Nsun4 and Mettl3 mediated translational reprogramming of Sox9 promotes BMSC chondrogenic differentiation.

The chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) has been used in the treatment and repair of cartilage defects; however, the in-depth regulatory mechanisms by which RNA modifications are involved in this process are still poorly understood. Here, we found that Sox9, a critical transcription factor that mediates chondrogenic differentiation, exhibited enhanced translation by ribosome sequencing in chondrogenic pellets, which was accompanied by increased 5-methylcytosine (m 5 C) and N6-methyladenosine (m 6 A) levels. Nsun4-mediated m 5 C and Mettl3-mediated m 6 A modifications were required for Sox9-regulated chondrogenic differentiation. Interestingly, we showed that in the 3'UTR of Sox9 mRNA, Nsun4 catalyzed the m 5 C modification and Mettl3 catalyzed the m 6 A modification. Furthermore, we found that Nsun4 and Mettl3 co-regulated the translational reprogramming of Sox9 via the formation of a complex. Surface plasmon resonance (SPR) assays showed that this complex was assembled along with the recruitment of Ythdf2 and eEF1α-1. Moreover, BMSCs overexpressing Mettl3 and Nsun4 can promote the repair of cartilage defects in vivo. Taken together, our study demonstrates that m 5 C and m 6 A co-regulate the translation of Sox9 during the chondrogenic differentiation of BMSCs, which provides a therapeutic target for clinical implications.