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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.

Show MeSH
Reporter gene constructs recapitulate the effect of C6 pyridinium ceramide. The alternative exons regulated by C6 pyridinium phosphate were cloned between two constitutively spliced insulin exons into a heterologous splice reporter construct. One microgram of the constructs was transfected into HEK293 cells and the cells were treated with PyrCer for 24 h. Representative semi-quantitative RT-PCR analysis is shown. The statistical evaluation is shown on the right and was performed as described for Figure 1.
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gkr1289-F2: Reporter gene constructs recapitulate the effect of C6 pyridinium ceramide. The alternative exons regulated by C6 pyridinium phosphate were cloned between two constitutively spliced insulin exons into a heterologous splice reporter construct. One microgram of the constructs was transfected into HEK293 cells and the cells were treated with PyrCer for 24 h. Representative semi-quantitative RT-PCR analysis is shown. The statistical evaluation is shown on the right and was performed as described for Figure 1.

Mentions: To determine whether the sequence elements that respond to PyrCer treatment are localized in the vicinity of the regulated exons, we introduced the alternatively spliced exons into reporter minigenes. We used a recently developed recombination technique that introduces the exon of interest between two constitutively spliced insulin exons (14). Depending on the intron length, the resulting reporter minigenes contained one to three exons cloned between the constitutive insulin exons. Their structures are schematically shown in Figure 2.Figure 2.


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)

Reporter gene constructs recapitulate the effect of C6 pyridinium ceramide. The alternative exons regulated by C6 pyridinium phosphate were cloned between two constitutively spliced insulin exons into a heterologous splice reporter construct. One microgram of the constructs was transfected into HEK293 cells and the cells were treated with PyrCer for 24 h. Representative semi-quantitative RT-PCR analysis is shown. The statistical evaluation is shown on the right and was performed as described for Figure 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1289-F2: Reporter gene constructs recapitulate the effect of C6 pyridinium ceramide. The alternative exons regulated by C6 pyridinium phosphate were cloned between two constitutively spliced insulin exons into a heterologous splice reporter construct. One microgram of the constructs was transfected into HEK293 cells and the cells were treated with PyrCer for 24 h. Representative semi-quantitative RT-PCR analysis is shown. The statistical evaluation is shown on the right and was performed as described for Figure 1.
Mentions: To determine whether the sequence elements that respond to PyrCer treatment are localized in the vicinity of the regulated exons, we introduced the alternatively spliced exons into reporter minigenes. We used a recently developed recombination technique that introduces the exon of interest between two constitutively spliced insulin exons (14). Depending on the intron length, the resulting reporter minigenes contained one to three exons cloned between the constitutive insulin exons. Their structures are schematically shown in Figure 2.Figure 2.

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