Stage-specific requirement for m⁶A RNA methylation during cardiac differentiation of pluripotent stem cells

Precise spatiotemporal control of gene expression patterns is critical for normal development. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with the ability of unlimited self-renewal and differentiation into any cell type, provide a unique tool for understanding the underlying mechanism of development and disease in a dish. N6-methyl-adenosine (m⁶A) modification is the most extensive internal mRNA modification, which regulates almost all aspects of mRNA metabolism and thus extensively participates in gene expression regulation. However, the role of m⁶A during cardiogenesis still needs to be fully elucidated. Here, we found that core components of m⁶A methyltransferase decreased during cardiomyocyte differentiation. Impeding m⁶A deposition, by either deleting the m⁶A methyltransferase Mettl3 or overexpressing m⁶A demethylase alkB homolog 5 (Alkbh5), at early stages of cardiac differentiation of mouse pluripotent stem cells, led to inhibition of cardiac gene activation and retardation of the outgrowth of embryoid bodies, whereas interfering m⁶A modification at later stages of differentiation had minimal effects. Consistently, stage-specific inhibition of METTL3 with METTL3 inhibitor STM2457 during human ESCs (hESCs) cardiac differentiation demonstrated a similarly pivotal role of METTL3 for the induction of mesodermal cells while dispensable function for later stages. In summary, our study reveals a stage-specific requirement of m⁶A on the cardiac differentiation of pluripotent stem cells and demonstrates that precise tuning of m⁶A level is critical for cardiac differentiation.