Missplicing downregulates expression of SF3B1 target genes. (A) Identification of erythroid-specific mutant SF3B1 targets based on the level of missplicing vs fold change of transcript expression during normal erythroid differentiation. Selected genes were misspliced in both SF3B1-mutant MDS patient cells and iPSC 5F-HPC derived erythroid cells. (B) Total level of missplicing of TMEM14C, PPOX, ABCB7, and MAP3K7 in normal bone marrow (BM), SF3B1-WT and mutant MDS patients, 5F-HPCs, and K562s. *P < .5, **P < .01, ***P < .001, ****P < .0001, 1-sided Mann-Whitney U test. 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 (C) The change in RNA expression (log fold) by RNA-seq in SF3B1-mutant compared with isogenic WT 5F-HPC-derived erythroid cells. (D) The efficiency of LUC translation in reporter assay with WT or mutant TMEM14C 5′UTR. Left: The schematic of WT and mutant TMEM14C 5′UTR and dual LUC 5′UTR reporter design. Right: The ratio of nano-LUC to firefly LUC fluorescence with WT or mutant TMEM14C 5′UTR. N = 6 independent experiments, *P = .015, Student t test. (E) Western blot analysis of TMEM14C, PPOX, MAP3K7, and ABCB7 protein levels at day 14 of erythroid differentiation of SF3B1-mutant and isogenic WT 5F-HPC derived erythroid cells. The expression was normalized to glyceraldehyde-3-phosphate dehydrogenase and shown as fold-change of SF3B1-mutant vs WT; 3 independent experiments, mean ± SD.