Formation and Abundance of 5-Hydroxymethylcytosine in RNA

RNA methylation is emerging as a regulatory RNA modification that could have important roles in the control and coordination of gene transcription and protein translation. Herein, we describe an in vivo isotope-tracing methodology to demonstrate that the ribonucleoside 5-methylcytidine (m5C) is subject to oxidative processing in mammals, forming 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C). Furthermore, we have identified hm5C in total RNA from all three domains of life and in polyA-enriched RNA fractions from mammalian cells. This suggests m5C oxidation is a conserved process that could have critical regulatory functions inside cells.