The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B

Abstract

Modulation of alternative pre-mRNA splicing is a potential approach to therapeutic targeting for a variety of human diseases. We investigated the mechanism by which digitoxin, a member of the cardiotonic steroid class of drugs, regulates alternative splicing. Transcriptome-wide analysis identified a large set of alternative splicing events that change after digitoxin treatment. Within and adjacent to these regulated exons, we identified enrichment of potential binding sites for the splicing factors SRp20 (SRSF3/SFRS3) and Tra2-β (SFRS10/TRA2B). We further find that both of these proteins are depleted from cells by digitoxin treatment. Characterization of SRp20 and Tra2-β splicing targets revealed that many, but not all, digitoxin-induced splicing changes can be attributed to the depletion of one or both of these factors. Re-expression of SRp20 or Tra2-β after digitoxin treatment restores normal splicing of their targets, indicating that the digitoxin effect is directly due to these factors. These results demonstrate that cardiotonic steroids, long prescribed in the clinical treatment of heart failure, have broad effects on the cellular transcriptome through these and likely other RNA binding proteins. The approach described here can be used to identify targets of other potential therapeutics that act as alternative splicing modulators.

FIGURE 1.

Digitoxin regulates a large set of alternative cassette exons that share potential binding sites for SRp20 and Tra2-β. (A) Representative RT-PCR gels for four high-confidence hits identified by HJAY. (B) Summary of exon inclusion levels for these exons across three independent experiments. Error bars, SEM exon inclusion (PSI). (C) For digitoxin repressed exons, Tra2-β binding sites are enriched. Plotted is mean rank of all tetramers that satisfy the given consensus binding sequence. The dashed line denotes the arithmetic mean tetramer rank of 128. Motifs with a significantly altered distribution of ranks (both high and low) are denoted by an asterisk and satisfy a Wilcoxon rank-sum P-value cutoff of 0.01. (D) The same analysis for digitoxin enhanced exons show an enrichment of SRp20 sites in the upstream intron.

FIGURE 2.

Digitoxin induces degradation of SRp20 and Tra2-β proteins. (A) Immunoblot analysis of digitoxin-treated HEK293 cells shows decreased expression of SRp20 and Tra2-β proteins. An unrelated splicing factor, PTB, remains unchanged. (B) Quantification of SRp20 and Tra2-β protein expression normalized to GAPDH and β-tubulin. Error bars, SE across three independent digitoxin treatments. (C) Immunoblot with pan-SR antibody reveals that other members of the SR family are largely unchanged after digitoxin treatment. (D) Co-incubation with MG-132 prevents depletion of SRp20 and Tra2-β and is quantified in E for three independent experiments. Error bars, SEM protein level.

FIGURE 3.

Regulation of digitoxin target exons by SRp20 and Tra2-β. (A) shRNA-mediated knockdown of SRp20 and Tra2-β in HEK293 cells. Number below each blot shows relative expression level normalized to GAPDH and β-tubulin. (B) OmniViewer analysis for changes in alternative cassette exons in the two knockdown conditions compared with control. (C) Representative RT-PCR gels for alternative splicing changes. (D) APP exon 8 is the second exon of a dual cassette exon pair with exon 7. It possesses a consensus SRp20 binding site and is repressed by both digitoxin and SRp20 knockdown, but not Tra2-β knockdown. (E) RIPK2 exon 2 contains two potential Tra2-β binding sites and is repressed by digitoxin treatment and Tra2-β knockdown, but is unaffected by SRp20 knockdown. (F) ZNF207 exon 9 contains two potential SRp20 binding sites and one potential Tra2-β binding site and is repressed by digitoxin and both knockdowns.

FIGURE 4.

SRp20 and Tra2-β overexpression reverses digitoxin effect on coregulated alternative exons. (A) Digitoxin-induced repression of ZNF207 exon 9 is reversed by SRp20 expression. (B) RIPK2 exon 2 repression is rescued by Tra2-β expression. (C,D) Digitoxin induced repression of APP exon 8 is rescued by expression of exogenous SRp20, while the same overexpression reduced digitoxin-induced enhancement of FN1 exon 33. (E,F) Flag-tagged overexpression of SRp20 and Tra2-β in HEK293 cells, respectively.