Conformational diversity of collided ribosomes determines stress signaling

Translating ribosomes are stalled by amino acid starvation and by chemical damage of the mRNA template, leading to ribosome collisions. These collisions serve as signaling platforms that promote quality control (QC) pathways that clear stalled ribosomes, and when sufficiently abundant, induce general stress responses, including the integrated stress response (ISR) mediated by the kinase GCN2 and the ribotoxic stress response (RSR) mediated by the kinase ZAK. Translational elongation inhibitors such as anisomycin (ANS), emetine (EME), and didemnin B (DDB) are commonly used to induce ribosome collisions in studying these responses. Here, we demonstrate that these three collision-inducing drugs, which target different stages of translational elongation, induce distinct signatures for QC, ISR and RSR activation. Collisions induced by EME, unlike those induced by ANS and DDB, are cleared inefficiently by ASCC3. Moreover, while ANS/DDB-induced collisions potently activate ZAK, EME-induced collisions barely activate. Finally, ANS at most modestly activates the ISR while EME exhibits no ISR activation. We use RNA-seq to define transcriptional programs induced by elongation inhibitors, demonstrating that RSR-dependent signaling is mediated by ribosome collisions whereas RSR-independent signaling is mediated by general translation inhibition. The cryo-EM structure of EME-induced collided ribosomes from human cells reveals the detailed mode of EME inhibition of the ribosome and a distinct overall disome conformation. Our biochemical data and structural comparison of different inhibitor-bound collided ribosomes support a model where EME induces collision states distinct from those induced by ANS and DDB. We conclude that these structural differences in collision geometry are the key determinants of differences in elongation inhibitor-induced QC and signaling activation, suggesting that distinct ribosome stalling events may result in different cellular responses.