Unresolved stalled ribosome complexes restrict cell-cycle progression after genotoxic stress

Abstract

During the translation surveillance mechanism known as ribosome-associated quality control, the ASC-1 complex (ASCC) disassembles ribosomes stalled on the mRNA. Here, we show that there are two distinct classes of stalled ribosome. Ribosomes stalled by translation elongation inhibitors or methylated mRNA are short lived in human cells because they are split by the ASCC. In contrast, although ultraviolet light and 4-nitroquinoline 1-oxide induce ribosome stalling by damaging mRNA, and the ASCC is recruited to these stalled ribosomes, we found that they are refractory to the ASCC. Consequently, unresolved UV- and 4NQO-stalled ribosomes persist in human cells. We show that ribosome stalling activates cell-cycle arrest, partly through ZAK-p38^MAPK signaling, and that this cell-cycle delay is prolonged when the ASCC cannot resolve stalled ribosomes. Thus, we propose that the sensitivity of stalled ribosomes to the ASCC influences the kinetics of stall resolution, which in turn controls the adaptive stress response.

Keywords: ASC-1 complex; RNA damage; RNA-binding protein; cell-cycle arrest; ribosome stalling; ribosome-associated quality control; ultraviolet light.

Graphical abstract

Figure 1

Bulky nucleic acid damage induces ASCC3 RNA binding (A) ASCC3 RNA binding using RIC in mock and UVB-irradiated MCF10A cells. (B) ASCC3 RNA binding in MCF10As exposed to nucleic acid damaging agents. (C) ASCC3 RNA binding in cytoplasmic (C) and nuclear (N) fractions after UVB treatment. Error bars represent the SD. ∗p < 0.05. (D) The effect of puromycin pre-treatment on ASCC3 RNA binding after UVB or 4NQO exposure. (E and F) In vivo RNA binding of ASCC3-F (WT) and ASCC3-F^AAA (AAA) in (E) or ASCC3-F (WT) and ASCC3-F^E612Q (EQ) in (F) in response to 4NQO. Error bars represent the SD. ∗∗p < 0.01. See also Figure S1.

Figure 2

The ASCC accumulates on UVB- and 4NQO-stalled ribosomes (A) Harringtonine run-off assays to measure the rate of polysome loss in HeLa cells treated with 4NQO or UVB. Error bars represent the SD. ∗∗p < 0.01, ∗∗∗p < 0.001. (B) Micrococcal nuclease assay to measure ribosome collisions in HeLa cells treated with 2-μM emetine (low), 0.2-μM anisomycin (low), 300-μM emetine (high), 75-μM anisomycin (high), 4NQO, or UVB for 15 min. (C) Distribution of the ASCC proteins after SDGC of lysates prepared from control or 4NQO-treated cells. (D and E) ASCC3 distribution after SDGC of lysates prepared from cells exposed to 4NQO. Lysates were treated with EDTA (+EDTA) in (D) or MNase (+MNase) in (E). See also Figure S2.

Figure 3

UVB- and 4NQO-stalled ribosomes persist in the cell (A) Ribosome collisions in HeLa cells treated with 4NQO, 0.6-μM anisomycin, or 0.6-μM emetine for 1 h. (B) ASCC3 RNA binding in HeLa cells treated with anisomycin (A), emetine (E), or 4NQO (N) as above. Error bars represent the SD. ∗p < 0.05. (C) ASCC3 distribution after SDGC of lysates prepared from cells treated as in (A). (D) Kinetic analysis of collided ribosomes in cells treated with 0.2-μM emetine or anisomycin, 4NQO, or UVB. (E and F) Collided ribosomes in HeLa cells recovering from exposure to 4NQO in (E) or UVB in (F).

Figure 4

UV- and 4NQO-stalled ribosomes are resistant to the ASCC (A) The effect of ASCC3 or ZNF598 depletion on the loss of collided ribosomes 1 h after treatment with emetine, anisomycin, 4NQO, or UVB. ASCC3 (A) and ZNF598 (Z) were efficiently depleted compared with the control (C). (B) The effect of ASCC3, ASCC2, ASCC1, or ASC-1 depletion on the loss of collided ribosomes 1 h after treatment with anisomycin. ASCC1 (1), ASCC2 (2), ASCC3 (3), or ASC-1 (A) were efficiently depleted compared with the control (C). (C and D) ASCC3 depletion in (C) or Pelo depletion in (D) has no effect on the loss of 4NQO-stalled ribosomes. See also Figure S3.

Figure 5

UVB and 4NQO induce ribosome stalling by damaging mRNA (A) Harringtonine run-off of UVB (U)- or 4NQO (N)-stalled ribosomes in ASCC3 (A3)-depleted cells. (B) UVB irradiation of HeLa cells, in which translation is blocked with hippuristanol (HPL), results in ribosome stalling when mRNA translation resumes. (C) The effect of MMS (M), UVB (U), 4NQO (N), anisomycin (A), emetine (E), or didemnin B (D) on eIF2α phosphorylation (upper). GCN2i prevents eIF2α phosphorylation in response to UVB, 4NQO, or MMS (middle). PERKi prevents eIF2α phosphorylation in response to thapsigargin (T), but not UVB, 4NQO, or MMS (lower). (D) Schematic of the UVPD reporter mRNA (noYY sequence, blue; pyrimidine tract, red; remaining coding sequence, gray). In vitro translation of untreated and UVB-damaged UVPD reporter mRNA. The noYY peptidyl-tRNA (noYY-tRNA) and full-length protein (FL) are indicated. (E) Ribosome collisions after MMS treatment. (F) Depletion of ASCC3 (A3) prevents the resolution of MMS-stalled ribosomes. See also Figure S4.

Figure 6

Unresolved stalled ribosomes cause cell-cycle arrest (A) Phosphorylation of p38^MAPK and JNK after treatment with anisomycin (ANS) or UVB. (B) Collided ribosomes at 1, 3, and 6 h after treatment with anisomycin in control or ASCC3-depleted cells. (C) ASCC3 depletion causes prolonged p38^MAPK phosphorylation in response to anisomycin. (D and E) p38^MAPK phosphorylation after UVB treatment in (D) or after anisomycin treatment of ASCC3-depleted cells in (E) depends on ZAK. (F) Cell-cycle distribution in control (C) and ASCC3-depleted cells (A) after treatment with anisomycin. Error bars represent the SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (G) The effect of anisomycin treatment in control and ASCC3-depleted cells on the G2 to M-phase transition measured by trapping the cells in mitosis with nocodazole (Noc). (H and I) The accumulation of ASCC3-depleted cells in G2 after anisomycin treatment is prevented by pre-exposure to a p38^MAPK inhibitor (p38i) in (H) or depletion of ZAK in (I). ∗∗p < 0.01, ∗∗∗p < 0.001. (J) ASCC3-depleted cells have a prolonged G1 arrest compared with control cells after anisomycin treatment. Error bars represent the SD. ∗∗p < 0.01. See also Figure S5.