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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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The effect of mutation of M2 and ISE on the hnRNPH and F-mediated regulation of PLP/DM20. (A) Representative RT-PCR analysis of the M2-MT derived PLP and DM20 products amplified from RNA isolated from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The increase in PLP/DM20 ratio induced by siF3+H3 and siF/H are statistically significant (P < 0.01). (B) Representative RT-PCR analysis of the ISEdel derived PLP and DM20 products amplified from RNA prepared from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The increase in PLP/DM20 ratio induced by siH3, siF3+H3 and siF/H is statistically significant (P < 0.01). (C) Representative RT-PCR analysis of the M2-MT/ISEdel derived PLP and DM20 products amplified from RNA prepared from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The changes in PLP/DM20 ratio induced by siF3+H3 and siF/H are statistically significant (P < 0.05).
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Figure 9: The effect of mutation of M2 and ISE on the hnRNPH and F-mediated regulation of PLP/DM20. (A) Representative RT-PCR analysis of the M2-MT derived PLP and DM20 products amplified from RNA isolated from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The increase in PLP/DM20 ratio induced by siF3+H3 and siF/H are statistically significant (P < 0.01). (B) Representative RT-PCR analysis of the ISEdel derived PLP and DM20 products amplified from RNA prepared from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The increase in PLP/DM20 ratio induced by siH3, siF3+H3 and siF/H is statistically significant (P < 0.01). (C) Representative RT-PCR analysis of the M2-MT/ISEdel derived PLP and DM20 products amplified from RNA prepared from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The changes in PLP/DM20 ratio induced by siF3+H3 and siF/H are statistically significant (P < 0.05).

Mentions: M2 and ISE contain G-rich sequences that have nearly identical composition, raising the possibility that a balance between these enhancers regulates PLP/DM20 ratio (Figure 5A). To evaluate their functional relationship, we asked whether M2 and ISE enhance the upstream 5′ splice site when they are exchanged. The G-rich core and flanking sequences of the 19-bp ISE contribute to the enhancer's function (15). In order to move the ISE and not change the distance of G sequences from the 5′ splice site, we replaced M2 and the five bases 5′ and four bases 3′ of M2 (19 nt, M2F) with the ISE and vice versa. We have made the following constructs: ISE-ISE, M2F-M2F and M2-MT/ISEdel (Figure 5A). The naturally occurring ISE deletion (ISEdel) reduces PLP 5′ splice site selection (15). PLP and DM20 PCR products were amplified in Oli-neu cells transfected with these constructs and with PLP-neo, M2-MT and ISEdel (Figure 5B). The PLP/DM20 ratio derived from ISE-ISE and M2F-M2F was lower than the ratio derived from the WT, suggesting that the ISE is a strong enhancer of DM20 5′ splice site and M2F is a weak enhancer of PLP 5′ splice site. The PLP/DM20 ratio derived from M2-MT/ISEdel was increased compared with WT, although it was not as high as that derived from M2-MT (Figures 5B and 9C). These data suggest that although the ISE and M2F can replace each other, they differ in the strength by which they enhance the 5′ splice site when removed from their natural position.


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)

The effect of mutation of M2 and ISE on the hnRNPH and F-mediated regulation of PLP/DM20. (A) Representative RT-PCR analysis of the M2-MT derived PLP and DM20 products amplified from RNA isolated from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The increase in PLP/DM20 ratio induced by siF3+H3 and siF/H are statistically significant (P < 0.01). (B) Representative RT-PCR analysis of the ISEdel derived PLP and DM20 products amplified from RNA prepared from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The increase in PLP/DM20 ratio induced by siH3, siF3+H3 and siF/H is statistically significant (P < 0.01). (C) Representative RT-PCR analysis of the M2-MT/ISEdel derived PLP and DM20 products amplified from RNA prepared from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The changes in PLP/DM20 ratio induced by siF3+H3 and siF/H are statistically significant (P < 0.05).
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Figure 9: The effect of mutation of M2 and ISE on the hnRNPH and F-mediated regulation of PLP/DM20. (A) Representative RT-PCR analysis of the M2-MT derived PLP and DM20 products amplified from RNA isolated from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The increase in PLP/DM20 ratio induced by siF3+H3 and siF/H are statistically significant (P < 0.01). (B) Representative RT-PCR analysis of the ISEdel derived PLP and DM20 products amplified from RNA prepared from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The increase in PLP/DM20 ratio induced by siH3, siF3+H3 and siF/H is statistically significant (P < 0.01). (C) Representative RT-PCR analysis of the M2-MT/ISEdel derived PLP and DM20 products amplified from RNA prepared from Oli-neu cells treated with siH3, siF3, siF3+H3, siF/H (35 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3). The changes in PLP/DM20 ratio induced by siF3+H3 and siF/H are statistically significant (P < 0.05).
Mentions: M2 and ISE contain G-rich sequences that have nearly identical composition, raising the possibility that a balance between these enhancers regulates PLP/DM20 ratio (Figure 5A). To evaluate their functional relationship, we asked whether M2 and ISE enhance the upstream 5′ splice site when they are exchanged. The G-rich core and flanking sequences of the 19-bp ISE contribute to the enhancer's function (15). In order to move the ISE and not change the distance of G sequences from the 5′ splice site, we replaced M2 and the five bases 5′ and four bases 3′ of M2 (19 nt, M2F) with the ISE and vice versa. We have made the following constructs: ISE-ISE, M2F-M2F and M2-MT/ISEdel (Figure 5A). The naturally occurring ISE deletion (ISEdel) reduces PLP 5′ splice site selection (15). PLP and DM20 PCR products were amplified in Oli-neu cells transfected with these constructs and with PLP-neo, M2-MT and ISEdel (Figure 5B). The PLP/DM20 ratio derived from ISE-ISE and M2F-M2F was lower than the ratio derived from the WT, suggesting that the ISE is a strong enhancer of DM20 5′ splice site and M2F is a weak enhancer of PLP 5′ splice site. The PLP/DM20 ratio derived from M2-MT/ISEdel was increased compared with WT, although it was not as high as that derived from M2-MT (Figures 5B and 9C). These data suggest that although the ISE and M2F can replace each other, they differ in the strength by which they enhance the 5′ splice site when removed from their natural position.

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