N-methyladenosine modification-mediated mRNA metabolism is essential for human pancreatic lineage specification and islet organogenesis.

Pancreatic differentiation from human pluripotent stem cells (hPSCs) provides promising avenues for investigating development and treating diseases. N-methyladenosine (mA) is the most prevalent internal messenger RNA (mRNA) modification and plays pivotal roles in regulation of mRNA metabolism, while its functions remain elusive. Here, we profile the dynamic landscapes of mA transcriptome-wide during pancreatic differentiation. Next, we generate knockout hPSC lines of the major mA demethylase ALKBH5, and find that ALKBH5 plays significant regulatory roles in pancreatic organogenesis. Mechanistic studies reveal that ALKBH5 deficiency reduces the mRNA stability of key pancreatic transcription factors in an mA and YTHDF2-dependent manner. We further identify that ALKBH5 cofactor α-ketoglutarate can be applied to enhance differentiation. Collectively, our findings identify ALKBH5 as an essential regulator of pancreatic differentiation and highlight that mA modification-mediated mRNA metabolism presents an important layer of regulation during cell-fate specification and holds great potentials for translational applications.