PLP alternative splicing and oligodendrocyte differentiation

描述（由申请人提供）：功能多样的蛋白质是通过分化少突胶质细胞中初级转录物的选择性剪接而产生的。 PLP 和 DM20 是通过外显子 3 中竞争性 5' 剪接位点的选择性选择而产生的。随着 PLP 成为主要同种型，分化的少突胶质细胞 (OL) 与祖细胞 (OPC) 中的 PLP/DM20 比率增加。改变 PLP 与 DM20 比例的突变会导致人类神经系统疾病 [Hobson et al., 2006;霍布森等人，2002]。这些突变之一是 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，而其他外显子序列则增强 PLP 5' 位点。尽管 ISE 和 M2 都富含 G，但其功能不同。许多 hnRNP 与 ISE 和 M2 结合，其中一些在 OL 中与 OPC 相比下调。我们假设 M2 增强了 OPC 中的 DM20 5' 位点，而 ISE 有利于 OL 中的 PLP 5' 位点，这是由于 hnRNP 的减少以及一般因素和细胞特异性因素平衡变化的结果。其他外显子 3B 序列偏向于 PLP 5' 位点，并且既具有通用性又具有细胞特异性。在目标 1 中，我们将在带有 ISE 缺失的新型敲入小鼠发育中的神经系统中的 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大脑、神经和非神经胶质组织中得到阐明。在目标 2 中，我们将通过绘制 G 序列和侧翼序列对控制 PLP/DM20 比率的贡献来描述 M2 在控制少突胶质细胞和非神经胶质细胞中 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，并在 OPC 和 OL 中检查它们的表达。在目标 3 中，我们将检查 PLP 5' 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。在目标 4 中，我们将通过 RNAi 敲低研究来检查 hnRNP 在控制 PLP/DM20 比率中的功能。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磷脂遗传性疾病的剪接改变具有广泛的相关性。

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.