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

Area of Science:

• Biochemistry
• Molecular Biology
• Regenerative Medicine

Background:

• Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is crucial for cartilage repair.
• The precise regulatory mechanisms of RNA modifications in chondrogenesis remain largely unknown.

Purpose of the Study:

• To investigate the role of RNA modifications, specifically 5-methylcytosine (m⁵C) and N6-methyladenosine (m⁶A), in the chondrogenic differentiation of BMSCs.
• To elucidate how these modifications regulate the translation of the key chondrogenic transcription factor, Sox9.

Main Methods:

• Ribosome sequencing to analyze Sox9 translation during chondrogenesis.
• RNA modification assays to quantify m⁵C and m⁶A levels.
• In vitro and in vivo experiments using BMSCs with manipulated Nsun4 and Mettl3 expression.
• Surface plasmon resonance (SPR) to study protein complex formation.

Main Results:

• Sox9 translation is enhanced during chondrogenesis, correlating with increased m⁵C and m⁶A levels.
• Nsun4-mediated m⁵C and Mettl3-mediated m⁶A modifications in the Sox9 3'UTR are essential for Sox9-regulated chondrogenesis.
• Nsun4 and Mettl3 form a complex with Ythdf2 and eEF1α-1 to co-regulate Sox9 translation.
• Overexpression of Nsun4 and Mettl3 in BMSCs promotes in vivo cartilage defect repair.

Conclusions:

• m⁵C and m⁶A RNA modifications synergistically control Sox9 translation during BMSC chondrogenesis.
• The Nsun4-Mettl3 complex plays a critical role in this regulatory process.
• Targeting these RNA modifications presents a promising therapeutic strategy for cartilage regeneration.