Novel dual methylation of cytidines in the RNA of mammals†
RNA modifications play critical roles in regulating a variety of physiological processes. Methylation is the most prevalent modification occurring in RNA. Three isomeric cytidine methylation modifications have been reported in RNA, including 3-methylcytidine (m3C), N4-methylcytidine (m4C), and 5-methylcytidine (m5C), in mammals. Aside from the single methylation on the nucleobase of cytidines, dual methylation modifications occurring in both the 2′ hydroxyl of ribose and the nucleobase of cytidines also have been reported, including N4,2′-O-dimethylcytidine (m4Cm) and 5,2′-O-dimethylcytidine (m5Cm). m4Cm has been found in the 16S rRNA of E. coli, while m5Cm has been found in the tRNA of terminal thermophilic archaea and mammals. However, unlike m4Cm and m5Cm, the presumed dual methylation of 3,2′-O-dimethylcytidine (m3Cm) has never been discovered in living organisms. Thus, the presence of m3Cm in RNA remains an open question. In the current study, we synthesized m3Cm and established a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to determine the dimethylation of cytidines, m3Cm, m4Cm and m5Cm. Under optimized analytical conditions, m3Cm, m4Cm and m5Cm can be clearly distinguished. Using the method, we discovered the existence of m3Cm in the RNA of mammals. The identified m3Cm is a novel modification that hasn't been reported in the three-domain system, including archaea, bacteria, and eukaryotes. We confirmed that m3Cm mainly existed in the small RNA (<200 nt) of mammals. In addition, we identified, for the first time, the presence of m4Cm in the 18S rRNA of mammalian cells. The stable isotope tracing monitored by mass spectrometry demonstrated that S-adenosyl-L-methionine was a methyl donor for all three dimethylations of cytidines in RNA. The discovery of m3Cm broadens the diversity of RNA modifications in living organisms. In addition, the discovery of m3Cm and m4Cm in mammals opens new directions in understanding RNA modification-mediated RNA processing and gene expression regulation.