Aberrant DNA N⁶‐methyladenine incorporation via adenylate kinase 1 is suppressed by ADAL deaminase‐dependent 2′‐deoxynucleotide pool sanitation

Abstract

Intracellular decay of N6‐methyladenine (m6A)‐containing RNA potentially induces aberrant N6‐methyl‐2′‐adenine (6mdA) misincorporation into DNA. Biophysically, misincorporated 6mdA may destabilize the DNA duplex in a manner similar to bona fide methylated 6mdA DNA, thereby affecting DNA replication and transcription. Utilizing heavy stable isotope labeling and ultrasensitive UHPLC–MS/MS assay, we demonstrate that intracellular m6A‐RNA decay does not generate free 6mdA species, nor lead to any misincorporated DNA 6mdA in most mammalian cell lines tested, unveiling the existence of a sanitation mechanism that prevents 6mdA misincorporation. Depletion of deaminase ADAL increases the levels of free 6mdA species, concomitant with the presence of DNA‐misincorporated 6mdA resulting from intracellular RNA m6A decay, suggesting that ADAL catabolizes 6mdAMP in vivo. Furthermore, we show that the overexpression of adenylate kinase 1 (AK1) promotes 6mdA misincorporation, while AK1 knockdown diminishes 6mdA incorporation, in ADAL‐deficient cells. We conclude that ADAL together with other factors (such as MTH1) contributes to 2′‐deoxynucleotide pool sanitation in most cells but compromised sanitation (e.g., in NIH3T3 cells) and increased AK1 expression may facilitate aberrant 6mdA incorporation. This sanitation mechanism may provide a framework for the maintenance of the epigenetic 6mdA landscape.

Randomly incorporated genomic N6‐methyldeoxyadenosine delays zygotic transcription initiation in a cnidarian

“…Next, we injected m6ATP into zygotes and observed higher level of 6mA in the nuclei of 32-cell embryos compared with ATP-injected or ATPuntreated embryos (Fig 5C), consistent with studies done in mammalian cells, showing that m6A ribonucleotides can be converted to 6mA deoxyribonucleotides and incorporated into the genome through replication that is conserved in eukaryotes(Musheev et al, 2020;Liu et al, 2021). Finally, we found that knocking down the Hydractinia Adal1, homologs of which convert m6A ribonucleotides into inosine in plants and mammals(Chen et al, 2018b;Chen et al, 2023), also delayed EU incorporation beyond the 64-cell stage(Fig EV3E). Since Adal1 only acts on monomeric m6A, this experiment provides further evidence for the RNA degradation scenario over demethylation.Taken together, we conclude that m6A-marked RNAs are degraded into nucleotide form.…”

A sequential, RNA-derived, modified adenosine pathway safeguards cellular metabolism

“…Recently, murine ADAL was shown to catalyze deamination of DNA-derived 2-deoxy- N 6 -methyladenosine monophosphate (6mdAMP), which might potentially induce aberrant epigenetic markers via modified N 6 -methyadenosine di- or triphosphoate 56 . Using in vitro kinase assays, we showed that m 6 AMP, m 6,6 AMP, and i 6 AMP are barely converted to m 6 AD/TP, m 6,6 AD/TP, and i 6 AD/TP by AK (Fig.…”

“…Extensive studies on m 6 A in recent years identified its intracellular role in RNA stability, translation, and microRNA biogenesis 14,53,54 , but the extracellular role of m 6 A has been elusive. The presence of m 6 AMP was reported in plants and mammals 31,55,56 , but it was simply considered an RNA degradation product. Since lysosome is one of the major RNA-degrading organelles, m 6 AMP will be quickly dephosphorylated to m 6 A by lysosomal phosphatase, and since m 6 AMP cannot be formed in ADK-deficient cells, it is likely that most intracellular m 6 AMP is generated by ADK-mediated phosphorylation of RNA catabolism-derived m 6 A.…”