The presence of an N1 methyl group on adenine bases in DNA and RNA was thought to be a form of damage. Results now show that it also occurs at specific sites in messenger RNAs, where it affects protein expression. See Article p.441
The fact that chemical modifications to DNA bases can alter gene expression without changing the nucleic-acid sequence has been known for more than a decade. But the key regulatory function of many such epigenetic modifications to messenger RNA molecules has been recognized only recently1,2,3. A simple mark — the methyl group — is widely observed in DNA and its associated histone proteins, and has been studied in mRNA in the forms of 5-methylcytosine and N6-methyladenosine. On page 441 of this issue, Dominissini et al.4 present a new member of the mRNA methyl-marked family, N1-methyladenosine, and propose that its presence in mRNAs has an influence on biological processes. What is surprising about this modification is that it has previously been described as a form of cellular damage5.
N1-methyladenosine (m1A) is unusual in having a positive charge at physiological pH (other bases are uncharged) and a methyl group that blocks the Watson–Crick base-pairing edge of adenine. The modification was previously documented in transfer RNA molecules, where it plays a crucial part in the formation of tertiary RNA structure. The methyl group forces the m1A base to pair with a non-Watson–Crick configuration6, and the positive charge has also been hypothesized to exert an electrostatic influence on protein interactions7. Dominissini et al. propose that this base modification may have similar biophysical effects in mRNAs: it could affect base-pairing interactions close to the site at which protein translation starts, and might alter RNA folding and electrostatic interactions.
m1A was previously known to be formed by the exposure of RNA molecules to alkylating agents, and under alkaline conditions it is converted to N6-methyladenosine (m6A) through the Dimroth rearrangement8 (Fig. 1). This is potentially a big problem in analysis, because m6A is a considerably more abundant modification in mRNA, and thus obscures the rarer m1A. Dominissini et al. found methods to suppress this confounding chemical instability of m1A. Through carefully controlled sample preparation, they reduced the incidence of the m1A-to-m6A rearrangement to less than 10%, and the use of an isotopically labelled internal control during mass-spectrometry analysis allowed them to accurately quantify m1A. These techniques enabled the researchers to identify the overall amounts of the modification in cells, and to map its locations in mRNAs.
The authors find that, although the N1 methylation of adenine occurs several times less often than N6 methylation, it is still found in thousands of mRNAs. They note an enrichment of m1A signals near the translational start site of these mRNAs, and most of the molecules analysed were methylated only once, suggesting that the group has a single role in a given RNA. In the same region, they found sequences rich in the bases guanine and cytosine; such GC-rich motifs correlate with thermodynamically stable secondary RNA structure, which could be involved in directing the placement of m1A. Moreover, the authors' mapping of m1A across all RNA transcripts in the cell (the transcriptome) revealed that the modification in mRNAs is associated with higher levels of proteins.
What role does m1A have in the cell? Dominissini et al. found that the fraction of mRNAs containing m1A correlates positively with the level of gene expression, and may affect changes in cellular metabolism. The data also suggest the presence of a greater number of alternative translation initiation sites in the methylated RNAs than in RNAs lacking this modification. Most notable was the finding that m1A-modified RNAs produced 1.7-fold higher protein levels than did non-methylated ones. Deeper analysis of the translation process led the authors to suggest that m1A is involved in the processes by which pre-mRNA molecules are 'spliced' to form mature mRNAs. Thus, influencing protein expression seems to be one of the strongest effects of this methyl mark.
Importantly, the m1A pattern in yeast RNAs differed from that in human and mouse RNAs: in yeast, the mark was distributed across the coding sequence without preferred locations, suggesting that more-sophisticated organisms have evolved m1A marking as a distinct pathway. Comparing mouse and human mRNAs, the authors observed that the positions of m1A modification showed 33% conservation, and that N1-methylation in 5′-untranslated regions and close to the start site exhibited even higher conservation. The researchers also demonstrated that the m1A level varies significantly between different mouse tissues. Furthermore, exposing cultured cells to different stress conditions resulted in changes in the m1A level, which suggests that m1A is a dynamic modification with a role in cellular stress responses and signalling processes.
A key question raised by these findings is how the cell differentiates between damage and intentional, stable cellular markers. When is m1A a 'bug', and when is it a useful feature? Methylation of adenine at position 1 is well documented to arise from exposure to chemical methylating agents9 (Fig. 1), and thus 1-methyladenine has been widely studied as a form of damage in DNA and RNA. At least one enzyme (ALKBH3) has been documented to remove this lesion and reduce its cytotoxicity10. A recent study that also documents m1A in non-damaged mRNAs shows explicitly that this 'repair' enzyme can remove much of this methyl mark11. It will be important to determine how the stable m1A mark is recognized as being distinct from that resulting from alkylation damage, and thus protected from immediate repair. And if m1A is truly dynamic, in the sense of being specifically placed and removed from existing RNA as a cellular switch, then what methylase enzyme puts the mark in place, and what enzymes — ALKBH3 or other — remove it?
Dominissini and colleagues' findings represent an intriguing step for epitranscriptomics. More work is needed to understand the mechanism by which m1A influences translation initiation and regulation, as well as the changes in its levels in stress responses. It will be exciting to see how this modification affects, and is affected by, RNA structure. Moreover, we need to know about the hypothesized proteins that are 'writers', 'readers' and 'erasers' of m1A and thus take part in the dynamics of its regulation. Finally, as yet another modified base has been identified in mRNAs, one wonders how many more exist: is this the final one, or are we just seeing the tip of the epitranscriptome iceberg?Footnote 1
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Edelheit, S., Schwartz, S., Mumbach, M. R., Wurtzel, O. & Sorek, R. PLoS Genet. 9, e1003602 (2013).
Dominissini, D. et al. Nature 530, 441–446 (2016).
Mishima, E. et al. J. Am. Soc. Nephrol. 25, 2316–2326 (2014).
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