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PLP/DM20 ratio is regulated by hnRNPH and F and a novel G-rich enhancer in oligodendrocytes.

Wang E, Dimova N, Cambi F - Nucleic Acids Res. (2007)

Bottom Line: Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not.Mutation of M2, but not ISE reduced the synergistic effect.We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Kentucky, Lexington, KY, USA.

ABSTRACT
Alternative splicing of competing 5' splice sites is regulated by enhancers and silencers in the spliced exon. We have characterized sequences and splicing factors that regulate alternative splicing of PLP and DM20, myelin proteins produced by oligodendrocytes (OLs) by selection of 5' splice sites in exon 3. We identify a G-rich enhancer (M2) of DM20 5' splice site in exon 3B and show that individual G triplets forming M2 are functionally distinct and the distal group plays a dominant role. G-rich M2 and a G-rich splicing enhancer (ISE) in intron 3 share similarities in function and protein binding. The G-rich sequences are necessary for binding of hnRNPs to both enhancers. Reduction in hnRNPH and F expression in differentiated OLs correlates temporally with increased PLP/DM20 ratio. Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not. Silencing hnRNPH and F increased the PLP/DM20 ratio more than hnRNPH alone, demonstrating a novel synergistic effect. Mutation of M2, but not ISE reduced the synergistic effect. Replacement of M2 and all G runs in exon 3B abolished it almost completely. We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.

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Analysis of G triplets within M2 in Oli-neu cells and primary oligodendrocytes. (A) Partial wild-type sequences of exon 3B are shown and M2 is underlined. Linker scan mutations of M2 and G triplets within M2 are shown. (B) Results of RT-PCR assay of PLP and DM20 from RNA isolated from differentiated Oli-neu cells transfected with wild-type PLP-neo (WT) and mutated constructs, M2-MT, M2-MT2, G2-MT, G3-MT and G2-G3-MT. The PLP/DM20 ratios ± SD are shown (n = 3). The upper and lower limits of accurate quantification of the PLP/DM20 ratio are 10 and 0.01. (C) PLP and DM20 PCR products amplified in RNA extracted from differentiated OLs transfected with WT and M2-MT, M2-MT2 and G3-MT, duplicate transfections are shown.
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Figure 3: Analysis of G triplets within M2 in Oli-neu cells and primary oligodendrocytes. (A) Partial wild-type sequences of exon 3B are shown and M2 is underlined. Linker scan mutations of M2 and G triplets within M2 are shown. (B) Results of RT-PCR assay of PLP and DM20 from RNA isolated from differentiated Oli-neu cells transfected with wild-type PLP-neo (WT) and mutated constructs, M2-MT, M2-MT2, G2-MT, G3-MT and G2-G3-MT. The PLP/DM20 ratios ± SD are shown (n = 3). The upper and lower limits of accurate quantification of the PLP/DM20 ratio are 10 and 0.01. (C) PLP and DM20 PCR products amplified in RNA extracted from differentiated OLs transfected with WT and M2-MT, M2-MT2 and G3-MT, duplicate transfections are shown.

Mentions: To identify novel regulatory sequences that control the PLP/DM20 ratio, we have systematically mutated PLP exon 3B in a PLP splicing construct and assessed in vivo splicing in transfected Oli-neu cells (Figure 1A). In published work, we transfected primary OLs (14–16). In the present study, we have used Oli-neu cells, immortalized OPCs that are induced to differentiate by dbcAMP (21,24). Because the efficiency of transfection in Oli-neu cells is 36% compared with 6% in primary OLs (data not shown), changes in plasmid-derived PLP/DM20 ratio are more easily quantified. We have validated the use of Oli-neu cells as a model for the OLs in splicing studies. We have established that various PLP-neo constructs previously tested in primary OLs expressed the same PLP and DM20 products when transfected into Oli-neu cells (data not shown). Expression results for some of the critical mutations characterized in the present study in Oli-neu cells were confirmed in primary OLs (Figure 3). Finally, we show that differentiated Oli-neu cells replicate the trend of changes in hnRNP's expression that we detect in differentiated OLs (Figure 7).


