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. Overall design: To identify ribosome pausing across the transcriptome, we used MS2 affinity pulldowns of 18S-tag constructs followed by ribosome profiling in different genetic backgrounds.

18S无功能核糖体RNA降解（18S nonfunctional rRNA decay, NRD）可识别并清除丧失翻译功能的18S核糖体RNA。目前，与无功能18S核糖体RNA的识别及周转过程相关的分子机制仍不明朗。尽管NRD已在酿酒酵母（Saccharomyces cerevisiae）中被发现并得到专门研究，但该质量控制通路是否存在于哺乳动物体内仍有待确认。本研究证实了18S NRD在哺乳动物细胞中的保守性。通过全基因组CRISPR遗传相互作用筛选，我们鉴定出无功能18S核糖体RNA触发的两类分子事件：一是由GCN2介导的整合应激反应（integrated stress response, ISR）激活，二是由RNF10介导的核糖体蛋白泛素化。选择性核糖体谱分析结果显示，无功能18S核糖体RNA会诱导翻译起始位点处出现翻译停滞。生化分析表明，整合应激反应的激活会限制翻译起始，减缓扫描过程中的43S预起始复合物与停滞在起始位点的无功能80S核糖体之间的碰撞。因此，整合应激反应可通过RNF10介导的泛素化过程，促进无功能18S核糖体RNA与40S核糖体蛋白的周转。综上，本研究结果揭示了一种动态反馈机制：细胞通过微调翻译起始过程，借助GCN2-RNF10信号轴完成核糖体功能质量监控。
整体实验设计：为鉴定转录组范围内的核糖体暂停位点，我们采用针对18S标签构建体的MS2亲和纯化沉淀技术，结合不同遗传背景下的核糖体谱分析。