Anisomycin-induced ribosome collisions follow a dose-dependent positional rule, distinct from the UV-induced collision landscape
Ribosome collisions trigger stress pathways such as the ribotoxic stress response (RSR), yet how different ribosome-impairing stimuli shape where collisions form on mRNAs remains unclear. Here, we combine monosome- and disome-resolved ribosome profiling with proteomics of collided-ribosome fractions to compare anisomycin- and UV-induced ribosome impairment in mouse embryonic stem cells. Low-dose anisomycin, a widely used collision-inducing paradigm, does not generate collisions dispersed along transcripts but a post-initiation collision zone, with stalled monosomes and disomes accumulating within the first ~70 nucleotides of coding sequences across most translated mRNAs. High-dose anisomycin, commonly considered collision-poor, produces the same pattern shifted closer to the start codon and reduced in amplitude. These observations support a single dose-dependent positional rule, in which drug occupancy determines how far ribosomes progress before arrest and ongoing initiation supplies trailing ribosomes. In contrast, 30 min after UV irradiation, collisions are detectable biochemically but appear more dispersed, with few recurrent hotspots and preferential accumulation on highly translated mRNAs. Collided-ribosome fractions share enrichment of the collision sensor EDF1 across conditions, whereas recruitment of ZNF598 and initiation factors is most evident after UV. Our results refine the interpretation of anisomycin-based collision paradigms and show that collision landscapes depend on the nature of the stimulus.