RNase L produces tRNA-derived RNAs that contribute to translation inhibition.

Ribonuclease L (RNase L) is an RNase which is activated by viral double-stranded RNAs (dsRNAs). RNase L cleaves not only viral RNAs but also host RNAs, including mRNAs and tRNAs, which contributes to innate immune defense against viruses. While it has been reported that RNase L-mediated bulk mRNA cleavage induces rapid translation repression independently of the integrated stress response, the significance of RNase L-mediated tRNA cleavage remains largely unknown. Here we show that RNase L cleaves various tRNA species in the anticodon loops, generating transfer RNA-derived RNAs (tDRs) similar to tRNA-derived stress-induced RNAs (tiRNAs) that are generated by a stress-responsive RNase angiogenin (ANG). Three tRNA species (tRNA(Leu), tRNA(SeC) (,) and tRNA(Ser)) were cleaved within the variable loops as well as in the anticodon loops by RNase L, generating noncanonical tDRs. As RNase L-induced 5'-tDR(Ala/Cys) were similar in length to 5'-tiRNA(Ala/Cys) that possess a translation inhibitory effect, we examined whether RNase L-induced 5'-tDR(Ala) also inhibited translation. In vitro translation analysis showed that RNase L-induced 5'-tDR(Ala) significantly inhibits mRNA translation like 5'-tiRNA(Ala), suggesting that the production of 5'-tDR(Ala) may be involved in the mechanism of RNase L-mediated stress response during viral infection. Our data shed new light on the potential roles of tDRs in innate immunity against viral infection.