Liver and epididymis exhibit alterations in specific tRNA modifications in response to dietary alterations. (A) Schematic of the experimental design. Three-week-old male mice were fed one of three diets for 7 weeks: low-protein (LP), high-fat (HF), or control (C). Liver and epididymis (Epi) tissues were collected for downstream analyses. (B) Volcano plot of sRNA (16–40 nt) modification levels in the epididymis relative to the liver. Modifications detected at significantly differential levels between the two tissues are labeled on the plot (modifications that showed log₂FC ≥ 0.585 and −log₁₀(P-value) > 1.3). The color scale represents the fold change. Dot size reflects the −log₁₀(P-value). n = 6 replicates. (C) Volcano plot showing significantly altered tRNA modifications (70–90 nt) in the epididymis relative to the liver (n = 6). (D–G) Volcano plots illustrating RNA modifications significantly altered by LP or HF diets in the liver and epididymis tissues (HF sample n = 3; control and LP sample n = 6). (H) Schematic of tissue-specific and diet-sensitive tRNA modifications identified by LC-MS/MS, annotated with positional information on the tRNA.