Determination of 5-methyluridine in messenger RNA of mammals

The discovery of dynamic and reversible modifications in messenger RNA (mRNA) is opening new directions in RNA modification-mediated regulation of biological processes. Methylation is the most prevalent modification occurring in mRNA and the methylation group is mainly decorated in the adenine, cytosine, and guanine base, or in the 2'-hydroxyl group of ribose. However, methylation of the uracil base (5-methyluridine, m5U) hasn't been discovered in mRNA of eukaryotes. In the current study, we established a method by N-cyclohexyl-N'-ß-(4-methylmorpholinium) ethylcarbodiimide p-toluenesulfonate (CMCT) labelling coupled with liquid chromatography-electrospray ionization-mass spectrometry analysis (LC-ESI-MS/MS) for the sensitive determination of uridine modifications in RNA. Our results demonstrated that the detection sensitivities of uridine modifications in RNA increased up to 1543 folds upon CMCT labelling. Using the developed method, we identified the distinct existence of m5U in mRNA of various mammalian cells and tissues. In addition, the stable isotope tracing monitored by mass spectrometry revealed that the methylation group of m5U originated from S-Adenosyl-L-methionine (SAM). Our study expanded the list of modifications occurring in mRNA of mammals. Future work on transcriptome-wide mapping of m5U will further uncover the functional roles of m5U in mRNA of mammals. Overall design: The isolated mRNA was processed using KAPA Stranded RNA-Seq Library Prep Kit (Illumina) for sequencing library construction following the manufacturer's protocol. RNA-seq was carried out on Illumina HiSeq 4000 (Illumina) platform.