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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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RNAi-mediated knock down of hnRNPH and F increases the PLP/DM20 ratio in Oli-neu cells. (A) Representative Western blot of cell extracts prepared from Oli-neu cells treated with siRNAs that target hnRNPH (siH1, H2, H3), hnRNPF (siF1, F2 and F3) and both H and F (siF/H). Mock are cells treated with negative control siRNA. After quantification of the bands, the values were corrected by actin, used as control for loading accuracy. The hnRNPH was reduced by 40, 70 and 50% in cells treated with siH1, siH2 and siH3 and 50% in cells treated with siH/F versus control (n = 2). The hnRNPF was reduced by 60 and 40% in cells treated with siF2 and siF3 and by 60% in cells treated with siF/H versus controls. (B) Representative RT-PCR analysis of the PLP-neo derived PLP and DM20 spliced products and the endogenous PLP and DM20 transcripts amplified from RNA isolated from Oli-neu cells treated with siH1, siH3, siF2, siF3, siF3 + H3 and siF/H (35 PCR cycles). The bar graph shows the PLP/DM20 ratios ± SD (n = 3). Mock are cells treated with control siRNA. Increase in PLP/DM20 ratio is statistically significant for siH3 (P < 0.05) and for siF3+H3 and siF/H (P < 0.01)-treated cells compared with mock-treated cells.
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Figure 8: RNAi-mediated knock down of hnRNPH and F increases the PLP/DM20 ratio in Oli-neu cells. (A) Representative Western blot of cell extracts prepared from Oli-neu cells treated with siRNAs that target hnRNPH (siH1, H2, H3), hnRNPF (siF1, F2 and F3) and both H and F (siF/H). Mock are cells treated with negative control siRNA. After quantification of the bands, the values were corrected by actin, used as control for loading accuracy. The hnRNPH was reduced by 40, 70 and 50% in cells treated with siH1, siH2 and siH3 and 50% in cells treated with siH/F versus control (n = 2). The hnRNPF was reduced by 60 and 40% in cells treated with siF2 and siF3 and by 60% in cells treated with siF/H versus controls. (B) Representative RT-PCR analysis of the PLP-neo derived PLP and DM20 spliced products and the endogenous PLP and DM20 transcripts amplified from RNA isolated from Oli-neu cells treated with siH1, siH3, siF2, siF3, siF3 + H3 and siF/H (35 PCR cycles). The bar graph shows the PLP/DM20 ratios ± SD (n = 3). Mock are cells treated with control siRNA. Increase in PLP/DM20 ratio is statistically significant for siH3 (P < 0.05) and for siF3+H3 and siF/H (P < 0.01)-treated cells compared with mock-treated cells.

Mentions: The coordinate decrease in the expression of hnRNP H, F and A1 in OLs suggests that these splicing factors may regulate the changes in the PLP/DM20 ratio. We have tested whether RNAi-mediated removal of either hnRNPH or F is sufficient to increase the PLP/DM20 ratio in undifferentiated Oli-neu cells in which the endogenous expression of H and F is high. Oli-neu cells were treated with siRNA that either target hnRNPH (siH1, H2, H3) or hnRNPF (siF1, F2 and F3) (Ambion, see Materials and Methods section) and hnRNPH and F levels were quantified by Western blot of cell lysates prepared 72 h after transfection. hnRNPH was reduced by 70% with siH2, 50% with siH3 and 40% with siH1 (Figure 8A). hnRNPF was reduced by 60% with siF2 and 40% with siF3, while was unaffected by siF1 (Figure 8A). Expression of hnRNP A1 and L was not changed by treatment with the siRNAs, confirming the specificity of the knock down (data not shown). Subsequent experiments were carried out with siH3, H1 and siF2, F3.Figure 8.


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)

RNAi-mediated knock down of hnRNPH and F increases the PLP/DM20 ratio in Oli-neu cells. (A) Representative Western blot of cell extracts prepared from Oli-neu cells treated with siRNAs that target hnRNPH (siH1, H2, H3), hnRNPF (siF1, F2 and F3) and both H and F (siF/H). Mock are cells treated with negative control siRNA. After quantification of the bands, the values were corrected by actin, used as control for loading accuracy. The hnRNPH was reduced by 40, 70 and 50% in cells treated with siH1, siH2 and siH3 and 50% in cells treated with siH/F versus control (n = 2). The hnRNPF was reduced by 60 and 40% in cells treated with siF2 and siF3 and by 60% in cells treated with siF/H versus controls. (B) Representative RT-PCR analysis of the PLP-neo derived PLP and DM20 spliced products and the endogenous PLP and DM20 transcripts amplified from RNA isolated from Oli-neu cells treated with siH1, siH3, siF2, siF3, siF3 + H3 and siF/H (35 PCR cycles). The bar graph shows the PLP/DM20 ratios ± SD (n = 3). Mock are cells treated with control siRNA. Increase in PLP/DM20 ratio is statistically significant for siH3 (P < 0.05) and for siF3+H3 and siF/H (P < 0.01)-treated cells compared with mock-treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: RNAi-mediated knock down of hnRNPH and F increases the PLP/DM20 ratio in Oli-neu cells. (A) Representative Western blot of cell extracts prepared from Oli-neu cells treated with siRNAs that target hnRNPH (siH1, H2, H3), hnRNPF (siF1, F2 and F3) and both H and F (siF/H). Mock are cells treated with negative control siRNA. After quantification of the bands, the values were corrected by actin, used as control for loading accuracy. The hnRNPH was reduced by 40, 70 and 50% in cells treated with siH1, siH2 and siH3 and 50% in cells treated with siH/F versus control (n = 2). The hnRNPF was reduced by 60 and 40% in cells treated with siF2 and siF3 and by 60% in cells treated with siF/H versus controls. (B) Representative RT-PCR analysis of the PLP-neo derived PLP and DM20 spliced products and the endogenous PLP and DM20 transcripts amplified from RNA isolated from Oli-neu cells treated with siH1, siH3, siF2, siF3, siF3 + H3 and siF/H (35 PCR cycles). The bar graph shows the PLP/DM20 ratios ± SD (n = 3). Mock are cells treated with control siRNA. Increase in PLP/DM20 ratio is statistically significant for siH3 (P < 0.05) and for siF3+H3 and siF/H (P < 0.01)-treated cells compared with mock-treated cells.
Mentions: The coordinate decrease in the expression of hnRNP H, F and A1 in OLs suggests that these splicing factors may regulate the changes in the PLP/DM20 ratio. We have tested whether RNAi-mediated removal of either hnRNPH or F is sufficient to increase the PLP/DM20 ratio in undifferentiated Oli-neu cells in which the endogenous expression of H and F is high. Oli-neu cells were treated with siRNA that either target hnRNPH (siH1, H2, H3) or hnRNPF (siF1, F2 and F3) (Ambion, see Materials and Methods section) and hnRNPH and F levels were quantified by Western blot of cell lysates prepared 72 h after transfection. hnRNPH was reduced by 70% with siH2, 50% with siH3 and 40% with siH1 (Figure 8A). hnRNPF was reduced by 60% with siF2 and 40% with siF3, while was unaffected by siF1 (Figure 8A). Expression of hnRNP A1 and L was not changed by treatment with the siRNAs, confirming the specificity of the knock down (data not shown). Subsequent experiments were carried out with siH3, H1 and siF2, F3.Figure 8.

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