APOBEC3C rewires RNA splicing and self-renewal in hematopoietic stem and progenitor cells

Introduction Inflammatory cytokine-responsive APOBEC3 cytosine deaminases promote antiviral defense through DNA and RNA editing and, when deregulated, drive somatic mutations and cancer progression. While APOBEC3A and APOBEC3B are well studied, the role of APOBEC3C remains less defined. However, our recent whole genome and whole transcriptome analyses of myeloproliferative neoplasm (MPN) derived hematopoietic stem and progenitor cells (HSPCs) revealed that APOBEC3C is highly expressed in this context, suggesting a context-specific function in hematopoiesis. This observation prompted us to investigate the effects of APOBEC3C, alongside other APOBEC family members, on HSPC biology, with an emphasis on their roles as drivers of clonal hematopoiesis (CH) and myeloid disorders in the context of aging and inflammation. Methods We collected healthy cord blood (CB) and aged bone marrow (ABM), and lentivirally transduced immunomagnetic bead-selected CD34 + cells with APOBEC3B, C, D, F, or G, or pCDH lentiviral backbone controls. Subsequently, we performed whole genome and transcriptome analyses and assessed C-to-T DNA mutations, C-to-U RNA edits and differential gene expression. As widespread changes in RNA splicing where observed as well as widespread changes in expression of genes implicated in CH upon lentiviral transduction of APOBEC3C, we also performed dual fluorescent splicing reporter assays, survival and self-renewal assays, and in vivo engraftment studies, respectively. Results We discovered that APOBEC3C and 3F, induce more DNA mutations, whereas APOBEC3B, 3C and 3G disrupt RNA splicing. We observed differential expression of genes crucial for hematopoiesis, such as ZRSR2, U2AF1, and SRSF2, as well as differential exon usage and enrichment of the spliceosome pathway. Additionally, we demonstrated that APOBEC3C induced substantially greater transcriptomic effects in ABM HSPCs compared to CB HSPCs. We observed a 10-fold increase in differential gene expression, including alterations in ribosomal genes (RPS19 and RPL5), alongside splicing factors (U2AF1, ZRSR2, and SF3B2) implicated in CH, MPN, and myelodysplastic syndrome (MDS) pathogenesis. Moreover, in ABM HSPCs, APOBEC3C was found to affect CH-associated transcripts, including DNMT3A, enhance ADAR1-mediated RNA editing, and increase self-renewal capacity. Consistent with these findings, APOBEC3C is elevated in JAK2 + MPN HSPCs, while its knockdown reduced JAK2⁺ MPN CD34⁺ self-renewal and ADAR1 reporter activity. Conclusion In conclusion, these findings demonstrate that APOBEC3C modulates RNA splicing and C-to-U RNA editing in CB derived HSPCs, with more pronounced effects in ABM derived HSPCs. In ABM, this also includes increased ADAR1p150 expression and elevated A-to-I editing, which combined with increased HSPC expansion, may contribute to the pathogenesis and progression of myeloid disorders.