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.
|Bachstetter, Adam D; Webster, Scott J; Van Eldik, Linda J et al. (2013) Clinically relevant intronic splicing enhancer mutation in myelin proteolipid protein leads to progressive microglia and astrocyte activation in white and gray matter regions of the brain. J Neuroinflammation 10:146|
|Bankston, Andrew N; Li, Wenqi; Zhang, Hui et al. (2013) p39, the primary activator for cyclin-dependent kinase 5 (Cdk5) in oligodendroglia, is essential for oligodendroglia differentiation and myelin repair. J Biol Chem 288:18047-57|
|Wang, Erming; Aslanzadeh, Vahid; Papa, Filomena et al. (2012) Global profiling of alternative splicing events and gene expression regulated by hnRNPH/F. PLoS One 7:e51266|
|Zhu, H; Zhao, L; Wang, E et al. (2012) The QKI-PLP pathway controls SIRT2 abundance in CNS myelin. Glia 60:69-82|
|Wang, Erming; Cambi, Franca (2012) MicroRNA expression in mouse oligodendrocytes and regulation of proteolipid protein gene expression. J Neurosci Res 90:1701-12|
|Wang, Erming; Mueller, William F; Hertel, Klemens J et al. (2011) G Run-mediated recognition of proteolipid protein and DM20 5' splice sites by U1 small nuclear RNA is regulated by context and proximity to the splice site. J Biol Chem 286:4059-71|
|Bargagna-Mohan, Paola; Paranthan, Riya R; Hamza, Adel et al. (2010) Withaferin A targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis. J Biol Chem 285:7657-69|
|Wang, Erming; Cambi, Franca (2009) Heterogeneous nuclear ribonucleoproteins H and F regulate the proteolipid protein/DM20 ratio by recruiting U1 small nuclear ribonucleoprotein through a complex array of G runs. J Biol Chem 284:11194-204|
|Wang, Erming; Dimova, Neviana; Sperle, Karen et al. (2008) Deletion of a splicing enhancer disrupts PLP1/DM20 ratio and myelin stability. Exp Neurol 214:322-30|
|Wang, Erming; Dimova, Neviana; Cambi, Franca (2007) PLP/DM20 ratio is regulated by hnRNPH and F and a novel G-rich enhancer in oligodendrocytes. Nucleic Acids Res 35:4164-78|