Abstract
The Drosophila circadian pacemaker consists of transcriptional feedback loops subjected to post-transcriptional and post-translational regulation. While post-translational regulatory mechanisms have been studied in detail, much less is known about circadian post-transcriptional control. Thus, we targeted 364 RNA binding and RNA associated proteins with RNA interference. Among the 43 hits we identified was the alternative splicing regulator P-element somatic inhibitor (PSI). PSI regulates the thermosensitive alternative splicing of timeless (tim), promoting splicing events favored at warm temperature over those increased at cold temperature. Psi downregulation shortens the period of circadian rhythms and advances the phase of circadian behavior under temperature cycle. Interestingly, both phenotypes were suppressed in flies that could produce TIM proteins only from a transgene that cannot form the thermosensitive splicing isoforms. Therefore, we conclude that PSI regulates the period of Drosophila circadian rhythms and circadian behavior phase during temperature cycling through its modulation of the tim splicing pattern.
Highlights
Circadian rhythms are the organism’s physiological and behavioral strategies for coping with daily oscillations in environment conditions
To identify novel genes that act at the post-transcriptional level to regulate circadian locomotor behavior, we screened 364 genes, which were annotated in either Flybase (FB2014_03, Thurmond et al, 2019) or the RNA Binding Protein Database (Cook et al, 2011) as RNA binding or involved in RNA associated processes, using period length as a readout of clock function (Supplementary file 1: RNA-associated proteins (RAPs) Screen Dataset)
When Psi is downregulated, the circadian pacemaker speeds up and behavior phase under temperature cycles is advanced by 3 hr, and these phenotypes appear to be predominantly caused by an abnormal tim splicing pattern
Summary
Circadian rhythms are the organism’s physiological and behavioral strategies for coping with daily oscillations in environment conditions. Per is regulated post-transcriptionally by the TWENTYFOUR-ATAXIN2 translational activation complex (Zhang et al, 2013; Lim et al, 2011; Lim and Allada, 2013a; Lee et al, 2017) This complex works by binding to per mRNA as well as the cap-binding complex and poly-A binding protein. They mediate post-transcriptional regulation at every level Many of these regulated events – including alternative splicing, splicing efficiency, mRNA stability, and translation – have been shown to function in molecular clocks. To obtain a broad view of the Drosophila circadian RAP landscape and its mechanism of action, we performed an RNAi screen targeting 364 of these proteins This led us to discover a role for the splicing factor P-element somatic inhibitor (PSI) in regulating the pace of the molecular clock through alternative splicing of tim
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