•  The RNase A superfamily is one of the most extensively studied enzyme families. However, their endogenous RNA substrates, recognition mechanisms, and physiological functions remain uncharacterized. We identify eosinophil-derived neurotoxin (EDN/RNase 2) as a principal endoribonuclease mediating tRNA cleavage in the asthmatic lung. In a mouse model of asthma, we observed a robust accumulation of tRNA halves in the lungs, coinciding with exclusive upregulation of EDN, but not other ribonucleases. In human lung epithelial cells, internalized EDN cleaves specific tRNAs to generate 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 and structural analyses reveal that EDN recognizes the anticodon loop of tRNA by anchoring the phosphate backbones, thereby orienting the cleavage site towards the catalytic center. This highly efficient cleavage is mediated by conserved elements, including Arg36 in EDN and U33 and C38 in the target tRNA. These findings identify a physiological RNA substrate for EDN and uncover a molecular mechanism linking RNase A superfamily activity to immune signaling.

•  Full length tRNAs can be cleaved into fragments, called tRNA-derived small RNAs by a set of RNAses, including Angiogenin, Dicer and ELAC. Here we will discuss the pattern of tDR biogenesis in the setting of different stress signals in different cell-types. We will focus on one such tDR called Asp-GTC-3’tDR that we have recently found to have an important role in regulating RNA autophagy in kidney cells. We will discuss the role of Angiogenin in cleaving the parent tRNA. Finally we will describe a novel RNA editing tool we have bioengineered that leverages the RNAse and targeting properties of Angiogenin to direct the cleavage and biogenesis of functional tDRs.

Time: 15:45-16:30_Dr. Saumya Das

• Extracellular RNAs are traditionally studied in association with extracellular vesicles. However, the majority of extracellular RNAs in cell-conditioned media and biofluids exist outside vesicles. By comparing extracellular RNA profiles in the presence or absence of RNase A inhibitors (RI), we found that cells initially release a diverse repertoire of RNAs and ribonucleoprotein complexes, including ribosomes and full-length tRNAs. Although these are rapidly degraded, certain extracellularly generated RNA fragments are remarkably stable and become selectively enriched. This is particularly striking for tRNAs, which are cleaved at the anticodon loop to yield highly stable nicked tRNAs that are largely invisible to conventional sequencing methods. Moreover, stable nonvesicular RNAs can spontaneously enter cells, where they are detected by both endosomal and cytosolic RNA sensors in an RI-dependent manner. Together, these findings reveal how extracellular RNases shape the extracellular RNA landscape and highlight the implications of these processes for RNA-based intercellular communication and immune surveillance.