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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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Related in: MedlinePlus

Mutation of a G-rich sequence activates inclusion of PLP exon 3B. Results of RT-PCR assay of PLP and DM20 from total RNA isolated from Oli-neu cells (30 PCR cycles) (A) and L cells (35 PCR cycles) (B) transfected with wild-type PLP-neo (WT) and M1-MT to M10-MT. The PLP/DM20 ratios ± SD are shown (n = 3). GAPDH is used for accuracy of RNA loading (25 PCR cycles). Control represents the untransfected cells. The increase in PLP/DM20 ratio with the M2-MT construct is statistically significant (P = 0.017 for Oli-neu cells and P < 0.002 for L cells).
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Figure 2: Mutation of a G-rich sequence activates inclusion of PLP exon 3B. Results of RT-PCR assay of PLP and DM20 from total RNA isolated from Oli-neu cells (30 PCR cycles) (A) and L cells (35 PCR cycles) (B) transfected with wild-type PLP-neo (WT) and M1-MT to M10-MT. The PLP/DM20 ratios ± SD are shown (n = 3). GAPDH is used for accuracy of RNA loading (25 PCR cycles). Control represents the untransfected cells. The increase in PLP/DM20 ratio with the M2-MT construct is statistically significant (P = 0.017 for Oli-neu cells and P < 0.002 for L cells).

Mentions: Following transfection and expression of PLP-neo constructs carrying the mutated M1–M10 (M1-MT–M10-MT) in Oli-neu cells differentiated in medium containing dbcAMP for 72 h, the PLP and DM20 products were simultaneously amplified by RT-PCR (Figure 2A). Replacement of a G-rich sequence in the 5′ end of exon 3B (M2) resulted in 5.4-fold increase in PLP/DM20 ratio compared with the WT (Figure 2A). In contrast, replacement of the other exon 3B sequences caused a reduction in the PLP/DM20 ratio, with sequences replaced in M1, M8 and M10 having less of an effect (Figure 2A). Similar changes in the PLP/DM20 ratio were detected in Oli-neu cells transfected with M1-MT/M10-MT and differentiated for 6 days (data not shown).


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)

Mutation of a G-rich sequence activates inclusion of PLP exon 3B. Results of RT-PCR assay of PLP and DM20 from total RNA isolated from Oli-neu cells (30 PCR cycles) (A) and L cells (35 PCR cycles) (B) transfected with wild-type PLP-neo (WT) and M1-MT to M10-MT. The PLP/DM20 ratios ± SD are shown (n = 3). GAPDH is used for accuracy of RNA loading (25 PCR cycles). Control represents the untransfected cells. The increase in PLP/DM20 ratio with the M2-MT construct is statistically significant (P = 0.017 for Oli-neu cells and P < 0.002 for L cells).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919487&req=5

Figure 2: Mutation of a G-rich sequence activates inclusion of PLP exon 3B. Results of RT-PCR assay of PLP and DM20 from total RNA isolated from Oli-neu cells (30 PCR cycles) (A) and L cells (35 PCR cycles) (B) transfected with wild-type PLP-neo (WT) and M1-MT to M10-MT. The PLP/DM20 ratios ± SD are shown (n = 3). GAPDH is used for accuracy of RNA loading (25 PCR cycles). Control represents the untransfected cells. The increase in PLP/DM20 ratio with the M2-MT construct is statistically significant (P = 0.017 for Oli-neu cells and P < 0.002 for L cells).
Mentions: Following transfection and expression of PLP-neo constructs carrying the mutated M1–M10 (M1-MT–M10-MT) in Oli-neu cells differentiated in medium containing dbcAMP for 72 h, the PLP and DM20 products were simultaneously amplified by RT-PCR (Figure 2A). Replacement of a G-rich sequence in the 5′ end of exon 3B (M2) resulted in 5.4-fold increase in PLP/DM20 ratio compared with the WT (Figure 2A). In contrast, replacement of the other exon 3B sequences caused a reduction in the PLP/DM20 ratio, with sequences replaced in M1, M8 and M10 having less of an effect (Figure 2A). Similar changes in the PLP/DM20 ratio were detected in Oli-neu cells transfected with M1-MT/M10-MT and differentiated for 6 days (data not shown).

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