METTL3-mediated mA modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition.

N6-methyladenosine (mA), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an mA transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated mA modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and mA modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose mA-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the mA reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated mA modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-mA-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.