Hu proteins as novel splicing regulators in neurons

娄华 标题：HU蛋白作为神经元中的新剪接调节因子 项目总结： 这个项目的长期目标是了解替代剪接如何在 哺乳动物中枢神经系统(CNS)。在上一次资助期间进行的研究 在神经元中建立了HU家族的副肿瘤性神经疾病(PND)抗原 可选的剪接调节器。最近，来自酵母双杂交筛查的初步数据 发现了Hu蛋白作为中介将转录与剪接联系起来的潜在作用。 具体地说，Huc与组蛋白H3和组蛋白脱乙酰酶HDAC2相互作用。重要的是，治疗 使用HDAC活性抑制剂的细胞的数量对Hu介导的替代有显著影响 剪接，为Huc-HDAC2相互作用的功能相关性提供了令人信服的证据 HU介导的选择性剪接。这项提议的中心目标是检验胡舒立 蛋白质通过与染色质结合直接相互作用以共转录的方式调节剪接 组蛋白H3和/或染色质重塑因子HDAC2。为了定义分子基础， 这些相互作用调节中枢神经系统神经元中前-mRNA剪接的机制，三 将追求具体的目标。在目标一中，将建立一个健壮的小鼠系统，用于派生 从小鼠ES细胞中获得同种中枢神经系统神经元，以研究神经元特异性的选择性剪接。这 系统将允许结合遗传和生化方法来研究剪接调控 神经细胞。在AIM II中，转录机制可能参与了Hu介导的 我们将研究神经元中选择性剪接的调节。HU-HDAC的特异性 将确定相互作用，并进行缺失和点突变分析，以确定 这些互动的性质。进一步的研究将测试Hu蛋白是否与 神经元中NF1等基因的启动子区域。在目标三中，胡适的功能后果-- 将确定HU介导的神经元选择性剪接中的HDAC/H3相互作用。使用外显子 23A对神经纤维瘤病I型前-mRNA为底物，转录延长的影响 将检查可选剪接的速率。这些研究将为我们提供对 控制组织特定的，特别是神经元特定的，替代的RNA剪接和 转录和剪接的耦合。将开发的中枢神经系统分化系统将 为神经元特异性剪接调控的研究提供了一个有价值的新的替代模型。

Lou, Hua Title: Hu proteins as novel splicing regulators in neurons Project Summary: 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, treatment of cells with an inhibitor of HDAC activity had a significant effect on Hu-mediated alternative splicing, providing compelling evidence for the functional relevance of the HuC-HDAC2 interaction in Hu-mediated alternative splicing. 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 a combined genetic and biochemical approached 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 Neurofibromatosis Type I 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.