Polyubiquitin architecture editing on collided ribosomes maintains persistent RQC activity.

In ribosome-associated quality control (RQC), K63-linked polyubiquitination of ribosomal protein uS10 on the stalled ribosome is crucial for recruiting the RQC-trigger (RQT) complex. However, the mechanisms governing the maintenance and recycling of polyubiquitin architecture on colliding ribosomes remain unclear. Here we demonstrate that two deubiquitinating enzymes (DUBs), Ubp2 and Ubp3, play key roles in editing and recycling polyubiquitin chains on yeast uS10, thereby contributing to the promotion of RQC activity. Specifically, Ubp2 eliminates K63-linked polyubiquitin chains from uS10 on the free 40S subunit for recycling, while Ubp3 predominantly cleaves K48-linked di-ubiquitin and K48/K63-mixed-linkage polyubiquitin chains from uS10 on the translating ribosomes. We further demonstrate that K48-linkage-containing ubiquitin chains on uS10 of the colliding ribosome act as a negative signal for the RQT-mediated ribosome dissociation process. Collectively, our findings provide insight into the ubiquitin code in RQC, and define positive functions of two DUBs in maintaining persistent RQC activity.