Low-Temperature Stress on the Expression of mettl3 and mettl14 and RNA m⁶A Modification in Larimichthys crocea

N6-methyladenosine (m⁶A), a predominant epigenetic modification of RNA in eukaryotes, exerts crucial regulatory functions during environmental adaptation. Nevertheless, the responsive profiles and functional mechanisms of m⁶A under cold stress remain poorly understood in teleost fish. In the present study, large yellow croaker (Larimichthys crocea) was selected as the experimental organism to characterize the alterations in global m⁶A methylation levels, together with the expression patterns of two methyltransferases Lc-mettl3 and Lc-mettl14 under cold stress, and to further explore their potential correlation with the mitogen-activated protein kinase (MAPK) signaling pathway. The core coding sequences (CDS) of Lc-METTL3 and Lc-METTL14 were cloned and subjected to comprehensive bioinformatic analyses. Real-time quantitative PCR (RT-qPCR) was applied to detect the tissue expression profiles as well as the transcriptional dynamics of the two genes under gradient temperatures and 8℃ cold stress with different exposure durations. Western blot was performed to quantify the protein abundance of Lc-METTL14, and the total hepatic m⁶A methylation level was biochemically measured. Differentially m⁶A-methylated genes were screened based on MeRIP-seq data, followed by KEGG pathway enrichment analysis. RT-qPCR was used to verify the transcriptional changes of hub genes within the MAPK signaling cascade. Both Lc-mettl3 and Lc-mettl14 were ubiquitously expressed in examined tissues of L. crocea, with the highest transcript abundance detected in the liver. Under cold stress, the hepatic mRNA levels of Lc-mettl3 and Lc-mettl14 declined gradually with the decrease in ambient temperature and the prolongation of cold exposure; the protein expression trend of Lc-METTL14 was consistent with its transcriptional variation. Global hepatic m⁶A methylation was significantly reduced upon cold treatment in a time-dependent manner. A large set of genes with altered m⁶A modification were identified via MeRIP-seq, which were significantly enriched in the MAPK signaling pathway and multiple metabolism-associated pathways. Four MAPK pathway genes (map2k7, map3k14a, map4k4, mapk8b) exhibited elevated m⁶A modification levels after cold stress, accompanied by gene-specific transcriptional responsive patterns. Cold stress repressed the expression of hepatic Lc-mettl3/Lc-mettl14 and decreased the global m⁶A methylation level in the liver, along with dynamic shifts in m⁶A modification and transcription of MAPK pathway genes. Collectively, our findings suggest that m⁶A-dependent post-transcriptional regulation participates in modulating the MAPK signaling pathway during cold stress response in L. crocea. This study provides novel theoretical insights into the epigenetic regulatory mechanism underlying cold acclimation in marine fish species.