Table3_Integrated Study of Transcriptome-wide m6A Methylome Reveals Novel Insights Into the Character and Function of m6A Methylation During Yak Adipocyte Differentiation.XLSX

Yak (Bos grunniens) is considered an iconic symbol of Tibet and high altitude, but they suffer from malnutrition during the cold season that challenges the metabolism of energy. Adipocytes perform a crucial role in maintaining the energy balance, and adipocyte differentiation is a complex process involving multiple changes in the expression of genes. N6-methyladenosine (m6A) plays a dynamic role in post-transcription gene expression regulation as the most widespread mRNA modification of the higher eukaryotes. However, currently there is no research existing on the m6A transcriptome-wide map of bovine animals and their potential biological functions in adipocyte differentiation. Therefore, we performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to determine the distinctions in m6A methylation and gene expression during yak adipocyte differentiation. In yak adipocyte and preadipocyte the content of m6A and m6A-associated enzymes was substantially different. In the two groups, a total of 14,710 m6A peaks and 13,388 m6A peaks were identified. For the most part, m6A peaks were enriched in stop codons, 3′-untranslated regions, and coding regions with consensus motifs of GGACU. The functional enrichment exploration displayed that differentially methylated genes participated in some of the pathways associated with adipogenic metabolism, and several candidate genes (KLF9, FOXO1, ZNF395, and UHRF1) were involved in these pathways. In addition to that, there was a positive association between m6A abundance and levels of gene expression, which displayed that m6A may play a vital role in modulating gene expression during yak adipocyte differentiation. Further, in the adipocyte group, several methylation gene protein expression levels were significantly higher than in preadipocytes. In short, it can be concluded that the current study provides a comprehensive explanation of the m6A features in the yak transcriptome, offering in-depth insights into m6A topology and associated molecular mechanisms underlying bovine adipocyte differentiation, which might be helpful for further understanding its mechanisms.

牦牛（Bos grunniens）被视作青藏高原与高海拔地区的标志性物种，但它们在寒冷季节常遭遇营养不良问题，这会对机体能量代谢造成严峻挑战。脂肪细胞在维持能量平衡中发挥关键作用，而脂肪细胞分化是一个复杂过程，涉及诸多基因表达的动态改变。N6-甲基腺嘌呤（N6-methyladenosine，m6A）作为高等真核生物中最普遍的mRNA修饰方式，在转录后基因表达调控中扮演动态调控角色。然而，目前尚无针对牛科动物的全转录组m6A图谱及其在脂肪细胞分化中潜在生物学功能的相关研究。因此，本研究通过甲基化RNA免疫沉淀测序（MeRIP-seq）与RNA测序（RNA-seq），解析牦牛脂肪细胞分化过程中m6A甲基化与基因表达的差异。结果显示，牦牛脂肪细胞与前脂肪细胞中的m6A含量及m6A相关酶的表达水平存在显著差异。两组样本共鉴定出14710个m6A峰与13388个m6A峰。m6A峰主要富集于终止密码子、3'非翻译区以及编码区，其保守基序为GGACU。功能富集分析表明，差异甲基化基因参与了多条与脂肪生成代谢相关的通路，且多个候选基因（KLF9、FOXO1、ZNF395及UHRF1）均参与其中。此外，m6A修饰丰度与基因表达水平呈正相关，这提示m6A可能在牦牛脂肪细胞分化过程中对基因表达调控发挥至关重要的作用。进一步研究发现，脂肪细胞组中多个甲基化相关基因的蛋白表达水平显著高于前脂肪细胞组。综上，本研究全面阐释了牦牛转录组中的m6A修饰特征，深入揭示了牛科动物脂肪细胞分化的m6A拓扑结构与相关分子机制，可为进一步阐明其作用机理提供重要参考。