子中的内含子和/或外显子的缺失来确定。 原始小基因及其在体内和体内转录物的研究 体外 3)参与剪接的反式因子的鉴定 的tau任选外显子的通用在体外互补 将提取物与来自神经母细胞瘤细胞的提取物或已经 特征剪接因子。 4)鉴定和表征 细胞因子的相互作用的结构域编码的 通过使用酵母双杂交系统的替代tau外显子。 cDNA脑 将检查文库与带有tau的靶质粒的结合 替代外显子。 组织特异性和发育概况 通过这种方法鉴定的新蛋白质将作为 初步确定其编码基因。 的 相互作用的生理相关性将通过体内试验进行验证。 或体外结合或共沉淀测定。 这项工作的结果 将有助于理解神经元的可塑性， 在发展过程中专业化。 从长远来看，这项研究将使 深入了解神经原性级联反应以及异常过程 常见于唐氏综合症和老年痴呆症。

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.