Little is known about the contribution of translational control to circadian rhythms. To address this issue and in
particular translational control by microRNAs (miRNAs), we knocked down the miRNA biogenesis pathway in
Drosophila circadian tissues. In combination with an increase in circadian-mediated transcription, this severely
affected Drosophila behavioral rhythms, indicating that miRNAs function in circadian timekeeping. To identify
miRNA–mRNA pairs important for this regulation, immunoprecipitation of AGO1 followed by microarray
analysis identified mRNAs under miRNA-mediated control. They included three core clock mRNAs—clock (clk),
vrille (vri), and clockworkorange (cwo). To identify miRNAs involved in circadian timekeeping, we exploited
circadian cell-specific inhibition of the miRNA biogenesis pathway followed by tiling array analysis. This
approach identified miRNAs expressed in fly head circadian tissue. Behavioral and molecular experiments show
that one of these miRNAs, the developmental regulator bantam, has a role in the core circadian pacemaker. S2 cell
biochemical experiments indicate that bantam regulates the translation of clk through an association with three
target sites located within the clk 39 untranslated region (UTR). Moreover, clk transgenes harboring mutated
bantam sites in their 39 UTRs rescue rhythms of clk mutant flies much less well than wild-type CLK transgenes.
Kadener S., Menet J., Sugino K., Horwich M.D., Weissbein U., Nawathean P., Vagin V., Zamore P., Nelson S. and Rosbash M. “A role for miRNAs in the Drosophila circadian clock”. Genes Dev. 23(8):2179 (2009).[Web]
The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. Here, we show that Clk mRNA is strongly regulated post-transcriptionally through its 3’UTR. Flies expressing Clk transgenes missing their normal 3’UTR, exhibited variable CLK-driven transcription and circadian behavior, as well as ectopic expression of CLK-target genes in the brain. Surprisingly, in these flies, the numbers of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. In addition, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall our results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila.
Lerner I.*, Bartok O.*, Afik S., Menet J., Wolfson V., Weissbein U., Haimovich D., Gafni C., Friedman N., Rosbash M. and Kadener S. “Clk post-transcriptional control denoises circadian transcription in time and space”. Nature Communications 6 (2015).[Web]