Novel dual methylation of cytidines in the RNA of mammals

Abstract

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.