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 (m⁷G) is unique owing to its presence in the 5′ cap structure. It remains unknown whether m⁷G is also present internally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal m⁷G 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 m⁷G modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5′-terminal m⁷G. By using this method, we found that internal m⁷G 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 m⁷G in mRNA than mammals. We also observed that exposure of rice to cadmium (Cd) stimulated marked diminution in the levels of m⁷G at both the 5′ cap and internal positions of mRNA, which was correlated with the Cd-induced elevated expression of m⁷G-decapping enzymes. Taken together, we reported here a strategy to distinguish internal and 5′-terminal m⁷G in mRNA, and by using this method, we demonstrated the prevalence of internal m⁷G modification in mRNA, which we believe will stimulate future functional studies of m⁷G on post-transcriptional gene regulation in eukaryotes.

中文翻译：

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差异酶处理结合质谱分析确定的mRNA中内部N 7-甲基鸟苷修饰的存在

mRNA上可逆化学修饰的最新发现开启了真核生物转录后基因调控的新时代。在真核生物的mRNA中鉴定出的15种修饰类型中，N 7-甲基鸟苷（m ⁷ G）由于在5'帽结构中的存在而具有独特性。尚不清楚m ⁷内部是否还存在m ⁷ G，这在很大程度上归因于缺乏适当的分析方法来区分mRNA内部的m ⁷ G和5'帽内部的m 7G。为了解决这一分析难题，我们开发了一种新的策略，将差异酶消化与液相色谱-串联质谱分析相结合，以量化这两种类型的m ⁷的水平。mRNA中的G修饰。特别是，我们发现S1核酸酶和磷酸二酯酶I对内部和5'端m ⁷ G表现出不同的活性。通过这种方法，我们发现内部m ⁷ G存在于培养的人细胞以及植物和植物的mRNA中。大鼠组织。此外，我们的结果表明，植物中mRNA的内部m ⁷ G含量高于哺乳动物。我们还观察到，水稻暴露于镉（Cd）刺激了m '的5'帽和内部位置处m ⁷ G的水平显着降低，这与Cd诱导的m ⁷表达升高相关G脱盖酶。综上所述，我们在这里报告了一种区分mRNA中内部和5'端m ⁷ G的策略，并且通过使用这种方法，我们证明了mRNA中内部m ⁷ G修饰的普遍性，我们相信这将刺激未来的功能研究。米⁷ G于真核生物中转录后基因调节。