Optimized tDR Sequencing Reveals Diversity and Heterogeneity in tRNA-Derived Fragment Landscapes in Mouse Tissues

Transfer RNA-derived small RNAs (tDRs) are increasingly being recognized as versatile regulators, yet their physiological landscape remains poorly charted. We analyzed tDR expression in seven adult mouse tissues to explore tissue-specific tDR enrichment using a tDR-optimized library preparation methodology. We catalogued 26,901 unique nuclear tDRs (ntDRs) and 5114 mitochondrial tDRs (mtDRs). Clustering analysis segregated the tissues, with the spleen and lungs forming a distinct immune cluster. Tissue-versus-all and pairwise differential analysis showed the spleen harboring unique ntDRs and mtDRs. Tissue-enriched tDRs arose from specific isoacceptor and isodecoder tRNAs, independent of mature tRNA abundance, suggesting selective biogenesis rather than bulk turnover. G-quadruplex prediction revealed a pronounced enrichment of potentially quadruplex-forming ntDRs in the kidneys, heart, and spleen, predominantly derived from i-tRFs and tRF3 fragments, suggesting structure-dependent functions in immune regulation. We also benchmarked our library strategy against the PANDORA-seq method. Despite comparable or lower sequencing depth, our method detected ~3-10-fold more unique ntDRs and we observed a clearer representation of tRF-3 fragments and greater isotype diversity. Our tissue atlas and improved tDR sequencing method reveal extensive tissue-specific heterogeneity in tDR biogenesis, sequencing, and structure, providing a framework for understanding the context-dependent regulatory roles of tDRs.