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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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C6 Ceramide changes alternative splicing patterns of several endogenous genes. RT-PCR analyses of RNA from cells treated for 24 h with C6 pyridinium ceramide. Two representative examples of untreated and cells treated with 10 µM C6 pyridinium ceramide (PyrCer) are shown. The exon–intron structure of the regulated genes is indicated schematically next to the ethidium bromide stained gels. Numbers indicate the length of the exons. A statistical analysis of three independent experiments is shown on the right. The P values are (A–D) 0.0001 and (E) 0.001. The percent exon inclusion was calculated by dividing the intensity of the band representing the regulated exon with the combined intensity of all bands. C is a negative control without the reverse transcription. The regulated exons were: for DRF1 65 nt; TIAF1 45 nt; Pol-B: 58 nt; tau: 93 nt; SYK: 69 nt and are indicated in gray. Primers are indicated by arrows.
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gkr1289-F1: C6 Ceramide changes alternative splicing patterns of several endogenous genes. RT-PCR analyses of RNA from cells treated for 24 h with C6 pyridinium ceramide. Two representative examples of untreated and cells treated with 10 µM C6 pyridinium ceramide (PyrCer) are shown. The exon–intron structure of the regulated genes is indicated schematically next to the ethidium bromide stained gels. Numbers indicate the length of the exons. A statistical analysis of three independent experiments is shown on the right. The P values are (A–D) 0.0001 and (E) 0.001. The percent exon inclusion was calculated by dividing the intensity of the band representing the regulated exon with the combined intensity of all bands. C is a negative control without the reverse transcription. The regulated exons were: for DRF1 65 nt; TIAF1 45 nt; Pol-B: 58 nt; tau: 93 nt; SYK: 69 nt and are indicated in gray. Primers are indicated by arrows.

Mentions: In total, 92 alternative splicing events were tested and 29 splicing events showed a significant change in their alternative splicing patterns. We validated these 29 events by performing RT-PCR with mRNA extracted from HEK293 cells treated with or without 10 µM C6 pyridinium ceramide. From these, we selected five exons with the most significant changes for further analysis. These exons were present in the DRF1, TIAF1, POL-β, TAU and SYK pre-mRNAs. As shown in Figure 1, treatment with 10 µM PyrCer promoted both inclusion and skipping of exons. As a negative control, we validated the absence of a PyrCer effect on the pre-mRNAs of CCNE1, ECT2, SHC1 and STM1 genes which showed no change in the primary screen (Supplementary Figure S3). Similar to other signal-dependent splicing systems (22), exon inclusion differs around 10–20% between the treated cells and controls. However, these differences are statistically significant, with P-values from the student's test <0.01.Figure 1.


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)

C6 Ceramide changes alternative splicing patterns of several endogenous genes. RT-PCR analyses of RNA from cells treated for 24 h with C6 pyridinium ceramide. Two representative examples of untreated and cells treated with 10 µM C6 pyridinium ceramide (PyrCer) are shown. The exon–intron structure of the regulated genes is indicated schematically next to the ethidium bromide stained gels. Numbers indicate the length of the exons. A statistical analysis of three independent experiments is shown on the right. The P values are (A–D) 0.0001 and (E) 0.001. The percent exon inclusion was calculated by dividing the intensity of the band representing the regulated exon with the combined intensity of all bands. C is a negative control without the reverse transcription. The regulated exons were: for DRF1 65 nt; TIAF1 45 nt; Pol-B: 58 nt; tau: 93 nt; SYK: 69 nt and are indicated in gray. Primers are indicated by arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351148&req=5

gkr1289-F1: C6 Ceramide changes alternative splicing patterns of several endogenous genes. RT-PCR analyses of RNA from cells treated for 24 h with C6 pyridinium ceramide. Two representative examples of untreated and cells treated with 10 µM C6 pyridinium ceramide (PyrCer) are shown. The exon–intron structure of the regulated genes is indicated schematically next to the ethidium bromide stained gels. Numbers indicate the length of the exons. A statistical analysis of three independent experiments is shown on the right. The P values are (A–D) 0.0001 and (E) 0.001. The percent exon inclusion was calculated by dividing the intensity of the band representing the regulated exon with the combined intensity of all bands. C is a negative control without the reverse transcription. The regulated exons were: for DRF1 65 nt; TIAF1 45 nt; Pol-B: 58 nt; tau: 93 nt; SYK: 69 nt and are indicated in gray. Primers are indicated by arrows.
Mentions: In total, 92 alternative splicing events were tested and 29 splicing events showed a significant change in their alternative splicing patterns. We validated these 29 events by performing RT-PCR with mRNA extracted from HEK293 cells treated with or without 10 µM C6 pyridinium ceramide. From these, we selected five exons with the most significant changes for further analysis. These exons were present in the DRF1, TIAF1, POL-β, TAU and SYK pre-mRNAs. As shown in Figure 1, treatment with 10 µM PyrCer promoted both inclusion and skipping of exons. As a negative control, we validated the absence of a PyrCer effect on the pre-mRNAs of CCNE1, ECT2, SHC1 and STM1 genes which showed no change in the primary screen (Supplementary Figure S3). Similar to other signal-dependent splicing systems (22), exon inclusion differs around 10–20% between the treated cells and controls. However, these differences are statistically significant, with P-values from the student's test <0.01.Figure 1.

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