Mutant SF3B1 missplicing during iPSC erythroid differentiation. (A) Relative expression of the indicated differentially spliced isoforms in SF3B1-WT and SF3B1-mutant cells. Plot restricted to competing 3′ splice sites, cassette exons, and retained introns. Normal samples include: bone marrow HPCs (BM), MDS without SF3B1 mutations (WT), K562 cells, and normal isogenic iPSC-derived 5F-HPCs. SF3B1-mutant samples include: SF3B1-mutant MDS, K562 SF3B1^K700E/+ cells, and SF3B1^G742D/+ iPSC-derived 5F-HPCs. Events were restricted to ≥20% missplicing, Bayes factor ≥5, and a minimum counts cutoff ≥20. MDS-D refers to SF3B1^K700E/+ patient data from Dolatshad et al⁴ and MDS-T refers to SF3B1^K700E/+ patient data from Taylor et al.²⁵ (B) The proportion of misspliced isoforms in 5F-HPCs: progenitor CD34⁺, proerythroblast CD71⁺GlyA⁻, and erythroblast CD71⁺GlyA⁺. Splicing events classified as arising from tandem 3′ UTRs (tutr), cassette or skipped exons (se), retained introns (ri), mutually exclusive exons (mxe), alternative usage of normally constitutively spliced junctions (cj), alternative retention of normally constitutively spliced introns (ci), alternative 5′ss (a5ss), and alternative 3′ss (a3ss). (C) The number of misspliced isoforms grouped by absolute value of missplicing at the 3 stages of erythroid differentiation of 5F-HPCs: progenitor CD34⁺, proerythroblast CD71⁺GlyA⁻, and erythroblast CD71⁺GlyA⁺. (D) Pearson correlation matrix of the level of missplicing between the 3 stages of erythroid differentiation of SF3B1^G742D/+ 5F-HPCs. ****P < .0001. (E) The level of missplicing of individual gene isoforms during erythroid differentiation of SF3B1^G742D/+ 5F-HPCs; included isoforms were detected at all the stages of differentiation. (F) Top 20 misspliced a3ss isoforms in SF3B1-mutant 5F-HPCs and 3 stages of erythroid cells: CD71⁺ proerythroblasts, CD71⁺GlyA⁺ erythroblasts, and K562s. Unless otherwise stated, for all panels, differential missplicing between SF3B1-mutant and WT cells is defined based on the absolute value of missplicing ≥20%, Bayes factor >5, and supported by >5 transcript counts. Prog, progenitor; Pro-ery, proerythroblast; Ery, erythroblast.