Contrôle post-traductionnel des protéines d'horloge dans l'oscillateur circadien de la drosophile – DROSOCLOCK

Circadian rhythms (period of about 24 h) are observed in most living organisms and are associated to many physiological and behavioral functions in metazoans. Such rhythms are driven by internal clocks that can be synchronized with the cycling environment through external cues such as temperature and light. In humans, the clock controls behavioral daily rhythms such as sleep-wake, alertness and intellectual performance and its alteration is involved in sleep disorders and depression. Our laboratory aims at understanding the functionning of the circadian oscillator, using the Drosophila brain clock as a model.
Transcriptional feedback loops appear to be the rule among circadian clocks, and interlocked dual loops have been identified in drosophila and mammals. In drosophila the CLOCK-CYCLE dimer activates the transcription of the period and timeless genes whose products negatively feed-back on the genes activation. However, recent data show that clock proteins oscillations can occur in the absence of transcriptional control and that these oscillations are sufficient to generate behavioral rhythmicity. It indeed appears that the core mechanisms of the circadian oscillator rely to a large extent on the post-translational control of the clock proteins stability. These mechanisms involve phosphorylation, ubiquitination and degradation of the major clock proteins PERIOD, TIMELESS and CLOCK. The overall goal of this project is to understand how these post-translational mechanisms define 24h clock proteins oscillations and participate to the differentiation of the clock neurons during brain development through the regulation of the CLOCK protein.
Our experiments will aim at characterizing how the E3 ubiquitin ligase encoded by the slmb gene controls the circadian degradation of the PER and TIM proteins and to define the molecular function of two new ubiquitin proteases. One is required for the sleep-activity rhythms of drosophila and may interact with SLMB. The other one is encoded by a gene isolated in a transcriptional microarray analysis, which displays a dual circadian control on both its mRNA and protein, similarly to the control of the per and tim genes. Since SLMB-independent degradation mechanisms participate to the core oscillator mechanism and its synchronisation by light-dark cycles, components of other E3 ubiquitin ligases were screened for defects in sleep-activity rhythms through the targeted expression of RNAi transgenes in the clock neurons. Based on experiments indicating that the CLK protein is required for the proper differentiation of the PDF-expressing neurons, we will investigate how its PAS domain partner, CYCLE, participates to the control of the CLK protein levels and how a new ubiquitin ligase specifically expressed in the clock neurons could participate to their differentiation and/or the control of the clock protein levels in these cells.