Single-cell transfer RNA sequencing reveals principles of translational regulation

Transfer RNAs (tRNAs) decode genetic instructions from messenger RNA (mRNA) into protein, yet their abundance and regulation across cell states remain largely undefined, as multimodal measures of cellular tRNAs have remained elusive. Here, we develop a massively parallel assay for single-cell translatome and RNA sequencing (sctRNA-seq), which profiles transcriptome-wide mRNA and tRNA abundance simultaneously and chart the tRNA composition of 364,841 cells. Through multimodal perturbation screening of protein-coding genes and tRNA loci, we reveal dynamics across the mRNA and tRNA transcriptomes, delineating distinct tRNA-linked modules regulated by metabolic and Pol III-mediated transcription. Further, we uncover a close correspondence between codon demand and tRNA-mediated anticodon composition during hematopoietic multilineage commitment and differentiation. Tri-modal profiling of mRNA, tRNA, and proteins from primary human T cells reveals tRNA-mediated regulation that partially explains the discordance between mRNA and protein levels, particularly for selenoproteins and transcription factors. Finally, we uncover a stress-responsive tRNA module defined by gene-internal promoter sequences. Together, our analyses resolve tRNAs as a previously undefined layer of translational control underlying cellular identity and cell-state transitions.