Angiogenin-induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly

Abstract

Angiogenin (ANG) is a secreted ribonuclease that cleaves tRNA to initiate a stress-response program in mammalian cells. Here we show that ANG inhibits protein synthesis and promotes arsenite- and pateamine A-induced assembly of stress granules (SGs). These effects are abrogated in cells transfected with the ANG inhibitor RNH1. Transfection of natural or synthetic 5'- but not 3'-tRNA fragments (tRNA-derived stress-induced RNAs; tiRNAs) induces the phospho-eukaryotic translation initiation factor 2alpha-independent assembly of SGs. Natural 5'-tiRNAs but not 3'-tiRNAs are capped with a 5'-monophosphate that is required for optimal SG assembly. These findings reveal that SG assembly is a component of the ANG- and tiRNA-induced stress response program.

FIGURE 1.

Effect of ANG on SG assembly. U2OS cells were cultured in media alone (No treat) (A) or media containing ANG (0.5 μg/ml) (B), SA (70 μm) (C), or ANG plus sodium arsenite (ANG + SA) (D) for 45 min before processing for immunofluorescence microscopy using anti-eIF4E (green), anti-G3BP (red), and eIF3b (blue). Insets, enlarged views of the boxed region stained for eIF4E (green), G3BP (red), and eIF3b (blue) and merged channels (from left to right). The yellow arrows point out cells with SGs. E, quantification of the percentage of cells with SGs. Data report the average percentage of SGs from three independent experiments in which 200 cells were counted in each experiment. Error bars, S.D. (n = 3). F, quantification of [³⁵S]methionine incorporation. Cells were incubated with metabolic labeling medium for 30 min, and then a fresh metabolic medium containing [³⁵S]methionine and wild type ANG or mutant P112L (0.5 μg/ml) in the presence or absence of 70 μm sodium arsenite or 15 nm of pateamine A was added to the cells for 1 h. [³⁵S]Methionine incorporation is reported as a percentage of that observed in untreated cells. The average percentage of [³⁵S]methionine incorporation was calculated from three independent experiments and plotted for each experimental condition. The error bars indicate the S.D. (n = 3).

FIGURE 2.

Effect of natural tiRNAs on SG assembly in U2OS cells. Shown is immunofluorescence microscopy of U2OS cells transfected with 750 nm control RNA (ctrlRNA) (A), natural 3′-tRNA (Nat 3′end) (B), or natural 5′-tRNA (Nat 5′end) (C) and stained with SG markers (anti-G3BP (red) and eIF3b (blue)) and a PB marker (anti-SK1-hedls (green)) at 7 h post-transfection. Insets, enlarged views showing individual and merged channels. The yellow arrows indicate cells with SGs. The white arrowheads indicate PBs detected in juxtaposition interaction with SGs. D, quantification of the percentage of cells with SGs. Cells that showed SGs were counted in a total number of 600 cells/experiment, and the average percentage of cells that showed SGs was calculated from three independent experiments and plotted for each experimental condition. Error bars, S.D. (n = 3).

FIGURE 3.

Natural 5′-tiRNAs possess 5′-monophosphates that enhance SG assembly. A, synthetic 5′-OH-5′-tiRNA^Ala (lanes 1 and 2), 5′-P-5′-tiRNA^Ala (lanes 3 and 4), or 5′-OH-3′-tiRNA^Ala (lanes 7 and 8) were used as controls for the specificity of 5′-monophosphate exoribonuclease treatments. The control synthetic RNAs, natural 5′-tiRNAs (lanes 5 and 6) and natural 3′-tiRNAs (lanes 9 and 10) were treated with 5′-monophosphate exoribonuclease (lanes 2, 4, 6, 8, and 10) or a buffer control (lanes 1, 3, 5, 7, and 9) for 30 min prior to separation on a TBE-urea gel and visualization using CYBR Gold. U2OS cells were transfected with 750 nm synthetic phosphorylated control RNA (P-ctrlRNA) (B) or phosphorylated 5′-tiRNAs (P-5′Val, P-5′Gly, or P-5′Ala) (C–E) and then stained with anti-Sk1-hedls (green), anti-G3BP (red), and eIF3b (blue) antibodies at 7 h after transfection. Insets, enlarged views showing individual and merged channels. The yellow arrows point out representative cells with SGs. The white arrowheads indicate PBs in juxtaposition interaction with SGs. F, quantification of the percentage of cells with SGs. Cells that showed SGs were counted in a total number of 600 cells/experiment, and the average percentage of cells that showed SGs was calculated from three independent experiments and plotted for each experimental condition. The error bars indicate the S.D. (n = 3).

