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

Figures

• 

  FIGURE 1.. RNase L-mediated tRNA cleavage induced by poly(I:C) transfection. (A, B) Poly(I:C) transfection induces tRNA cleavage in U2OS/A549 cells. (A) SYBR Gold staining and (B) northern blotting for tRNAHis-GTG, tRNAPro-TGG, and tRNAGln-CTG. (C, D) RNASEL knockdown decreases the amount of poly(I:C)-induced tDRs in A549 cells. (C) SYBR Gold staining and (D) northern blotting for tRNAHis-GTG and tRNAPro-TGG. RNase L expression levels evaluated by western blotting are also shown. Red arrowheads indicate 5′-tDRs generated by RNase L-mediated cleavage.

• 

  FIGURE 2.. RNase L cleaves various tRNA species into tDRs in A549 cells. RNase L activated by poly(I:C) transfection induces tDRs originating from various tRNA species. (A) SYBR Gold staining and (B) northern blotting. Red and blue arrowheads indicate 5′-tDRs likely generated by cleavage in the anticodon loops and variable loops, respectively. Asterisk indicates 5′-tDR derived from pre-tRNATyr-GTA (Akiyama et al. 2022).

• 

  FIGURE 3.. RNase L can target variable loops of tRNAs. To identify how the longer 5′-tDRs were generated from tRNALeu-CAG, tRNASec-TCA, and tRNASer-GCT, northern blotting was performed using both 5′ and 3′ probes for these tRNAs. The sequences around anticodon loops and variable loops in these tRNAs are shown. Predicted cleavage sites in anticodon loops and variable loops are indicated by red and blue arrowheads, respectively. (VL) Variable loop.

• 

  FIGURE 4.. RNase L-induced 5′-tDRAla shows a translation inhibitory effect. (A–D) Comparison between RNase L-induced 5′-tDRAla and ANG-induced 5′-tiRNAAla. (A) Northern blotting for tRNAAla-AGC. (B) Predicted cleavage sites of tRNAAla-AGC-2-1 by ANG/RNase L. Red and blue arrowheads indicate the predicted sites of ANG-mediated and RNase L-mediated cleavage, respectively. (C, D) Estimation of the length of RNase L-induced 5′-tDRAla and ANG-induced 5′-tiRNAAla. Mobility of the tDRs was compared with that of synthetic 5′-tDRsAla (32 and 33 nt). (C) SYBR Gold staining and (D) northern blotting for tRNAAla-AGC. (E) 5′-tDRAla (33 nt) as well as 5′-tiRNAAla (32 nt) inhibit translation of mRNA reporters in vitro. Means and standard deviation were obtained from three independent experiments. Mean values and P-values are shown. (F, G) 5′-tDRAla as well as 5′-tiRNAAla can form G-quadruplex structure. (F) SYBR Gold staining and (G) northern blotting for tRNAAla-AGC.

• 

  Yoshika Takenaka.

• 

  Asuka Yamada.