mRNA m(6)A plays opposite role in regulating UCP2 and PNPLA2 protein expression in adipocytes

Background/objective: N-6-methyladenosine (m(6)A) modification of mRNA plays an important role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Subjects/methods: Using Jinhua and Landrace pigs as fat and lean models, we presented a comprehensive transcriptome-wide m(6)A profiling in adipose tissues from these two pig breeds. Two differentially methylated genes were selected to explore the mechanisms of m(6)A-mediated regulation of gene function. Results: The ratio of m(6)A/A in the layer of backfat (LB) was significantly higher in Landrace than that in Jinhua. Transcriptome-wide m(6)A profiling revealed that m(6)A modification on mRNA occurs in the conserved sequence motif of RRACH and that the pig transcriptome contains 0.53-0.91 peak per actively expressed transcript. The relative density of m(6)A peaks in the 3'UTR were higher than in 5'UTR. Genes with common m(6)A peaks from both Landrace (L-LB) and Jinhua (J-LB) were enriched in RNA splicing and cellular lipid metabolic process. The unique m(6)A peak genes (UMGs) from L-LB were mainly enriched in the extracellular matrix (ECM) and collagen catabolic process, whereas the UMGs from J-LB are mainly involved in RNA splicing, etc. Lipid metabolism processes were not significantly enriched in the UMGs from L-LB or J-LB. Uncoupling protein-2 (UCP2) and patatin-like phospholipase domain containing 2 (PNPLA2) were two of the UMGs in L-LB. Synonymous mutations (MUT) were conducted to reduce m(6)A level of UCP2 and PNPLA2 mRNAs. Adipogenesis test showed that UCP2-MUT further inhibited adipogenesis, while PNPLA2-MUT promoted lipid accumulation compared with UCP2-WT and PNPLA2-WT, respectively. Further study showed m(6)A negatively mediates UCP2 protein expression and positively mediates PNPLA2 protein expression. m(6)A modification affects the translation of PNPLA2 most likely through YTHDF1, whereas UCP2 is likely neither the target of YTHDF2 nor the target of YTHDF1. Conclusion: Our data demonstrated a conserved and yet dynamically regulated m(6)A methylome in pig transcriptomes and provided an important resource for studying the function of m(6)A epitranscriptomic modification in obesity development.