Single-protein/RNA imaging reveals ZNF598 as a limiting factor in resolving collided ribosomes

Abstract

Ribosome-associated protein quality control (RQC) is a surveillance system that identifies and processes aberrant mRNAs with collided ribosomes. ZNF598 plays a key role by ubiquitinating the 40S subunit of collided ribosomes. However, how ZNF598 distinguishes stalled from transient ribosome collisions remains unclear. To address this, we developed a method to visualize the binding of a single protein to a specific mRNA while simultaneously determining its translation status. By endogenously tagging ZNF598 with HaloTag, we observed its strong interaction with RQC reporter mRNAs. We discovered that multiple ZNF598s engage with a single RQC mRNA, suggesting that ZNF598 recognizes more than just the leading collided ribosome in a queue. Overexpressing ZNF598 increased the ribosomal clearance rate, indicating that it is a rate-limiting factor for RQC. Interestingly, a subset of supposedly “normal” mRNAs may be damaged and targeted by ZNF598, underscoring the importance of RQC to maintain the proteome quality even in unstressed conditions. Under global UV-induced RNA damage, ZNF598 recruitment to the reporter RQC mRNA diminished, highlighting its role as a limiting factor in managing widespread ribosome collisions.