外源N^(6)-甲基腺嘌呤掺入对细胞mRNA表达的影响

Effects of Exogenous N^(6)-methyladenosine Incorporation on the Expression of Cellular mRNA Transcripts

N^(6)-甲基腺嘌呤(m^(6)A)是一种真核细胞中最广泛存在且可逆的内源mRNA修饰,它在决定RNA命运中起着至关重要的作用.本文发现外源m^(6)A可以通过补救合成途径掺入到细胞mRNA,并评估了外源m^(6)A掺入mRNA后所产生的生物学效应.首先,发现用m^(6)A核苷处理HeLa细胞会显著改变细胞的形态和活力;然后,合成了同位素标记的d_(3)-m^(6)A(N^(6)-甲基-d_(3)-腺苷),并采用质谱法检测了不同处理时间下细胞mRNA中d_(3)-m^(6)A的掺入率;随后,使用RNA测序(RNA-seq)研究外源m^(6)A掺入的生物学效应.结果表明,掺入外源m^(6)A后的细胞有数千个基因产生差异表达,并且这些差异表达的基因在核糖体生物发生、mRNA代谢过程和细胞形态发生分化等途径中显著富集.研究结果表明,外源m^(6)A可以通过代谢途径掺入细胞内源mRNA中,并可以影响细胞基因表达.

N^(6)-methyladenosine(m^(6)A)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 m^(6)A into cellular mRNA through salvage pathway and evaluated its resultant biological effects.First,we found that feeding of HeLa cells with m^(6)A nucleoside significantly altered cell morphology and viability.We then synthesized isotope-labelled d_(3)-m^(6)A(N^(6)-methyl-d_(3)-adenosine)and examined the d_(3)-m^(6)A 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 m^(6)A 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 m^(6)A could be incorporated into mRNAs through a metabolic pathway to influence cellular gene expression.