Effects of Exogenous N6-methyladenosine Incorporation on the Expression of Cellular mRNA Transcripts

doi:10.7503/cjcu20220173

Abstract

Abstract:

N⁶-methyladenosine（m⁶A） is the most prevalent and reversible internal mRNA modification in eukaryotic cells， and plays an essential role in RNA fate determination. In this work， we studied the exogenous incorporation of m⁶A into cellular mRNA through salvage pathway and evaluated its resultant biological effects. First， we found that feeding of HeLa cells with m⁶A nucleoside significantly altered cell morphology and viability. We then synthesized isotope-labelled d₃-m⁶A（N⁶-methyl-d₃-adenosine） and examined the d₃-m⁶A incorporation rate in cellular mRNA along with incubation time using mass spectrometry. Next， RNA sequencing（RNA-seq） was used to study the biological effects of exogenous m⁶A incorporation. Finally， thousands of genes were found to be differentially expressed， and were dramatically enriched in pathways such as ribosome biogenesis， mRNA metabolic process and cell morphogenesis differentiation. All these results suggested that exogenous m⁶A could be incorporated into mRNAs through a metabolic pathway to influence cellular gene expression.

Key words: m⁶A Modification, mRNA Methylation, d₃-m⁶A Isotope labelling, mRNA Metabolic labelling

CLC Number:

O652

TrendMD:

ZHANG Qingyi, CAO Jie, SHU Xiao, LIU Jianzhao. Effects of Exogenous N⁶-methyladenosine Incorporation on the Expression of Cellular mRNA Transcripts[J]. Chem. J. Chinese Universities, 2023, 44(3): 20220173.

Figures/Tables 4

Fig.1 Cell morphology of HeLa cells treated with m6A at different concentrations for 12 h（A） DMSO as control；（B） 50 μmol/L m6A；（C） 500 μmol/L m6A；（D） 1 mmol/L m6A.

Fig.2 Mass spectrometry characterization of d3⁃m6A/A ratio in mRNA of HeLa cells（A） and cytotoxicity of d3⁃m6A in HeLa cells（B）（A） The cells were treated with 500 μmol/L or 1 mmol/L d3⁃m6A for different time intervals；（B） the cells were treated with 500 μmol/L d3⁃m6A for different time intervals. Data are expressed as mean±SD， n=3.

Fig.3 RNA⁃seq analysis showing the abundance change of mRNA transcripts under the metabolic incorporation of d3⁃m6A into mRNA of HeLa cells（A） The number of differentially expressed genes in samples under the metabolic feeding of d3-m6A for different time intervals versus control（Ctrl） without treatment. P1： 500 μmol/L d3-m6A， 24 h treatment； P7： 500 μmol/L d3-m6A， 168 h treatment. UP： up-regulated genes； DOWN： down-regulated genes. （B） The percentage of m6A-containing target genes among the total differentially expressed genes of the samples. （C） The heatmap and cluster analyses of differentially expressed genes in the samples.

Fig.4 Gene ontology analysis of differentially expressed genes in d3⁃m6A⁃treated HeLa cells for different time intervals（A） P1-UP；（B） P1-DOWN；（C） P7-UP；（D） P7-DOWN. P1： 500 μmol/L d3-m6A， 24 h treatment； P7： 500 μmol/L d3-m6A， 168 h treatment； UP： up-regulated genes； DOWN： down-regulated genes.

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Effects of Exogenous N⁶-methyladenosine Incorporation on the Expression of Cellular mRNA Transcripts

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