C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1.
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.
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.
Chiranthani
C
Sumanasekera
Olga
O
Kelemen
Monique
M
Beullens
Brandon E
BE
Aubol
Joseph A
JA
Adams
Manjula
M
Sunkara
Andrew
A
Morris
Mathieu
M
Bollen
Athena
A
Andreadis
Stefan
S
Stamm
D017398:Alternative Splicing / Q000187:drug effects*; D001665:Binding Sites; D002518:Ceramides / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005091:Exons; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors*; D011726:Pyridinium Compounds / Q000494:pharmacology*; D012322:RNA Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism
C539200:2-N-(6'-(1''-pyridinium)hexanoyl)sphingosine; D002518:Ceramides; D004791:Enzyme Inhibitors; D011726:Pyridinium Compounds; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D054645:Protein Phosphatase 1