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Hu proteins as novel splicing regulators in neurons

Hu 蛋白作为神经元中新型剪接调节因子

基本信息

批准号：
8492172
负责人：
HUA LOU
金额：
$ 28.1万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8492172
关键词：
Affect Alternative Splicing Antibodies Antigens Autoimmune Process Binding Biochemical Biochemical Genetics Biological Process Cell Culture System Cells Chromatin Chromatin Remodeling Factor Complex Coupling Data Deletion Mutation Derivation procedure Development Disease Epitopes Exons Family Funding Gene Targeting Genes Genetic Genetic Transcription Genomics Goals HDAC2 gene Histone Deacetylase Histone H3 Histones Individual Knock-in Mouse Link Location Measures Mediating Mediator of activation protein Modeling Modification Molecular Mus Mutation Analysis NF1 gene Nature Neuraxis Neurodegenerative Disorders Neurofibromatosis 1 Neuroglia Neurologic Neuronal Differentiation Neurons Nuclear Extract Pathogenesis Play Point Mutation Population Process Promoter Regions Proteins RNA Interference RNA Splicing Regulation Role Sequence Analysis Signal Transduction Specificity Syndrome System Testing Time Tissues Transcription Elongation Undifferentiated Yeasts base cell type chromatin immunoprecipitation embryonic stem cell exon skipping histone deacetylase 2 insight interest mRNA Precursor mutant nervous system development novel novel strategies public health relevance research study tool yeast two hybrid system

项目摘要

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.

描述（由申请人提供）：本项目的长期目标是了解哺乳动物中枢神经系统（CNS）中选择性剪接是如何调节的。在上一个资助期进行的研究确定了神经元中副肿瘤性神经疾病（PND）抗原的Hu家族作为选择性剪接调节剂。最近，来自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体的潜在作用。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，Hu蛋白与参与延伸的RNAPII相关，并且Hu蛋白的表达与选择性剪接外显子23 a周围的1型神经纤维瘤病（NF 1）基因的内部区域中较高水平的乙酰化H3和H4相关。该提议的中心目标是检验Hu蛋白通过与染色质结合的组蛋白H3和/或染色质重塑因子HDAC 2直接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神经元中前mRNA剪接的机制的分子基础，将追求三个具体目标。目的一是建立一个稳定的小鼠系统，从小鼠ES细胞中分离出同源的中枢神经系统神经元，研究神经元特异性选择性剪接。该系统将使我们能够将联合收割机遗传学和生物化学方法结合起来研究神经细胞中的剪接调节。在目标II中，将检查胡介导的调节神经元中的选择性剪接的转录机制的潜在参与。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NF 1和其他基因的启动子区域相关。在目的III中，将确定神经元中Hu-HDAC/H3相互作用在Hu介导的选择性剪接中的功能后果。使用NF 1前体mRNA的外显子23 a作为底物，将检查转录延伸率对可变剪接的影响。这些研究将为控制组织特异性，特别是神经元特异性的RNA剪接以及转录和剪接的耦合机制提供基本的见解。本研究建立的中枢神经系统神经元分化系统为研究神经元特异性剪接调控提供了一种新的有价值的模型。