mRNA m5C controls adipogenesis by promoting CDKN1A mRNA export and translation

5-Methylcytosine (m⁵C) is a type of RNA modification that exists in tRNAs and rRNAs and was recently found in mRNA. Although mRNA m⁵C modification has been reported to regulate diverse biological process, its function in adipogenesis remains unknown. Here, we demonstrated that knockdown of NOL1/NOP2/Sun domain family member 2 (NSUN2), a m⁵C methyltransferase, increased lipid accumulation of 3T3-L1 preadipocytes through accelerating cell cycle progression during mitotic clonal expansion (MCE) at the early stage of adipogenesis. Mechanistically, we proved that NSUN2 directly targeted cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNA, a key inhibitory regulator of cell cycle progression, and upregulated its protein expression in an m⁵C-dependent manner. Further study identified that CDKN1A was the target of Aly/REF export factor (ALYREF), a reader of m⁵C modified mRNA. Upon NSUN2 deficiency, the recognition of CDKN1A mRNA by ALYREF was suppressed, resulting in the decrease of CDKN1A mRNA shuttling from nucleus to cytoplasm. Thereby, the translation of CDKN1A was reduced, leading to the acceleration of cell cycle and the promotion of adipogenesis. Together, these findings unveiled an important function and mechanism of the m⁵C modification on adipogenesis by controlling cell cycle progression, providing a potential therapeutic target to prevent obesity.