Methyltransferase complex subunit METTL3 maintains genome stability of erythroid cells via MTHFD1-mediated nucleotide biosynthesis
Linlin Zhang, Huizhi Zhao, Shihui Wang, Xueting Wu, Donghao Liu, Hengchao Zhang, Qianqian Yang, Ying Cheng, Xiuyun Wu, Jiangwei Zhao, Shijie Zhang, Huan Zhang, Haojian Zhang, Qiaozhen Kang, Lixiang Chen, Xiuli An, Xiaoli Qu
Linlin Zhang, Huizhi Zhao, Shihui Wang, Xueting Wu, Donghao Liu, Hengchao Zhang, Qianqian Yang, Ying Cheng, Xiuyun Wu, Jiangwei Zhao, Shijie Zhang, Huan Zhang, Haojian Zhang, Qiaozhen Kang, Lixiang Chen, Xiuli An, Xiaoli Qu
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Research Article Cell biology Hematology

Methyltransferase complex subunit METTL3 maintains genome stability of erythroid cells via MTHFD1-mediated nucleotide biosynthesis

Abstract

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, its functional role in erythropoiesis remains poorly understood. In this study, we found that deletion of Mettl3 using the 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 colony-forming unit–erythroid cells and cell-cycle arrest of erythroblasts. Integrated m6A-seq and RNA-seq analysis along with biochemical studies identified Mthfd1, a key enzyme involved in nucleotide biosynthesis, as a Mettl3 direct target gene. Furthermore, deletion of Mettl3 led to decreased expression of Mthfd1, accompanied by a shortage of nucleotides deoxythymidine monophosphate and inosine monophosphate, in erythroid cells. Additionally, inhibition of METTL3 in human erythroid cells led to similar phenotypic and molecular changes, indicating a conserved role of METTL3 in human and murine erythropoiesis. Our findings have identified an 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 important insights into the regulatory mechanisms of erythropoiesis and may have implications for underlying the mechanisms of anemias.

Authors

Linlin Zhang, Huizhi Zhao, Shihui Wang, Xueting Wu, Donghao Liu, Hengchao Zhang, Qianqian Yang, Ying Cheng, Xiuyun Wu, Jiangwei Zhao, Shijie Zhang, Huan Zhang, Haojian Zhang, Qiaozhen Kang, Lixiang Chen, Xiuli An, Xiaoli Qu

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Figure 5

Integrated SLIM-seq/RNA-seq identified Mthfd1 as a Mettl3 target regulating erythroid nucleotide biosynthesis.

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Integrated SLIM-seq/RNA-seq identified Mthfd1 as a Mettl3 target regulat...
(A) Integrated analysis of downregulated m6A-modified mRNAs in control and Mettl3-deficient CFU-EKit-lo cells. (B) GO enrichment analysis of the 129 overlapping genes from (A). (C) Significance ranking of DEGs in purine-containing compound biosynthetic processes. (D) Expression levels of Mthfd1 in BM CFU-E cells from RNA-seq by TPM (n = 3/group). (E) Expression levels of Mthfd1 in BM ProEs from RNA-seq by TPM (n = 3/group). (F) Integrative Genomics Viewer snapshots of SLIM-seq read coverage for the Mthfd1 transcriptome in control CFU-E cells, comparing Input and IP groups. (G) Measurement of m6A modification levels on Mthfd1 in control and Mettl3-deficient CFU-E cells by MeRIP-qPCR (n = 3/group). (H) Schematic representation of mRNA half-life measurements in actinomycin D–treated erythroid progenitor CFU-E cells. (I) mRNA half-life of Mthfd1 in sorted CFU-E cells, measured after actinomycin D treatment at 0, 1, 2, and 4 hours. (J) Dual-luciferase reporter assay identifying METTL3-regulated Mthfd1 transcripts. The experiment used pMIR-firefly and TK–Renilla luciferase vectors, with WT and Mut sequences inserted at the 3′UTR of Fluc (n = 4/group). (K) Schematic diagram of MTHFD1-mediated biosynthesis pathway of dTMP and IMP. (L) Targeted liquid chromatography–tandem mass spectroscopy quantification of dTMP and IMP levels in erythroid cells (n = 3/group). Data are presented as the mean ± SD. Comparisons between 2 groups were performed using an unpaired 2-tailed Student’s t test. A 2-way ANOVA with Tukey’s post hoc test was used to calculate statistical significance among multiple groups. **P < 0.01, ***P < 0.001. FC, fold change; PS, penicillin-streptomycin; SCF, stem cell factor.