Stage-specific requirement for METTL3-dependent m6A modification during dental pulp stem cell differentiation
Haiyun Luo, Wenjing Liu, Yachuan Zhou, Yanli Zhang, Junrong Wu, Ruolan Wang, Longquan Shao
Abstract
Abstract 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.