Dissecting ubiquitin-mediated circadian clock regulation in Arabidopsis via high-throughput yeast screening

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 ubiquitin ligases without the domains linking to ubiquitin conjugation, or called 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 PLANT U-BOX 18 (PUB18) physically interacts with the central clock regulators LATE ELONGATED HYPOCOTYL (LHY) and JUMONJI DOMAIN-CONTAINING 5 (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.