Evolutionary analysis and immune-induced expression and m⁶A modification of RIPK1 and RIPK2 in the miiuy croaker

Receptor Interacting Protein Kinases (RIPKs), including RIPK1 and RIPK2, are key mediators of inflammatory signaling and inflammatory cell death. In this study, we identified the RIPK1 and RIPK2 genes in the miiuy croaker (Miichthys miiuy) and analyzed their evolutionary conservation and structural characteristics using bioinformatics approaches, highlighting their potential immune functions. Notably, research on the role of N6-methyladenosine (m⁶A) modification in regulating RIPKs remains limited. Here, methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed significant m⁶A enrichment near the stop codons of RIPK1 and RIPK2 of Miichthys miiuy, which was further confirmed by MeRIP-PCR. Expression analyses showed that RIPK1 and RIPK2 were markedly upregulated after poly(I:C) and LPS stimulation. Interestingly, poly(I:C) increased the m⁶A modification level of RIPK1, whereas LPS reduced the m⁶A level of RIPK2. Moreover, treatment with the methylation inhibitor cycloleucine further elevated their expression. These findings suggest that m⁶A modification may participate in fine-tuning the immune regulatory networks involving RIPK1 and RIPK2, potentially modulating their expression dynamics in response to diverse immune stimuli. This work provides valuable insights into the epigenetic regulation of RIPK family members and offers a foundation for exploring m⁶A-mediated control of fish innate immunity.