C6 pyridinium ceramide influences alternativepre-mRNA splicing by inhibiting proteinphosphatase-1Chiranthani Sumanasekera1, Olga Kelemen1, Monique Beullens2, Brandon E. Aubol3,
Joseph A. Adams3, Manjula Sunkara1, Andrew Morris1, Mathieu Bollen2,
Athena Andreadis4 and Stefan Stamm1,*
1Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA,2Laboratory of Biosignaling and Therapeutics, Department of Molecular Cell Biology, University of Leuven,B-3000 Leuven, Belgium, 3University of California, San Diego La Jolla, CA 92093 and 4UMASS Medical School,55 Lake Avenue North, Worcester, MA 01655-0106, USA
Received August 5, 2011; Revised December 6, 2011; Accepted December 14, 2011
Alternative pre-mRNA processing is a centralelement of eukaryotic gene regulation. The cellfrequently alters the use of alternative exons inresponse to physiological stimuli. Ceramides arelipid-signaling molecules composed of sphingosineand a fatty acid. Previously, water-insoluble cera-mides were shown to change alternative splicingand decrease SR-protein phosphorylation byactivating protein phosphatase-1 (PP1). To gainfurther mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effectof C6 pyridinium ceramide (PyrCer) on alternativesplice site selection. PyrCer is a water-solubleceramide analog that is under investigation as acancer drug. We found that PyrCer binds to the PP1catalytic subunit and inhibits the dephosphorylationof several splicing regulatory proteins containing theevolutionarily conserved RVxF PP1-binding motif(including PSF/SFPQ, Tra2-beta1 and SF2/ASF). Incontrast to natural ceramides, PyrCer promotesphosphorylation of splicing factors. Exons that areregulated by PyrCer have in common suboptimalsplice sites, are unusually short and share two 4-ntmotifs, GAAR and CAAG. They are dependent onPSF/SFPQ, whose phosphorylation is regulated byPyrCer. Our results indicate that lipids can influencepre-mRNA processing by regulating the phosphoryl-ation status of specific regulatory factors, which ismediated by protein phosphatase activity.
All polymerase-II transcripts undergo pre-mRNAprocessing and at least 95% of the transcriptional unitsare alternatively spliced (1). The exact regulation of alter-native splicing events is physiologically important, asevidenced by an increasing number of diseases that arecaused by the selection of the wrong splice site (2). Theproper recognition of exons is regulated by the transientformation of protein complexes on the pre-mRNA thatidentify a sequence for its recognition by the spliceosome.The signals on the pre-mRNA that mark an exon for itsinclusion in the mRNA are highly degenerate, most likelyto avoid interference with coding requirements. Therefore,exons are recognized by the formation of a complexbetween the pre-mRNA and various hnRNPs and SRproteins. Since these proteins generally bind to RNAwith low selectivity, a higher specificity is achieved bysimultaneous binding of the proteins to each other.The interaction between these proteins is regulated in
part by their phosphorylation status. For example,protein phosphatase-1 (PP1)-mediated dephosphorylationpromotes the interaction between Tra2-beta1 andSF2/ASF (also called SRSF1) in vitro (3). Reversible phos-phorylation of SR proteins and hnRNPs is achieved by aninterplay between protein kinases and phosphatases.Several SR proteins contain an evolutionarily highlyconserved PP1 binding element (RVxF) in theirRNA recognition motif which allows PP1 to influencethe phosphorylation status of proteins bound to pre-mRNA. In addition to the interaction between individualSR proteins, the affinity between components of thespliceosome, such as U1-70K and proteins that associatewith pre-mRNA, including PSF/SFPQ (4), SRp38 (5) and
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Published online 30 December 2011 Nucleic Acids Research, 2012, Vol. 40, No. 9 40254039doi:10.1093/nar/gkr1289
The Author(s) 2011. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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SF2/ASF (6) is influenced by their phosphorylation status.This suggests that PP1 activity can regulate the recogni-tion of pre-mRNA substrates by the spliceosome andinfluences the selection of alternative exons by alteringprotein affinities. In agreement with this model, achange in PP1 activity has been shown to alter splice siteselection in vivo (3,7,8).Ceramides are a class of sphingolipids that are
composed of sphingosine and a fatty acid moiety. In thecell, ceramide can be generated by sphyingomyelinhydrolysis by sphingomyelinase or by de novo synthesis.Sphingomyelin and sphingomyelinase are both present inthe nucleus, which led to the suggestion that they partici-pate in nuclear signaling (9). Natural ceramides containlong alkyl groups (C1426) and are therefore hydropho-bic. It has previously been shown that endogenousceramides alter bcl-x splicing via a purine-rich splicingenhancer (10). To improve delivery and water solubility,cationic-ceramide analogs containing pyridinium moietieswere synthesized. Owing to their ability to induce apop-tosis, these water-soluble ceramide analogs have beentested as anti-cancer drugs (11,12). Despite their usage inclinical studies, the effect of these pyridinium ceramides onalternative splicing has not been determined.Here, we investigate the role of a water-soluble
ceramide analog, C6 pyridinium ceramide (PyrCer), inalternative splicing. We found that PyrCer treatmentinhibits the dephosphorylation of splicing regulatoryproteins by PP1, which is opposite to the activationreported for water-insoluble short and long-chainceramides (13). In addition to SR proteins known to beaffected by natural ceramides, PyrCer treatment increasesthe phosphorylation of other proteins involved in splicing,such as PSF/SFPQ, SAP155 and UAP56. It changes thealternative splicing patterns of exons that are short anddependent on splicing enhancers. These findings suggestthat the dephosphorylation of splicing factors by PP1 isa molecular link between lipids and alternative splice siteselection. The difference between PyrCer and naturalceramides suggest that subclasses of lipids have distincitveeffects on splice site selection.
