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

Area of Science:

• Molecular Biology
• Cellular Biology
• Genetics

Background:

• Ribosome-associated protein quality control (RQC) is crucial for cellular proteostasis.
• ZNF598 is a key E3 ubiquitin ligase in the RQC pathway, targeting collided ribosomes.
• The mechanism by which ZNF598 distinguishes between transient and stalled ribosome collisions is not well understood.

Purpose of the Study:

• To investigate how ZNF598 recognizes and binds to collided ribosomes on aberrant mRNAs.
• To determine the role of ZNF598 as a rate-limiting factor in the RQC pathway.
• To explore the involvement of ZNF598 in maintaining proteome quality under normal and stress conditions.

Main Methods:

• Developed a novel method to visualize single ZNF598 binding to specific mRNAs.
• Simultaneously assessed mRNA translation status using endogenously tagged ZNF598 with HaloTag.
• Quantified ZNF598 recruitment dynamics under varying cellular conditions, including UV-induced RNA damage.

Main Results:

• ZNF598 exhibits strong binding to RQC reporter mRNAs, with multiple ZNF598 molecules engaging a single mRNA.
• Overexpression of ZNF598 accelerates ribosomal clearance, confirming its rate-limiting role in RQC.
• ZNF598 targets a subset of non-aberrant mRNAs, suggesting a broader role in proteome surveillance.
• UV-induced RNA damage reduces ZNF598 recruitment, indicating its limited capacity during widespread stress.

Conclusions:

• ZNF598 recognizes more than just the leading collided ribosome, potentially sensing mRNA or ribosome queue characteristics.
• ZNF598 is a critical rate-limiting factor for efficient RQC.
• ZNF598 plays a vital role in maintaining proteome integrity even under unstressed conditions by targeting potentially damaged mRNAs.