A novel tRF-Gly is associated with obesity development through post-transcriptional regulation of lipid metabolism

Obesity, characterized by excessive fat accumulation, represents a global health crisis closely linked to metabolic disorders such as type 2 diabetes, hypertension, and atherosclerosis. tRNA-derived small RNAs (tsRNAs) have recently emerged as important epigenetic regulators, yet their roles in fat deposition remain poorly characterized. This study aims to identify tsRNAs that influence fat accumulation and to elucidate their molecular mechanisms, with a focus on tRF‑Gly‑GCC‑037 (tRF‑Gly) as a candidate regulator of adipocyte differentiation.

Visceral adipose tissue was collected from obese and lean pigs for comprehensive tRF and tiRNA sequencing. Differential expression analysis identified tRF‑Gly as a highly abundant candidate in obese samples. Functional assays in 3T3‑L1 preadipocytes included both overexpression and knockdown of tRF‑Gly, followed by lipid accumulation measurements and assessment of key adipogenic markers (CEBPα and PPARγ) by quantitative real-time PCR (qRT‑PCR) and western blot. Mechanistically, dual‑luciferase reporter assays, RNA immunoprecipitation (RIP), and nuclear-cytoplasmic protein fractionation were performed to examine how tRF‑Gly modulates the RAC1/JNK2/β‑catenin signaling axis.

tRF‑Gly was significantly upregulated in visceral adipose tissue from obese pigs and ranked among the most abundant tsRNAs. Overexpression of tRF‑Gly in 3T3‑L1 cells and in C57BL/6 mice promoted lipid accumulation and increased CEBPα and PPARγ expression, whereas tRF‑Gly knockdown reduced lipid deposition. Mechanistically, tRF-Gly was suggested to bind RAC1 mRNA with AGO3 involvement, leading to RAC1 silencing. Consistently, RAC1 knockdown phenocopied the adipogenic effects of tRF-Gly, whereas RAC1 overexpression reversed these effects. Furthermore, RAC1 deficiency disrupted the RAC1/JNK2/β‑catenin complex, impaired β‑catenin nuclear translocation, and suppressed Wnt/β‑catenin signaling.

Our findings demonstrate that tRF‑Gly functions as a key regulator of fat accumulation. By silencing RAC1 via AGO3, tRF‑Gly disrupts RAC1/JNK2/β‑catenin complex assembly, prevents β‑catenin nuclear translocation, and downregulates Wnt/β‑catenin signaling, thereby promoting lipid deposition. This study uncovers a novel epigenetic mechanism by which tRF‑Gly controls fat accumulation and suggests that targeting tRF‑Gly may represent a therapeutic strategy for obesity and related metabolic disorders.