Dissecting ubiquitin-mediated circadian clock regulation in Arabidopsis via high-throughput yeast screening
Journal
Journal of Experimental Botany
Start Page
eraf536
ISSN
0022-0957
1460-2431
Date Issued
2025-12-16
Author(s)
Abstract
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, termed 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.
Subjects
Arabidopsis
circadian clock
E3 ubiquitin ligase
F-box protein
JMJD5
LHY
PUB18
U-box protein
ubiquitination
Publisher
Oxford University Press (OUP)
Type
journal article