The RNA repertoire of Srsf3-null MKs reflects the failure in activating the maturation program. (A) Volcano plots depicting differentially expressed genes (DEGs) between control 8N and ≥16N MKs. The data points marked with blue and red denote significantly down- and upregulated genes (FDR <0.05 and FC >2), respectively. (B) Volcano plots depicting DEGs between Pf4-Srsf3^Δ/Δ 8N and ≥16N MKs as in panel A. (C) Venn diagram comparing DEGs in control and Pf4-Srsf3^Δ/Δ MKs upon 8N to ≥16N transition. (D) Volcano plot depicting DEGs between control and Pf4-Srsf3^Δ/Δ ≥16N MKs as in panel A. (E) Expression of genes encoding proteins central for MK structure and function in control and Pf4-Srsf3^Δ/Δ ≥6N MKs. FDR <0.05 unless otherwise noted. (F) Significantly enriched GO terms (Biological Process) among DEGs between control and Pf4-Srsf3^Δ/Δ ≥16N MKs (FDR <0.05, FC >2). The x-axis depicts percent genes and -log₁₀ (FDR) of each category. (G) Percentage of RNAs with SRSF3 RNA binding sites (iCLIP peaks) as identified in mouse pluripotent stem cells³³ within RNAs induced during MK maturation (left) or differentially expressed between control and Pf4-Srsf3^Δ/Δ ≥16N MKs (right). (H) RNA immunoprecipitation (IP) using anti-GFP antibody in MEG-01 megakaryoblast cell lines expressing SRSF3-GFP or only GFP. The y-axis denotes the enrichment of SRSF3 RNA binding over input as measured by RT-qPCR (n = 3). Neg. is a nontarget and SRSF3 a known-target control to demonstrate the specificity of the RNA-IP. (I) Quantification of RNAs in the nuclear and cytoplasmic fractions of MEG-01 cells following SRSF3 deletion by CRISPR/Cas9 gene editing. The data are presented as a ratio of nuclear and cytoplasmic mRNA abundance (n = 3). *P ≤ .05. Ctrl, MEG-01 cells targeted with scrambled control guide RNA; FC, fold change; FDR, false discovery rate; KO, MEG-01 cells targeted with SRSF3 guide RNA; ns, not significant.