College of Animal Sciences, Zhejiang University, Key Laboratory of Animal Nutrition & Feed Sciences, Ministry of Agriculture, Zhejiang Provincial Laboratory of Feed and Animal Nutrition, Hanghou, 310058, Zhejiang, P. R. China.
Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, Chinese Academy of Sciences, 100101, Beijing, China.
Int J Obes (Lond). 2018 Nov;42(11):1912-1924. doi: 10.1038/s41366-018-0027-z. Epub 2018 Feb 27.
BACKGROUND/OBJECTIVE: N-methyladenosine (mA) 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 mA profiling in adipose tissues from these two pig breeds. Two differentially methylated genes were selected to explore the mechanisms of mA-mediated regulation of gene function.
The ratio of mA/A in the layer of backfat (LB) was significantly higher in Landrace than that in Jinhua. Transcriptome-wide mA profiling revealed that mA 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 mA peaks in the 3'UTR were higher than in 5'UTR. Genes with common mA peaks from both Landrace (L-LB) and Jinhua (J-LB) were enriched in RNA splicing and cellular lipid metabolic process. The unique mA 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 mA 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 mA negatively mediates UCP2 protein expression and positively mediates PNPLA2 protein expression. mA 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 mA methylome in pig transcriptomes and provided an important resource for studying the function of mA epitranscriptomic modification in obesity development.
背景/目的：mRNA 中的 N6-甲基腺苷（mA）修饰在调节脂肪生成中起着重要作用。然而，其潜在机制在很大程度上仍然未知。
研究对象/方法：我们以金华猪和长白猪为脂肪和瘦肉模型，在这两个猪品种的脂肪组织中进行了全面的转录组范围的 mA 谱分析。选择两个差异甲基化基因来探索 mA 介导的基因功能调控机制。
长白猪背部脂肪层（LB）的 mA/A 比值明显高于金华猪。全转录组 mA 谱分析表明，mA 修饰发生在 RRACH 保守序列基序上，猪转录组中每个活跃表达的转录本含有 0.53-0.91 个峰。3'UTR 中的 mA 峰相对密度高于 5'UTR。来自长白猪（L-LB）和金华猪（J-LB）的共同 mA 峰基因在 RNA 剪接和细胞脂质代谢过程中富集。来自 L-LB 的独特 mA 峰基因（UMG）主要富集在外泌体和胶原蛋白代谢过程中，而来自 J-LB 的 UMG 主要参与 RNA 剪接等过程。脂质代谢过程在 L-LB 或 J-LB 的 UMG 中没有明显富集。解偶联蛋白 2（UCP2）和类脂肪酶结构域包含蛋白 2（PNPLA2）是 L-LB 中的两个 UMG。进行同义突变（MUT）以降低 UCP2 和 PNPLA2 mRNA 的 mA 水平。脂肪生成试验表明，与 UCP2-WT 和 PNPLA2-WT 相比，UCP2-MUT 进一步抑制脂肪生成，而 PNPLA2-MUT 促进脂质积累。进一步的研究表明，mA 负调节 UCP2 蛋白表达，正调节 PNPLA2 蛋白表达。mA 修饰很可能通过 YTHDF1 影响 PNPLA2 的翻译，而 UCP2 既不是 YTHDF2 的靶标，也不是 YTHDF1 的靶标。
我们的数据证明了猪转录组中存在保守且动态调节的 mA 甲基组，并为研究 mA 表遗传修饰在肥胖发展中的功能提供了重要资源。
