Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling
Fig 1
Dual luciferase splicing reporter assay (DLR assay) to identify splicing modulators.
(a) Minigene containing titin PEVK exons 4–13. Boxes indicate exons, blue lines mature transcripts generated with and without RBM20. (b) PCR products of alternative transcripts produced from the PEVK minigene by RBM20. RBM20 increases the amount of transcripts lacking all alternative exons or only retaining alternative exon 5. (c) Quantitative PCR to relate inclusion of PEVK exon 8 to the constitutive exon 13 presented as percent spliced in (PSI). RBM20 reduces inclusion of exon 8 by ~70% (N = 3). (d) Dual luciferase splicing reporter with firefly luciferase (FLuc) integrated into exon 8 and renilla luciferase (RLuc) downstream of exon 13. The FLuc/RLuc ratio reflects the inclusion ratio of alternative exon 8. (e) RBM20 shifts alternative splicing of the dual luciferase reporter (DLR) construct to exclude all alternative exons. (f, g) Quantitative PCR (N = 3) and FLuc/RLuc activity (N = 8) produce similar readouts with increased sensitivity of the luciferase based assay. ***P<0.001 versus CTRL (Dunnett’s post-test). Data are presented as mean ±SD.