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 (Ψ), 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 Ψ sites to three Ψ writer enzymes. Both Ψ-Ψ and Ψ-charging have strong positive crosstalks. Applying polysome tRNA profiling, we identify differential tRNA isodecoder utilization and tRNA Ψ 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.