M1A within cytoplasmic mRNAs at single nucleotide resolution: A reconciled transcriptome-wide map

Following synthesis, RNA can be modified with over 100 chemically distinct modifications, and in recent years it was shown that processing, localization, stability and translation of mRNAs can be impacted by an increasing number of these modifications. A modification that recently gained attention is N1-methyladenosine (m1A), which is present across all three domains of life. Recently, two studies - one of them ours - developed conceptually similar approaches to map m1A in a transcriptome-wide manner and at single nucleotide resolution. Surprisingly, the two studies diverged quite substantially in terms of their estimates of the abundance, whereabouts and stoichiometry of this modification within internal sites in cytosolic mRNAs: One study reported it to be a very rare modification, present at very low stoichiometries, and invariably catalyzed by TRMT6/61A. The other found it to be present at >470 sites, in dozens of which at relatively high levels, and in the vast majority of cases these sites were highly unlikely to be substrates of TRMT6/61A, suggesting that additional methyltransferases are active on cytosolic mRNAs. Here we aim to reconcile the contradictions between the two studies, primarily by reanalyzing and re-annotating the set of sites identified in the latter study. We find that the vast majority of sites detected in this study originate from duplications, misannotations, mismapping, SNPs, sequencing errors, and a set of sites originating from the very first transcribed base ( TSS sites). We raise concerns as to whether the TSS sites truly reflect m1A originating from the first transcribed base. We find that only 53 of the sites detected in this study likely reflect bona-fide internal modifications of cytoplasmically encoded mRNA molecules. The vast majority of these are likely to be TRMT6/TRMT61A substrates, and are typically modified at low to undetectable levels. We conclude that within cytosolic mRNAs, m1A is a rare internal modification where it is typically catalyzed at ultra-low stoichiometries via TRMT6/TRMT61A. Our findings offer a clear and consistent view on the abundance and whereabouts of this modification, and lays out key directions for future studies in the field.