Decoding human tRNA modifications and crosstalk by enhanced single-read analysis

tRNA modifications regulate gene expression and protein synthesis. Human tRNAome contains ~40 chemical modification types distributed on average at 13 sites in cytosolic tRNA and 6 sites in mitochondrial tRNA. tRNA modifications display dynamic patterns dependent on cell type and cell state, underscoring the need for advancing methodologies to assess their variations and crosstalks transcriptome-wide. Here, we develop enhanced single-read analysis of tRNA crosstalks (eSLAC), an integrative platform that combines the NGS sequencing method of multiplex small RNA sequencing (MSR-seq), expanded capability to measure pseudouridine (?), 5-formylcytidine (f5C), and N4-acetylcytidine (ac4C), and a single-read analysis pipeline that examines the modification-modification and modification-charging crosstalks. We achieve assessments of over 60% of all human tRNA modification sites and assign ? sites to three ? writer enzymes. Both ?-? and ?-charging have strong positive crosstalks. Applying polysome tRNA profiling, we identify differential tRNA isodecoder utilization and tRNA ? variations in the E-site versus A and P sites on the polysome. Our approach establishes a foundation for revealing the interconnectedness and the functional complexity of the tRNA modome. Overall design: Wild-type (WT) cells were analyzed with no treatment (NT), bisulfite treatment (BS), or picoborane treatment (PB) to assess baseline and chemically-induced modification signatures. To dissect the role of specific pseudouridine synthases, bisulfite-treated samples from PUS1, TRUB1, and PUS7 knockout (KO) lines were included. Polysome-associated and input (total) RNA fractions from WT cells were also analyzed with and without bisulfite treatment to evaluate ribosome-associated tRNA modifications. All conditions were performed with biological replicates to ensure robustness in differential modification detection.