The integrated stress response finetunes 18S nonfunctional rRNA decay (Ribo-Seq)

18S nonfunctional rRNA decay (NRD) detects and eliminates translationally nonfunctional 18S rRNA. The underlying mechanisms associated with the detection and turnover of nonfunctional 18S rRNA remain elusive. While NRD has been identified and exclusively studied in Saccharomyces cerevisiae, it is unclear whether this quality control pathway exists in mammals. Here we demonstrate the conservation of 18S NRD in mammalian cells. Using genome-wide CRISPR genetic interaction screens, we identify two molecular events triggered by nonfunctional 18S rRNA— activation of the integrated stress response (ISR) and ubiquitination of ribosomal proteins elicited by GCN2 and RNF10, respectively. Selective ribosome profiling reveals nonfunctional 18S rRNA induces translation arrest at start sites. Biochemical analyses show that activation of the ISR limits translation initiation, attenuating collisions between scanning 43S preinitiation complexes and nonfunctional 80S ribosomes arrested at start sites. Thus, the ISR facilitates the turnover of nonfunctional 18S rRNA and 40S ribosomal proteins by RNF10-mediated ubiquitination. Altogether, these results establish a dynamic feedback mechanism by which cells finetune translation initiation to enable ribosome functionality surveillance through the GCN2-RNF10 axis.