a, Scatter plots comparing differential splicing (ΔPSI) between patients in the TCGA UVM cohort with SF3B1 mutations or wild-type SF3B159 (x axis) and patients from a myelodysplastic syndromes cohort with SF3B1 mutations or wild-type SF3B127 (y axis). Events were classified as alternative 3′ splice sites or skipped exons. b, Rank plot for the −log 10 (FDR) associated with each sgRNA in our CRISPR–Cas9 positive-selection screen. sgRNAs targeting the positive control (Pten) and Brd9 are highlighted. For the probe-level (per-sgRNA) analysis, we fitted a negative binomial generalized log-linear model and performed a likelihood ratio test. FDR values were computed using the Benjamini–Hochberg method. c, Read counts for sgRNAs targeting the positive control (Pten) or Brd9. D0 and D7 indicate days following withdrawal of IL-3. d, Heat map summarizing the results of a competition assay to measure the effect of each indicated sgRNA on the growth of Cas9-expressing Ba/F3 cells. Cell growth was computed with respect to cells treated with a non-targeting (control) sgRNA and the percentages of GFP+ cells on day 14 were normalized to the percentages on day 2. The illustrated values correspond to the mean computed value over n = 3 biological replicates. Rpa3 sgRNAs were used as a negative control. e, As in d, but for 32Dcl3 cells. f, As in d, but for the indicated melanoma and pancreatic ductal adenocarcinoma cells. g, As in d, but for RN2 cells. h, Sequence conservation of the BRD9 poison exon locus as estimated by phyloP60. Conservation and repetitive element annotation is from the UCSC Genome Browser43. i, RT–PCR analysis of Brd9 poison exon inclusion using whole bone marrow cells from Mx1-cre Sf3b1WT/WT (WT/WT) and Mx1-cre Sf3b1K700E/WT (K700E/WT) mice. Three weeks after pIpC treatment, RT–PCR was performed with mouse primers corresponding to those used to assay BRD9 poison exon inclusion in human cells. Representative images from n = 2 technically independent replicates. j, RT–PCR analysis to confirm mutant-SF3B1-dependent inclusion of the BRD9 poison exon in isogenic NALM-6 cell lines engineered to contain the indicated mutations. SF3B1K700K is a wild-type control for genome engineering. Representative images from n = 2 technically independent replicates. k, Western blot for Flag, SF3B1 and BRD9 in K562 cells overexpressing N-terminally Flag-tagged wild-type SF3B1 or SF3B1K700E cDNAs, or an empty vector; this panel corresponds to the cells evaluated in m. Representative images from n = 2 biologically independent replicates. l, Western blot for SF3B1 in K562 cells treated with doxycycline-inducible SF3B1-targeting shRNAs or a non-targeting control shRNA (shRen); this panel corresponds to cells evaluated in m. Representative images from n = 2 technically independent replicates. m, RT–PCR illustrating the specificity of BRD9 poison exon inclusion for SF3B1-mutated cells in the indicated cell lines. These include K562 cells treated with control shRNA (shRen) or SF3B1-targeting shRNAs (the columns labelled ‘K562 knock-down’); knock-in of the SF3B1K700K, SF3B1K700E or SF3B1K666N mutation into the endogenous locus of SF3B1 (the columns labelled ‘K562 knock-in’); or overexpression of wild-type SF3B1 or SF3B1K700E cDNA (the columns labelled ‘K562 cDNA’). The two right-most lanes show acute myeloid leukaemia cell lines with wild-type SF3B1 (MV4;11) or a naturally occurring endogenous SF3B1K700E mutation (HNT34 cells; the columns labelled ‘leukaemia cell lines’). Representative images from n = 3 biologically independent experiments. n, As in m, but for the indicated pancreatic ductal adenocarcinoma cell lines (left), UVM cell lines (centre) and a cohort of patients with chronic lymphocytic leukaemia (right). CFPAC1 and MIA PaCa2 cells lack SF3B1 mutations; Panc05;04 cells carry SF3B1Q699H/K700E; UPMD1 and MEL270 cells lack SF3B1 mutations; MEL202 and UPMD2 cells carry SF3B1R625G and SF3B1Y623H mutations, respectively. Sample identifiers for patients with chronic lymphocytic leukaemia correspond to the genotypes shown in Supplementary Table 7. Representative images from n = 2 technically independent experiments (left and centre) and n = 3 biologically independent experiments (right). o, RNA-seq read coverage plots of the BRD9 poison exon locus from patient samples with the indicated SF3B1 genotypes. All SF3B1-mutated samples exhibit BRD9 poison exon inclusion. p, As in o, but for the indicated tissues from healthy donors (from BodyMap 2.0). q, qRT–PCR measurement of the half-lives of the poison exon inclusion (left) and exclusion (right) isoforms in isogenic K562 SF3B1K700E cells treated with the indicated shRNAs and actinomycin D to inhibit transcription. NMD inhibition via UPF1 knockdown stabilizes the inclusion, but not exclusion, isoform. Red arrows indicate primers used to specifically detect the two isoforms. n = 2 biologically independent experiments and n = 2 technically independent experiments for the inclusion isoform; n = 3 technically independent experiments for the exclusion isoform. P value was calculated by two-sided t-test at 8 h. r, Bar graph illustrating the estimated poison exon inclusion isoform half-life in the indicated conditions from the data in q. Error bars, mean + s.d. n = 2 biologically independent experiments and n = 2 technically independent experiments. P value was calculated by two-sided t-test. s, As in r, but for the exclusion isoform. Error bars, mean + s.d. n = 3 technically independent experiments. P value was calculated by two-sided t-test. t, As in q, but for NALM-6 SF3B1K700E cells. n = 3 technically independent experiments for the inclusion isoform and the exclusion isoform. P value was calculated by two-sided t-test at 8 h. u, As in r, but for NALM-6 SF3B1K700E cells. n = 3 technically independent experiments. Error bars, mean + s.d. P value was calculated by two-sided t-test. v, As in s, but for NALM-6 SF3B1K700E cells. Error bars, mean + s.d. n = 3 technically independent experiments. P value was calculated by two-sided t-test. w, Western blot for BRD9 in NALM-6 cells with or without knock-in of an SF3B1 mutation. Actin, loading control. Representative images from n = 3 biologically independent experiments. x, Rank plot of BRD9 poison exon inclusion (scale of 0 to 1; top) and box plot of gene expression (inset) for patients stratified by SF3B1 mutational status (data are from cohorts of patients with myelodysplastic syndromes or UVM, as in Fig. 1a). SF3B1 mutations were strongly associated with high poison exon inclusion and low BRD9 expression. Boxes illustrate 1st and 3rd quartiles, with whiskers extending to 1.5× interquartile range. P value computed with one-sided Mann–Whitney U-test. Source data