Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate

Abstract

Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.

Keywords: SAPK; UV radiation; ZAK; amino acid starvation; integrated stress response; ribosome collisions.

Figure 1.. Phosphorylation of SAPKs (p38/JNK) in response to ribosome collisions

(A) Schematic of ribosome collisions on treatment with varying concentrations of elongation inhibitor: untreated (unt), intermediate (int), or high dose. (B) Polysome profiles from RNaseA- digested lysates of HEK293 cells treated with ANS at 0 (black), 1 (orange), or 100 mg/L (grey). (C) Immunoblots for phosphorylation of p38 and JNK in HEK293 cells treated with ANS (0.001–100 mg/L, 15min) compared to untreated. (D) p38 phosphorylation in HeLa cells treated with EME (0.048–48 mg/L, 15min). (E) p38 phosphorylation induced by intermediate doses of ANS (1 mg/L, 15min) in HEK293 cells with or without pretreating with high doses of EME (48 mg/L, 5min). (F) p38 phosphorylation in HeLa cells transfected with pcDNA5 (Vec), A₀ or A₆₀ reporter with schematic depicting the sequence features of the reporter.

Figure 2.. Ribosome collisions induce ZAtα phosphorylation

(A) Schematic depicting two isoforms of ZAK (top). p38 phosphorylation (uppermost blot) in WT MCF10A, ZAK KO, or ZAK KO complemented with ZAKα, ZAKa-K45M, or ZAKβ under ANS treatment (1 mg/L, 15min) (bottom); control blots for total p38 and expression of ZAKα and ZAKβ. * denotes non-specific bands. (B) Schematic depicting C-terminal deletions of ZAKα (top). p38 phosphorylation in MCF10A cells expressing only FALG-tagged full-length ZAKa, a(1–720), or a(1–649). (C-D) Immunoblots of Phos-tag gels for ZAKα phosphorylation in HeLa-ZAKα-FLAG cells treated with ANS at 0, 1, or 100 mg/L (15min) (C) or with EME at 0, 0.24 or 48 mg/L (15min) (D), using FLAG antibody. Fraction of ZAKα shifted through phosphorylation is indicated. (E) ZAKα phosphorylation in MCF10A cells expressing only ZAKα or ZAKα-K45M. (F) Immunoblots for ZAKα and p38 phosphorylation in HeLa cells expressing endogenous ZAKα and FLAG-tagged ZAKα (WT or K45M mutant). Bottom most panel shows relative levels of endogenous and exogenous ZAKα after phosphatase treatment.

Figure 3.. ZAtα associates with colliding ribosomes

(A) Representative polysome profile (top) from MCF10A ZAK KO cells complemented with ZAKα, ZAKα-K45M, or ZAKβ. Fractions were analyzed by immunoblotting. (B) Similar to (A), with MCF10A ZAK KO cells complemented by FLAG-tagged ZAKα, α(1–720), or α(1–649). (C) Polysome profiles from HeLa cells treated with an intermediate (1 mg/L, orange) or high (100 mg/L, gray) dose of ANS compared to untreated sample (black). (D) Polysome profiles from HeLa cells with indicated treatment after RNase digestion. Fractions were immunoblotted with indicated antibodies. (E) Polysome profiles from ZAKα-FLAG HeLa cells with indicated treatment following RNase digestion. Fractions were analyzed by Phos-tag gels and immunoblotted using antibodies against FLAG (ZAKa) or RPL4. Fraction of ZAKα phosphorylation in monosome and disome fractions is indicated. (F) Average disome occupancies aligned at stop codons across the transcriptome from Mock IP (black) or ZAKα IP (magenta). Average normalized density (solid line) with standard deviation (shaded area) across two replicates. (G) p38 phosphorylation in WT or ZNF598 KO HEK293 cells treated with ANS (0.1–100 mg/L).

Figure 4.. Ribosome collisions activate GCN2-mediated eIF2α phosphorylation

(A) Immunoblots for phosphorylation of eIF2α and mTOR in MCF10A cells treated with ANS (0.1–100 mg/L, 0.5 h). Total eIF2α and β-actin as loading controls. (B) eIF2α phosphorylation induced by intermediate doses of ANS (0.5 mg/L, 0.5 h) in MCF10A cells pretreated with GCN2 inhibitor (A-92), PERK inhibitor (GSK 2606414), or p38 inhibitor (BIRB 796). (C) eIF2α phosphorylation induced by ANS in WT or ZAK KO MCF10A cells. (D) eIF2α phosphorylation in WT, ZAK KO, or ZAK KO MCF10A complemented with ZAKα, ZAKα -K45M, or ZAKβ under ANS treatment (0.5 mg/L, 0.5 h). (E) Polysome profiles from DSP-crosslinked WT and ZAK KO MCF10A cells. Fractions were analyzed by immunoblotting with indicated antibodies.

Figure 5.. Glutamine starvation induces ribosome collision-mediated ZAKα activation

(A) p38 phosphorylation in HeLa cells grown in rich or glutamine-depleted media pretreated with vehicle (DMSO) or ISRIB compared to stimulation by intermediate-dose ANS (1 mg/L, 15min). (B) p38 phosphorylation in WT or ZAK KO MCF10A cells in media with or without glutamine. (C) Immunoblots of a Phos-tag gel for ZAKα phosphorylation in ZAKα-FLAG HeLa cells grown in media containing 2, 0.02 or 0 mM glutamine pretreated with DMSO or ISRIB. (D) eIF2α phosphorylation induced by glutamine starvation in WT or ZAK KO MCF10A cells pretreated with DMSO or ISRIB.

Figure 6.. UV irradiation induces ribosome collision-mediated ZAKα activation

(A) Polysome profiles from RNase-digested lysates of HeLa cells treated with UV compared to untreated. (B) Size distribution of ribosome footprints (RPFs) from UV-irradiated HeLa cells compared to untreated cells. (C) Scatter plot of codon-specific ribosome occupancies for short RPFs comparing UV-irradiated to untreated cells. Orange dots indicate codons containing two adjacent pyrimidines. (D) Metacodon analysis aligning at all CUU codons comparing UV- irradiated to untreated cells. Arrows indicate colliding ribosomes upstream of the paused ribosomes at CUU codons. (E) Phosphorylation of p38, JNK, and eIF2a in WT or ZAK KO MCF10A cells at indicated time points after UV irradiation. (F) Phosphorylation of endogenous ZAKa, p38, JNK, and eIF2α in HeLa cells transfected with untreated or UV-irradiated EGFP mRNA.

Figure 7.. Molecular gauge for ribosome collisions determines cell fate

(A) Model for ribosome collision-mediated stress response signaling through ZAKα to activate GCN2 and SAPKs (p38/JNK), leading to translation initiation block and apoptosis, respectively. Short-lived and long-lived colliding disomes are in gray and magenta, respectively. (B) Proposed model of cellar gauge for ribosome collisions that mounts measured cellular responses under stress. In unstressed cells, basal levels of ribosome collisions are handled by RQC and NGD machineries (left). Intermediate levels of collisions induced by cellular stresses trigger ZAKα- dependent GCN2-mediated translation initiation block to reduce further collisions, promoting cell survival (middle), whereas “dangerous” levels of collisions initiate ZAKα-dependent apoptosis (right).