Variable presence of 5-methylcytosine in commercial RNA and DNA

Schmid, Katharina; Thüring, Kathrin; Keller, Patrick; Ochel, Antonia; Kellner, Stefanie; Helm, Mark

doi:10.1080/15476286.2015.1076612

Cited by 15 publications

(13 citation statements)

References 33 publications

(32 reference statements)

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“…However, practically, many modified nucleotides remain commercially unavailable, which necessitates (often) complicated chemical synthesis by expert laboratories. Furthermore, commercially available (unmodified) RNAs appear to contain trace amounts of modified nucleotides [58], which might affect experimental outcomes when testing synthetic or synthetically modified tsRNAs for structure-function relationships.…”

Section: Discussionmentioning

confidence: 99%

Production and purification of endogenously modified tRNA-derived small RNAs

et al. 2020

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2020): Production and purification of endogenously modified tRNA-derived small RNAs, RNA Biology, ABSTRACT During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question as to how such limited molecule numbers could exert measurable biological impact. While the molecular function of individual tsRNAs is likely mediated through association with other molecules, addressing the interactome of specific tsRNAs has only been attempted by using synthetic RNA sequences. Since tRNAs carry post-transcriptional modifications, tsRNAs are likely modified but the extent of their modifications remains largely unknown. Here, we developed a biochemical framework for the production and purification of specific tsRNAs using human cells. Preparative scale purification of tsRNAs from biological sources should facilitate experimentally addressing as to how exactly these small RNAs mediate the multitude of reported molecular functions. ARTICLE HISTORY

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Section: Discussionmentioning

confidence: 99%

Production and purification of endogenously modified tRNA-derived small RNAs

et al. 2020

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“…N 6 -methyldeoxyadenosine (m 6 dA) content in the S. cerevisiae DNA digest was calibrated by a nucleoside standard (Sigma Aldrich, Steinheim, Germany). Quantification of 5-methyldeoxycytidine (m 5 dC) was achieved via a chemically synthesized triple deuterated internal standard as described recently3031.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive DNA methylation analysis of the Aedes aegypti genome

Falckenhayn

Carneiro

Amarante

et al. 2016

Sci Rep

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Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation.

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“…for inadvertent measurement of contaminating methyltransferase activities. Methods that do confirm the identity of a given modified nucleoside by biochemical or biophysical means have shifted from the use of thin-layer chromatography (TLC, 20 ) to high performance liquid chromatography coupled to mass spectrometry (LC-MS, 21 ). Moreover, powerful methods have emerged that use the specific chemical properties of m 5 C to detect its occurrence in a transcriptome-wide sequence context.…”

Section: Part 1: Phylogeny Structure and Biochemistry Of Dnmt2mentioning

confidence: 99%

Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation

et al. 2016

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A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics.

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Variable presence of 5-methylcytosine in commercial RNA and DNA

Cited by 15 publications

References 33 publications

Production and purification of endogenously modified tRNA-derived small RNAs

Production and purification of endogenously modified tRNA-derived small RNAs

Comprehensive DNA methylation analysis of the Aedes aegypti genome

Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation

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