Mechanistic insights into vascular biology via methyltransferase-like 3-driven N ⁶-adenosine methylation of RNA

N6-methyladenosine (m6A) has been identified as the most abundant modification on eukaryote messenger RNA (mRNA). Although the rapid development of high-throughput sequencing technologies has enabled insight into the biological functions of m6A modification, the function of m6A during vertebrate embryogenesis remains poorly understood. Here we show that m6A determines cell fate during the endothelial-to-haematopoietic transition (EHT) to specify the earliest haematopoietic stem/progenitor cells (HSPCs) during zebrafish embryogenesis. m6A-specific methylated RNA immunoprecipitation combined with high-throughput sequencing (MeRIP–seq) and m6A individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing (miCLIP–seq) analyses reveal conserved features on zebrafish m6A methylome and preferential distribution of m6A peaks near the stop codon with a consensus RRACH motif. In mettl3-deficient embryos, levels of m6A are significantly decreased and emergence of HSPCs is blocked. Mechanistically, we identify that the delayed YTHDF2-mediated mRNA decay of the arterial endothelial genes notch1a and rhoca contributes to this deleterious effect. The continuous activation of Notch signalling in arterial endothelial cells of mettl3-deficient embryos blocks EHT, thereby repressing the generation of the earliest HSPCs. Furthermore, knockdown of Mettl3 in mice confers a similar phenotype. Collectively, our findings demonstrate the critical function of m6A modification in the fate determination of HSPCs during vertebrate embryogenesis. Show more

Haematopoietic stem cells (HSCs) maintain balanced self-renewal and differentiation, but how these functions are precisely regulated is not fully understood. N6-methyladenosine (m6A) mRNA methylation has emerged as an important mode of epitranscriptional gene expression regulation affecting many biological processes. We show that deleting the m6A methyltransferase, Mettl3, from the adult haematopoietic system led to an accumulation of HSCs in the bone marrow and marked reduction of reconstitution potential due to a blockage of HSC differentiation. Interestingly, deleting Mettl3 from myeloid cells using Lysm-cre did not impact myeloid cell number or function. m6A sequencing revealed 2,073 genes with significant m6A modification in HSCs. Myc was identified as a direct target of m6A in HSCs. Mettl3-deficient HSCs failed to up-regulate MYC expression upon stimulation to differentiate and enforced expression of Myc rescued differentiation defects of Mettl3-deficient HSCs. Our results revealed a key role of m6A in governing HSC differentiation. Show more