Formation and Abundance of 5‐Hydroxymethylcytosine in RNA

Abstract

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.

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

“…In RNA, the analogous oxidation of m 5 C to hm 5 C has been reported by catalytic domains of mammalian TET enzymes (Fu et al , 2014) and the homologous Drosophila protein dTET (Delatte et al , 2016). f 5 C was detected as minor oxidation product in total cellular RNA by mass spectrometry‐based isotope tracing (Huber et al , 2015), but the enzymes generating this modification have remained unknown. The observation that oxidation products of m 5 C have been detected in RNA from all domains of life, including organisms that do not contain homologous TET enzymes, suggests that m 5 C can be metabolically oxidised by enzymes other than those of the TET family.…”

RNA 5-Methylcytosine Analysis by Bisulfite Sequencing

“…Given that the concentration of sodium bisulfite in commercially available kits has been optimized to guarantee high deamination rates, the possibility to "over-deaminate" and thereby lose m 5 C residues in RNA is likely, which will negatively impact both on the discovery of low m 5 C levels, especially in presently unknown RNAs as well as on the quantification of m 5 C at known positions. In addition, also hm 5 C, an oxidation product of m 5 C, has been detected in RNA (Fu et al, 2014;Huber et al, 2015;Rácz, Juhász, Király, & Lásztity, 1982). Although the amount of hm 5 C in RNA is very low, its presence in specific RNAs could cause cytosine calls after RNA-BisSeq because bisulfite sequencing does not allow distinguishing m 5 C from hm 5 C, unless performed under specific conditions (Booth et al, 2012).…”

Post-transcriptional modifications in development and stem cells

“…Methylation of mitochondrial tRNA Met by NSUN3 is required for the subsequent formation of 5-formylcytosine (f 5 C) at the same tRNA position (Nakano et al, 2016;Van Haute et al, 2016). Recent findings also suggest that TET-mediated oxidation reverses cytosine-5 methylation in vitro and 5-hydroxymethylcytosine (hm 5 C) has been detected in polyAenriched RNA in vivo (Delatte et al, 2016;Fu et al, 2014;Huber et al, 2015).…”