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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 ( less then 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 m
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