Characterization of the m ⁶ A Epitranscriptome in Fibroblast Senescence

A critical developmental process affecting aging and age-associated disease, cell senescence is characterized by persistent growth arrest and adaptive gene expression patterns. A common RNA modification, N6-methyladenosine (m6A), regulates gene expression profiles but its impact on senescence has not been studied globally. Here, we elucidated the m6A landscape in proliferating and senescent human fibroblasts using epitranscriptomic microarray and m6A-crosslinking and immunoprecipitation followed by sequencing (CLIP-Seq) analyses. Our findings revealed that while global m6A levels remain stable between proliferating and senescent cells, several transcripts display altered methylation patterns, particularly in senescence-associated pathways like cell cycle, DNA repair, and inflammation. We observed that m6A deposition shifted toward 3′-untranslated regions (UTRs) in senescent cells, although the m6A site modified (the DRACH motif) was unchanged, and we found a positive correlation between m6A levels and transcript abundance. Interestingly, m6A was particularly enriched in mRNAs encoding senescence-associated secretory phenotype (SASP) factors, and silencing METTL3, a major m6A methyltransferase, reduced the stability of certain SASP mRNAs such as IL6 mRNA, suggesting a selective function of m6A in fine-tuning the senescent transcriptome. These findings provide insights into the epitranscriptomic regulation of senescence, and highlight m6A as a potential intervention target in age-related conditions influenced by senescent cells.