Structural determinants for tRNA selective cleavage by RNase 2/EDN

tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of tRFs population. By comparing wild-type and knock-out macrophage cell lines our previous work (Lu L, et al. CMLS, 2022, 79: 209) revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening synthetic tRNAs, single-mutant variants and anticodon-loop DNA/RNA hairpins. By sequencing the tRFs products, we identified the cleavage selectivity by recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Knowledge of RNase 2 specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases. To identify the corresponding tRFs degraded by RNase 2 in vitro, we used a crude method which mimicked next-generation sequencing technology to process our in vitro samples including the teminal treatment on tRFs with enzymes to enable the adaptors to ligate onto the fragments