CULLIN-3 controls TIMELESS oscillations in the Drosophila circadian clock

PLoS Biol. 2012;10(8):e1001367. doi: 10.1371/journal.pbio.1001367. Epub 2012 Aug 7.

Abstract

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the PERIOD (PER) and TIMELESS (TIM) proteins accumulate during the night, inhibit the activity of the CLOCK (CLK)/CYCLE (CYC) transcriptional complex, and are degraded in the early morning. The control of PER and TIM oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. SLMB, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated PER. We show here that CULLIN-3 (CUL-3) is required for the circadian control of PER and TIM oscillations. Expression of either Cul-3 RNAi or dominant negative forms of CUL-3 in the clock neurons alters locomotor behavior and dampens PER and TIM oscillations in light-dark cycles. In constant conditions, CUL-3 deregulation induces behavioral arrhythmicity and rapidly abolishes TIM cycling, with slower effects on PER. CUL-3 affects TIM accumulation more strongly in the absence of PER and forms protein complexes with hypo-phosphorylated TIM. In contrast, SLMB affects TIM more strongly in the presence of PER and preferentially associates with phosphorylated TIM. CUL-3 and SLMB show additive effects on TIM and PER, suggesting different roles for the two ubiquitination complexes on PER and TIM cycling. This work thus shows that CUL-3 is a new component of the Drosophila clock, which plays an important role in the control of TIM oscillations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Behavior, Animal / physiology*
  • Blotting, Western
  • Circadian Clocks*
  • Cullin Proteins / genetics
  • Cullin Proteins / metabolism*
  • Drosophila / genetics
  • Drosophila / metabolism*
  • Drosophila / physiology
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Gene Expression Regulation
  • Locomotion
  • Multiprotein Complexes / metabolism
  • Neurons / metabolism
  • Period Circadian Proteins / metabolism
  • Phosphorylation
  • Protein Stability
  • Proteolysis
  • RNA Interference
  • RNA, Messenger / analysis
  • RNA, Messenger / metabolism
  • Ubiquitination

Substances

  • Cul3 protein, Drosophila
  • Cullin Proteins
  • Drosophila Proteins
  • Multiprotein Complexes
  • PER protein, Drosophila
  • Period Circadian Proteins
  • RNA, Messenger
  • tim protein, Drosophila

Grants and funding

This work was supported by Agence Nationale de la Recherche “DrosoClock” and “ClockGene”, “Equipe FRM” program of Fondation pour la Recherche Médicale, European Union 6th Framework Programme “EUCLOCK”. AD was supported by Ministère de l'Enseignement Supérieur et de la Recherche and FR by Institut National de la Santé et des Etudes et Recherches Médicales. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.