Stage-specific requirement for METTL3-dependent m(6)A modification during dental pulp stem cell differentiation

BACKGROUND: N(6)-methyladenosine (m(6)A) is the most prevalent epigenetic modification in eukaryotic messenger RNAs and plays a critical role in cell fate transition. However, it remains to be elucidated how m(6)A marks functionally impact the transcriptional cascades that orchestrate stem cell differentiation. The present study focuses on the biological function and mechanism of m(6)A methylation in dental pulp stem cell (DPSC) differentiation. METHODS: m(6)A RNA immunoprecipitation sequencing was utilized to assess the m(6)A-mRNA landscape during DPSC differentiation. Ectopic transplantation of DPSCs in immunodeficient mice was conducted to verify the in vitro findings. RNA sequencing and m(6)A RNA immunoprecipitation sequencing were combined to identify the candidate targets. RNA immunoprecipitation and RNA/protein stability of Noggin (NOG) were evaluated. The alteration in poly(A) tail was measured by 3′-RACE and poly(A) tail length assays. RESULTS: We characterized a dynamic m(6)A-mRNA landscape during DPSC mineralization with increasing enrichment in the 3′ untranslated region (UTR). Methyltransferase-like 3 (METTL3) was identified as the key m(6)A player, and METTL3 knockdown disrupted functional DPSC differentiation. Moreover, METTL3 overexpression enhanced DPSC mineralization. Increasing m(6)A deposition in the 3′ UTR restricted NOG expression, which is required for DPSC mineralization. This stage-specific m(6)A methylation and destabilization of NOG was suppressed by METTL3 knockdown only in differentiated DPSCs. Furthermore, METTL3 promotes the degradation of m(6)A-tagged NOG by shortening the poly(A) tail length in the differentiated stage. CONCLUSIONS: Our results address an essential role of dynamic m(6)A signaling in the temporal control of DPSC differentiation and provide new insight into epitranscriptomic mechanisms in stem cell-based therapy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-022-03814-9.