ABSTRACT

N6-methyladenosine (m6A) is an abundant epitranscriptomic mark that regulates gene expression to execute cellular developmental programmes and environmental adaptation. Fusaric acid (FA) is a mycotoxin that contaminates agricultural foods and exerts toxicity in humans and animals; however, its epitranscriptomic effects are unclear. We investigated the effect of FA on global m6A RNA methylation and mRNA expression levels of key m6A regulatory genes in C57BL/6 mouse livers. C57BL/6 mice (n = 6/group) were orally administered 0.1 M phosphate-buffered saline (PBS) or 50 mg/kg FA. Mice were euthanized 24 h after oral administration, livers were harvested, and RNA was isolated. RNA samples were assayed for global m6A levels using an m6A RNA Methylation Quantification Kit. The mRNA expression of m6A regulators i.e. writers, erasers, and readers were measured by qRT-PCR. FA increased global m6A RNA methylation (p < 0.0001) in mouse livers. FA increased the expression of METTL3 (p = 0.0143) and METTL14 (p = 0.0281), and decreased the expression of FTO (p = 0.0036) and ALKBH5 (p = 0.0035). The expression of YTHDF2 (p = 0.0007), YTHDF3 (p = 0.0061), and YTHDC2 (p = 0.0258) were increased by FA in mouse livers. This study shows that the liver m6A epitranscriptome can be modified by FA exposure in an in vivo model and can be useful for identifying the molecular mechanisms whereby m6A RNA modifications influence the toxicological outcomes of FA exposure.

摘要

N6-甲基腺苷 (m6A) 是一种丰富的表观转录组标记，可调节基因表达以执行细胞发育程序和环境适应。镰刀菌酸（FA）是一种霉菌毒素，污染农业食品并对人和动物产生毒性；然而，其表观转录组效应尚不清楚。我们研究了 FA 对 C57BL/6 小鼠肝脏中关键 m6A 调节基因的全局 m6A RNA 甲基化和 mRNA 表达水平的影响。C57BL/6 小鼠 ( n= 6/组) 口服 0.1 M 磷酸盐缓冲盐水 (PBS) 或 50 mg/kg FA。口服给药后24小时对小鼠实施安乐死，收获肝脏并分离RNA。使用 m6A RNA 甲基化定量试剂盒测定 RNA 样品的整体 m6A 水平。m6A 调节因子（即写入器、擦除器和读取器）的 mRNA 表达通过 qRT-PCR 测量。FA 增加了小鼠肝脏中的全局 m6A RNA 甲基化 ( p < 0.0001)。FA 增加了METTL3 ( p = 0.0143) 和METTL14 ( p = 0.0281) 的表达，降低了FTO ( p = 0.0036) 和ALKBH5 ( p= 0.0035)。FA 在小鼠肝脏中增加了YTHDF2 ( p = 0.0007)、YTHDF3 ( p = 0.0061) 和YTHDC2 ( p = 0.0258) 的表达。该研究表明，肝脏 m6A 表转录组可以通过 FA 暴露在体内模型中进行修饰，并且可用于确定 m6A RNA 修饰影响 FA 暴露毒理学结果的分子机制。