P-bodies control circadian rhythms by regulating the translational repression and decay of period mRNAs.

Circadian clocks are cell-autonomous timekeepers that regulate 24-hour oscillations in gene expression through conserved transcription-translation feedback loops. Although the core components of these clocks are well-characterized, other regulators remain underexplored. To address this, we used BioID-based proximity labeling to identify proteins near the Period repressor in Drosophila melanogaster clock neurons. Surprisingly, among approximately 700 high-confidence hits, genes linked to processing bodies (p-bodies), such as pacman (pcm) and maternal expression at 31B (me31b), showed significant enrichment. Fluorescence microscopy revealed that p-body condensates associate with Period protein during the late activation phase of the circadian cycle. We found that per mRNAs localize to p-bodies, promoting their translational repression. Disruption of p-body proteins, including the 5'–3' exoribonuclease Pcm or the DEAD-box RNA helicase Me31b, in clock neurons caused persistent accumulation of PER/TIM nuclear condensates, eliminating the delay between activation and repression phases and disrupting ~24-hour circadian rhythms. Additionally, p-body proteins are essential for the decay of core clock mRNAs during the repression phase. Incorporating the Period 3' untranslated region (UTR) into reporter transcripts was sufficient to recruit them to p-bodies, reducing both transcript and protein levels. Thus, p-bodies play a critical dual role in translational repression and mRNA decay, indispensable for maintaining 24-hour circadian rhythms. These findings provide novel insights into circadian regulation and underscore broader principles of post-transcriptional control in gene expression. Overall design: polyA RNA sequencing of Drosophila melanogaster adult whole head lysate (tim-GAL4>UAS-RpL3-3XFLAG). For each biological replicate, two samples were collected and sequenced: 1) INPUT, a portion of lysate collected immediately after head homogenization, 2) FLAG, co-immunoprecipitated RNA prepared from the remaining lysate using anti-FLAG magnetic beads.