The splicing reporter system that we describe here has several advantageous properties that make it a powerful tool for the discovery of inhibitors and modulators of splicing and splicing-dependent processes in cells. Our assay system was designed to overcome two major potential problems. First, given the essential role of splicing in gene expression, prolonged exposure to splicing inhibitors would be toxic to cells and preclude their discovery. We therefore reasoned that the reporter needed to be highly sensitive and robust and provide real-time readout of changes in splicing before general toxic effects interfere. We achieved this by incorporating destabilizing elements in both the luciferase protein and the mRNA encoding it, generating a rapid-response reporter with a half-life of less than 4 h. Second, many compounds could affect the enzymatic activity or stability of the reporter or steps in its expression other than splicing, such as transcription and translation. To address this, we constructed a firefly luciferase reporter gene that was the same except for its lack of an intron and eliminated compounds that showed an effect on both Luc and Luc-I. The assay is suitable for large-scale screens for chemical and genetic modifiers of splicing in 384- and 1,536-well formats. This strategy opens up the possibility of discovering splicing inhibitors that would likely not be identified by other recently described cell-based assays (34
). Furthermore, the short time frame of the assay helps minimize secondary effects of compounds and increases the likelihood of capturing direct effects on splicing.
Screening with this assay, using a strong intron commonly used for constitutive splicing studies, identified several inhibitors. Importantly, each showed differential effects on constitutive and alternative splicing. Both clotrimazole and flunarizine inhibited splicing of many introns but had distinct profiles. Of the introns tested, each compound inhibited some that the other did not; some were inhibited by both, and some showed different degrees of inhibition for the two compounds. This suggests that although both clotrimazole and flunarizine inhibit the splicing of the reporter, they do so by different modes of action. We note that neither compound inhibited splicing in vitro
of several test introns. Thus, unlike spliceostatin A and pladienolide (24
), clotrimazole and flunarizine do not inhibit components of the basal splicing machinery such as U2 snRNP, which is surprising because the β-globin/immunoglobulin intron used in the screen is considered to be independent of splicing factors due to its strong (constitutive) splice sites (51
). These findings also underscore the capacity of cell-based screens to reveal regulatory mechanisms that are not recapitulated in an in vitro
system. Our observations suggest that entirely constitutive splicing may not exist in cells and that all introns are regulated. The compounds that we identified should make it possible to discover the pathways and factors involved in this unexpected aspect of regulation of constitutively spliced introns.
Clotrimazole is widely used without prescription for treatment of fungal infections. In mammalian cells, it inhibits cytochrome P450, interferes with cellular calcium homeostasis (1
), and inhibits proliferation of cancer and vascular endothelial cells (4
). Flunarizine is a calcium channel blocker and vasodilator that is used to treat neurological disorders, including vertigo, migraine, and epilepsy (22
). It has a protective effect on neurons from serum and nerve growth factor deprivation, oxidative stress, and axotomy (25
). Clotrimazole and flunarizine have not previously been shown to have an effect on splicing. The mechanism by which these compounds inhibit splicing is not presently known, and it is thus not clear whether this is related to their clinical efficacy. It is noted, however, that both affect calcium physiology. Several anticancer drugs have been recently shown to affect alternative splicing of a group of apoptosis-related test genes (52
). However, the drug treatments were for 24 h, and thus, the effects on splicing could be an adaptive response rather than a direct effect. Some of these were included in the library of compounds that we screened but did not show specific activity in our assay.
Our results demonstrate that chlorhexidine had a weaker effect on constitutive splicing than clotrimazole and flunarizine. However, it was highly effective in modulating alternative splicing. Chlorhexidine is a widely used, prescription-free disinfectant and topical anti-infective agent, most commonly used in mouthwash. In addition to its broad-spectrum antibacterial activity, chlorhexidine is active against yeast and some lipid-enveloped viruses, including HIV (40
). It has also been reported to induce an inflammatory reaction (45
) and tissue necrosis (16
). Several mechanisms have been suggested for this induced toxicity, including inhibition of mitochondrial activity, protein and DNA synthesis, endoplasmic reticulum stress, an increase in intracellular calcium, and induction of oxidative stress (19
). However, chlorhexidine has not previously been known to have an effect on splicing.
Because our data demonstrated that chlorhexidine affected alternative splicing of many exons, including those of RON, caspase 9, and HIV Tat2-3, which are known to be regulated by SR proteins, we studied its effect on this major class of splicing factors. SR proteins play critical roles in both constitutive and alternative splicing (5
). Phosphorylation of the RS domain of SR proteins regulates their activity in alternative splicing by modulating their protein-protein interactions, RNA binding, cellular localization, and stability (8
). We found marked reductions in the phosphorylation states of SRp75, SRp55, and SRp30 after chlorhexidine treatment. Other SR proteins, such as SRp20, were not affected. It has been shown that the relative stoichiometry of SR proteins on a given pre-mRNA is complex and depends on their phosphorylation state (50
) and that disruption of this stoichiometry leads to not only negative but also positive outcomes on splicing (17
). Since chlorhexidine did not affect the phosphorylation of all SR proteins to the same level, it is impossible to predict the outcome of chlorhexidine treatment on a given splicing event. For example, while inhibition of μC3-C4 splicing could be due to the inability of a dephosphorylated SR protein(s), such as ASF/SF2, to bind and enhance splicing, the increased splicing efficiency of Tat2-3 could be explained in light of previous data showing that SC35 can bind to an exonic splicing silencer and repress splicing of Tat2-3 (38
). Thus, the release of SC35 from this pre-mRNA or the disruption of the balance between SC35 and ASF/SF2 could lead to enhanced splicing of Tat2-3 pre-mRNA. Several kinases that phosphorylate SR proteins and regulate their activity have been described, particularly SRPKs (21
) and Clks (3
). Other potential SR protein kinases have also been reported but remain less characterized, including pre-mRNA processing mutant 4 (PRP4) (30
) and topoisomerase I (48
). Our studies show that chlorhexidine is a selective inhibitor of Clk4 and Clk3 and to a lesser extent Clk2. Clk1, on the other hand, is only weakly inhibited by chlorhexidine. This activity profile is different from that of a previously described Clk inhibitor, TG003, which mainly targets Clk1 and Clk4 (42
). While all Clks can phosphorylate any SR protein in vitro
, it was not previously known whether the individual Clks have activities toward different SR proteins in cells. Our findings suggest that by inhibiting Clk4 and Clk3, chlorhexidine decreases or alters the phosphorylation signature of a subset of SR proteins, resulting in a change in the splicing pattern of a distinct group of exons. Further characterization of these splicing events should provide a better understanding of the functional relationship between phosphorylation of specific SR proteins by different Clks and their role in regulating alternative splicing. Chlorhexidine is therefore a valuable tool with which to dissect these mechanisms.
Exon array analysis of clotrimazole-, flunarizine-, or chlorhexidine-treated cells showed that each compound affects a distinct set of splicing events. Closer analysis of the transcripts that were affected by at least two compounds showed that a large fraction of these transcripts were affected on different exons or in opposite directions, indicating that each compound regulates splicing by a unique mechanism. The effect of the clinically used drugs clotrimazole, flunarizine, and chlorhexidine on splicing is unexpected and off target at concentration ranges in which they show pharmacologic activity. This effect may provide a molecular basis for their toxicity as well as their activity and should help design more-specific next-generation drugs. The screening method that we describe will provide a wealth of additional reagents for studying the mechanism and regulation of splicing. With slight modifications, the same reporter can be used to screen for effectors of specific introns and of other splicing-dependent processes.