MATERIALS AND METHODS
Cell culture and transfection
HEK 293T cells were grown in DMEM (Invitrogen)supplemented with 10% (v/v) fetal bovine serum(Invitrogen) for 24 h to 80% confluence prior to ceramidetreatment and to 6070% confluence prior to calciumphosphate transfections. Transfection of the minigenesand RT-PCR analyses was performed as described (14).
Four microliters of HeLa nuclear extract (Dundee CellProducts) was incubated in 1mM MnCl2 and 1 PNKbuffer (70mM TrisHCl pH 7.6, 10mM MgCl2, 5mMdithiothreitol). Twenty micromolar of C6 pyridiniumceramide (D-erythro-2-N-[60-(1-pyridinium)-hexanoyl]-sphingosine bromide) (Avanti Polar Lipids), or 20 mM1-(2-oxopropyl) pyridinium bromide (Sigma-Aldrich)
was added. After 5min at 37C, 4 mCi of [g-32P] ATP(7000Ci/mmol, MP Biochemical) was added. The 50 mlreactions were then incubated for 30min and thereaction was stopped by adding 6 SDS sample buffer(0.375M TrisHCl pH 6.8, 30% glycerol, 12% SDS,12% 2-b mercaptoethanol and 0.075% bromophenolblue) followed by 10% SDSPAGE. Proteins that weredifferentially phosphorylated in the ceramide lanes wereexcised from the gel and analyzed by MALDI TOF/TOF.
Minigenes were constructed as described (14). The primersfor the minigenes and the insert sequences are shown inSupplementary Figure S1.
Hydrolysis of p-nitrophenylphosphate (p-NPP) wasperformed in 25 ml reactions containing 1 phosphatebuffer (0.1M sodium acetate, 0.05M bisTris, 0.05MTrisHCl, 2mM dithiothreitol, at pH 8.0), 50mMpNPP, 1mM MnCl2 and 0.1U of purified PP1 enzymemix (Upstate, #14-110) at 37C for 30min. A PP1 unitreleases 1 nmol of phosphate/min from 15 mM phosphor-ylase at 30C. The reaction was terminated by the additionof 100 ml of 0.25M NaOH and absorbance was measuredat 410 nm. The activity of PP1 on Phosphorylase b wasmeasured as previously described (15).
C6 pyridinium ceramide treatment of HeLa nuclearextracts
Ten micrograms of HeLa nuclear extract (NE) wasincubated with 4 mCi [g32P] ATP for 30min at 37Cin 1 PNK buffer (New England Bio Labs) plus or10 mM C6 pyridinium ceramide in a 50 ml final volume.Radiolabeled-NE proteins were then subjected toimmunoprecipitation overnight with 2 mg of indicatedantibodies, 10 ml proteins A/G beads (Santa Cruz) and200 ml RIPA-rescue buffer containing phosphatase andprotease inhibitors. Immunoprecipitates were subjectedto SDSPAGE, proteins were transferred on to nitrocel-lulose membranes and analyzed by phosphor-imaging.The membranes were then blotted with the indicatedantibodies for total protein expression. Representative32P signals and western blot results of triplicate experi-ments for SF2/ASF, Tra2-beta1, PSF, UAP56 andSAP155 are shown.
Protein expression and purification
Recombinant proteins were overexpressed in Escherichiacoli BL21 (DE3) cells. Expression was induced with IPTGin a final concentration of 0.4mM for 4 h at room tem-perature. The