5'-tiRNA-Lys maintains intestinal epithelial homeostasis by EWSR1-dependent suppression of miR-125a and autophagy activation.

The intestinal epithelium relies on autophagy to maintain barrier integrity and homeostasis, and dysregulation of this process has been implicated in inflammatory bowel disease (IBD). While tRNA-derived small RNAs (tsRNAs) are emerging as regulators of cellular stress responses, their roles in intestinal autophagy remain poorly understood. Here, we identify that 5'-tiRNA-Lys, a tsRNA derived from mature tRNA-Lys, is significantly upregulated in the inflamed intestinal epithelium of IBD patients. Overexpression of 5'-tiRNA-Lys in mice ameliorates dextran sulfate sodium (DSS)-induced colitis. Mechanistically, 5'-tiRNA-Lys enhances autophagy in intestinal epithelial cells (IECs), as evidenced by elevated LC3-II level and autophagosome formation. RNA pull-down and immunoprecipitation assays reveal that 5'-tiRNA-Lys directly binds to the RNA-binding protein EWSR1 via its RNA recognition motif, disrupting EWSR1's interaction with the Drosha/DGCR8 microprocessor complex. This interference specifically suppresses the maturation of miR-125a, a microRNA that targets the autophagy-promoting gene UVRAG. Consequently, 5'-tiRNA-Lys increases UVRAG expression, thereby enhancing autophagic activity. Our findings reveal that 5'-tiRNA-Lys modulates autophagy through EWSR1-mediated miR-125a processing, which in turn affects intestinal inflammation, highlighting the potential of 5'-tiRNA-Lys as a therapeutic target for IBD.