3’tRNA ^Asp(GTC) -derived fragment links inflammation to post-transcriptional reprogramming in chondrocytes during osteoarthritis

Abstract Objective Transfer RNA-derived fragments (tRFs) belong to an emerging class of small non-coding RNAs that dynamically respond to metabolic stressors and drive different pathological processes, yet their role in osteoarthritis (OA) remains poorly explored. We aimed to define the tRF landscape in OA and investigate the function of 3’tRF Asp(GTC) in chondrocyte stress adaptation and translational control. Methods Ex vivo , cartilage specimens from OA patients (n=6) and healthy donors (n=7) were analyzed by small RNA sequencing to define disease-associated tRF signatures. In vitro , primary chondrocytes derived from OA patients were treated with lipopolysaccharide (LPS) to mimic inflammatory environment of OA, used for small RNA sequencing (n=3) and validation analysis (n=6). Functional studies in C28/I2 chondrocytes included antisense oligonucleotide-mediated 3’tRF Asp(GTC) inhibition, AGO2-RNA immunoprecipitation (RIP), polysome profiling, stress granule (SG) immunofluorescence, and differential protein analysis. Computational target prediction and pathway enrichment were used to explore tRF-mediated regulatory networks. Results Both OA cartilage and LPS-treated chondrocytes displayed upregulation of 3’tRF Asp(GTC) and 5’tRF Glu(CTC) , indicating a shared inflammatory tRF signature. Predicted targets of upregulated tRFs were enriched in stress-adaptive, proteostasis, and translational control pathways, whereas downregulated tRFs modulated mitochondrial processes. Silencing 3’tRF Asp(GTC) inhibited LPS-induced COX2 and MMP13 expression, prevented ER stress, and blocked SG assembly. RIP confirmed selective recruitment of 3’tRF Asp(GTC) into AGO2 complexes. Polysome profiling revealed association with 40S ribosomal subunit, mediating translational arrest and influencing selective mRNA expression. Conclusion 3’tRF Asp(GTC) emerges as a regulator linking inflammation to translational control and SG dynamics in OA. tRFs thus could represent novel therapeutic targets in OA disease.