FIGURE 4.

Emetine inhibits P-5′-tiRNA^Ala-induced SGs. A–D, immunofluorescence microscopy of non-transfected cells treated with 200 μm SA for 60 min (A), 200 μm SA for 60 min followed by 50 μg/ml emetine (SA + Em) treatment for another 60 min (B), or cells transfected with 750 nm P-5′-tiRNA^Ala (C) and then treated with 50 μg/ml emetine (P-5′Ala + Em) (D) for 60 min. Insets, enlarged views showing individual and merged channels. The yellow arrows point out representative cells with SGs. E, quantification of the percentage of cells with SGs. The percentage of cells that showed SGs were quantified by counting 200 cells.

FIGURE 5.

5′-tiRNAs enhance arsenite-induced SG assembly. A and B, quantification of the percentage of cells with SGs in control U2OS cells cultured in the absence (No treat) or presence (SA) of 70 μm SA or cells transfected with 750 nm natural 5′-tiRNAs (Nat 5′end) or 3′-tiRNAs (Nat 3′end) or synthetic tiRNAs (P-ctrlRNA, P-3′tiRNA^Ala, P-5′tiRNA^Ala) for 7 h and then treated with 70 μm SA (3′end + SA, 5′end + SA, P-ctrlRNA (P-ctrlRNA + SA), P-3′tiRNA^Ala (P-3′Ala + SA), or P-5′-tiRNA^Ala (P-5′-Ala + SA)) for 60 min. Cells were stained with SG markers (anti-eIF3b, anti-G3BP, and anti-eIF4G). The percentage of cells with SGs was quantified by counting 200 cells/experiment. Data show the average percentage of SGs from three independent experiments. Error bars, S.D. (n = 3).

FIGURE 6.

Effect of phospho-eIF2α on ANG- and/or P-5′-tiRNA^Ala. A, quantification of the percentage of cells with SGs in untransfected U2OS cells (No RNA) or cells transfected with control siRNA (siD0) or with HRI siRNA (siHRI) and then mock-transfected or transfected with P-5′-tiRNA^Ala (P-5′Ala; 750 nm) for 7 h. The percentage of cells with SGs was quantified by counting 500 cells/experiment. Data show the average percentage of SGs from three independent experiments. Error bars, S.D. (n = 3). B, Western blot analysis of phospho-eIF2α in U2OS cells transfected with either D0 or HRI siRNAs and then treated or not treated with 200 μm SA for 60 min. Lane 1, control knockdown cells (siD0); lane 2, control knockdown cells treated with 200 μm SA (siD0 + SA); lane 3, control knockdown cells transfected with P-5′-tiRNA^Ala (siD0 + P-5′Ala); lane 4, HRI knockdown cells (siHRI); lane 5, HRI knockdown cells treated with 200 μm SA (siHRI + SA); lane 6, HRI knockdown cells transfected with P-5′-tiRNA^Ala (siHRI + P-5′Ala). C, Western blot analysis of phospho-eIF2α in untreated cells (No treat; lane 1) or in U2OS cells treated with either ANG (0.5 μg/ml; lane 2), SA (70 μm; lane 3), ANG plus sodium arsenite (ANG + SA; lane 4), pateamine A (PatA; 15 nm; lane 5), ANG plus pateamine A (ANG + PatA; lane 6), ANG mutant + SA (P112L + SA; lane 7), P112L + PatA (lane 8), or P112L (lane 9). Phospho-eIF2α was detected with polyclonal antibodies specific for eIF2α phosphorylated on serine 52 (upper panels). Actin (lower panels) was used as a loading control.

FIGURE 7.

Effect of RNH1 on ANG-mediated promotion of SG assembly. Quantification of the percentage of cells with SGs in U2OS cells transfected with either FLAG-LSM1 or FLAG-RNH1 and then treated with 60 μm SA in the presence or absence of ANG for 45 min. Cells were stained with SG markers (anti-FLAG, anti-G3BP, and anti-eIF3b). SGs were counted only in 40–50 cells overexpressing either protein. The average percentage of cells with SG was calculated from three independent experiments, expressed as -fold increase over ANG alone, and plotted for each experimental condition. The error bars indicate the S.D. (n = 3).