In vivo structure profiling reveals human cytosolic and mitochondrial tRNA structurome and interactome in response to stress.

Transfer RNA (tRNA) is the most abundant cellular RNA family in terms of copy numbers. It not only folds into defined structures but also has complex cellular interaction networks involving aminoacyl-tRNA synthetases, translation factors, and ribosomes. The human tRNAome is comprised of chromosomal-encoded tRNAs with a large sequence diversity and mitochondrial-encoded tRNAs with A/U-rich sequences and noncanonical tertiary interactions. How tRNA folding and interactions in a eukaryotic cell respond to stress is poorly understood. Here, we develop DM-DMS-MaPseq, which utilizes in vivo dimethyl-sulfate (DMS) chemical probing and mutational profiling (MaP) coupled with demethylase (DM) treatment in transcriptome-wide tRNA sequencing to profile structures and the cellular interactions of human chromosomal and mitochondrial-encoded tRNAs. We found that tRNAs maintain stable structures in vivo, but the in vivo DMS profiles are vastly different from those in vitro, which can be explained by their interactions with cellular proteins and the ribosome. We also identify cytosolic and mitochondrial tRNA structure and interaction changes upon arsenite treatment, a type of oxidative stress that induces translational reprogramming, which is consistent with global translation repression in both compartments. Our results reveal variations of tRNA structurome and dynamic interactome that have functional consequences in translational regulation.