Characterization of the m⁶A 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.