Hu proteins as novel splicing regulators in neurons

卢华 Hu蛋白作为神经元中的新型剪接调节剂 项目摘要： 该项目的长期目标是了解选择性剪接在细胞中是如何调节的。 哺乳动物中枢神经系统（CNS）。上一个供资期间开展的研究 建立了神经元中副肿瘤性神经疾病（PND）抗原的Hu家族， 可变剪接调节子。最近，酵母双杂交筛选的初步数据 揭示了Hu蛋白作为连接转录与剪接的介质的潜在作用。 具体而言，HuC与组蛋白H3和组蛋白脱乙酰酶HDAC 2相互作用。重要的是，治疗 HDAC活性抑制剂的细胞对Hu介导的替代性 剪接，为HuC-HDAC 2相互作用的功能相关性提供了令人信服的证据， Hu-mediated alternative splicing.这一提议的中心目标是检验胡锦涛的假设， 蛋白质通过直接与染色质结合物相互作用， 组蛋白H3和/或染色质重塑因子HDAC 2。为了确定 这些相互作用调节CNS神经元中前mRNA剪接的机制， 将追求具体目标。在目标I中，将建立一个稳健的小鼠系统，用于衍生 同源CNS神经元从小鼠ES细胞研究神经元特异性选择性剪接。这 系统将允许一种遗传和生物化学相结合的方法来研究剪接调控， 神经元细胞在目的II中，转录机制在Hu介导的 将检查神经元中可变剪接的调节。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 这些相互作用的性质。进一步的研究将测试Hu蛋白是否与 NF 1启动子区和神经元中的其他基因。在目标III中，胡- 将确定神经元中Hu介导的可变剪接中的HDAC/H3相互作用。使用外显子 以神经纤维瘤病I型前体mRNA的23 a为底物， 将检查选择性剪接的比率。这些研究将提供基本的见解， 控制组织特异性，特别是神经元特异性，可变RNA剪接的机制， 转录和剪接的偶联。将要开发的CNS神经元分化系统将 作为一个有价值的新的替代模型，在研究神经元特异性剪接调控。

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.