PLP alternative splicing and oligodendrocyte differentiation

PLP选择性剪接和少突胶质细胞分化

• 批准号: 7564053
• 负责人: FRANCA CAMBI
• 金额: $ 32.05万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-03 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7564053
• 关键词: 5' Splice Site; Alternative Splicing; Binding; Biochemical; Brain; Cells; Coupled; Data; Demyelinating Diseases; Disease; Elements; Enhancers; Equilibrium; Exons; Fibroblasts; Future; Generations; Genes; Genetic; Grant; Human; In Vitro; Inborn Genetic Diseases; Inherited; L Cells; Maps; Mus; Mutation; Myelin; Myelin Proteolipid Protein; Nerve; Nervous system structure; Neurodegenerative Disorders; Neuroglia; Nuclear; Oligodendroglia; Patients; Protein Binding; Protein Isoforms; Proteins; RNA Interference; RNA Splicing; Regulation; Regulatory Element; Rest; Role; Scanning; Site; Testing; Tissues; Transcript; Transcriptional Activation; cell injury; in vivo; innovation; knock-down; nervous system disorder; novel; progenitor

DESCRIPTION (provided by applicant): Functionally diverse proteins are generated by alternative splicing of primary transcripts in differentiating oligodendrocytes. PLP and DM20 are generated through the alternative selection of competing 5' splice sites in exon 3. As PLP becomes the predominant isoform, the PLP/DM20 ratio increases in differentiated oligodendrocytes (OL) versus progenitors (OPC). Mutations that alter the ratio of PLP to DM20 cause neurological disorders in humans [Hobson et al., 2006; Hobson et al., 2002]. One of these mutations is a deletion of a G-rich intronic enhancer (ISE) of the PLP 5' site. In the preliminary studies, we show that exon 3B contains sequences that regulate the PLP/DM20 ratio. A G-rich sequence (M2) is an enhancer of DM20 5' site, while the other exonic sequences enhance the PLP 5' site. Although both are G-rich, the ISE and M2 are functionally distinct. A number of hnRNP's bind to the ISE and M2 and some of them are down regulated in OL versus OPC. We hypothesize that M2 enhances the DM20 5' site in OPC, while the ISE favors the PLP 5' site in OL as a result of decrease in hnRNP's and changes in the balance of general and cell-specific factors. Other exon 3B sequences favor the PLP 5' site and are both general and cell-specific. In Aim 1, we will characterize the ISE's function within the full context of the PLP gene in the developing nervous system of a novel knockin mouse that carries a deletion of the ISE. The cell-specific and differentiation-dependent function of the ISE will be elucidated in the brain, nerves and non-glial tissues. In Aim 2, we will characterize the role of M2 in controlling the PLP/DM20 ratio in oligodendrocytes and non-glial cells by mapping the contribution of the G-sequences and flanking sequences to controlling the PLP/DM20 ratio. The proteins that bind to M2 and to ISE will be identified in biochemical studies and their expression will be examined in OPC and OL. In Aim 3 we will examine enhancers of the PLP 5' site and define their role in general, cell-specific and differentiation-dependent regulation of PLP/DM20 ratio. In Aim 4 we will examine the function of hnRNP's in controlling the PLP/DM20 ratio with knock down studies by RNAi. These studies have broad relevance to oligodendrocyte differentiation, generation of transcript diversity, and alterations of splicing that causes inherited disorders of myelin.

描述（由申请人提供）：功能多样的蛋白质是通过区分少突胶质细胞中的主要转录本的替代剪接而产生的。 PLP和DM20是通过外显子3中的替代选择5'剪接位点的替代选择而产生的。随着PLP成为主要同工型，PLP/DM20比在分化的少突胶质细胞（OL）与祖细胞（OPC）中增加了PLP/DM20。改变PLP与DM20的比例的突变会导致人类神经系统疾病[Hobson等，2006; Hobson等，2002]。这些突变之一是删除PLP 5'位点的富含G富含G的内含子增强子（ISE）。在初步研究中，我们表明外显子3B包含调节PLP/DM20比的序列。富含G的序列（M2）是DM20 5'位点的增强子，而其他外显子序列增强了PLP 5'位点。尽管两者都是富含G的，但ISE和M2在功能上是不同的。许多HNRNP与ISE和M2结合，其中一些在OL与OPC中受到调节。我们假设M2增强了OPC中的DM20 5'位点，而ISE则有利于OL中的PLP 5'位点，这是由于HNRNP的降低以及一般和细胞特异性因素平衡的变化。其他外显子3B序列有利于PLP 5'位点，并且既通用又是细胞特异性的。在AIM 1中，我们将在PLP基因的完整环境中表征ISE的功能，在一种新型敲蛋白小鼠的神经系统中，它带有ISE的删除。 ISE的细胞特异性和分化依赖性功能将在大脑，神经和非腹膜组织中阐明。在AIM 2中，我们将通过映射G序列和侧翼序列对控制PLP/DM20比的贡献来表征M2在控制PLP/DM20比率中的作用。与M2和ISE结合的蛋白质将在生化研究中鉴定，其表达将在OPC和OL中进行检查。在AIM 3中，我们将检查PLP 5'位点的增强子，并定义其在PLP/DM20比的一般，细胞特异性和分化依赖性调节中的作用。在AIM 4中，我们将研究HNRNP在控制PLP/DM20比的功能，并通过RNAi的敲除研究。这些研究与少突胶质细胞分化，转录本多样性的产生以及引起髓磷脂遗传疾病的剪接的改变具有广泛的相关性。