High-Amplitude Circadian Rhythms in Drosophila Driven by Calcineurin-Mediated Post-translational Control of sarah

摘要

Post-translational control is a crucial mechanism for circadian timekeeping. Evolutionarily conserved kinases and phosphatases have been implicated in circadian phosphorylation and the degradation of clock-relevant proteins, which sustain high-amplitude rhythms with 24-hr periodicity in animal behaviors and physiology. Here, we report a novel clock function of the heterodimeric Ca²⁺/calmodulin-dependent phosphatase calcineurin and its regulator sarah () in Drosophila. Genomic deletion of the locus dampened circadian locomotor activity rhythms in free-running constant dark after entrainment in light–dark cycles. Poor rhythms in mutant behaviors were accompanied by lower expression of two oscillating clock proteins, PERIOD (PER) and TIMELESS (TIM), at the post-transcriptional level. RNA interference-mediated depletion in circadian pacemaker neurons was sufficient to phenocopy loss-of-function mutation in . On the other hand, a constitutively active form of the catalytic calcineurin subunit, Pp2B-14D^ACT, shortened circadian periodicity in locomotor behaviors and phase-advanced PER and TIM rhythms when overexpressed in clock neurons. Heterozygous deletion induced behavioral arrhythmicity in Pp2B-14D^ACT flies, whereas overexpression rescued short periods in these animals. Finally, pharmacological inhibition of calcineurin in either wild-type flies or clock-less S2 cells decreased the levels of PER and TIM, likely by facilitating their proteasomal degradation. Taken together, these data suggest that negatively regulates calcineurin by cell-autonomously titrating calcineurin-dependent stabilization of PER and TIM proteins, thereby sustaining high-amplitude behavioral rhythms in Drosophila.