Utilization of Arabidopsis E3 ubiquitin decoys high-throughput yeast screen platform to dissect the ubiquitin-mediated circadian clock regulation

Protein ubiquitination, mediated by E3 ubiquitin ligases, is a critical regulatory mechanism of eukaryotic cellular processes, including circadian clock function. However, identifying E3–substrate pairs remains technically challenging due to substrate instability and the genetic redundancy of E3s. To overcome these limitations, we developed a high-throughput yeast two-hybrid E3 decoy screening platform, enabling systematic mapping of E3–substrate interactions. Using a library of 283 Arabidopsis F-box and U-box E3 decoys, we screened 21 core circadian clock regulators and identified 77 potential E3–substrate interaction pairs involving 56 E3s and 16 clock proteins. Focusing on high-confidence hits, we demonstrated that PUB18 physically interacts with the central clock regulators LHY and JMJD5 and promotes their ubiquitination in planta. Genetic analyses further revealed that PUB18 and its homolog PUB19 function redundantly in circadian clock regulation. This study establishes the E3 decoy yeast two-hybrid platform as a versatile and scalable tool for dissecting ubiquitination networks in broad biological processes.