Unique Ada deamination-mediated [ 15 N 5 ]-rA labeling. In this work, one heavy stable isotope-labeling strategy involved the use of the nucleoside [15N5]-rA. [15N5]-rA could be utilized for RNA synthesis, as indicated by the observation of [15N5]-rA in RNA. Interestingly, in addition to RNA [15N5]-rA, we also observed a new but major form, [15N4]-rA, in RNA. Likely, the presence of RNA [15N4]-rA should be associated with the deamination activity of Ada. Indeed, after inhibiting Ada activity using the specific inhibitor DCF, we observed an increase in RNA [15N5]-rA (data not shown). The observations support that the formation of [15N4]-rA is associated with the deamination activity of Ada. Essentially, we showed an Ada deamination-mediated labeling strategy that is unique for Ada-containing mammalian cells.
Noteworthy, in [15N5]-rA-treated cells, we observed efficient labeling of mRNA m6A, too. Consistent with the observation of the two forms of labeled rA ([15N4]- and [15N5]-rA) in RNA, we also observed two forms of labeled RNA m6A ([15N4]- and [15N5]-m6A). Upon Ada inhibition, we observed an increase in [15N5]-m6A and a concomitant decrease in [15N4]-m6A (Fig. S3C). As the METTL3/METTL14 methyltransferase complex is solely responsible for mediating the N6-methylation of mRNA adenine48, the labeled mRNA m6A should result from the methylation of [15N4]-rA and [15N5]-rA in mRNA by the METTL3/METTL14 complex. Indeed, upon the depletion of Mettl3, mRNA m6A lost, and we did not observe any labeled m6A in mRNA (data not shown). Taken together, intracellular RNA m6A can be traced using heavy stable isotope labeled rA.
Intracellular RNA m6A degradation cannot induce misincorporated DNA i6mA. By the use of the efficient [15N5]-rA labeling strategy, intracellular RNA m6A can be efficiently labeled. If the degraded RNA m6A could be reformulated to generate 6mdATP via purine salvage pathway, we should observe labeled DNA 6mA. However, we did not observe any labeled DNA 6mA over 7–50 days of treatment, indicating the absence of misincorporated DNA i6mA. Although it was reported that DNA misincorporation occurred at a delayed phase (approximately 5-day delay) in comparison to RNA labeling41, our observation on the absence of labeled DNA 6mA (or misincorporated i6mA) over 50 days of treatment proves that the m6A nucleotides generated from RNA m6A decay are not reutilized in DNA replication. By the use of the second strategy involved [D3]-L-methionine labeling, we also observed the labeled RNA m6A ([D3]-m6A), but consistently did not observe any labeled DNA 6mA after 7 days of treatment. Notably, both labeling strategies showed high labeling efficiency (> 60%). Collectively, our data strongly support that intracellular RNA m6A decay cannot induce any misincorporated DNA i6mA at least in the tested cells.
Checkpoint, ADAL, and AK1. Given the capacity of DNA polymerases to incorporate 6mdATP13 and the absence of the labeled DNA 6mA (also misincorporated i6mA herein), we infer that the degradation products of RNA m6A cannot be ultimately converted into 6mdATP via the purine salvage pathway. This inference drives us to propose that a checkpoint functioning similar to that in the cell cycle49,50 and in the immunological response51–53 exists for suppressing DNA misincorporation along with RNA m6A degradation. Essentially, we identified two players in this proposed checkpoint, ADAL and AK1.
The depletion of the deaminase Adal by either knockdown or knockout resulted in the presence of the labeled DNA 6mA. In contrast, the depletion of the mRNA m6A demethylase FTO or ALKBH5 did not break the checkpoint, nor did overexpression of the kinases involved in the purine salvage pathway alone. All these findings support the idea that ADAL plays a pivotal role in the checkpoint. Mechanistically, ADAL simultaneously catabolizes the methylated nucleotides m6AMP and 6mdAMP to the nontoxic nucleotides IMP and dIMP, respectively, reducing the misincorporation of N6-methyladenine via DNA polymerase; thus, the hydrolysis of both m6AMP and 6mdAMP by ADAL contributes critically to the blockade of replication-dependent 6mA incorporation. Moreover, the catabolizing activity of ADAL toward 6mdAMP is 2.14-fold as high as that toward m6AMP. Of note, here we showed, for the first time, the activity of ADAL in catabolizing 6mdAMP. Collectively, our data indicate that ADAL functions as a key checkpoint enzyme to block the catabolic conversion of intracellular RNA m6A to genomic DNA 6mA by catabolizing both m6AMP and 6mdAMP, particularly the latter.
In addition to the deaminase ADAL, adenylate kinase 1 (AK1) was identified as a protein involved in the checkpoint. Overexpression of AK1 did not induce any detectable labeling of DNA i6mA in the presence of sufficient ADAL but increased the labeled DNA i6mA in the absence of ADAL. This finding suggests that the phosphorylation of 6mdAMP into 6mdADP is a much slower process than the hydrolysis of 6mdAMP mediated by ADAL. In other words, the preferential substrate for AK1 is not 6mdAMP but dAMP, which is required for DNA synthesis. Overexpression of AK1 can partially compensate for the slow phosphorylation of 6mdAMP by AK1. The slow phosphorylation process would provide a time window long enough for ADAL to catabolize the generated 6mdAMP completely.
