Clk mRNA turnover de-noises circadian transcription and behavior in time and space. Sebastian Kadener¹, Lerner Immanuel¹, Bartok Osnat¹, Afik Shaked¹, Friedman Nir². 1) Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel; 2) Computer Sciences Department and Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.

   Most organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. These clocks keep time through a complex transcriptional-translational negative feedback loop. Circadian clocks are extraordinarily robust systems, although the mechanisms that contribute to this robustness are still unknown. Here we demonstrated that Clk mRNA turnover is exceptionally high and that this is a key mechanism underlying the robustness of the circadian system. We show that while Clk is transcribed at high levels, mature Clk RNA molecules are quickly degraded in a 3 UTR-depending way through a polyA-shortening mechanism. This regulation is key to buffer stochastic changes in transcription that could result in ectopic expression of clk in time or space. We hypothesize that clk high turnover rate restricts the translatability of Clk mRNA to few hours during the circadian cycle. This is important since we also show that although CLK protein levels are constant throughout the day, CLK protein localization is strongly regulated. In order to test the importance of this mechanism for normal timekeeping, we generated flies carrying a Clk genomic construct in which the Clk 3 UTR has been replaced with a control (SV40) 3 UTR. Indeed, a Clk genomic rescue that has lost the post-transcriptional control rescues Clk mutants at much lower degree than control genomic contructs. In addition, these flies exhibit high levels of ectopic CLK and CLK-CYC targets in non-circadian cells in the fly brain. This ectopic expression is more pronounced under perturbation (i.e. temperature changes) and leads to decrease fertility, higher mortality and developmental phenotypes. Therefore our study shows that post-transcriptional regulation of Clk is essential for proper circadian-cell determination and hence for developmental robustness.