Stage-specific requirement for METTL3-dependent m6A modification during dental pulp stem cell differentiation

Abstract

Background N6-methyladenosine (m6A) 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 m6A marks functionally impact the transcriptional cascades that orchestrate stem cell differentiation. The present study focuses on the biological function and mechanism of m6A methylation in dental pulp stem cell (DPSC) differentiation. Methods m6A RNA immunoprecipitation sequencing was utilized to assess the m6A-mRNA landscape during DPSC differentiation. Ectopic transplantation of DPSCs in immunodeficient mice was conducted to verify the in vitro findings. RNA sequencing and m6A 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 m6A-mRNA landscape during DPSC mineralization with increasing enrichment in the 3′ untranslated region (UTR). Methyltransferase-like 3 (METTL3) was identified as the key m6A player, and METTL3 knockdown disrupted functional DPSC differentiation. Moreover, METTL3 overexpression enhanced DPSC mineralization. Increasing m6A deposition in the 3′ UTR restricted NOG expression, which is required for DPSC mineralization. This stage-specific m6A methylation and destabilization of NOG was suppressed by METTL3 knockdown only in differentiated DPSCs. Furthermore, METTL3 promotes the degradation of m6A-tagged NOG by shortening the poly(A) tail length in the differentiated stage. Conclusions Our results address an essential role of dynamic m6A signaling in the temporal control of DPSC differentiation and provide new insight into epitranscriptomic mechanisms in stem cell-based therapy.

METTL3‐Mediated m6A Modification Regulates the Osteogenic Differentiation through LncRNA CUTALP in Periodontal Mesenchymal Stem Cells of Periodontitis Patients

“…However, m6A methylation modification in oral nonneoplastic diseases has been relatively poorly studied. Luo et al [54] mineralization. Inhibition of ALKBH5 or FTO promotes resistance of human oral epithelial cells to herpes simplex virus type I infection by increasing m6A methylation modification [55].…”

Cloning of the RNA m6A Methyltransferase 3 and Its Impact on the Proliferation and Differentiation of Quail Myoblasts

“…Poly(A) RNA sequencing libraries were generated and paired-end (PE150) sequenced using an Illumina Novaseq 6000 System (LC Bio, Hangzhou, China) according to standard protocols. Clean reads were mapped to the Coturnix japonica reference genome (https://www.ncbi.nlm.nih.gov/genome/113?genome_assembly_id=265191, accessed on 20 June 2022) and subjected to subsequent analysis following previously described methods [24]. Gene set enrichment analysis (GSEA) was conducted as previously described [25].…”

Negative Regulation of LINC01013 by METTL3 and YTHDF2 Enhances the Osteogenic Differentiation of Senescent Pre‐Osteoblast Cells Induced by Hydrogen Peroxide

“…[24] METTL3 is a crucial component of the m6A writer complex and contributes significantly to the regulation of m6A methylation levels, impacting cell survival, stem cell maintenance, and cell fate decisions. [25,26] METTL3 lossof-function impaired bone formation [27] and exhibited accelerated senescence. [28] However, the function of METTL3 in osteogenic differentiation of hFOB1.19 cells remains unknown.…”