Li, M.; Zhou, Y.; Huang, H.; Ren, M.; Hu, W.; Liu, X.; Zhang, Y.

Circadian clocks rely on evolutionarily conserved transcriptional-translational feedback loops to align physiology and behavior with the 24 hour day-night cycle. Post translational modifications, especially ubiquitination, critically control core clock protein stability and circadian pace. In both fruit flies and mammals, the abundance of key pacemaker protein PERIOD (PER/PER2) must be tightly regulated to sustain circadian rhythms. However, the canonical phosphorylation dependent pathway cannot fully account for turnover of PER in Drosophila. Here we identify Yippee as a critical regulator of PER level through a genetic screen of downregulating ubiquitination pathways. Knockdown of Yippee in circadian pacemaker neurons significantly lengthens the circadian period by ~2 hours and elevates PER levels. Indeed, YIPPEE physically interacts with PER in fly head. Mechanistically, its mammalian homolog YPEL5 binds PER2, promotes K63 linked polyubiquitination and proteasomal degradation of PER2 independently of FASP region phosphorylation. Lastly, downregulating YPEL5/Ypel5 prolongs circadian periods in human U2OS cells and mice. Our findings reveal an evolutionarily conserved mechanism in which Yippee/YPEL5 regulates circadian rhythms by mediating ubiquitin dependent degradation of PER/PER2.