A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators

Abstract

Alternative splicing has emerged as a promising therapeutic target in a number of human disorders. However, the discovery of compounds that target the splicing reaction has been hindered by the lack of suitable high-throughput screening assays. Conversely, the effects of known drugs on the splicing reaction are mostly unclear and not routinely assessed. We have developed a two-color fluorescent reporter for cellular assays of exon inclusion that can accommodate nearly any cassette exon and minimizes interfering effects from changes in transcription and translation. We used microtubule-associated protein tau (MAPT) exon 10, whose missplicing causes frontotemporal dementia, to test the reporter in screening libraries of known bioactive compounds. These screens yielded several compounds that alter the splicing of the exon, both in the reporter and in the endogenous MAPT mRNA. One compound, digoxin, has long been used in the treatment of heart failure, but was not known to modulate splicing. The positive compounds target different signal transduction pathways, and microarray analysis shows that each compound affects the splicing of a different set of exons in addition to MAPT exon 10. Our results identify currently prescribed cardiotonic steroids as modulators of alternative splicing and demonstrate the feasibility of screening for drugs that alter exon inclusion.

Fig. 1.

Reporter design and testing. (A) Schematic of the reporter design used here, which accommodates exons without translation initiation codons and without a 3′-terminal adenosine. The initiation codon for GFP is split between the first and third exons of the reporter. This split results in GFP synthesis when the alternative exon is skipped and RFP synthesis when it is included. (B) Fluorescence microscopy of cells expressing three constructs, each with a different exon inclusion level. The exposures for the GFP and RFP fluorescence were constant across the three images. (Insets) The exon inclusion levels as detected by RT-PCR with the exon-included (+) and the exon-skipped (−) isoforms labeled.

Fig. 2.

Dynamic range of the assay. (A) The MAPT exon 10 reporter was compared to two single-intron reporters designed to produce only GFP (pFlare 5A) or RFP (pFlare 5G). (B) Bar plot showing the RFP/GFP ratio for the three reporters. The fluorescence intensity for the two proteins was measured on cell lysates from transient transfections and corrected for background fluorescence as described in SI Materials and Methods.

Fig. 3.

Biomol library screen. LOWESS-smoothed scatter plots of the log-ratio vs. the log-expression for each compound (orange dots) and the DMSO controls (blue dots). Compounds that change splicing change the log-ratio of the RFP/GFP fluorescence intensities. Compounds that affect overall gene expression change the log-expression but not the log-ratio.

Fig. 4.

Dose–response curves. Cells carrying the MAPT exon 10 reporter were treated with increasing amounts of digoxin, tyrphostin-9, and 5-iodotubercidin. (A) Effect of the drugs on the RFP/GFP ratio. (B) Effect of the drugs on the splicing of the MAPT exon 10 reporter relative to the DMSO control as determined by RT/PCR. Tyrphostin-9 caused increased exon inclusion at concentrations >400 nM, 5-iodotubercidin caused increased exon inclusion at concentrations between 800 nM and 1.6 μM and exon skipping at concentrations >3 μM, and digoxin caused increased exon inclusion at concentrations >50 nM.

Fig. 5.

Digoxin affects the use of downstream translation initiation codons. (A) Schematic diagrams of the two single-intron reporters used to test the effect of the drugs on the translation efficiency. MAPT ΔI2 was derived from the MAPT exon 10 reporter by deleting the second intron. It produces exon 10 containing mRNA equivalent to that generated by the MAPT exon 10 reporter and will result exclusively in RFP expression. The pFlare5A reporter produces exclusively mRNA equivalent to the exon-skipped isoform from the MAPT exon 10 reporter. (B) Dose–response curves of the RFP/GFP fluorescence in cells transiently cotransfected with the two reporters and treated with increasing amounts of digoxin, tyrphostin-9, and 5-iodotubercidin for 16–20 h.

Fig. 6.

Effect of digoxin, tyrphostin-9, and 5-iodotubercidin on the splicing of the endogenous MAPT exon 10. Illustrated is RT-PCR showing the splicing of the endogenous MAPT exon 10 in SHSY-5Y cells. (A) Tyrphostin-9 (1.6 μM) and digoxin (100 nM) cause increased exon 10 inclusion in the mature transcript, whereas 5-iodotubercidin (1.6 μM) results in increased exon skipping compared with the DMSO controls. (B) Quantification of the splice variants shown in A.