Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry , Wuhan University , Wuhan 430072 , People's Republic of China.
ACS Chem Biol. 2018 Dec 21;13(12):3243-3250. doi: 10.1021/acschembio.7b00906. Epub 2018 Nov 29.
The recent discovery of reversible chemical modifications on mRNA has opened a new era of post-transcriptional gene regulation in eukaryotes. Among the 15 types of modifications identified in mRNA of eukaryotes, N7-methylguanosine (mG) is unique owing to its presence in the 5' cap structure. It remains unknown whether mG is also present internally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal mG in mRNA from that in the 5' cap. To address this analytical challenge, we developed a novel strategy of combining differential enzymatic digestion with liquid chromatography-tandem mass spectrometry analysis to quantify the levels of these two types of mG modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5'-terminal mG. By using this method, we found that internal mG was present in mRNA of cultured human cells as well as plants and rat tissue. In addition, our results showed that plants contain higher levels of internal mG in mRNA than mammals. We also observed that exposure of rice to cadmium (Cd) stimulated marked diminution in the levels of mG at both the 5' cap and internal positions of mRNA, which was correlated with the Cd-induced elevated expression of mG-decapping enzymes. Taken together, we reported here a strategy to distinguish internal and 5'-terminal mG in mRNA, and by using this method, we demonstrated the prevalence of internal mG modification in mRNA, which we believe will stimulate future functional studies of mG on post-transcriptional gene regulation in eukaryotes.
最近在真核生物的 mRNA 上发现了可逆转的化学修饰，这为真核生物的转录后基因调控开辟了一个新时代。在真核生物 mRNA 中鉴定出的 15 种修饰类型中，m7G（N7-甲基鸟苷）因其存在于 5' 帽结构中而独具特色。目前尚不清楚 mG 是否也存在于 mRNA 内部，这主要归因于缺乏一种合适的分析方法来区分 mRNA 内部的 mG 与 5' 帽中的 mG。为了解决这个分析上的挑战，我们开发了一种将差异酶消化与液相色谱-串联质谱分析相结合的新策略，以定量测量这两种 mG 修饰在 mRNA 中的水平。特别是，我们发现 S1 核酸酶和磷酸二酯酶 I 对内部和 5' 端 mG 具有不同的活性。通过使用这种方法，我们发现培养的人类细胞以及植物和大鼠组织的 mRNA 中存在内部 mG。此外，我们的结果表明，植物的 mRNA 中内部 mG 的含量高于哺乳动物。我们还观察到，水稻暴露于镉（Cd）会刺激 mRNA 5' 帽和内部位置的 mG 水平显著降低，这与 Cd 诱导的 mG 脱帽酶表达升高有关。总之，我们在这里报告了一种区分 mRNA 内部和 5' 端 mG 的策略，并使用该方法证明了 mRNA 内部 mG 修饰的普遍性，我们相信这将激发未来对真核生物转录后基因调控中 mG 的功能研究。
