The m⁶A methyltransferase METTL3 regulates antimicrobial peptide expression via the Toll pathway in Eriocheir...

The RNA N⁶-methyladenosine (m⁶A) methylation, catalyzed by methyltransferase-like 3 (METTL3), has emerged as a pivotal epitranscriptomic regulator in immune processes. However, its functional role in crustacean immunity remains poorly understood. In this study, we identified and characterized a METTL3 homolog-designated EsMETTL3-from the Chinese mitten crab Eriocheir sinensis. EsMETTL3 contains a highly conserved MT-A70 domain and shares significant sequence identity in this catalytic region across diverse species. Expression analysis revealed that EsMETTL3 is ubiquitously expressed across all tested tissues, with relatively low basal levels observed in hemocytes. Notably, its transcription in hemocytes was significantly upregulated following infection with Vibrio parahaemolyticus. Functional experiments demonstrated that bacterial infection elevates global m⁶A levels in hemocytes in an EsMETTL3-dependent manner. Knockdown of EsMETTL3 or pharmacological inhibition of m⁶A methylation led to a marked reduction in the expression of key antimicrobial peptides (AMPs), including Crustin1, Crustin2, Lysozyme, and DWD. Further mechanistic analyses revealed that EsMETTL3-mediated m⁶A methylation modulates the Toll signaling pathway by enhancing pelle expression and suppressing cactus expression, thereby promoting nuclear translocation of the transcription factor Dorsal. Our findings establish a critical role for EsMETTL3-mediated m⁶A methylation in regulating crab innate immunity through fine-tuning the Toll/Dorsal pathway and AMP production, providing novel insights into the epitranscriptomic regulation of immune responses in crustaceans.