Defining the Role of METTL5 in m6A Deposition: No Evidence for mRNA Methylation In Vivo

RNA methylations have emerged as key regulators of all aspects of RNA function, including RNA stability and translation. The most abundant internal mRNA modification is N6-methyladenosine (m6A). So far, two enzymes (METTL3 and METTL16) have been identified that can deposit m6A on mRNAs. In addition, we and others previously identified METTL5 as an enzyme that catalyses m6A on rRNAs. Our previous work revealed intriguing metabolic phenotypes of METTL5 knockout models. Interestingly, previous in vitro studies suggested an additional activity of METTL5 on mRNA.

In our recent work, we addressed the question of whether METTL5 can deposit m6A on mRNAs in vivo and thus regulate mRNA function. For this, we investigated the impact of METTL5 loss on m6A profiles in mESCs and applied Oxford Nanopore (ONT)-based direct mRNA sequencing and m6A calling.

Applying this approach to METTL5 knockout cell lines, we found no evidence for METTL5-mediated m6A deposition on mRNA in vivo, demonstrating that its catalytic activity is indeed restricted to rRNA. Thus, our quantitative m6A profiling provides clarity on a key question regarding the enzymes responsible for catalysing m6A deposition on mRNA, a central regulator of RNA function.

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