Existence of Internal N7‑Methylguanosine Modification in mRNA Determined by Differential Enzyme Treatment Coupled with Mass Spectrometry Analysis

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.