RNA editing of H19 shapes hematopoietic stem cell function and differentiation via cell cycle regulation

Abstract

Abstract Adenosine-to-inosine (A-to-I) RNA editing is a key post-transcriptional modification involved in immune response and cell fate determination. Our previous RNA editome analysis of hematopoietic stem and progenitor cells (HSPCs) identified H19 as a prominent editing target specifically enriched in hematopoietic stem cells (HSCs). H19 is a maternally imprinted long non-coding RNA known to regulate embryonic HSC development and maintain adult HSC quiescence; however, the role of H19 RNA editing in HSC biology remains undefined. We validated that this RNA editing event occurs in the first exon of H19, within the seed sequence of the embedded miRNA miR-675-3p, in mouse long-term HSCs (LT-HSCs). Analysis of public GEO datasets confirmed conservation between humans and mice and showed increased editing under pathological stress, including aging, viral infection, hypoxia-related disease, and interferon stimulation in vitro. Functionally, RNA editing enhanced H19 abundance, promoted miR-675-3p biogenesis, and altered its target repertoire. Absolute quantification revealed that the majority (87.9%) of miR-675-3p molecules in LT-HSCs are edited. Notably, edited miR-675-3p targeted a gene set distinct from its unedited isoform, indicating a shift in regulatory output. To characterize the developmental dynamics of this editing event, we profiled both H19 expression and RNA editing levels in HSCs from the fetal to adult stages. Single-cell RNA sequencing analyses revealed that HSCs with higher editing levels display downregulation of genes related to cell cycle regulation. Consistently, analyses using single-cell HSC datasets and Ki67-eGFP reporter mice confirmed that quiescent HSCs exhibit both elevated H19 expression and increased H19 RNA editing levels compared to cycling HSCs, suggesting a link between RNA editing and cell cycle regulation. To assess the in vivo function, we generated two mouse models with targeted mutations at the endogenous H19 locus: H19-Guanosine (G; fully edited mimic) and H19-Thymine (T; editing-resistant, 0% edited). Compared to H19-A (~50% edited) controls, H19-T mice exhibited reduced myeloid differentiation potential at 8 weeks of age. In competitive transplantation assays, the fully-edited H19-G mice showed a modestly higher engraftment rate at 16 weeks post-secondary transplantation (72.9% vs 55.3%) compared to H19-A mice, associated with an increased proportion of common myeloid progenitors. In contrast, the editing-resistant H19-T mice displayed impaired long-term engraftment (12.3% vs 55.3%), likely due to reduced progenitor cell output. Bulk RNA-seq of Lineage-c-Kit+Sca-1+ cells collected 4 months post-transplantation revealed upregulated genes associated with immune responses, and downregulated genes enriched in translation-related terms. Given that HSC maintenance may be mediated through the combined effects of the edited H19 transcript and its downstream edited miR-675-3p, we performed RNA pulldown assays using different H19 variants to compare the proteins bound by H19-G versus H19-A, and predicted the target genes of the edited versus non-edited isoforms of miR-675. These analyses identified a distinct protein interaction profile and downstream targets implicated in HSC self-renewal and differentiation. Together, our findings indicate that RNA editing of H19 is indispensable for HSC maintenance. This study reveals a novel RNA editing–dependent mechanism governing HSC quiescence and self-renewal across development and stress conditions.