Fig 2: Transferrin receptor two is a Negative Regulator of Human Erythropoiesis.(A) Quantitative RT-PCR and (B) Western blot showing the expression of TFR2 in human CD34+ cells five days post-infection with the respective lentiviral shRNAs targeting TFR2 (TFR2 sh1 and sh2) and a control luciferase gene (shLUC). (C) Representative FACS plots of erythroid cell surface markers CD71 (transferrin receptor) and CD235a (Glycophorin A) expression at various time points during erythroid differentiation. Percentages in each quadrant are represented as mean and standard deviation of 3 independent experiments (D) Hoechst staining showing more enucleated cells after TFR2 knockdown at day 21 of erythroid culture. (E) Representative histogram plots showing increased expression of CD235a (Glycophorin A) after TFR2 knockdown (F) Enhanced pSTAT5 response after TFR2 knockdown in UT7/EPO cells.

Fig 3: Defects in diverse ribosomal proteins and eukaryotic initiation factors impair GATA1 translation(a, b, c) Western blots are shown from cells obtained 5 days following infection with shRNAs targeting RPL11, RPL5, and RPS24. Arrowheads indicate the major protein forms of GATA1 full length and GATA1 short.(d) Protein levels after increased concentrations of eIF4E-eIF4G interaction inhibitor 4EGI-1 are used to treat K562 erythroid cells (48 hours of treatment). Arrowheads indicate the major protein forms of GATA1 full length and GATA1 short.(e) Erythropoiesis (as measured by CD235a+ cells) and myeloid cell production (as measured by CD11b+ or CD41a+ cells) following treatment of primary hematopoietic cells with 4EGI-1 (48 hours of treatment).(f) Protein levels after increased concentrations of 4EGI-1 are used to treat CD34-derived primary erythroid cells (48 hours of treatment). Arrowheads indicate the major protein forms of GATA1 full length and GATA1 short.

Fig 4: Global disruption of GATA1 transcriptional activity in DBA patients and rescue of ribosomal protein haploinsufficiency with exogenous GATA1(a) Mean gene expression values of DBA and control patient samples are plotted on a scatter plot with a linear regression shown in red. The coefficient of determination is shown.(b) Relative GATA1 mRNA levels are plotted from DBA and control samples.(c, d) Enrichment profiles from GSEA comparing the relative expression of genes in DBA patients versus controls and examining the distribution of GATA1 target genes in this data (shown as black bars).(e) Representative FACS plots on transduction of bone marrow mononuclear cells from a DBA patient with GATA1 or HMD (empty) control lentiviruses.(f) The ratio of erythroid (CD235a+) to non-erythroid (CD235a-) -cells is shown after transduction of GATA1 for three independent DBA patients. The data are shown as the mean ± the standard deviation (n = 3). (** p < 0.01; *** p < 0.001).(g) Representative FACS forward scatter histogram plots (measuring cell size) of cultured DBA patient cells transduced with control or GATA1 lentivirus. The mean forward scatter intensity is shown ± the standard deviation (n = 3).(h) Representative cytospin images of cultured DBA patient cells transduced with control or GATA1 lentivirus. Transduced cells were sorted based on GFP expression and stained with May-Grünwald-Giemsa staining. Scale bar = 10 µm.(i) Gene expression analysis by RT-qPCR in DBA patient cells transduced with GATA1 relative to the empty vector control. The data are shown as the mean ± the standard deviation (for 3 independent samples). (Normalized to ß-actin; *** p < 0.001)

Fig 5: SF3A2 is a Key regulator of Human Erythropoiesis and Modulates Erythropoiesis Defects in a Murine Model of MDS.(A) Quantitative RT-PCR and (B) Western blot showing the expression of SF3A2 in human CD34+ cells five days post-infection with the respective lentiviral shRNAs targeting SF3A2 (sh1-4) and a control luciferase gene (shLUC). (C) Growth curves showing that downregulation of SF3A2 results in reduced total cell numbers during erythroid differentiation from three independent experiments. (D) Representative FACS plots of erythroid cell surface markers CD71 (transferrin receptor) and CD235a (Glycophorin A) expression at various time points during erythroid differentiation. Percentages in each quadrant are represented as mean and standard deviation of three independent experiments (E) Altered splicing events identified by RNA-Seq analysis of stage matched erythroid cells (shSF3A2 vs. shLUC). Overlapping changes observed in SF3B1 mutant BM cells from MDS patients (Obeng et al) (Figure 6—source data 5 and 6). Differentially expressed genes and pathway analysis available in Figure 6—source data 1–4. (F) Lineage negative bone marrow cells from wildtype (WT) and Sf3b1K700E mice were infected with shRNAs targeting murine Sf3a2 gene co-expressing a reporter GFP gene. Percentage of Ter119+ CD71+ erythroid cells within the GFP compartment after 48 hr in erythroid differentiation. (G) Total cell numbers of GFP+ erythroid cells after 48 hr in erythroid differentiation.10.7554/eLife.44080.022Figure 6—source data 1.Table containing the DESeq2 output for differentially expressed genes in cells undergoing SF3A2 knockdown or control shRNA treatment.10.7554/eLife.44080.023Figure 6—source data 2.Table containing the DESeq2 output for differentially expressed genes in MDS patients with and without mutations in SF3B1.10.7554/eLife.44080.024Figure 6—source data 3.Tables containing the GO component (Table 1) and function (Table 2) enrichments calculated using GOrilla for cells undergoing SF3A2 knockdown or control shRNA treatment.10.7554/eLife.44080.025Figure 6—source data 4.Tables containing the GO component (Table 1) and function (Table 2) enrichments calculated using GOrilla for MDS patient samples with and without mutations in SF3B1.10.7554/eLife.44080.026Figure 6—source data 5.Tables containing the differential splicing analysis for cells undergoing SF3A2 knockdown or control shRNA treatment.Categories of splice mutations presented in each table are alternative 3’ splice sites, alternative 5’ splice sites, mutually exclusive exons, retrained introns, and skipped exons, respectively.10.7554/eLife.44080.027Figure 6—source data 6.Tables containing the differential splicing analysis for MDS patient patient samples with and without mutations in SF3B1.Categories of splice mutations presented in each table are alternative 3’ splice sites, alternative 5’ splice sites, mutually exclusive exons, retrained introns, and skipped exons, respectively.