Prokaryotic RNA N1-Methyladenosine Erasers Maintain tRNA m1A Modification Levels in Streptomyces venezuelae

tRNA modifications help maintain tRNA structure and facilitate translation and stress response. Found in all three kingdoms of life, m¹A tRNA modification occurs in the T loop of many tRNAs, stabilizes tertiary tRNA structure, and impacts translation. M¹A in the T loop is reversible by three mammalian demethylase enzymes, which bypasses the need of turning over the tRNA molecule to adjust its m¹A levels in cells. However, no prokaryotic tRNA demethylase enzyme has been identified that acts on endogenous RNA modifications. Using Streptomyces venezuelae as a model organism, we confirmed the presence and quantitative m¹A tRNA signatures using mass spectrometry and high-throughput tRNA sequencing. We identified two RNA demethylases that can remove m¹A in tRNA and validated the activity of a previously annotated tRNA m¹A writer. Using single-gene knockouts of these erasers and the m¹A writer, we found dynamic changes of m¹A levels in many tRNAs under stress conditions. Phenotypic characterization highlighted changes in their growth and altered antibiotic production. Our identification of the first prokaryotic tRNA demethylase enzyme paves the way for investigating new mechanisms of translational regulation in bacteria.