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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 G1, G4 and G5 on the hnRNPH and F-mediated regulation of PLP/DM20. (A) Partial sequences of PLP exon 3B/intron 3 (WT) and of mutations of M2/ISE and G1, G4 and G5 in exon 3B (M2-MT/ISEdel-G1-G4-G5MT) are shown. (B) Representative RT-PCR analysis of the M2-MT/ISEdel-G1-G4-G5MT derived PLP and DM20 products amplified from RNA isolated from Oli-neu cells treated with siF3+H3, siF/H (35 PCR cycles). Mock are cells transfected with M2-MT/ISEdel-G1-G4-G5MT and treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3).
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Figure 10: The effect of mutation of G1, G4 and G5 on the hnRNPH and F-mediated regulation of PLP/DM20. (A) Partial sequences of PLP exon 3B/intron 3 (WT) and of mutations of M2/ISE and G1, G4 and G5 in exon 3B (M2-MT/ISEdel-G1-G4-G5MT) are shown. (B) Representative RT-PCR analysis of the M2-MT/ISEdel-G1-G4-G5MT derived PLP and DM20 products amplified from RNA isolated from Oli-neu cells treated with siF3+H3, siF/H (35 PCR cycles). Mock are cells transfected with M2-MT/ISEdel-G1-G4-G5MT and treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3).

Mentions: We next determined whether the other G-rich sequences present in exon 3B may mediate the increase in PLP/DM20 ratio derived from M2-MT/ISEdel after knock down of hnRNPH and F. We have replaced G runs in M1 (G1), M6 (G4) and M8 (G5) with T's in the M2-MT/ISEdel (Figure 10A) and determined the PLP/DM20 ratio derived from this construct in transfected Oli-neu cells (Figure 10B). The PLP/DM20 ratio derived from M2-MT/ISEdel/G1-G4-G5-MT was 0.22 which is similar to the ratio derived from the WT (Figure 2A). The PLP/DM20 ratio derived from M2-MT/ISEdel-G1-G4-G5MT in Oli-neu cells treated with siF3 + siH3 did not change compared with mock treated cells (0.27 versus 0.22), while ∼1.6-fold increase was detected after treatment with siF/H (0.4 versus 0.22) (Figure 10B). The data show that mutations of G1, G4 and G5 in addition to M2 almost completely abolish the PLP/DM20 increase induced by knock down of hnRNPH/F and suggest that some or all of these G runs participate in mediating the synergistic effect.Figure 10.


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 G1, G4 and G5 on the hnRNPH and F-mediated regulation of PLP/DM20. (A) Partial sequences of PLP exon 3B/intron 3 (WT) and of mutations of M2/ISE and G1, G4 and G5 in exon 3B (M2-MT/ISEdel-G1-G4-G5MT) are shown. (B) Representative RT-PCR analysis of the M2-MT/ISEdel-G1-G4-G5MT derived PLP and DM20 products amplified from RNA isolated from Oli-neu cells treated with siF3+H3, siF/H (35 PCR cycles). Mock are cells transfected with M2-MT/ISEdel-G1-G4-G5MT and treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 10: The effect of mutation of G1, G4 and G5 on the hnRNPH and F-mediated regulation of PLP/DM20. (A) Partial sequences of PLP exon 3B/intron 3 (WT) and of mutations of M2/ISE and G1, G4 and G5 in exon 3B (M2-MT/ISEdel-G1-G4-G5MT) are shown. (B) Representative RT-PCR analysis of the M2-MT/ISEdel-G1-G4-G5MT derived PLP and DM20 products amplified from RNA isolated from Oli-neu cells treated with siF3+H3, siF/H (35 PCR cycles). Mock are cells transfected with M2-MT/ISEdel-G1-G4-G5MT and treated with control siRNA. The bar graph shows the PLP/DM20 ratios ± SD (n = 3).
Mentions: We next determined whether the other G-rich sequences present in exon 3B may mediate the increase in PLP/DM20 ratio derived from M2-MT/ISEdel after knock down of hnRNPH and F. We have replaced G runs in M1 (G1), M6 (G4) and M8 (G5) with T's in the M2-MT/ISEdel (Figure 10A) and determined the PLP/DM20 ratio derived from this construct in transfected Oli-neu cells (Figure 10B). The PLP/DM20 ratio derived from M2-MT/ISEdel/G1-G4-G5-MT was 0.22 which is similar to the ratio derived from the WT (Figure 2A). The PLP/DM20 ratio derived from M2-MT/ISEdel-G1-G4-G5MT in Oli-neu cells treated with siF3 + siH3 did not change compared with mock treated cells (0.27 versus 0.22), while ∼1.6-fold increase was detected after treatment with siF/H (0.4 versus 0.22) (Figure 10B). The data show that mutations of G1, G4 and G5 in addition to M2 almost completely abolish the PLP/DM20 increase induced by knock down of hnRNPH/F and suggest that some or all of these G runs participate in mediating the synergistic effect.Figure 10.

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