Barry, M. L.; Abu-Shumays, R. L.; Barnes, L. E.; Shaw, E. A.; Reinsch, J. L.; Vaaler, A. L.; Basham, Z. D.; Jain, M.; Koutmou, K. S.; Garcia, D. M.

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 even in a widely used model organism, the budding yeast Saccharomyces cerevisiae, the positions of all pseudouridines in tRNA have not been completely annotated. Using Nanopore direct RNA sequencing (DRS), an established method for detecting RNA pseudouridylation positions, we sequenced cytosolic tRNA from eight pseudouridine synthase (PUS) knockout S. cerevisiae strains, including deletion strains of Pus1, Pus3, and Pus7. Analysis of these data verified thirty-four existing pseudouridine annotations and predicted eleven previously unannotated pseudouridine sites. Our analysis 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. LC-MS/MS and primer extension assays, however, indicated no change in the abundance of these modifications with the loss of Pus3. Our analysis underscores the need for caution in interpreting DRS-based signal changes, particularly in modification-dense regions. Combining existing modification annotations for the thirty-one isoacceptors in the Modomics database with our dataset that added annotations for the remaining eleven isoacceptors, we created a map of all detected pseudouridines, and the enzymes responsible for their catalysis, across the forty-two S. cerevisiae cytosolic tRNA isoacceptors.