Rapid-response splicing reporter screens identify differential regulators of constitutive and alternative splicing

Abstract

Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phosphorylate serine-arginine-rich (SR) protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing and uncover differential regulation of constitutively spliced introns.

FIG. 1.

Characterization of the splicing reporter. (A) Schematic diagram of the intron-containing (Luc-I) and intronless (Luc) luciferase reporters. Solid yellow bars denote exons and the black line depicts the inserted intron. 5′ and 3′ untranslated regions (UTR) are indicated. The destabilizing sequences (DS) are CL1 and PEST for protein DS and five tandem repeats of the AUUUA sequence for RNA. “ORF” represents the open reading frame of luciferase. (B) Expression of the luciferase reporters in cells. The luciferase assay was performed 24 h after transfection of HeLa cells. Error bars denote the standard deviations for three independent experiments. (C) Half-life measurement of reporter protein and mRNA. Luc and Luc-I stably expressing cells were treated with cycloheximide (CHX) or actinomycin D (ActD) for 4 h, followed by a luciferase assay. Data are presented as percent activities compared to the level for DMSO-treated cells. Error bars denote the standard deviations for three independent experiments.

FIG. 2.

High-throughput screen for splicing modulators. (A) Workflow for HTS. Cells stably expressing Luc-I were screened with a collection of >23,000 compounds for 4 h (see Materials and Methods). Hits were counterscreened on Luc cells, and compounds that passed the selectivity step and showed a well-behaved dose response were further characterized as illustrated in the scheme. QPCR, quantitative PCR. (B) Scatter plot of a representative control plate of Luc-I cells in a 1,536-well format. Four columns on the plate were treated with ActD or CHX. The last four columns (Control) did not contain any cells and reflect the background/noise of the assay. The calculated Z′ value was 0.6, and the CV was 11%.

FIG. 3.

Splicing inhibitors identified from HTS inhibit luciferase protein and mRNA expression. Luc-I cells were treated with various concentrations of clotrimazole, flunarizine, and chlorhexidine for 4 h. Luciferase activity, measured in light units, reflects the amount of luciferase protein produced (blue bars). To quantify the amount of spliced luciferase mRNA, real-time qPCR was performed at the IC₅₀ of each compound (red bars). The spliced mRNA measurements were normalized to those of intronless Luc in order to eliminate effects on transcription or mRNA stability. Data are presented as percentages of the level for DMSO-treated cells, and error bars denote the standard deviations for three independent experiments.

FIG. 4.

Splicing modulators have distinct effects on constitutive and alternative splicing of endogenous genes in cells. (A) RT-PCR analysis of total RNA extracted from HeLa cells treated with clotrimazole, flunarizine, and chlorhexidine for 6 h. Results are shown for constitutive pre-mRNA splicing of endogenous coilin intron 2 and SR protein-regulated alternative splicing of exon 11 of RON and exons 3 to 6 of caspase 9. (B) Real-time qPCR analysis of the samples shown in panel A. For each endogenous gene, a set of primers was designed to distinguish between exon-exon junction (spliced) and exon-intron junction (unspliced). Coilin intron 2 and ILF2 introns 4 and 5 are major (U2 dependent), whereas CCDC56 intron 1 and IK intron 14 are minor (U12 dependent). Three independent replicates were used for each treatment, and data are presented relative to the level for DMSO, which was set to 1. (C) Real-time qPCR analysis of introns from various endogenous genes, as described for panel A. For each transcript, the intron tested is indicated as a number after the gene name. Each bar represents the average of results from three measurements, and data are presented relative to the level for DMSO, which was set to 1. All qPCR measurements were normalized to the level for β-actin.

FIG. 5.

In vitro effects of splicing modulators. (A) Constitutive splicing of CDC14-15 pre-mRNA was analyzed in the presence of 50 μM clotrimazole, flunarizine, or chlorhexidine for 90 min. Splicing intermediates are depicted to the left of the gel, and molecular size markers are indicated to the right of the gel. Fully spliced mRNA is indicated with a red arrow and corresponds to 140 nucleotides. (B) SR protein-regulated splicing of HIV Tat2-3, HβΔ6, and μC3-C4 pre-mRNAs was analyzed in the presence of DMSO or 50 and 100 μM chlorhexidine in vitro. Splicing intermediates are depicted to the left of the gels, and molecular size markers are indicated to the right of the gels. Red arrows point to the altered levels of the spliced mRNA, which are 371, 367, and 271 nucleotides for Tat2-3, HβΔ6, and μC3-C4, respectively.

FIG. 6.

Chlorhexidine inhibits SR protein phosphorylation. (A) Total protein extracts from cells treated with 0, 10, and 20 μM chlorhexidine or TG003 for 6 h were separated by SDS-PAGE, and phosphorylated SR proteins were detected by Western blotting using the SR protein phospho-specific monoclonal antibody (1H4). Quantification of the phosphorylated SR proteins is depicted in the histogram. Values are presented relative to the level for DMSO, which was set to 1 (dashed line). (B) The ability of known recombinant SR protein kinases to phosphorylate an SR-rich substrate was tested in vitro in the presence of either DMSO or increasing concentrations of chlorhexidine. Activity in the presence of DMSO was set to 100%.

FIG. 7.

Differential effects of splicing modulators on alternative splicing. Heat maps of representative transcripts from the exon array are shown, with the gene structure depicted below the heat map.