Loss of METTL3 Mediated m6A RNA Modification Results in Double-Stranded RNA Induced Innate Immune Response and Hematopoietic Failure

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 naive 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 (CUTR 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.