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

Ana C. De La Cruz,Garrett Tisdale,Emily Nakayama,Zhiyuan Huang,Niladri Sinha,Rachel Green,Bin Wu

Published 2025 in EMBO Journal

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. Ribosomal quality control (RQC) ubiquitin ligase ZNF598 distinguishes transient ribosome collisions from permanent ones. Here, simultaneous imaging of protein-mRNA binding dynamics and translation status visualizes ZNF598 interactions with defective mRNAs in live cells. A new method allows visualization of single-protein/mRNA interaction and translation in live cells. Endogenous tagging of ZNF598 reveals its strong interaction with RQC reporter mRNAs. Multiple ZNF598 proteins are bound to a single RQC mRNA. ZNF598 is a limiting factor in disassembling collided ribosomes. A new method allows visualization of single-protein/mRNA interaction and translation in live cells. Endogenous tagging of ZNF598 reveals its strong interaction with RQC reporter mRNAs. Multiple ZNF598 proteins are bound to a single RQC mRNA. ZNF598 is a limiting factor in disassembling collided ribosomes. Simultaneous visualization of mRNA-protein interactions and translation status in live cells suggests that the ZNF598 E3 recognizes more than just the leading ribosome in a queue.

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