PLP/DM20 ratio is regulated by hnRNPH and F and a novel G-rich enhancer in oligodendrocytes.

Wang E, Dimova N, Cambi F - Nucleic Acids Res. (2007)

Analysis of G triplets within M2 in Oli-neu cells and primary oligodendrocytes. (A) Partial wild-type sequences of exon 3B are shown and M2 is underlined. Linker scan mutations of M2 and G triplets within M2 are shown. (B) Results of RT-PCR assay of PLP and DM20 from RNA isolated from differentiated Oli-neu cells transfected with wild-type PLP-neo (WT) and mutated constructs, M2-MT, M2-MT2, G2-MT, G3-MT and G2-G3-MT. The PLP/DM20 ratios ± SD are shown (n = 3). The upper and lower limits of accurate quantification of the PLP/DM20 ratio are 10 and 0.01. (C) PLP and DM20 PCR products amplified in RNA extracted from differentiated OLs transfected with WT and M2-MT, M2-MT2 and G3-MT, duplicate transfections are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Analysis of G triplets within M2 in Oli-neu cells and primary oligodendrocytes. (A) Partial wild-type sequences of exon 3B are shown and M2 is underlined. Linker scan mutations of M2 and G triplets within M2 are shown. (B) Results of RT-PCR assay of PLP and DM20 from RNA isolated from differentiated Oli-neu cells transfected with wild-type PLP-neo (WT) and mutated constructs, M2-MT, M2-MT2, G2-MT, G3-MT and G2-G3-MT. The PLP/DM20 ratios ± SD are shown (n = 3). The upper and lower limits of accurate quantification of the PLP/DM20 ratio are 10 and 0.01. (C) PLP and DM20 PCR products amplified in RNA extracted from differentiated OLs transfected with WT and M2-MT, M2-MT2 and G3-MT, duplicate transfections are shown.
Mentions: To identify novel regulatory sequences that control the PLP/DM20 ratio, we have systematically mutated PLP exon 3B in a PLP splicing construct and assessed in vivo splicing in transfected Oli-neu cells (Figure 1A). In published work, we transfected primary OLs (14–16). In the present study, we have used Oli-neu cells, immortalized OPCs that are induced to differentiate by dbcAMP (21,24). Because the efficiency of transfection in Oli-neu cells is 36% compared with 6% in primary OLs (data not shown), changes in plasmid-derived PLP/DM20 ratio are more easily quantified. We have validated the use of Oli-neu cells as a model for the OLs in splicing studies. We have established that various PLP-neo constructs previously tested in primary OLs expressed the same PLP and DM20 products when transfected into Oli-neu cells (data not shown). Expression results for some of the critical mutations characterized in the present study in Oli-neu cells were confirmed in primary OLs (Figure 3). Finally, we show that differentiated Oli-neu cells replicate the trend of changes in hnRNP's expression that we detect in differentiated OLs (Figure 7).

Bottom Line: Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not.Mutation of M2, but not ISE reduced the synergistic effect.We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Kentucky, Lexington, KY, USA.

ABSTRACT
Alternative splicing of competing 5' splice sites is regulated by enhancers and silencers in the spliced exon. We have characterized sequences and splicing factors that regulate alternative splicing of PLP and DM20, myelin proteins produced by oligodendrocytes (OLs) by selection of 5' splice sites in exon 3. We identify a G-rich enhancer (M2) of DM20 5' splice site in exon 3B and show that individual G triplets forming M2 are functionally distinct and the distal group plays a dominant role. G-rich M2 and a G-rich splicing enhancer (ISE) in intron 3 share similarities in function and protein binding. The G-rich sequences are necessary for binding of hnRNPs to both enhancers. Reduction in hnRNPH and F expression in differentiated OLs correlates temporally with increased PLP/DM20 ratio. Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not. Silencing hnRNPH and F increased the PLP/DM20 ratio more than hnRNPH alone, demonstrating a novel synergistic effect. Mutation of M2, but not ISE reduced the synergistic effect. Replacement of M2 and all G runs in exon 3B abolished it almost completely. We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.

Show MeSH
Related in: MedlinePlus