miRNAs Are Required for Generating a Time Delay Critical for the Circadian Oscillator

Background: Circadian clocks coordinate an organism's activities and regulate metabolic homeostasis in relation to daily environmental changes, most notably light/dark cycles. As in other organisms, the timekeeping mechanism in mammals depends on a self-sustaining transcriptional negative feedback loop with a built-in time delay in feedback inhibition. Although the time delay is essential for generating a slow, self-sustaining negative feedback loop with a period close to 24 hr, the exact mechanisms underlying the time delay are not known. Results: Here, we show that RNAi mediated by microRNAs (miRNAs) is an essential mechanism in generating the time delay. In Dicer-deficient (and thus miRNA-deficient) cells and mice, circadian rhythms were dramatically shortened (by similar to 2 hr), although the rhythms remained robust. The period shortening was caused by faster PERI and PER2 translation in the Dicer-deficient cells. We also identified three specific miRNAs that regulate Per expression and showed that knockdown of these miRNAs in wild-type cells also shortened the circadian period. Conclusions: Consistent with the canonical function of miRNAs as translational modulators of target genes and their widespread roles in cell physiology, circadian rhythms are also modulated by miRNA-mediated RNAi acting on posttranscriptional regulation of key clock genes. Our present study definitively shows that RNAi is an important modulator of circadian rhythms by controlling the pace of PER synthesis and presents a novel layer of regulation for the clock.