ABSTRACT: Transfer RNAs (tRNAs) and their derived small RNAs (tDRs) vary across mammalian tissues, but the . We applied Ordered Two-Template Relay sequencing (OTTR-seq) to profile small RNAs in 19 tissues from adult mice, quantifying mature tRNAs and tDRs at isodecoder resolution and inferring select base‑modification signatures from reverse‑transcriptase misincorporations. We observe tissue‑biased expression of many isodecoders and identify tDRs with greater tissue specificity than their parent tRNAs. Modification‑associated mismatch profiles are largely consistent across tissues, with notable isodecoder‑specific differences at canonical sites (e.g., A34→I, G37→m1G/wybutosine, C47d→m3C). Because RT‑based signatures do not capture all modifications without chemical pretreatments, our modification inferences represent a subset of the tRNA epitranscriptome. These data provide an isodecoder‑resolved reference for tissue‑biased tRNA and tDR regulation in mice and a framework for exploring disease‑associated remodeling of the tRNA pool. Recent studies have begun to illustrate that transfer RNA (tRNA) expression, processing, and modification can vary among different mammalian tissues and cell types. The full scope of these differences and the regulatory mechanisms that modulate production of a wide variety of tRNAs and tDRs (tRNA-derived small RNAs) across tissues, cell types, and changing cellular conditions is poorly understood. Dysregulation of these transcripts has increasingly been implicated as a critical factor in disease progression; thus, a deeper understanding of the homeostatic variance across somatic tissues is vital. Here, we systematically profile the relative changes in abundance and key sites of base modification within tRNAs, tDRs, and other small RNAs using a powerful small RNA sequencing method, Ordered Two-Template Relay sequencing (OTTR-seq), across a panel of 19 mouse tissues. By analyzing a diverse range of healthy tissues, we can better recognize tissue-biased regulation of tRNAs and identify tDRs that exhibit significantly greater tissue specificity than their corresponding source tRNAs. Furthermore, we leveraged reverse transcriptase-associated base misincorporations to construct an isodecoder-specific map of select nucleotide modifications across all mouse tRNAs. Differentially modified isodecoders can alter their translation functionality, stability, and basis for production of unique tDR profiles. This dataset provides the most comprehensive view to date of the dynamic relationships between tRNAs, their modifications, and tDRs in a mammalian model, offering an important knowledgebase for understanding tissue-specific regulation and the role of these molecules in disease.
