All polymerase-II transcripts undergo pre-mRNA processing and at least 95% of the transcriptional units are alternatively spliced (1
). The exact regulation of alternative splicing events is physiologically important, as evidenced by an increasing number of diseases that are caused by the selection of the wrong splice site (2
). The proper recognition of exons is regulated by the transient formation of protein complexes on the pre-mRNA that identify a sequence for its recognition by the spliceosome. The signals on the pre-mRNA that mark an exon for its inclusion in the mRNA are highly degenerate, most likely to avoid interference with coding requirements. Therefore, exons are recognized by the formation of a complex between the pre-mRNA and various hnRNPs and SR proteins. Since these proteins generally bind to RNA with low selectivity, a higher specificity is achieved by simultaneous 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 dephosphorylation promotes the interaction between Tra2-beta1 and SF2/ASF (also called SRSF1) in vitro
). Reversible phosphorylation of SR proteins and hnRNPs is achieved by an interplay between protein kinases and phosphatases. Several SR proteins contain an evolutionarily highly conserved PP1 binding element (RVxF) in their RNA recognition motif which allows PP1 to influence the phosphorylation status of proteins bound to pre-mRNA. In addition to the interaction between individual SR proteins, the affinity between components of the spliceosome, such as U1-70K and proteins that associate with pre-mRNA, including PSF/SFPQ (4
), SRp38 (5
) and SF2/ASF (6
) is influenced by their phosphorylation status. This suggests that PP1 activity can regulate the recognition of pre-mRNA substrates by the spliceosome and influences the selection of alternative exons by altering protein affinities. In agreement with this model, a change in PP1 activity has been shown to alter splice site selection in vivo
Ceramides are a class of sphingolipids that are composed of sphingosine and a fatty acid moiety. In the cell, ceramide can be generated by sphyingomyelin hydrolysis by sphingomyelinase or by de novo
synthesis. Sphingomyelin and sphingomyelinase are both present in the nucleus, which led to the suggestion that they participate in nuclear signaling (9
). Natural ceramides contain long alkyl groups (C14–26) and are therefore hydrophobic. It has previously been shown that endogenous ceramides alter bcl-x splicing via a purine-rich splicing enhancer (10
). To improve delivery and water solubility, cationic-ceramide analogs containing pyridinium moieties were synthesized. Owing to their ability to induce apoptosis, these water-soluble ceramide analogs have been tested as anti-cancer drugs (11
). Despite their usage in clinical studies, the effect of these pyridinium ceramides on alternative splicing has not been determined.
Here, we investigate the role of a water-soluble ceramide analog, C6 pyridinium ceramide (PyrCer), in alternative splicing. We found that PyrCer treatment inhibits the dephosphorylation of splicing regulatory proteins by PP1, which is opposite to the activation reported for water-insoluble short and long-chain ceramides (13
). In addition to SR proteins known to be affected by natural ceramides, PyrCer treatment increases the phosphorylation of other proteins involved in splicing, such as PSF/SFPQ, SAP155 and UAP56. It changes the alternative splicing patterns of exons that are short and dependent on splicing enhancers. These findings suggest that the dephosphorylation of splicing factors by PP1 is a molecular link between lipids and alternative splice site selection. The difference between PyrCer and natural ceramides suggest that subclasses of lipids have distincitve effects on splice site selection.