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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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PSF, SF2/ASF and Tra2-beta1 bind to motifs enriched in PyrCer dependent exons. (A) Gel retardation assay with TIAF1 and control probes. (B) Gel retardation assay with POL-B and control probes. (C) Sequence of the RNA probes. The GAAR motif is underlined, the CAAG motif is underlined with zigzag. The SF2/ASF site is indicated by a dotted superscript. The concentration of all oligonucleotides was 0.4 µM in all reactions in a total volume of 10 µl. The protein concentrations for Tra2-beta1 were 0, 2.4, 3.6, 4.8 and 7.2 µM in lanes 1–5, respectively. The concentrations for PSF/SFPQ were 0, 0.5, 1.1, 2.1 and 3.2 µM in lanes 1–5, respectively. The concentration for SF2/ASF was 0, 1.1, 2.8, 4.0 and 5.7 µM and in lanes 1–5, respectively.
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gkr1289-F5: PSF, SF2/ASF and Tra2-beta1 bind to motifs enriched in PyrCer dependent exons. (A) Gel retardation assay with TIAF1 and control probes. (B) Gel retardation assay with POL-B and control probes. (C) Sequence of the RNA probes. The GAAR motif is underlined, the CAAG motif is underlined with zigzag. The SF2/ASF site is indicated by a dotted superscript. The concentration of all oligonucleotides was 0.4 µM in all reactions in a total volume of 10 µl. The protein concentrations for Tra2-beta1 were 0, 2.4, 3.6, 4.8 and 7.2 µM in lanes 1–5, respectively. The concentrations for PSF/SFPQ were 0, 0.5, 1.1, 2.1 and 3.2 µM in lanes 1–5, respectively. The concentration for SF2/ASF was 0, 1.1, 2.8, 4.0 and 5.7 µM and in lanes 1–5, respectively.

Mentions: We next asked whether some of the regulated exons bind directly to PSF/SFPQ, SF2/ASF and Tra2-beta1, proteins that are dephosphorylated by PP1 and predicted to bind to PyrCer-reponsive exons. We purified PSF/SFPQ, SF2/ASF and Tra2-beta1 from bacteria and performed in vitro gel retardation assays using probes corresponding to the parts of POL-B and TIAF1 exons that contain the GAAR and CAAR motifs. As controls, we used arteficial oligonucleotides where these motifs were deleted. As shown in Figure 5, all three proteins bound to TIAF1 exon sequences. However Tra2-beta1 did not bind to the POL-B-exon RNA. This was surprising, since the exon containes one AGAA binding motif that is the high affinity binding site for Tra2-beta1 (34). This suggests that an unknown sequence context contributes to Tra2-beta1 binding. The binding of Tra2-beta1, SF2/ASF and PSF to tau exon 10 has been reported earlier (35–37).Figure 5.


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)

PSF, SF2/ASF and Tra2-beta1 bind to motifs enriched in PyrCer dependent exons. (A) Gel retardation assay with TIAF1 and control probes. (B) Gel retardation assay with POL-B and control probes. (C) Sequence of the RNA probes. The GAAR motif is underlined, the CAAG motif is underlined with zigzag. The SF2/ASF site is indicated by a dotted superscript. The concentration of all oligonucleotides was 0.4 µM in all reactions in a total volume of 10 µl. The protein concentrations for Tra2-beta1 were 0, 2.4, 3.6, 4.8 and 7.2 µM in lanes 1–5, respectively. The concentrations for PSF/SFPQ were 0, 0.5, 1.1, 2.1 and 3.2 µM in lanes 1–5, respectively. The concentration for SF2/ASF was 0, 1.1, 2.8, 4.0 and 5.7 µM and in lanes 1–5, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1289-F5: PSF, SF2/ASF and Tra2-beta1 bind to motifs enriched in PyrCer dependent exons. (A) Gel retardation assay with TIAF1 and control probes. (B) Gel retardation assay with POL-B and control probes. (C) Sequence of the RNA probes. The GAAR motif is underlined, the CAAG motif is underlined with zigzag. The SF2/ASF site is indicated by a dotted superscript. The concentration of all oligonucleotides was 0.4 µM in all reactions in a total volume of 10 µl. The protein concentrations for Tra2-beta1 were 0, 2.4, 3.6, 4.8 and 7.2 µM in lanes 1–5, respectively. The concentrations for PSF/SFPQ were 0, 0.5, 1.1, 2.1 and 3.2 µM in lanes 1–5, respectively. The concentration for SF2/ASF was 0, 1.1, 2.8, 4.0 and 5.7 µM and in lanes 1–5, respectively.
Mentions: We next asked whether some of the regulated exons bind directly to PSF/SFPQ, SF2/ASF and Tra2-beta1, proteins that are dephosphorylated by PP1 and predicted to bind to PyrCer-reponsive exons. We purified PSF/SFPQ, SF2/ASF and Tra2-beta1 from bacteria and performed in vitro gel retardation assays using probes corresponding to the parts of POL-B and TIAF1 exons that contain the GAAR and CAAR motifs. As controls, we used arteficial oligonucleotides where these motifs were deleted. As shown in Figure 5, all three proteins bound to TIAF1 exon sequences. However Tra2-beta1 did not bind to the POL-B-exon RNA. This was surprising, since the exon containes one AGAA binding motif that is the high affinity binding site for Tra2-beta1 (34). This suggests that an unknown sequence context contributes to Tra2-beta1 binding. The binding of Tra2-beta1, SF2/ASF and PSF to tau exon 10 has been reported earlier (35–37).Figure 5.

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