Source of the unlabeled DNA 6mA. As shown in this work, in mES cells treated with heavy stable isotope tracers, we found large proportions of the labeled m6A in both mRNA and total RNA, but no labeled genomic 6mA was detected. However, trace amounts of the unlabeled 6mA were detected. This finding is consistent with our recent work13. The observed unlabeled DNA 6mA was formed through polymerase-dependent misincorporation13,41. However, the source for generating the unlabeled 6mA was not known yet. In this work, we clearly showed that the observed DNA 6mA did not originate from intracellular RNA m6A, as described above. Because more than three-quarters of the m6A modifications were labeled, it is impossible that only unlabeled RNA m6A was incorporated into genomic DNA, and the labeled m6A could not be incorporated via the purine salvage pathway. The absence of the labeled DNA 6mA also excludes an origin from methylase-deposited methylation. We speculate that the observed DNA i6mA originated from catabolism of extracellular RNA m6A. Indeed, we detected urinary m6A nucleosides at high abundance (data not published), suggesting the existence of free m6A in extracellular spaces in humans. Meanwhile, by the extracellular exposure of heavy stable isotope-labeled m6A nucleoside, we clearly showed that exogenous m6A nucleoside can induce misincorporated DNA 6mA and such a process is also restricted by ADAL-pivoted checkpoint. It is reasonable that, under physiological conditions, both intracellular and extracellular RNA m6A are tightly regulated by the catabolism checkpoint. Biochemically, the N6-methyladenine modification on the DNA template disturbs base pairing54,55, hinders new chain extension, affects replication24 and transcription23, and regulates related gene expression22,54. Therefore, a strict catabolism checkpoint of N6-methyladenine is required to maintain genome integrity.
Biological implications. The expression of ADAL varies in different cells. It is expected that misincorporated i6mA could be observed in cells that express ADAL at low levels. Musheev et al41 reported that C2C12 and especially NIH 3T3 cells had exceptionally high levels of misincorporated i6mA, 13–500 i6mA per million dA. However, by our experiments on C2C12 and NIH 3T3 cells, we observed i6mA at a level of 100 folds lower (Fig. S6). We did not detect misincorporated i6mA in 293T and mES cells. Collectively, the misincorporated i6mA must be strictly controlled.
Our observation on the blockade of DNA 6mA misincorporation strongly suggests that the epigenetic DNA 6mA landscape should be tightly maintained in the tested cells. Essentially, the findings on epigenetic mark DNA 6mA remain elusive due to the neck-of-bottle of analytical technologies for 6mA detection, the contamination of coexisting bacteria carrying abundant 6mA, poor LC-MS/MS skill, and a lack of reliable 6mA sequencing technology56. However, it is still very surprising that misincorporated i6mA must be strictly controlled, while epigenetic pr6mA is rarely found in human cells. These conflicting observations may hint that true epigenetic 6mA (methylase-deposited 6mA) should have extremely important functions and appear in every cell in certain scenarios, e.g., responses to certain stimuli. Otherwise, the mammalian cells should not set such a strict bar for misincorporation. Interestingly, as the most important and abundant epigenetic mark, misincorporation of 5mC must also be strictly controlled57–59. In contrast, DNA 5-hydroxymethylcytosine (5hmC) is found only in limited cells at moderate abundance and can be misincorporated into genomic DNA60. On the other hand, low expression of Adal may shorten the survival of cancer patients (Fig. 6). These data and underlying logics allow us to infer that the ADAL-pivoted checkpoint and the suppression of i6mA misincorporation have biological importance, at least in tumor development and therapy.
RNA m6A checkpoint in the purine salvage pathway. In the free nucleotide pool, free m6AMP can be recycled by rephosphorylation via nucleotide kinases and further reduced to N6-methyldeoxyadenosine diphosphate (6mdADP) by ribonucleotide reductase. 6mdADP is next transformed into 6mdATP, which is utilized by DNA polymerases, forming DNA i6mA. Our results indicated that the substantially labeled RNA m6A in mES cells was not transformed and incorporated into genomic DNA. Taken together, our findings led us to propose a catabolic checkpoint of N6-methyladenine along with the purine pathway (Fig. S7). This checkpoint functions to block potential DNA 6mA misincorporation via ADAL-mediated hydrolysis of both m6AMP and 6mdAMP, AK1 mediated restriction on the phosphorylation of 6mdAMP, and other unknown steps.
In this work, by exploiting unique heavy stable isotope labeling technology coupled with sensitive UHPLC-MS/MS analysis, we demonstrated that ADAL catabolizes m6AMP and, more efficiently, 6mdAMP to nontoxic nucleotides and blocks DNA 6mA misincorporation. In addition, adenylate kinase 1 also contributes to the suppression of DNA 6mA misincorporation by limiting the rate of 6mdAMP phosphorylation, which may provide adequate time for ADAL to catabolize 6mdAMP. Therefore, ADAL and AK1 together suppress DNA 6mA misincorporation and maintain genomic epigenetic integrity.