Determination of RNA Hydroxylmethylation in Mammals by Mass Spectrometry Analysis

RNA molecules harbor diverse chemical modifications that play important regulatory roles in a variety of biological processes. Up to date, more than 150 modifications have been identified in various RNA species. Most of these modifications occurring in nucleic acids are the methylation of nucleic acids. It has been demonstrated that many of these methylation are reversible and undergo dynamic demethylation. Previous studies established that the demethylation of the two most important and prevalent modifications of 5-methylcytidine (m⁵C) and N⁶-methyladenosine (m⁶A) in nucleic acids is through the hydroxylation of m⁵C and m⁶A, forming 5-hydroxymethylcytidine (hm⁵C) and N⁶-hydroxymethyladenosine (hm⁶A), respectively. This indicates the hydroxylation of the methylated nucleosides may be a general pathway for the demethylation of nucleic acid methylation. However, few other hydroxylmethylation modifications have yet to be reported in existence in mammals. In the current study, we developed a neutral enzymatic digestion method for the mild digestion of nucleic acids, followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. With the established method, we reported the existence of a new hydroxylmethylated nucleosides, N²-hydroxymethylguanosine (hm²G), in mammalian RNA. In addition, we found that the contents of hm²G, as well as N-methylguanosine (m²G), showed significant differences between thyroid carcinoma tissues and tumor-adjacent normal tissues, indicating that m²G and hm²G in RNA may play certain roles in the carcinogenesis of thyroid carcinoma. Collectively, our study suggests that RNA hydroxylmethylation may be a new prevalent group of modifications existing in RNA, which expands the diversity of nucleic acid modifications and should exert regulatory functions in living organisms.