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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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Biochemical analysis of proteins that bind to M2F and ISE in Oli-neu extracts. (A) RNA templates used in RNA affinity precipitations. The natural G-rich sequences and the mutated poly-U sequences are underlined. (B) Upper panel: Western blot analysis of PCNA levels in each RNA affinity precipitate. One-tenth of each RNA/protein mixture prior to streptavidin beads precipitation was separated by SDS-PAGE and probed with an antibody to PCNA. Lower panel: representative silver stained gel of RNA affinity precipitates with biotinylated RNA templates containing wild-type (M2F and ISE), poly-U (M2F-MT and ISE-MT) and Oli-neu extracts (n = 2) (see Materials and Methods section). Controls are precipitates without nuclear extracts (no NE) and without RNA template (no RNA). The asterisks indicate protein bands that are uniquely present in precipitates with either M2F or ISE. The block arrows indicate the protein bands that were analyzed by LC/MS/MS and their identity is shown. (C). Western blot analysis of hnRNPA1, F, H and L in the RNA affinity precipitates (see Materials and Methods Section). Precipitates without nuclear extracts (no NE) and without RNA template (no RNA) are used as controls. Western blot of Oli-neu and HeLa nuclear extracts (9 μg) were used as control for the reactivity of the antibody. PCNA and QKI5 antibodies, used as control of the specificity of RNA affinity precipitates, detect a band in the nuclear extracts, but not in the RNA affinity precipitates.
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Figure 6: Biochemical analysis of proteins that bind to M2F and ISE in Oli-neu extracts. (A) RNA templates used in RNA affinity precipitations. The natural G-rich sequences and the mutated poly-U sequences are underlined. (B) Upper panel: Western blot analysis of PCNA levels in each RNA affinity precipitate. One-tenth of each RNA/protein mixture prior to streptavidin beads precipitation was separated by SDS-PAGE and probed with an antibody to PCNA. Lower panel: representative silver stained gel of RNA affinity precipitates with biotinylated RNA templates containing wild-type (M2F and ISE), poly-U (M2F-MT and ISE-MT) and Oli-neu extracts (n = 2) (see Materials and Methods section). Controls are precipitates without nuclear extracts (no NE) and without RNA template (no RNA). The asterisks indicate protein bands that are uniquely present in precipitates with either M2F or ISE. The block arrows indicate the protein bands that were analyzed by LC/MS/MS and their identity is shown. (C). Western blot analysis of hnRNPA1, F, H and L in the RNA affinity precipitates (see Materials and Methods Section). Precipitates without nuclear extracts (no NE) and without RNA template (no RNA) are used as controls. Western blot of Oli-neu and HeLa nuclear extracts (9 μg) were used as control for the reactivity of the antibody. PCNA and QKI5 antibodies, used as control of the specificity of RNA affinity precipitates, detect a band in the nuclear extracts, but not in the RNA affinity precipitates.

Mentions: To further examine M2F and ISE, we analyzed proteins that bind to M2F and ISE in differentiated Oli-neu nuclear extracts by RNA-affinity precipitations. Biotinylated 19 bp RNA oligoribonucleotides containing the M2F and ISE sequences and templates in which the Gs were replaced by poly-U, M2F-MT and ISE-MT (Figure 6A) were incubated with nuclear extracts. A reaction without the oligoribonucleotide was used as control for non-specific binding to the matrix. The UV-crosslinked protein–RNA complexes were precipitated with streptavidin–agarose beads and the proteins were separated by 10% SDS-PAGE and visualized by silver staining. Representative RNA-affinity precipitates (n = 2) are shown in Figure 6B.Figure 6.


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)

Biochemical analysis of proteins that bind to M2F and ISE in Oli-neu extracts. (A) RNA templates used in RNA affinity precipitations. The natural G-rich sequences and the mutated poly-U sequences are underlined. (B) Upper panel: Western blot analysis of PCNA levels in each RNA affinity precipitate. One-tenth of each RNA/protein mixture prior to streptavidin beads precipitation was separated by SDS-PAGE and probed with an antibody to PCNA. Lower panel: representative silver stained gel of RNA affinity precipitates with biotinylated RNA templates containing wild-type (M2F and ISE), poly-U (M2F-MT and ISE-MT) and Oli-neu extracts (n = 2) (see Materials and Methods section). Controls are precipitates without nuclear extracts (no NE) and without RNA template (no RNA). The asterisks indicate protein bands that are uniquely present in precipitates with either M2F or ISE. The block arrows indicate the protein bands that were analyzed by LC/MS/MS and their identity is shown. (C). Western blot analysis of hnRNPA1, F, H and L in the RNA affinity precipitates (see Materials and Methods Section). Precipitates without nuclear extracts (no NE) and without RNA template (no RNA) are used as controls. Western blot of Oli-neu and HeLa nuclear extracts (9 μg) were used as control for the reactivity of the antibody. PCNA and QKI5 antibodies, used as control of the specificity of RNA affinity precipitates, detect a band in the nuclear extracts, but not in the RNA affinity precipitates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Biochemical analysis of proteins that bind to M2F and ISE in Oli-neu extracts. (A) RNA templates used in RNA affinity precipitations. The natural G-rich sequences and the mutated poly-U sequences are underlined. (B) Upper panel: Western blot analysis of PCNA levels in each RNA affinity precipitate. One-tenth of each RNA/protein mixture prior to streptavidin beads precipitation was separated by SDS-PAGE and probed with an antibody to PCNA. Lower panel: representative silver stained gel of RNA affinity precipitates with biotinylated RNA templates containing wild-type (M2F and ISE), poly-U (M2F-MT and ISE-MT) and Oli-neu extracts (n = 2) (see Materials and Methods section). Controls are precipitates without nuclear extracts (no NE) and without RNA template (no RNA). The asterisks indicate protein bands that are uniquely present in precipitates with either M2F or ISE. The block arrows indicate the protein bands that were analyzed by LC/MS/MS and their identity is shown. (C). Western blot analysis of hnRNPA1, F, H and L in the RNA affinity precipitates (see Materials and Methods Section). Precipitates without nuclear extracts (no NE) and without RNA template (no RNA) are used as controls. Western blot of Oli-neu and HeLa nuclear extracts (9 μg) were used as control for the reactivity of the antibody. PCNA and QKI5 antibodies, used as control of the specificity of RNA affinity precipitates, detect a band in the nuclear extracts, but not in the RNA affinity precipitates.
Mentions: To further examine M2F and ISE, we analyzed proteins that bind to M2F and ISE in differentiated Oli-neu nuclear extracts by RNA-affinity precipitations. Biotinylated 19 bp RNA oligoribonucleotides containing the M2F and ISE sequences and templates in which the Gs were replaced by poly-U, M2F-MT and ISE-MT (Figure 6A) were incubated with nuclear extracts. A reaction without the oligoribonucleotide was used as control for non-specific binding to the matrix. The UV-crosslinked protein–RNA complexes were precipitated with streptavidin–agarose beads and the proteins were separated by 10% SDS-PAGE and visualized by silver staining. Representative RNA-affinity precipitates (n = 2) are shown in Figure 6B.Figure 6.

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