Limits...
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.

Show MeSH
M2 is an enhancer of DM20 5′ splice site selection. (A) PLP-neo construct and primers used for PCR amplification. Partial sequences of the natural exon 3B (WT) and mutated constructs are shown, underlined are mutations at the DM20 and PLP 5′ splice site and the M2-MT. (B) PLP and DM20 PCR products (35 PCR cycles) from WT and DM20 G>T and DM20 G>T-M2-MT amplified with primers 1 and 2 in RNA extracted from transfected Oli-neu cells. (C) PLP and DM20 PCR products (35 PCR cycles) derived from WT, c.453+2T>C and c.453T>C-M2-MT amplified with primers 1 and 2 in RNA extracted from Oli-neu cells. Plasmid-derived PLP+DM20 PCR product was amplified with primers 3 and 4 and represents the total plasmid-derived PLP/DM20 transcript. Endogenous PLP+DM20 PCR product amplified with primers 1 and 4 is the control for RNA loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1919487&req=5

Figure 4: M2 is an enhancer of DM20 5′ splice site selection. (A) PLP-neo construct and primers used for PCR amplification. Partial sequences of the natural exon 3B (WT) and mutated constructs are shown, underlined are mutations at the DM20 and PLP 5′ splice site and the M2-MT. (B) PLP and DM20 PCR products (35 PCR cycles) from WT and DM20 G>T and DM20 G>T-M2-MT amplified with primers 1 and 2 in RNA extracted from transfected Oli-neu cells. (C) PLP and DM20 PCR products (35 PCR cycles) derived from WT, c.453+2T>C and c.453T>C-M2-MT amplified with primers 1 and 2 in RNA extracted from Oli-neu cells. Plasmid-derived PLP+DM20 PCR product was amplified with primers 3 and 4 and represents the total plasmid-derived PLP/DM20 transcript. Endogenous PLP+DM20 PCR product amplified with primers 1 and 4 is the control for RNA loading.

Mentions: Total RNA was extracted with RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated with the DNA-free Kit (Ambion, Austin, TX, USA). Total RNA (0.5 μg) was reverse transcribed using random hexamer primers (BD Biosciences, Palo Alto, CA, USA). The PLP and DM20 PCR products and PLP+DM20 product derived from PLP-neo were amplified and quantitated as described (16) (Figures 1A and 4A).


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)

M2 is an enhancer of DM20 5′ splice site selection. (A) PLP-neo construct and primers used for PCR amplification. Partial sequences of the natural exon 3B (WT) and mutated constructs are shown, underlined are mutations at the DM20 and PLP 5′ splice site and the M2-MT. (B) PLP and DM20 PCR products (35 PCR cycles) from WT and DM20 G>T and DM20 G>T-M2-MT amplified with primers 1 and 2 in RNA extracted from transfected Oli-neu cells. (C) PLP and DM20 PCR products (35 PCR cycles) derived from WT, c.453+2T>C and c.453T>C-M2-MT amplified with primers 1 and 2 in RNA extracted from Oli-neu cells. Plasmid-derived PLP+DM20 PCR product was amplified with primers 3 and 4 and represents the total plasmid-derived PLP/DM20 transcript. Endogenous PLP+DM20 PCR product amplified with primers 1 and 4 is the control for RNA loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: M2 is an enhancer of DM20 5′ splice site selection. (A) PLP-neo construct and primers used for PCR amplification. Partial sequences of the natural exon 3B (WT) and mutated constructs are shown, underlined are mutations at the DM20 and PLP 5′ splice site and the M2-MT. (B) PLP and DM20 PCR products (35 PCR cycles) from WT and DM20 G>T and DM20 G>T-M2-MT amplified with primers 1 and 2 in RNA extracted from transfected Oli-neu cells. (C) PLP and DM20 PCR products (35 PCR cycles) derived from WT, c.453+2T>C and c.453T>C-M2-MT amplified with primers 1 and 2 in RNA extracted from Oli-neu cells. Plasmid-derived PLP+DM20 PCR product was amplified with primers 3 and 4 and represents the total plasmid-derived PLP/DM20 transcript. Endogenous PLP+DM20 PCR product amplified with primers 1 and 4 is the control for RNA loading.
Mentions: Total RNA was extracted with RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated with the DNA-free Kit (Ambion, Austin, TX, USA). Total RNA (0.5 μg) was reverse transcribed using random hexamer primers (BD Biosciences, Palo Alto, CA, USA). The PLP and DM20 PCR products and PLP+DM20 product derived from PLP-neo were amplified and quantitated as described (16) (Figures 1A and 4A).

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