Live-cell imaging reveals kinetic determinants of quality control triggered by ribosome stalling

Translation of problematic mRNA sequences induces ribosome stalling, triggering quality-control events, including ribosome rescue and nascent polypeptide degradation. To define the timing and regulation of these processes, we developed a SunTag-based reporter to monitor translation of a problematic sequence (poly[A]) in real time on single mRNAs. Although poly(A)-containing mRNAs undergo continuous translation over the timescale of minutes to hours, ribosome load is increased by ∼3-fold compared to a control, reflecting long queues of ribosomes extending far upstream of the stall. We monitor the resolution of these queues in real time and find that ribosome rescue is very slow compared to both elongation and termination. Modulation of pause strength, collision frequency, and the collision sensor ZNF598 reveals how the dynamics of ribosome collisions and their recognition facilitate selective targeting for quality control. Our results establish that slow clearance of stalled ribosomes allows cells to distinguish between transient and deleterious stalls. [Display omitted] •Ribosome stalling on poly(A) results in long queues of collided ribosomes•Slow rescue of stalled ribosomes confers selective targeting for quality control•Rescue efficiency depends on collision frequency and ribosome queue length•The E3 ubiquitin ligase ZNF598 accelerates rescue of stalled ribosomes Goldman et al. monitor ribosome stalling on defective mRNAs and subsequent rescue by the cellular quality-control machinery by using live-cell single-molecule microscopy. They uncover the determinants of selective and efficient rescue of stalled ribosomes, including stall duration, collision frequency, and sensing by the E3 ubiquitin ligase ZNF598.