Son maintains innate immune homeostasis in HSCs via splicing and retroelement control Free

Hematopoietic stem cells (HSC) maintain blood homeostasis during steady state and stress through a balanced regulation of self-renewal and differentiation. While transient activation of interferon stimulated gene (ISG) is critical for HSC function during development and stress, chronic inflammation impairs HSC integrity. However, the intrinsic mechanisms that suppress aberrant inflammatory signaling in HSCs remain elusive and further studies are necessary for restoring HSC function in settings such as aging and cancer.

We have identified a m⁶A-SON axis that governs HSC fate and controls inflammation (Cheng*, Luo* and Izzo* et al., 2019, Cell reports; Luo* and Lopez* et al., 2023, Cell Stem Cell). m⁶A is the most abundant RNA modification, and its loss leads to increased double-stranded RNA (dsRNA), ISG activation and HSC dysfunction. SON is a key m⁶A target, whose loss accounts for the observed defects. SON is a core component of the nuclear speckles that controls gene regulation through RNA processing. Importantly, individuals with heterozygous SON loss of function mutations (ZTTK syndrome), exhibit developmental delay and hematological symptoms including bone marrow failure. However, the molecular function of SON in HSCs and how it controls inflammation remains unclear.

To investigate SON function in vivo, we generated the Mx1-cre Son conditional knockout mice. SON loss resulted in a severe reduction in bone marrow cellularity (WT 168.2 million vs Son cKO 73.71 million, p<0.0001) and reduced platelet count (WT 408 k/ul vs Son cKO 74.5 k/ul, p<0.0001). SON loss also resulted in depletion of myeloid progenitors (WT 2.51million vs Son cKO 1.01 million, p=0.0405) and an expansion of phenotypic HSCs (WT 11.21k vs Son cKO 38.4k, p=0.0312), indicating an early differentiation block. Functionally, SON cKO HSCs failed to engraft recipient mice (WT 26.4% vs Son cKO 3.8%, p<0.000001), suggesting that SON is essential for HSC maintenance.

SON loss led to a 2-fold increase in dsRNA accumulation (p=0.0094), suggesting innate immune activation in SON cKO HSCs. Bulk RNA sequencing revealed 94 upregulated genes and 68 downregulated genes between SON cKO and WT HSCs (padj<0.05). Top enriched pathway among the upregulated genes upon SON loss is the interferon signaling (padj=8.46E-06) and RIG-I ligand pathways (padj=1.19E-11), indicating robust ISG induction. Major contributors to dsRNA accumulation include the derepression of endogenous retroelements (EREs) and aberrant RNA splicing (i.e retained introns). SON cKO hematopoietic stem and progenitor cells (HSPCs) exhibited increased ERE expression, including endogenous retroviruses (ERVs) and LINE elements (upregulated vs downregulated: 6 vs 0 in HSC; 72 vs 18 in MPP1; 108 vs 45 in MPP2; 70 vs 50 in MPP4, padj<0.05). We also observed widespread splicing abnormalities. In total, 6,330 splicing alterations were identified in SON-deficient HSCs: 50% intron retention, 25% exon skipping, and 25% alternative splice site usage (padj<0.05). Nearly 30% of upregulated genes, including numerous ISGs, exhibited differential splicing. To assess SON direct RNA binding targets, we performed SON CLIP-seq and identified 4337 genes as SON direct binding transcripts (padj<0.05). CLIP peak analysis showed that SON binds to preferentially to proximal introns and 3′ splice sites, with ~10% of ISG upregulation attributable to direct SON binding. These data suggest that ISG upregulation following SON loss may result both indirectly from dsRNA-mediated innate immune activation and directly from aberrant splicing of ISG transcripts.

Together, our finding highlights SON controls innate immune response in HSCs through splicing control of ISGs and repression of EREs. Notably, SON loss resulted in elevated levels of serum inflammatory cytokines, implicating HSC-intrinsic inflammation as a contributor to systemic inflammation.