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C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1.

Sumanasekera C, Kelemen O, Beullens M, Aubol BE, Adams JA, Sunkara M, Morris A, Bollen M, Andreadis A, Stamm S - Nucleic Acids Res. (2011)

Bottom Line: To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection.We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF).Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.

ABSTRACT
Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

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Splicing regulators with a PP1 binding site regulate ceramide responsive exons. The reporter genes shown in Figure 2 were co-transfected with constructs expressing SF2/ASF or Tra2-beta1. RNA was isolated and analyzed by RT-PCR (A–E). The statistical analysis of at least three independent experiments is shown on the right. Note that no PyrCer was added in these experiments. (F) Detection of GFP-SF2/ASF and GFP-Tra2-beta1 by Western Blot analysis.
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gkr1289-F7: Splicing regulators with a PP1 binding site regulate ceramide responsive exons. The reporter genes shown in Figure 2 were co-transfected with constructs expressing SF2/ASF or Tra2-beta1. RNA was isolated and analyzed by RT-PCR (A–E). The statistical analysis of at least three independent experiments is shown on the right. Note that no PyrCer was added in these experiments. (F) Detection of GFP-SF2/ASF and GFP-Tra2-beta1 by Western Blot analysis.

Mentions: As shown in Figure 7, all exons respond to SF2/ASF and Tra2-beta1, which supports the bioinformatic prediction. It is noticeable that SF2/ASF promotes exon skipping in most cases, and Tra2-beta1 promotes skipping of an exon in the TIAF1 pre-mRNA, which is in contrast to their usual function as splicing activators. This unusual effect of SF2/ASF on tau exon 10 has been previously observed (39). The effect of tra2-beta1 on the POL-B alternative exons was much weaker than on TIAF1, which correlates with the strong binding of tra2-beta1 in vitro to the TIAF1 pre-mRNA and our inability to detect binding of tra2-beta1 to POL-B (Figure 5). Both SF2/ASF and Tra2-beta1 are direct substrates for PP1 and PP1-mediated dephosphorylation that promotes their interaction (3) and it is possible that PyrCer affects exons that are dependent on both factors.Figure 7.


C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1.

Sumanasekera C, Kelemen O, Beullens M, Aubol BE, Adams JA, Sunkara M, Morris A, Bollen M, Andreadis A, Stamm S - Nucleic Acids Res. (2011)

Splicing regulators with a PP1 binding site regulate ceramide responsive exons. The reporter genes shown in Figure 2 were co-transfected with constructs expressing SF2/ASF or Tra2-beta1. RNA was isolated and analyzed by RT-PCR (A–E). The statistical analysis of at least three independent experiments is shown on the right. Note that no PyrCer was added in these experiments. (F) Detection of GFP-SF2/ASF and GFP-Tra2-beta1 by Western Blot analysis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351148&req=5

gkr1289-F7: Splicing regulators with a PP1 binding site regulate ceramide responsive exons. The reporter genes shown in Figure 2 were co-transfected with constructs expressing SF2/ASF or Tra2-beta1. RNA was isolated and analyzed by RT-PCR (A–E). The statistical analysis of at least three independent experiments is shown on the right. Note that no PyrCer was added in these experiments. (F) Detection of GFP-SF2/ASF and GFP-Tra2-beta1 by Western Blot analysis.
Mentions: As shown in Figure 7, all exons respond to SF2/ASF and Tra2-beta1, which supports the bioinformatic prediction. It is noticeable that SF2/ASF promotes exon skipping in most cases, and Tra2-beta1 promotes skipping of an exon in the TIAF1 pre-mRNA, which is in contrast to their usual function as splicing activators. This unusual effect of SF2/ASF on tau exon 10 has been previously observed (39). The effect of tra2-beta1 on the POL-B alternative exons was much weaker than on TIAF1, which correlates with the strong binding of tra2-beta1 in vitro to the TIAF1 pre-mRNA and our inability to detect binding of tra2-beta1 to POL-B (Figure 5). Both SF2/ASF and Tra2-beta1 are direct substrates for PP1 and PP1-mediated dephosphorylation that promotes their interaction (3) and it is possible that PyrCer affects exons that are dependent on both factors.Figure 7.

Bottom Line: To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection.We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF).Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.

ABSTRACT
Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

Show MeSH