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(申请人提供)：在分化少突胶质细胞的过程中，初级转录本的选择性剪接产生了功能多样化的蛋白质。PLP和DM20是通过外显子3竞争的5‘剪接位点的交替选择而产生的。随着PLP成为主要的异构体，分化的少突胶质细胞(OL)和祖细胞(OPC)中PLP/DM20的比率增加。改变PLP和DM20比率的突变会导致人类的神经疾病[Hobson等人，2006；Hobson等人，2002]。其中一个突变是PLP 5‘位点富含G的内含子增强子(ISE)的缺失。在初步研究中，我们发现外显子3B包含调节PLP/DM20比率的序列。富含G的序列(M2)是DM20 5‘位点的增强子，其他外显子序列是PLP 5’位点的增强子。虽然两者都是G丰富的，但ISE和M2在功能上是不同的。一些hnRNP与ISE和M2结合，其中一些在OL和OPC中下调。我们假设M2增强了OPC中的DM20 5‘位点，而ISE由于hnRNP的减少以及一般和细胞特异性因子平衡的变化而有利于OL中的PLP 5’位点。其他外显子3B序列偏爱PLP5‘位点，既是一般性的，也是细胞特异性的。在目标1中，我们将在PLP基因的完整背景下表征ISE在一种新的敲击小鼠神经系统发育中的功能，该小鼠携带ISE缺失。ISE的细胞特异性和分化依赖功能将在大脑、神经和非胶质组织中阐明。在目标2中，我们将通过定位G序列和侧翼序列对控制PLP/DM20比率的贡献来表征M2在控制少突胶质细胞和非胶质细胞中PLP/DM20比率中的作用。结合M2和ISE的蛋白质将在生化研究中确定，它们的表达将在OPC和OL中检测。在目标3中，我们将研究PLP 5‘位点的增强子，并确定它们在PLP/DM20比率的一般、细胞特异性和分化依赖的调节中的作用。在目标4中，我们将通过RNAi下调研究来研究hnRNP在控制PLP/DM20比率中的作用。这些研究与少突胶质细胞分化、转录多样性的产生以及导致髓鞘遗传性疾病的剪接改变有广泛的相关性。