RNA Binding Proteins & Alternative Splicing

描述（由申请人提供）： 该提案描述了一个多方面的计划，以促进我们对神经系统中RNA剪接的调节的理解。选择性剪接是产生哺乳动物蛋白质组多样性的主要驱动力，但其机制、调控网络以及对神经元功能和形态发生的反应却知之甚少。神经系统是组织特异性选择性剪接事件的丰富来源，其改变参与细胞通信和连接模式的蛋白质的功能。目的1将使用生物化学方法来表征NMDA R1受体转录物的CI和NI盒外显子的脑区域特异性剪接所涉及的机制。相关实验将确定RNA结合蛋白NAPOR在这些机制中的积极和消极作用的潜在基础，并将确定协作因子如PTB和hnRNP K的作用。RNAi基因沉默方法和原代神经元培养物将用于目标2，以表征NAPOR和PTB在参与突触功能的转录物剪接中的作用。补充实验将检查RNAi治疗对神经元中NMDA受体功能的影响。在这一目标的其他实验将监测的行为选择性剪接模式在原代皮层文化中的离子通道活性的变化，并在神经元分化的过程中。目的3将产生NAPOR缺陷型转基因小鼠，以确定其蛋白产物在剪接水平上的功能作用。还将描述对发育、生长和行为的影响。NAPOR缺陷小鼠将用于大规模分析大脑和原代培养物中的选择性剪接。这些实验将涉及大规模的mRNA和蛋白质表达谱，以确定参与NAPOR依赖性剪接途径的转录谱。选择性剪接异常与神经退行性疾病、精神疾病、神经肌肉疾病以及多种癌症有关。该研究项目将促进我们对神经元特异性剪接的潜在机制和可塑性的理解，并提供这些机制如何在人类疾病中失败的见解。

DESCRIPTION (provided by applicant): This proposal describes a multifaceted plan to advance our understanding of the regulation of alternative RNA splicing in the nervous system. Alternative splicing is a major driving force generating proteomic diversity in mammals, yet its mechanisms, regulatory networks, and responsiveness to neuronal function and morphogenesis are poorly understood. The nervous system is a rich source of tissue-specific alternative splicing events, which alter the functions of proteins involved in cell communication and connection patterns. Biochemical approaches will be used in Aim 1 to characterize the mechanisms involved in the brain region-specific splicing of the CI and NI cassette exons of the NMDA R1 receptor transcript. Related experiments will determine the underlying basis for the positive and negative roles of the RNA binding protein, NAPOR, in these mechanisms, and will determine the roles of collaborating factors, such as PTB and hnRNP K. RNAi gene silencing approaches and primary neuronal cultures will be used in Aim 2 to characterize the roles of NAPOR and PTB in the splicing of transcripts involved in synaptic functions. Complementary experiments will examine the effects of the RNAi treatments on NMDA receptor function in neurons. Additional experiments in this aim will monitor the behavior of alternative splicing patterns in the primary cortical cultures in response to changes in ion channel activity, and during the course of neuronal differentiation. NAPOR-deficient transgenic mice will be generated in Aim 3 to determine the functional roles of its protein products at the level of splicing. Effects on development, growth and behavior will also be characterized. NAPOR-deficient mice will be applied toward a large-scale analysis of alternative splicing in the brain and in primary cultures. These experiments will involve large-scale mRNA and protein expression profiling to identify the spectrum of transcripts involved in NAPOR-dependent splicing pathways. Abnormalities in alternative splicing are associated with neurodegenerative, psychiatric and neuromuscular diseases, as well as various cancers. This research project will advance our understanding of the underlying mechanisms and plasticity of neuron-specific splicing, and provide insights into how these mechanisms fail in human disease.