Characterizing a new role for timeless in the generation of robust and plastic circadian rhythms

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Project Summary Circadian clocks organize cellular, physiological and behavioral timing in 24-hour cycles. Understanding how circadian rhythms are generated, maintained and adapted to changing conditions is key. Indeed, several diseases such as cancer and depression are associated with a misalignment between the circadian clock and the external environment. The current model postulates that circadian oscillators keep time by complex transcriptional and post-transcriptional feedback loops. The circadian component encoded by timeless (tim) has a special place at the center of the circadian clock architecture. TIM is essential for circadian rhythms and, at the same time, constitutes an entry point for external signals, such as light and temperature, into the core circadian machinery. Recently, we uncovered the existence of several mRNA and protein isoforms generated from tim by alternative processing. These isoforms are subjected to different types of regulation and encode proteins with different properties, suggesting that the mechanisms mediating tim function are more complex than previously though. Importantly, we found that temperature strongly regulates the relative levels of the different RNA isoforms produced at a given time, likely by acting directly on tim pre-mRNA processing. Importantly, we have found that elimination of one isoform (named tim-s) results in abnormal locomotor activity and circadian rhythms, demonstrating the importance of this regulation. This proposal aims to unravel the importance of tim alternative RNA processing for the robustness and plasticity of the circadian system. In order to do so, we will first generate flies in which the alternative splicing of tim is locked into one state. This will allow us to determine the functionality of the different isoforms. We will follow by characterizing the mode of action of these tim isoforms. Last but not least, we will determine the mechanisms by which temperature regulates tim alternative splicing. In sum, this project will illuminate the mechanism of tim alternative splicing and elucidate the functions of the different tim and per mRNA and protein isoforms.