Investigation of TRMT61B methyltransferase activity on mRNA and its effects on translation

Despite recent advances in technology to map RNA chemical modifications transcriptome-wide, the distribution of N1-methyladenosine (m1A) in messenger RNA (mRNA) remains contested, hindering a clear understanding of its function. Additionally, the enzyme(s) that installs the majority of reported mRNA m1A sites has yet to be identified. In this study, we characterized TRMT61B, an m1A methyltransferase known to methylate mitochondrial RNAs, but whose sequence preferences have been underexplored. By integrating cellular overexpression of TRMT61B and in vitro methylation of a synthetic pool of diverse human mRNA sequences, we identified a preference for a YMRA consensus motif in single-stranded RNA regions. In these experiments, TRMT61B methylated thousands of novel human mRNA sites, revealing activity on cytosolic mRNAs. We used these novel m1A-modifiable sequences to test the effects of m1A on translation of luciferase reporters and on ribosome recruitment to modified transcripts in the pool. We found that m1A addition can significantly affect translation and ribosome recruitment, but that these effects vary by transcript. Taken together, our results inform future studies of TRMT61B and mRNA modifications, and emphasize that studies of m1A regulation of mRNA must be carried out and interpreted in a highly context-aware manner.