Mettl3 maintains genome stability of erythroid cells via Mthfd1-mediated nucleotide biosynthesis

N6-methyladenosine (m6A) is a prevalent modification of mammalian mRNA. Increasing evidence has documented diverse roles of m6A in normal cell physiology and diseases. However, our knowledge on the role of the m6A in erythropoiesis is very limited. A previous study implicated the role of METTL3, the catalytic enzyme for m6A modification, in human erythropoiesis in vitro. But the function role of METTL3 in vivo has yet to investigated. In this study, we found that deletion of Mettl3 using EpoR-Cre mouse led to microcytic/hypochromic anemia due to defective erythropoiesis along with impaired hemoglobin biosynthesis. Mechanically, Mettl3 deficiency disrupted nucleotide biosynthesis which induced DNA damage, leading to apoptosis of CFU-E cells and cell cycle arrest of erythroblasts. Integrated m6A sequencing and RNA-seq analysis along with biochemical studies identified Mthdf1, a key enzyme involved in nucleotide biosynthesis, as a Mettl3 direct target gene. Furthermore, deletion of Mettl3 led to decreased expression of Mthdf1 accompanied by a shortage of nucleotides dTMP and IMP in erythroid cells. Additionally, inhibition of METTL3 in human erythroid cells led to similar phenotypic and molecular changes, indicating conserved role of METTL3 in human and murine erythropoiesis. Our findings have identified a novel Mettl3-m6A-Mthfd1 axis that plays a critical role in erythropoiesis by maintaining genome stability of erythroid cells via regulation of nucleotide biosynthesis. These findings provide novel insights into the regulatory mechanisms of erythropoiesis.