Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.

核糖体在 mRNA 翻译过程中经常停滞，导致质量控制途径的上下文依赖性激活以维持蛋白质稳态。然而，特异性响应 A 位点封闭的停滞核糖体的监视机制尚未确定。我们发现延伸因子 1α (eEF1A) 抑制剂 ternatin-4 会触发停滞核糖体上 eEF1A 的泛素化和降解。使用化学遗传方法，我们揭示了一个包含两种 E3 连接酶 RNF14 和 RNF25 的信号网络，它们是 eEF1A 降解所需的。定量蛋白质组学揭示了 eEF1A 和一组离散核糖体蛋白的 RNF14 和 RNF25 依赖性泛素化。核糖体碰撞传感器 GCN1 通过与 RNF14 结合发挥重要作用，RNF14 直接泛素化 eEF1A。核糖体蛋白 RPS27A/eS31 的位点特异性、RNF25 依赖性泛素化提供了第二个重要的信号输入。我们的研究结果阐明了泛素信号网络，该网络可监测核糖体 A 位点并促进停滞翻译因子（包括 eEF1A 和终止因子 eRF1）的降解。