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Formation and abundance of 5-hydroxymethylcytosine in RNA.

Huber SM, van Delft P, Mendil L, Bachman M, Smollett K, Werner F, Miska EA, Balasubramanian S - Chembiochem (2015)

Bottom Line: Herein, we describe an in vivo isotope-tracing methodology to demonstrate that the ribonucleoside 5-methylcytidine (m(5)C) is subject to oxidative processing in mammals, forming 5-hydroxymethylcytidine (hm(5)C) and 5-formylcytidine (f(5)C).Furthermore, we have identified hm(5)C in total RNA from all three domains of life and in polyA-enriched RNA fractions from mammalian cells.This suggests m(5)C oxidation is a conserved process that could have critical regulatory functions inside cells.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW (UK).

No MeSH data available.


Levels of m5C and hm5C across different model organisms given in amounts relative to the sum of (modified) cytosine residues.[20]
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fig01: Levels of m5C and hm5C across different model organisms given in amounts relative to the sum of (modified) cytosine residues.[20]

Mentions: We detected hm5C in all samples studied (Figure 1), thus confirming RNA C5-hydroxymethylation as a common RNA modification. Notably, hm5C was found in species that do not exhibit detectable hm5dC in their DNA and lack TET homologues in their genomes, such as C. elegans and A. thaliana.[22] This suggests that, in such organisms, the formation of hm5C in RNA must occur through a non-TET mechanism. As noted by Fu et al., their observation that TET- ES cells in which TET1, TET2, and TET3 are genetically deleted still exhibit significant hm5C levels in RNA also supports the idea that hm5C in RNA can form from pathways that do not involve TET enzymes.[15] Comparison of m5C oxidation into hm5C across the different species revealed varying levels (Figure 2). This could also indicate that RNA hydroxymethylation is the result of different enzymatic transformations. It will ultimately be essential to map the position of hm5C to specific RNAs in order to understand its function. As a step towards elucidating hm5C location, we explored the abundance of hm5C in RNA classes other than tRNA and rRNA. Specifically, we isolated mRNA and lncRNAs from HEK293T cells that contain a polyA tail by RNA pulldown using polyT magnetic beads. Subsequent analysis of this fraction by LC-MS/MS allowed us to measure hm5C (Figure S6.2 in the Supporting Information). In these samples, the extent of C5-hydroxymethylation in polyA RNA was 40 times higher than in total RNA (Table S6.5). Currently, the function of m5C in mRNA and lncRNA is not clearly understood. However, the presence and levels of hm5C as an oxidative m5C metabolite in polyA RNA suggests that hm5C and m5C might be part of a dynamic regulatory mechanism. Further studies are needed to assess the functional roles of m5C and hm5C in RNA.


Formation and abundance of 5-hydroxymethylcytosine in RNA.

Huber SM, van Delft P, Mendil L, Bachman M, Smollett K, Werner F, Miska EA, Balasubramanian S - Chembiochem (2015)

Levels of m5C and hm5C across different model organisms given in amounts relative to the sum of (modified) cytosine residues.[20]
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4471624&req=5

fig01: Levels of m5C and hm5C across different model organisms given in amounts relative to the sum of (modified) cytosine residues.[20]
Mentions: We detected hm5C in all samples studied (Figure 1), thus confirming RNA C5-hydroxymethylation as a common RNA modification. Notably, hm5C was found in species that do not exhibit detectable hm5dC in their DNA and lack TET homologues in their genomes, such as C. elegans and A. thaliana.[22] This suggests that, in such organisms, the formation of hm5C in RNA must occur through a non-TET mechanism. As noted by Fu et al., their observation that TET- ES cells in which TET1, TET2, and TET3 are genetically deleted still exhibit significant hm5C levels in RNA also supports the idea that hm5C in RNA can form from pathways that do not involve TET enzymes.[15] Comparison of m5C oxidation into hm5C across the different species revealed varying levels (Figure 2). This could also indicate that RNA hydroxymethylation is the result of different enzymatic transformations. It will ultimately be essential to map the position of hm5C to specific RNAs in order to understand its function. As a step towards elucidating hm5C location, we explored the abundance of hm5C in RNA classes other than tRNA and rRNA. Specifically, we isolated mRNA and lncRNAs from HEK293T cells that contain a polyA tail by RNA pulldown using polyT magnetic beads. Subsequent analysis of this fraction by LC-MS/MS allowed us to measure hm5C (Figure S6.2 in the Supporting Information). In these samples, the extent of C5-hydroxymethylation in polyA RNA was 40 times higher than in total RNA (Table S6.5). Currently, the function of m5C in mRNA and lncRNA is not clearly understood. However, the presence and levels of hm5C as an oxidative m5C metabolite in polyA RNA suggests that hm5C and m5C might be part of a dynamic regulatory mechanism. Further studies are needed to assess the functional roles of m5C and hm5C in RNA.

Bottom Line: Herein, we describe an in vivo isotope-tracing methodology to demonstrate that the ribonucleoside 5-methylcytidine (m(5)C) is subject to oxidative processing in mammals, forming 5-hydroxymethylcytidine (hm(5)C) and 5-formylcytidine (f(5)C).Furthermore, we have identified hm(5)C in total RNA from all three domains of life and in polyA-enriched RNA fractions from mammalian cells.This suggests m(5)C oxidation is a conserved process that could have critical regulatory functions inside cells.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Chemistry, Lensfield Road, Cambridge, CB2 1EW (UK).

No MeSH data available.