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PLP alternative splicing and oligodendrocyte differentiation

PLP选择性剪接和少突胶质细胞分化

基本信息

批准号：
7992399
负责人：
FRANCA CAMBI
金额：
$ 31.41万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-01-03 至 2012-12-31
项目状态：
已结题

项目摘要

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由分化少突胶质细胞的初级转录物的选择性剪接产生的。PLP和DM20是通过外显子3上竞争的5'剪接位点的选择性选择产生的。随着PLP成为主要亚型，分化少突胶质细胞（OL）与祖细胞（OPC）的PLP/DM20比值增加。改变PLP与DM20比例的突变会导致人类神经系统疾病[Hobson等人，2006；霍布森等人，2002]。其中一个突变是plp5 '位点的富g内含子增强子（ISE）的缺失。在初步研究中，我们发现外显子3B包含调控PLP/DM20比值的序列。富g序列（M2）是DM20 5′位点的增强子，而其他外显子序列则是PLP 5′位点的增强子。虽然两者都富含g，但ISE和M2在功能上是不同的。许多hnRNP结合ISE和M2，其中一些在OL中与OPC相比下调。我们假设M2增强了OPC中的dm205 ‘位点，而ISE则增强了OL中的plp5 ’位点，这是由于hnRNP的减少以及一般因子和细胞特异性因子平衡的改变。其他外显子3B序列有利于plp5 '位点，并且是一般的和细胞特异性的。在Aim 1中，我们将在一种携带ISE缺失的新型敲入小鼠发育中的神经系统中，在PLP基因的完整背景下描述ISE的功能。ISE的细胞特异性和分化依赖性功能将在脑、神经和非胶质组织中得到阐明。在Aim 2中，我们将通过绘制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被识别，它们的表达将在OPC和OL中被检测。在Aim 3中，我们将研究plp5 '位点的增强子，并定义它们在PLP/DM20比率的一般、细胞特异性和分化依赖性调节中的作用。在Aim 4中，我们将通过RNAi敲低研究来研究hnRNP在控制PLP/DM20比率方面的功能。这些研究与少突胶质细胞分化、转录物多样性的产生以及导致髓磷脂遗传性疾病的剪接改变具有广泛的相关性。