Extended Data Fig. 4: Branchpoint manipulation and combinatorial 3’ss mutations enhance SF3B1 mutation-dependent splicing.
From: Synthetic introns enable splicing factor mutation-dependent targeting of cancer cells
(a) Diagrams of modifications in each intron relative to synMTERFD3i1-150. Deletions are specified as open intervals. Branchpoints were inserted in between the indicated positions. (b) Relative viability of K562 cells expressing the indicated constructs, measured in the full screen. Relative viability estimated as fold-change in representation of each construct, measured by full-length intron sequencing from genomic DNA, at day 8 for GCV-treated relative to untreated samples. GCV concentration, 100 ug/mL. Vector is hPGK-PuroR-P2A-HSV-TK. Data represented as mean ± s.d; s.d. estimated as sample proportion s.d. over counts. n = 6 replicates (n = 3 independent collections x n = 2 independent sequencing strategies). (c) Relative viability of K562 cells expressing the indicated constructs, measured in cells expressing each construct individually. Viability estimates from these single-construct experiments are concordant with estimates from parallelized screening in (b); note that fold-changes are greater in this experiment because of its longer duration (11 vs. 8 days). Relative viability measured by ATP after 11 days of treatment and normalized to PBS-treated samples. GCV concentration, 100 ug/mL. Vector is hPGK-PuroR-P2A-HSV-TK. Data represented as mean ± s.d. n = 3 biologically independent experiments. (d) RT-PCR demonstrating mutation-dependent excision of the synthetic intron in the experiments from (c). (e) RT-PCR demonstrating mutation-dependent excision of the synMTERFD3i1-150 synthetic intron or control synthetic introns in K562 cells with or without knockin of SF3B1K700E or SF3B1K666N, or MEL202 cells with an endogenous SF3B1R625G mutation. Experiment repeated independently n = 3 times with similar results. (f) RT-PCR of a series of synthetic introns in SF3B1R625G-mutant MEL202 cells. Experiment repeated independently n = 2 times with similar results. (g) RT-PCR of HSV-TK interrupted by synMTERFD3i1-150-v6700 or control synthetic introns in uveal melanoma cell lines that are wild-type or mutant for SF3B1. Experiment repeated independently n = 3 times with similar results. (h) Relative viability of the cells in (g) following treatment with 1 µg/mL GCV. Data represented as mean ± s.d. n = 3 biologically independent experiments. Uncropped gels are available as source data.
