mRNA m⁶A plays opposite role in regulating UCP2 and PNPLA2 protein expression in adipocytes.

Abstract

N⁶-methyladenosine (m⁶A) modification of mRNA plays an important role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Using Jinhua and Landrace pigs as fat and lean models, we presented a comprehensive transcriptome-wide m⁶A profiling in adipose tissues from these two pig breeds. Two differentially methylated genes were selected to explore the mechanisms of m⁶A-mediated regulation of gene function. The ratio of m⁶A/A in the layer of backfat (LB) was significantly higher in Landrace than that in Jinhua. Transcriptome-wide m⁶A profiling revealed that m⁶A 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 m⁶A peaks in the 3'UTR were higher than in 5'UTR. Genes with common m⁶A peaks from both Landrace (L-LB) and Jinhua (J-LB) were enriched in RNA splicing and cellular lipid metabolic process. The unique m⁶A 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 m⁶A 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 m⁶A negatively mediates UCP2 protein expression and positively mediates PNPLA2 protein expression. m⁶A modification affects the translation of PNPLA2 most likely through YTHDF1, whereas UCP2 is likely neither the target of YTHDF2 nor the target of YTHDF1. Our data demonstrated a conserved and yet dynamically regulated m⁶A methylome in pig transcriptomes and provided an important resource for studying the function of m⁶A epitranscriptomic modification in obesity development.