Hu proteins as novel splicing regulators in neurons

描述（由申请人提供）：该项目的长期目标是了解哺乳动物中枢神经系统（CNS）中如何调节选择性剪接。在之前的资助期内进行的研究确定了神经元中副肿瘤神经疾病（PND）抗原的Hu家族作为替代剪接调节因子。最近，酵母双杂交筛选的初步数据揭示了Hu蛋白作为连接转录和剪接的介质的潜在作用。具体来说，HuC与组蛋白H3和组蛋白去乙酰化酶HDAC2相互作用。重要的是，Hu蛋白与RNAPII相关，参与延伸，Hu蛋白的表达与1型神经纤维瘤病（NF1）基因内部区域中高水平的乙酰化H3和H4相关，这些区域围绕着可选剪接的外显子23a。本研究的中心目标是验证Hu蛋白通过直接与染色质结合组蛋白H3和/或染色质重塑因子HDAC2相互作用以共转录方式调节剪接的假设。为了确定这些相互作用调节CNS神经元前mrna剪接的机制的分子基础，将追求三个特定的目标。在目的1中，将建立一个健壮的小鼠系统，用于从小鼠胚胎干细胞中衍生同质中枢神经系统神经元，以研究神经元特异性选择性剪接。该系统将使我们能够结合遗传和生化方法来研究神经细胞中的剪接调节。在目的II中，转录机制在hu介导的神经元选择性剪接调节中的潜在参与将被检查。将确定Hu-HDAC相互作用的特异性，并进行缺失和点突变分析以确定这些相互作用的性质。进一步的研究将测试Hu蛋白是否与神经元中NF1的启动子区和其他基因相关。在目的III中，将确定Hu-HDAC/H3相互作用在hu介导的神经元选择性剪接中的功能后果。以NF1 pre-mRNA的23a外显子为底物，研究转录延伸率对选择性剪接的影响。这些研究将为控制组织特异性，特别是神经元特异性，选择性RNA剪接以及转录和剪接耦合的机制提供基本见解。所建立的中枢神经系统神经元分化系统将为研究神经元特异性剪接调控提供一个有价值的新的替代模型。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how alternative splicing is regulated in the mammalian central nervous system (CNS). Studies carried out during the previous funding period established the Hu family of paraneoplastic neurologic disease (PND) antigens in neurons as alternative splicing regulators. More recently, preliminary data from a yeast two-hybrid screen uncovered a potential role for Hu proteins as mediators that link transcription with splicing. Specifically, HuC interacts with histone H3 and histone deacetylase HDAC2. Importantly, Hu proteins associate with RNAPII engaged in elongation and expression of Hu proteins correlates with higher level of acetylated H3 and H4 in an internal region of the Neurofibromatosis Type 1 (NF1) gene surrounding the alternatively spliced exon 23a. The central goal of this proposal is to test the hypothesis that Hu proteins regulate splicing in a co-transcriptional manner by directly interacting with chromatin bound histone H3 and/or the chromatin remodeling factor HDAC2. To define the molecular basis of the mechanisms through which these interactions regulate pre-mRNA splicing in CNS neurons, three specific aims will be pursued. In aim I, a robust mouse system will be established for the derivation of homogeneous CNS neurons from mouse ES cells to study neuron-specific alternative splicing. This system will allow us to combine genetic and biochemical approaches to investigate splicing regulation in neuronal cells. In aim II, the potential involvement of the transcription machinery in Hu-mediated regulation of alternative splicing in neurons will be examined. The specificity of the Hu-HDAC interaction will be determined and deletion and point mutational analysis will be carried out to define the nature of these interactions. Further studies will test whether Hu proteins are associated with the promoter region of NF1 and other genes in neurons. In aim III, the functional consequences of the Hu-HDAC/H3 interactions in Hu-mediated alternative splicing in neurons will be determined. Using exon 23a of the NF1 pre-mRNA as the substrate, the effect of transcription elongation rate on alternative splicing will be examined. These studies will provide fundamental insights into the mechanisms that control tissue-specific, particularly neuron-specific, alternative RNA splicing and coupling of transcription and splicing. The CNS neuronal differentiation system to be developed will serve as a valuable new alternative model in studies of neuron-specific splicing regulation. PUBLIC HEALTH RELEVANCE: Hu proteins are autoimmune antigens of a neurodegenerative disease called Hu syndrome. The proposed studies will investigate how these proteins regulate expression of neuron-specific proteins. These studies will provide not only significant insights into the role of Hu proteins in the development and function of neurons, but also important hints of pathogenesis of the Hu syndrome.