DNA methylation is an epigenetic modification at the genomic level that regulates gene expression by modulating transcriptional activity. The DNA methyltransferase (DNMT) family serves as the core functional genes responsible for DNA methylation modification. Our previous studies have revealed that mitochondrial genomes undergo methylation in the ridgetail white prawn, Exopalaemon carinicauda in response to starvation stress. To explore the regulatory mechanism of mitochondrial genome methylation in crustaceans, E. carinicauda was used as the experimental material in this study. The full-length cDNAs of dnmt1, dnmt2 and dnmt3b were successfully cloned using RACE technology, and RNA interference assays were performed to investigate their effects on mitochondrial DNA methylation in E. carinicauda. The results demonstrated that dnmt1 and dnmt2 exerted no significant regulatory effects on mitochondrial DNA methylation, whereas silencing of dnmt3b mediated differential regulation of mitochondrial DNA methylation: it significantly increased the methylation level of the 16# region and markedly decreased the methylation level of the 21# region. Subcellular localization results showed that dnmt1 and dnmt3b were expressed in both the nucleus and cytoplasm of muscle tissue, with predominant cytoplasmic distribution. The encoded proteins DNMT1 and DNMT3B co-localized with mitochondria in muscle tissue. Furthermore, DNA methylation modification in E. carinicauda exhibited distinct tissue specificity: the intestinal tissue possessed the highest methylation level, followed by hepatopancreas, gill, heart, muscle and eyestalk. In this study, four proteins interacting with DNMT3B, including HIF1β, PROPO, MYHC and ECR, were screened and identified via pull-down assays, and the regulatory relationships between the genes encoding these interacting proteins and dnmt3b were preliminarily explored. This study firstly identifies dnmt3b as a key gene governing mitochondrial DNA methylation in E. carinicauda, which mediates organismal stress responses under starvation.

Keywords Exopalaemon carinicauda, mtDNA methylation, DNMT3B, starvation stress, epigenetic modification