biostudies-literatureDe La Cruz ACHHS | National Institutes of HealthHHS | National Institutes of Health (NIH)Howard Hughes Medical InstituteNSF | National Science Foundation Graduate Research Fellowship ProgramNINDS NIH HHSNational Science Foundation (NSF)NSF | National Science Foundation Graduate Research Fellowship Program (GRFP)NIGMS NIH HHSNational Science Foundation5215-5232https://www.ebi.ac.uk/biostudies/studies/S-EPMC12436648biostudies-literatureUnknown44(18)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 rThe EMBO journalSingle-protein/RNA imaging reveals ZNF598 as a limiting factor in resolving collided ribosomes.PMC12436648MCB1817447T32 GM135131T32 GM007445DGE1746891R01GM138770T32-GM135131T32-GM007445RF1NS113820RF1 NS113820Tisdale GGreen RHuang ZNakayama ESinha NKWu BDe La Cruz ACfalseSingle-protein/RNA imaging reveals ZNF598 as a limiting factor in resolving collided ribosomes.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 r2025-01-01T00:00:00Z2025 Sep2026-04-08T19:55:04.199Z2026-04-08T14:36:22.346ZS-EPMC124366484075070010.1038/s44318-025-00523-z