Barnes, L. E. | bioRxiv Science

bioRxiv2026

Mapping the rRNA methylome reveals contributions of methyltransferases to ribosome function and antibiotic sensitivity

Ribosomal RNA (rRNA) folds into a complex structure used as the macromolecular core for protein synthesis. Chemical modification of rRNA contributes to ribosome structure, function, and susceptibility to antibiotics. Despite their importance, the enzymes responsible for specific rRNA modifications remain unknown in most species. In this work, we integrate genetics and biochemistry with sequencing and mass spectrometry to uncover enzymes responsible for rRNA methylation events in Bacillus subtilis. We characterize 17 enzymes responsible for 20 methylation modifications on the 16S and 23S rRNAs, 11 of which are encoded by previously uncharacterized genes. For each rRNA methyltransferase, we define the modification identity, location, and we determine the impact of loss of cognate rRNA methylation on ribosome biogenesis and antibiotic sensitivity. Our findings demonstrate that loss of nearly half of the 17 genes studied results in alterations to ribosome assembly or antibiotic sensitivity underscoring the importance of chemical modifications to ribosome function.

biochemistry↗

bioRxiv2026

Pseudouridylation landscape across 42 S. cerevisiae cytosolic tRNA isoacceptors via Nanopore direct RNA sequencing

Pseudouridine is the most abundant RNA base modification due to its prevalence in tRNA and rRNA, where it serves as a key modulator of structure and function. Yet, in the widely used model organism, the budding yeast Saccharomyces cerevisiae, tRNA pseudouridine sites have not been comprehensively annotated. Here, we performed two high-throughput methods established for detecting tRNA pseudouridylation positions: Nanopore direct RNA sequencing (DRS) and 2-bromoacrylamide-assisted cyclization sequencing (BACS). Using DRS, we sequenced cytosolic tRNA from seven pseudouridine synthase (PUS) knockout S. cerevisiae strains, including deletion strains of Pus1, Pus3, and Pus7. Analysis of these data verified thirty-two existing pseudouridine annotations and predicted an additional ten previously unannotated pseudouridine sites. Our analysis also revealed DRS signal changes at several non-uridine sites with the loss of a PUS, including apparent changes in modification abundances at position 37 upon deletion of Pus3. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and primer extension assays, however, indicated no change in the abundance of these modifications with the loss of Pus3, underscoring the need for caution in interpreting DRS-based signal changes. Using BACS, we confirmed the ten DRS-predicted novel sites, while further detecting eleven additional previously unannotated pseudouridine sites. Combining existing modification annotations from the Modomics database with our DRS and BACS datasets, we created a map of all detected pseudouridines --totaling 126 sites, including 21 novel sites--and the enzymes responsible for their catalysis, across the forty-two nuclear-encoded S. cerevisiae tRNA isoacceptors.

molecular biology↗