RNase 2/EDN cleaves tRNA anticodon loops to generate immunoactive RNAs

The RNase A superfamily, one of the most extensively studied enzyme families, remains incompletely defined with respect to its endogenous RNA substrates, recognition mechanisms, and physiological functions. Here, we identify eosinophil-derived neurotoxin (EDN/RNase 2) as a principal endoribonuclease mediating tRNA cleavage in the asthmatic lung. Inhalation of house dust mite (HDM) in mice induces robust accumulation of tRNA halves in the lung, coinciding with exclusive upregulation of EDN, but not other ribonucleases. In human lung epithelial cells, internalized EDN cleaves specific tRNAs and generates multiple species of tRNA halves, including immunostimulatory species that activate Toll-like receptor 7 (TLR7). EDN also promotes the release of extracellular vesicles enriched in these RNAs, implicating them in cytokine production during asthma pathogenesis. Biochemical analyses and structural simulations reveal that EDN recognizes the anticodon loop of tRNA by anchoring the phosphate backbones flanking the cleavage site, thereby precisely positioning the C34-A35 phosphodiester bond for catalysis. Furthermore, the highly efficient cleavage by EDN is mediated by conserved elements, including Arg36 in EDN and U33 and C38 in the target tRNA. Together, these findings establish the endogenous RNA substrate of EDN and uncover a molecular mechanism that links RNase A superfamily activity to immune response.