Abstract
Transfer RNAs (tRNAs) are central to protein synthesis and are increasingly recognized as dynamic regulators of gene expression whose abundance and chemical modifications are subject to precise biological control. Here, we systematically investigate how two distinct dietary interventions, low-protein and high-fat diets, reshape the tRNA landscape across multiple mouse tissues, using RNA mass spectrometry and Ordered Two-Template Relay sequencing (OTTR-seq) to comprehensively profile cytosolic and mitochondrial tRNAs at single-nucleotide resolution. We reveal pronounced tissue-specific biases in tRNA isodecoder expression, including the unexpected presence of full-length cytosolic tRNAs in mature sperm with a distinct isotype composition. In somatic tissues such as liver and heart, dietary conditions alter both tRNA abundance and key modifications known to regulate decoding efficiency, whereas in reproductive tissues diet primarily affects the abundance of select tRNAs with comparatively limited changes in modification profiles. We further demonstrate that mitochondrial tRNAs are subject to diet-responsive changes in both abundance and modification status, and that even subtle differences in dietary fat composition are sufficient to alter tRNA modification signatures. Together, these findings establish the tRNA epitranscriptome as a sensitive and tissue-specific sensor of nutritional state, and provide a resource for understanding how dietary cues interface with translational regulation in somatic and reproductive tissues.