Selective Biogenesis and Structural Diversity Shape tRNA-Derived Fragment Landscapes across Mouse Organs

Transfer RNA derived small RNAs (tDRs) are increasingly recognized as versatile regulators, yet their physiological landscape remains poorly charted. We re-analyzed a small-RNA-seq dataset generated with tDR-optimized library preparation from seven adult mouse tissues to explore tissue specific tDRs enrichment. We catalogued 26901 unique nuclear tDRs (ntDRs) and 5114 mitochondrial tDRs (mtDRs). Library sizes and subclass composition varied markedly: heart was mtDRs-rich, whereas spleen produced the fewest reads but the greatest diversity. Clustering analysis segregated tissues, with spleen and lung forming a distinct immune cluster. Tissue-versus-all differential analysis showed the spleen harboring unique ntDRs and mtDRs. The brain preferentially accumulated 5'tiRNAs, while other organs were enriched for i-tRFs or tRF3s. Enriched fragments in different tissues originated from specific isoacceptors and isodecoder tRNAs independent of mature tRNA abundance, implying selective biogenesis rather than bulk turnover. G-quadruplex prediction revealed pronounced enrichment of potentially quadruplex-forming ntDRs in the spleen, predominantly Gly-, Asp- and Val-derived i-tRFs/tRF3s, suggesting structure-dependent functions in immune regulation. Collectively, our atlas reveals extensive tissue-specific heterogeneity in tDR biogenesis, sequence and structure, providing a framework for deciphering context-dependent regulatory roles of tDRs.