How P-bodies Influence Our Biological Clocks
Circadian clocks are cell-autonomous timekeepers that orchestrate ~24-hour rhythms in the
expression of a large number of genes, thereby controlling much of our physiology and
behavior, including sleep-wake cycles, and metabolism. These clocks are based on negative
transcription translation delayed feedback loops. Specifically, in Drosophila, this clock regulation
involves four core clock proteins: activators CLOCK (CLK) and CYCLE (CYC), and repressors
PERIOD (PER) and TIMELESS (TIM). CLK/CYC homodimers activate transcription by binding
to E-boxes of genes, including per and timeless. After a time delay, PER and TIM enter the
nucleus where they inhibit CLK/CYC, suppressing their own transcription as well as that of other
clock-regulated genes.
While past studies used traditional techniques to study PER nuclear complexes in mammalian
cells, we have adopted a novel BioID-based proximity labeling method to identify in vivo PER
partners in Drosophila clock neurons across the circadian cycle. By creating a unique fly line
with endogenous PER tagged using the miniTurbo enzyme (biotin ligase) and employing 10plex tandem mass tag (TMT) isobaric labeling, we successfully mapped PER's interactions
throughout the circadian rhythm. Surprisingly, we found that several components of Processing
bodies (P-bodies) such as Pcm/Xrn1 (5'-3' exonuclease), Edc3 (Enhancer of decapping-3), and
Me31B/DDX6 (DEAD-box family RNA helicase) interact with PER protein during the activation
phase. P-bodies, which are dynamic, membrane-less ribonucleoprotein granules in the
cytoplasm, function by selectively sequestering RNAs for either decay or delayed translation.
Yet, the potential role of P-bodies in post-transcriptionally regulating key clock mRNAs has not
been explored.
Here, using imaging and sequencing based methods we show that Period and Timeless mRNAs
are localized to P-bodies specifically during the activation phase, and this localization is required
for translational repression of PER and TIM proteins during the activation phase. Loss of me31B
or pcm causes PER and TIM to continuously accumulate in the nucleus, disrupting rhythms in
gene expresssion and extending the circadian cycle to 27 hours. Taken together, P-bodies play
a crucial role in repressing the translation of core clock proteins, ensuring their timely nuclear
entry and maintaining the regular ~24-hour circadian rhythms. We are currently investigating the
molecular mechanisms that govern localization of Period mRNAs to P-bodies.