Prokaryotic N1-methyladenosine erasers maintain tRNA m1A modification levels in Streptomyces venezuelae.

tRNA modifications help maintain tRNA structure and facilitate stress responses. Found in all three kingdoms of life, m1A tRNA modification occurs in the T loop of many tRNAs and stabilizes tertiary tRNA structure and impacts translation. M1A in T loop is known to be reversible by three mammalian demethylase enzymes, which bypasses the need of turning over the tRNA molecule to adjust their m1A levels in cells. However, no prokaryotic tRNA demethylase enzyme has been identified. 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 confirmed the activity of a previously annotated tRNA m1A writer. Using single gene knockouts of these erasers and the m1A writer, we subjected these strains to stress conditions and found dynamic changes to m1A levels in many tRNAs. Phenotypic characterization highlighted changes to their growth and altered antibiotic production. Our identification of the first prokaryotic tRNA demethylase enzyme paves the way for investigating new mechanisms in global translational regulation in bacteria. To investigate the changes to m1A modification levels across tRNA populations in Streptomyces venezuelae. Streptomyces venezuelae strains were grown in control and stress conditions, after which RNA was collected from each condition and underwent MSR-seq. Generated libraries were sequenced using Illumina technology. After initial processing of reads, libraries were aligned to a curated tRNA reference file. Associated tRNA properties (modification, charging and abundance) were calculated using a custom pipeline using Python and R scripts.