Combining Nanopore direct RNA sequencing with molecular genetics and mass spectrometry for analysis of T-loop base modifications across 42 yeast tRNA isoacceptors

Transfer RNAs (tRNAs) contain dozens of chemical modifications. These modifications are critical for maintaining tRNA tertiary structure and optimizing protein synthesis. Here we advance the use of Nanopore direct RNA-sequencing (DRS) to investigate the synergy between modifications that are known to stabilize tRNA structure. We sequenced the 42 cytosolic tRNA isoacceptors from wild-type yeast and five tRNA-modifying enzyme knockout mutants. These data permitted comprehensive analysis of three neighboring and conserved modifications in T-loops: 5-methyluridine (m5U54), pseudouridine (?55), and 1-methyladenosine (m1A58). We validated our results using direct measurements of chemical modifications by mass spectrometry. We observed concerted T-loop modification circuits—the potent influence of ?55 for subsequent m1A58 modification on more tRNA isoacceptors than previously observed. We also observed a novel condition-specific increase in m1A58 modification on some tRNAs upon nutrient depletion. We developed a global and isoacceptor-specific classification strategy to predict the status of T-loop modifications from a user-input tRNA DRS dataset, applicable to other conditions and tRNAs in other organisms. Finally, we designed a modified library preparation strategy for forthcoming direct ionic current analysis. These advancements demonstrate how orthogonal technologies combined with genetics enable precise detection of modification landscapes of individual, full-length tRNAs, at transcriptome-scale.