The mechanism by which methylase METTL3 affects intramuscular fat deposition

N⁶-methyladenosine (m⁶A) is a critical epigenetic modification that regulates lipid metabolism; however, its role in adipogenesis in chickens remains unclear. Here, we demonstrated that the m⁶A methylase METTL3 shows significant differences at the key stage of chicken development, and its expression in the breast muscle is significantly higher than in other tissues. Furthermore, METTL3 expression gradually decreases during the differentiation of both intramuscular preadipocytes (IMPA) and abdominal preadipocytes (APA). Gain- and loss-of-function assays demonstrated that METTL3 overexpression significantly inhibited the proliferation and differentiation of IMPA and APA cells, whereas METTL3 knockdown exerted the opposite effect. Combined with m⁶A sequencing (m⁶A-seq), we identified significant enrichment of m⁶A modification within the 3' untranslated region (3'UTR) of PCYT1A mRNA, indicating it as a direct modification target of METTL3. Mechanistically, METTL3 knockdown reduced m⁶A methylation of PCYT1A, consequently enhancing PCYT1A mRNA stability and expression. This upregulation of PCYT1A promoted adipogenic differentiation in both IMPA and APA. Our study elucidates the functional mechanism by which METTL3-mediated m⁶A modification regulates lipid deposition through modulating PCYT1A expression, thereby identifying novel molecular targets for investigating adipogenesis in chickens.