The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor.

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption.

Drosophila CLOCK protein is under posttranscriptional control and influences light-induced activity.

In the Drosophila circadian clock, daily cycles in the RNA levels of dclock (dClk) are antiphase to those of period (per). We altered the timing/levels of dClk expression by generating transgenic flies whereby per circadian regulatory sequences were used to drive rhythmic transcription of dClk. The results indicate that posttranscriptional mechanisms make substantial contributions to the temporal changes in the abundance of the dCLK protein. Circadian regulation is largely unaffected in the transgenic per-dClk flies despite higher mean levels of dCLK. However, in per-dClk flies the duration of morning activity is lengthened in light-dark cycles and light pulses evoke longer lasting bouts of activity. Our findings suggest that, in addition to a role in generating circadian rhythms, dCLK modulates the direct effects of light on locomotion.

Central and peripheral circadian oscillators in Drosophila.

Drosophila circadian oscillators comprise interlocked period (per)/timeless (tim) and Clock (Clk) transcriptional/translational feedback loops. Within these feedback loops, CLOCK (CLK) and CYCLE (CYC) bind E-box elements to activate per and tim transcription, and we now show that at the same time CLK-CYC repress Clk by activating the transcriptional repressor vrille (vri), thus accounting for the opposite cycling phases of these transcripts and identifying vri as the negative component of the Clk-feedback-loop. The core oscillator mechanism is assumed to be the same for oscillators in different tissues. However, we have shown that CRYPTOCHROME (CRY) has a light-independent function in the oscillator that controls olfaction rhythms, suggesting that CRY may function within the oscillator mechanism itself as it does in mammals. These olfaction rhythms require the function of 'peripheral' oscillators which are distinct from the 'central' lateral neuron (LN) oscillators that mediate locomotor activity rhythms. Preliminary results show that antennal oscillator cells are sufficient and LNs are not necessary for olfaction rhythms, indicating that unlike the situation in mammals, the central oscillator has little impact on the olfaction rhythm oscillator under these conditions.