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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 pyridinium ceramide binds to protein phosphatase-1 in vivo. (A) Detection of PyrCer and C18 ceramide bound to PP1. HEK293 cells were treated with 0 and 10 µM C6 ceramide for 14 h and PP1 was isolated by immunoprecipitation with a pan-PP1 antibody. From the immunoprecipitates, bound lipids were extracted with chloroform and analyzed by mass-spectrometry. The mass-spectrometry signal was normalized to the amount of immunoprecipitated PP1, which was detected by western blot. The antiserum precipitates the catalytic subunit of PP1. (B) Detection of PyrCer bound to PP1 variants. (C) EGFP-tagged constructs expressing PP1 gamma, PP1 alpha, Tra2-beta1-RATA were transfected into HEK293 cells and immunoprecipitated with an anti EGFP antiserum. PyrCer bound to the immunoprecipitates was determined by mass-spectrometry and normalized to the signal obtained by western blot using anti EGFP.
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gkr1289-F4: C6 pyridinium ceramide binds to protein phosphatase-1 in vivo. (A) Detection of PyrCer and C18 ceramide bound to PP1. HEK293 cells were treated with 0 and 10 µM C6 ceramide for 14 h and PP1 was isolated by immunoprecipitation with a pan-PP1 antibody. From the immunoprecipitates, bound lipids were extracted with chloroform and analyzed by mass-spectrometry. The mass-spectrometry signal was normalized to the amount of immunoprecipitated PP1, which was detected by western blot. The antiserum precipitates the catalytic subunit of PP1. (B) Detection of PyrCer bound to PP1 variants. (C) EGFP-tagged constructs expressing PP1 gamma, PP1 alpha, Tra2-beta1-RATA were transfected into HEK293 cells and immunoprecipitated with an anti EGFP antiserum. PyrCer bound to the immunoprecipitates was determined by mass-spectrometry and normalized to the signal obtained by western blot using anti EGFP.

Mentions: The inhibition of PP1 by PyrCer suggests that the two molecules interact with each other. To explore this interaction in vivo, we developed a mass-spectrometric assay for PyrCer detection. In brief, cells were treated with pyridinium ceramide for 24 h to allow PyrCer and PP1 to interact in vivo. Next, cells were washed and lysed, PP1-bound lipids were isolated with antibodies against PP1 and lipids were extracted from immune-complexes with chloroform, separated with HPLC and detected by ion trap triple quadrupole mass spectrometry (Supplementary Figure S5A). This method gives a linear response to PyrCer as shown in Supplementary Figure S5B and S5C. To test whether C6 ceramide binds to PP1 in vivo, we immunoprecipitated endogenous PP1 from cells that were treated with with or without 10 µM PyrCer using a pan-PP1 antibody (Figure 4A and B). The ceramide signal obtained from the mass-spectrometry was normalized to the amount of PP1 present in the immunoprecipitations (Figure 4B).Figure 4.


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 pyridinium ceramide binds to protein phosphatase-1 in vivo. (A) Detection of PyrCer and C18 ceramide bound to PP1. HEK293 cells were treated with 0 and 10 µM C6 ceramide for 14 h and PP1 was isolated by immunoprecipitation with a pan-PP1 antibody. From the immunoprecipitates, bound lipids were extracted with chloroform and analyzed by mass-spectrometry. The mass-spectrometry signal was normalized to the amount of immunoprecipitated PP1, which was detected by western blot. The antiserum precipitates the catalytic subunit of PP1. (B) Detection of PyrCer bound to PP1 variants. (C) EGFP-tagged constructs expressing PP1 gamma, PP1 alpha, Tra2-beta1-RATA were transfected into HEK293 cells and immunoprecipitated with an anti EGFP antiserum. PyrCer bound to the immunoprecipitates was determined by mass-spectrometry and normalized to the signal obtained by western blot using anti EGFP.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1289-F4: C6 pyridinium ceramide binds to protein phosphatase-1 in vivo. (A) Detection of PyrCer and C18 ceramide bound to PP1. HEK293 cells were treated with 0 and 10 µM C6 ceramide for 14 h and PP1 was isolated by immunoprecipitation with a pan-PP1 antibody. From the immunoprecipitates, bound lipids were extracted with chloroform and analyzed by mass-spectrometry. The mass-spectrometry signal was normalized to the amount of immunoprecipitated PP1, which was detected by western blot. The antiserum precipitates the catalytic subunit of PP1. (B) Detection of PyrCer bound to PP1 variants. (C) EGFP-tagged constructs expressing PP1 gamma, PP1 alpha, Tra2-beta1-RATA were transfected into HEK293 cells and immunoprecipitated with an anti EGFP antiserum. PyrCer bound to the immunoprecipitates was determined by mass-spectrometry and normalized to the signal obtained by western blot using anti EGFP.
Mentions: The inhibition of PP1 by PyrCer suggests that the two molecules interact with each other. To explore this interaction in vivo, we developed a mass-spectrometric assay for PyrCer detection. In brief, cells were treated with pyridinium ceramide for 24 h to allow PyrCer and PP1 to interact in vivo. Next, cells were washed and lysed, PP1-bound lipids were isolated with antibodies against PP1 and lipids were extracted from immune-complexes with chloroform, separated with HPLC and detected by ion trap triple quadrupole mass spectrometry (Supplementary Figure S5A). This method gives a linear response to PyrCer as shown in Supplementary Figure S5B and S5C. To test whether C6 ceramide binds to PP1 in vivo, we immunoprecipitated endogenous PP1 from cells that were treated with with or without 10 µM PyrCer using a pan-PP1 antibody (Figure 4A and B). The ceramide signal obtained from the mass-spectrometry was normalized to the amount of PP1 present in the immunoprecipitations (Figure 4B).Figure 4.

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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