Effects of Exogenous N6-methyladenosine (m6A) Incorporation on the Expression of Cellular mRNA Transcripts

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. 500 μM d3-m6A was added into HeLa cells and incubated for 24 h and until passage 7 (168 h), total RNAs were extracted and subjected to library construction