Teodorowicz, W.; Gurzeler, L.-A.; Muehlemann, O.

Various ribosome-associated quality control pathways safeguard translation fidelity by detecting and resolving aberrant translation events. Recently, a pathway that senses stalled ribosomes with an occluded A-site has been described. The small molecules NVS1.1 and Ternatin-4 induce ribosome stalling by trapping eRF1 and eEF1A1, respectively, within the ribosomal A-site, thereby promoting their ubiquitination and proteasomal degradation. In addition to the previously identified factors GCN1, RNF14, and RNF25, we demonstrate that the deubiquitinase USP9X is required for efficient proteasomal degradation of A-site-trapped proteins. Catalytic inactivation of USP9X abolishes K6-linked ubiquitin chains that serve as signals for VCP-mediated extraction. As a result, loss of K6-linked ubiquitination prevents efficient extraction of eRF1 from stalled ribosomes and impairs proteasomal degradation. Clearance of eRF1 engages a feedback mechanism that enforces translational shutdown through 4EHP (eIF4E2), whereas impaired degradation prevents translational repression.