An RNA-Based Dual-Fluorescence Reporter System Reveals Cell Type-Specific and Temporal Dynamics of -1 Programmed Ribosomal Frameshifting.

Translational recoding events, such as programmed ribosomal frameshifting (PRF), are critical regulatory processes that enable the production of multiple proteins from a single mRNA transcript in a finely tuned manner. PRF plays a pivotal role in the gene expression of many viruses, making it an attractive therapeutic target. While the cis-acting elements that drive many recoding events are well-characterised, the impact of the cellular environment on these elements remains poorly understood. To address this gap, we developed an RNA-based dual-fluorescence reporter system that enables real-time monitoring of translational recoding events across a wide range of cell types, including physiologically relevant cell types. This system permits for the quantification of the kinetics of in-trans modulation of frameshift efficiency, providing crucial insights into how cellular context influences recoding events. We validated the system's ability to accurately monitor the frameshift efficiency of signals from SARS-CoV-2, HIV-1 and the human gene PEG10 in live cells, revealing striking differences in frameshift efficiency between cell types, specific to each frameshift cassette. Importantly, this platform offers a robust foundation for large-scale drug screening in physiologically relevant cell types. It not only facilitates the identification of compounds that modulate translational recoding but also enables the assessment of drug entry and activity across different cell types. This dual capability is essential for evaluating the potential efficacy of therapeutic candidates and ensuring that drug effects are accurately measured in biologically meaningful contexts.