Fig 2: A proposed model of the regulation of stress-induced tiRNA production. Under normal conditions, ANG (and other RNase A superfamily enzymes) is inhibited by RNH1. Once cells are exposed to stress, the RNases become activated through RNH1 dissociation, cleaving tRNAs into tiRNAs. A part of tiRNAs is repaired to intact tRNAs before separation. Under the condition where oxidative stress coexists, the repair of tRNAs is repressed due to inhibition of the RTCB ligase complex, resulting in increased levels of separated tiRNAs that contribute to cell protection against adverse conditions. We propose that the net amount of tiRNA production is estimated by two factors, a trigger (RNH1 dissociation) and a booster (RTCB inhibition).

Fig 3: tiRNAs can be repaired to full-length tRNAs by RtcB in vitro. Gel-purified tiRNAs were incubated with bacterial RtcB. (A) SYBR Gold staining and (B) Northern blotting for tRNAGly-GCC and tRNASer-GCT. The lengths of both tRNAGly-GCC and tRNASer-GCT are also shown. tRNASer-GCT is longer than tRNAGly-GCC because of its long variable loop. Ligation products are indicated by arrowheads. Red arrowheads indicate the ligation products that showed the same mobility as that of the corresponding mature tRNA in total RNA. Note that two bands were detected by the probe for 5'-side (position 1–21) of tRNASer-GCT, in contrast to a single band detected by the probe targeting 3'-side (position 50–82) of tRNASer-GCT, which suggests that this shorter band (indicated by a blue arrowhead) consists of 5'-tiRNASer-GCT and 3'-tiRNAs derived from non-serine tRNAs.

Fig 4: Stress-induced tiRNA production is regulated by both RNH1 dissociation and RTCB inhibition. (A–D) Evaluation of stress-induced RNH1 dissociation from angiogenin. (A) Schematic model of ANG-His pulldown. When ANG was inactivated by RNH1, RNH1 is co-immunoprecipitated with ANG-His. Under the condition where ANG is activated, RNH1 is not co-immunoprecipitated because it dissociates from ANG. (B) Efficiency of immunoprecipitation. Overexpressed ANG-His was concentrated by immunoprecipitation. Under non-stress condition, RNH1 is co-immunoprecipitated with ANG-His. Note that RTCB was not co-immunoprecipitated with ANG-His, suggesting that RTCB does not interact with ANG or RNH1. (C) Overexpressed ANG-His is enzymatically functional. Overexpression of ANG-His enhanced sodium arsenite-induced tiRNA production. 5'-tiRNAGly-GCC is indicated by a red arrowhead. Note that ANG-His overexpression does not induce tiRNA production under non-stress conditions, suggesting that ANG-His is completely inhibited by RNH1 under basal conditions. (D) ANG-His pulldown under various stress stimuli. (E) The net amount of tiRNAs is regulated by both RNH1 dissociation and RTCB inhibition. RNH1 dissociation (trigger), RTCB inhibition (booster) and the net amounts of tiRNAs are evaluated by ANG-bound RNH1, pre-tRNATyr-derived fragment and 5'-tiRNAGly-GCC, respectively. (F–H) The effect of N-acetyl-L-cysteine treatment (at 5 mM) on tiRNA production. (F) SYBR Gold staining and (G) Northern blotting for tRNAGly-GCC and tRNATyr-GTA. 5'-tiRNAGly-GCC is indicated by a red arrowhead, pre-tRNATyr-GTA-derived fragments generated due to RTCB inhibition is indicated by a blue arrowhead. (H) ANG-His pulldown. Note that N-acetyl-L-cysteine treatment did not prevent RNH1 dissociation, suggesting that sodium arsenite-induced RNH1 dissociation is independent of oxidative stress. NC, negative control; SA, sodium arsenite; Mena., menadione; NAC, N-acetyl-L-cysteine.

Fig 5: RTCB ligase complex negatively regulates tiRNA production. (A) shRNA-mediated knockdown of RTCB. (B,C) RTCB knockdown enhances ANG-mediated tiRNA production. U2OS cells were incubated with 0.5 µg/mL ANG for 2 h. (B) SYBR Gold staining and (C) Northern blotting for tRNAGly-GCC and tRNAiMet-CAT. Relative amounts of 5'-tiRNAs calculated by densitometry are also shown (n = 3 each). (D) RTCB knockdown boosts constitutively generated tiRNAs in RNH1 knockout cells. Northern blotting for tRNAGly-GCC and tRNAiMet-CAT, and relative amounts of 5'-tiRNAs are shown (n = 3 each). Red arrowheads indicate tiRNAs.