Quantitative profiling and dynamics of mRNA modifications in Escherichia coli

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. In contrast to the large number of eukaryotic mRNA modifications that have been described, N6-methyladenosine (m6A) is the only modification of bacterial mRNA identified to date. Here, we used a gel electrophoresis-based RNA separation method and quantitatively analyzed the mRNA-specific modification profile of Escherichia coli using mass spectrometry. In addition to m6A, we provide evidence for the presence of 7-methylguanosine (m7G), and we found first hints for 5-methylcytidine (m5C), N6,N6-dimethyladenosine (m6,6A), 1-methylguanosine (m1G), 5-methyluridine (m5U), and pseudouridine (Ψ) in the mRNA of E. coli, which implies that E. coli has a complex mRNA modification pattern. Furthermore, we observed changes in the abundance of some mRNA modifications during the transition of E. coli from the exponential growth to the stationary phase as well as upon exposure to stress. This study reveals a previously underestimated level of regulation between transcription and translation in bacteria.

Quantitative profiling and dynamics of mRNA modifications in Escherichia coli

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. In contrast to the large number of eukaryotic mRNA modifications that have been described, N6-methyladenosine (m6A) is the only modification of bacterial mRNA identified to date. Here, we used a gel electrophoresis-based RNA separation method and quantitatively analyzed the mRNA-specific modification profile of Escherichia coli using mass spectrometry. In addition to m6A, we provide evidence for the presence of 7-methylguanosine (m7G), and we found first hints for 5-methylcytidine (m5C), N6,N6-dimethyladenosine (m6,6A), 1-methylguanosine (m1G), 5-methyluridine (m5U), and pseudouridine (Ψ) in the mRNA of E. coli, which implies that E. coli has a complex mRNA modification pattern. Furthermore, we observed changes in the abundance of some mRNA modifications during the transition of E. coli from the exponential growth to the stationary phase as well as upon exposure to stress. This study reveals a previously underestimated level of regulation between transcription and translation in bacteria.

Quantitative profiling and dynamics of mRNA modifications in Escherichia coli

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. In contrast to the large number of eukaryotic mRNA modifications that have been described, N6-methyladenosine (m6A) is the only modification of bacterial mRNA identified to date. Here, we used a gel electrophoresis-based RNA separation method and quantitatively analyzed the mRNA-specific modification profile of Escherichia coli using mass spectrometry. In addition to m6A, we provide evidence for the presence of 7-methylguanosine (m7G), and we found first hints for 5-methylcytidine (m5C), N6,N6-dimethyladenosine (m6,6A), 1-methylguanosine (m1G), 5-methyluridine (m5U), and pseudouridine (Ψ) in the mRNA of E. coli, which implies that E. coli has a complex mRNA modification pattern. Furthermore, we observed changes in the abundance of some mRNA modifications during the transition of E. coli from the exponential growth to the stationary phase as well as upon exposure to stress. This study reveals a previously underestimated level of regulation between transcription and translation in bacteria.

UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model

Background Epimedin C, one of the main active ingredients of Epimedium, has been reported to have potential hepatotoxicity. However, the mechanism of Epimedin C-induced liver injury has not been studied. mRNA methylation, mainly including N6-methyladenosine and N5-methylcytidine, is implicated in the regulation of many biological processes and diseases. The study of quantifying mRNA methylation alterations in Epimedin C-induced liver injury mice may contribute to clarify the mechanism of its hepatotoxicity. Therefore, an analysis method needs to be established to determine nucleoside and methyl-nucleoside levels in liver mRNA. Methods An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously determine six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in liver mRNA. Besides, the Epimedin C-induced liver injury mouse model was studied by intragastrical administration Epimedin C at a daily dose of 10 or 40 mg/kg for 4 weeks. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer. Results In this method, calibration curves of the six nucleosides showed good linearity over their concentration ranges. The linear ranges were 40–20,000 pg/mL for adenosine, cytidine, N6-methyladenosine and N5-methylcytidine, 0.2–100 ng/mL for guanosine, and 2–1000 ng/mL for uridine. Epimedin C-induced liver injury mouse model was successfully established,which could be proved by the elevation of serum aminotransferase levels, and the increased inflammatory cell infiltration as well as vacuolar degeneration in liver. The N6-methyladenosine and N5-methylcytidine levels, and the ratios of N6-methyladenosine to adenosine and N5-methylcytidine to cytidine of the mice liver mRNA were all significantly increased after Epimedin C treatment. Conclusion The established method was successfully applied to the determination of six nucleosides levels in liver mRNA of the Epimedin C-induced liver injury mice model and the control group. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study will facilitate the mechanism research on the hepatotoxicity of Epimedin C.

Microarray analysis of genes with differential expression of m6A methylation in lung cancer

Purpose: N6-methyladenosine (m6A) is among the most abundant mRNA modifications in eukaryote. The aim of this study was to investigate function of m6A mRNA methylation in lung cancer and the underlying mechanism. Methods: Microarray analysis was performed to detect the differences in RNA expression between cancerous and adjacent non-cancerous tissue samples. The target mRNAs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Hierarchical clustering of RNAs was conducted to identify distinct m6A methylation or expression patterns between the samples. Results: In this study, some differentially expressed genes (DEGs) of mRNAs were identified, including up-regulated SPP1 and down-regulated pRB. Functional enrichment analysis revealed that while differential hypermethylation was related to cell cycle, intracellular part and protein binding, the main pathway involved herpes simplex virus 1 infection related to down-regulated AKT, Araf1 and BCL2A1. In the meantime, sexual reproduction, cohesin complex and portein C-terminus binding was functionally linked to differential hypomethylation, while fluid shear stress and atherosclerosis were identified as the main pathways related to up-regulated GST and CNP. Conclusions: We showed that lung cancer development involved differential expression of SPP1 and pRB mRNA, as well as m6A mRNA methylation in AKT, APAF1, BCL2A1, GST and CNP genes.