Hematopoietic stem cell (HSC) self-renewal and lineage output are orchestrated by multiple regulatory layers, including RNA modifications. N6-methyladenosine (m6A) is an abundant modification found in RNAs which affects the translation and stability of modified transcripts. The effects of m6A are determined by m6A writers (install m6A), erasers (remove m6A) and readers (recognize m6A). In embryonic stem cells, deletion of the m6A writer METTL3 enforces a naïve pluripotent state. This raises the question of whether m6A RNA methylation analogously regulates stem cell self-renewal and differentiation in somatic stem cells such as HSCs.
Using a Vav-Cre/Mettl3 (VCM3) hematopoietic-specific knockout mouse model, we show that loss of the RNA m6A writer METTL3 in fetal HSCs results in hematopoietic failure and perinatal lethality. At E14.5 (FL) hematopoiesis loss of Mettl3/m6A results in hematopoietic failure with expansion of Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) that are defective in the production of progenitors and mature blood cells, as evidenced by failure to rescue lethally irradiated congenic recipient mice in transplant experiments. The relative defect I hematopoiesis was further demonstrated by competitive transplant experiments, in which transplanted KO FL were consistently out-competed by WT FL. Interestingly, BrdU/7AAD labeling reveals a significant proliferative defect with reduced BrdU uptake in VCM3 KO FL cells, and specifically in Lin-c-Kit+Sca-1- (LK) progenitor cells.
RNA-seq analysis of FL LSK cells reveals that loss of m6A results in upregulation of multiple 2'-5'-oligoadenylate synthetase (OAS) family genes. Interestingly, the majority of OAS family genes are not m6A modified in several m6A sequencing data sets. We therefore hypothesized that the OAS genes might be regulated at the transcriptional level. We performed cleavage under targets and release using nuclease (CUT&RUN) analysis and found that OAS family genes are transcriptionally activated, as evidenced by significant increase in H3K4 trimethylation (H3K4me3) at their respective promoter regions.
The OAS family genes are activated by the presence of double stranded RNA (dsRNA), which can arise either endogenously or as a pathogen-associated trigger of the innate immune system in the context of viral infection. The dsRNA response includes three major response mechanisms. First, OAS genes facilitate RNase L dimerization, which mediates cleavage of cellular tRNA and rRNA, resulting in translation and proliferation arrest. Second, activation of the PKR/eIF2a pathway also results in translation arrest. Lastly, activation of the MDA-5/RIG-I/MAVS response induces interferon signaling, which further limits cellular proliferation.
Staining of VCM3 WT & KO FL cells with the dsRNA-specific J2 antibody directly demonstrated significant accumulation of dsRNA in KO FL cells. We further demonstrated activation of the OAS/RNaseL axis in KO cells by quantification of tRNA-His-36 cleavage, which acts as a sensitive marker of RNase L activity. Phosphorylation of both PKR and eIF2a in KO cells demonstrated enhanced activity of this pathway. Lastly, we found a significant upregulation of interferon pathway genes in KO cells, and demonstrated that CRISPR mediated knockout of Mavs in VCM3 KO FL cells diminishes the activity of interferon response genes and rescues the Mettl3 KO phenotype in colony forming unit (CFU) assays. These results suggest that in the absence of m6A, endogenous dsRNA formation is enhanced, resulting in an associated inflammatory response which partially accounts for the hematopoietic defect observed in Mettl3 KO mice.
In conclusion, our study suggests a novel, protective role for the m6A RNA modification in preventing endogenous dsRNA formation and aberrant activation of a detrimental innate immune response during mammalian hematopoietic development.
Fan:IsoPlexis: Other: served on the scientific discovery board, Patents & Royalties; Singleron Biotechnologies: Other: served on the Scientific Advisory Board; BioTechne: Other: served on the Scientific Advisory Board. Flavell:Rheos Biomedicines: Equity Ownership; GSK: Consultancy; Artizan Biosciences: Equity Ownership; Troy: Equity Ownership; SMOC: Equity Ownership; Zai labs: Consultancy.
Asterisk with author names denotes non-ASH members.