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 (m5C) and N6-methyladenosine (m6A) levels. Nsun4-mediated m5C and Mettl3-mediated m6A modifications were required for Sox9-regulated chondrogenic differentiation. Interestingly, we showed that in the 3'UTR of Sox9 mRNA, Nsun4 catalyzed the m5C modification and Mettl3 catalyzed the m6A 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 m5C and m6A co-regulate the translation of Sox9 during the chondrogenic differentiation of BMSCs, which provides a therapeutic target for clinical implications.