An RNAi Screen To Identify Protein Phosphatases That Function Within theDrosophilaCircadian Clock

Abstract

Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans.

Proteomic analysis of Drosophila CLOCK complexes identifies rhythmic interactions with SAGA and Tip60 complex component NIPPED-A

“…DBT is a component of PER-TIM repression complexes that is found in CLK-GFP complexes coincident with PER (Supplementary Data S1 ). PPD3 function in the clock was tested previously, but RNAi knockdown of Ppd3 did not alter circadian behavior 45 . The remaining GFP-CLK interactors, Prl-1 , Nipped-A and Ubc6 , were characterized in more detail.…”

“…Many other proteins are enriched in GFP-CLK complexes (Supplementary Data S1 ), and a small subset of these proteins associate with GFP-CLK at specific phases of the diurnal cycle. Of these proteins, DBT plays a prominent role in PER phosphorylation and degradation 33 , 64 , 65 and PPD3 has no detectable impact on clock function 45 . RNAi knock down of Prl-1 lengthened circadian period and decreased rhythm strength, while knocking down Nipped-A lengthened circadian period (Table 1 ), consistent with previous work 20 .…”

RNAi-Mediated Reverse Genetic Screen IdentifiedDrosophilaChaperones Regulating Eye and Neuromuscular Junction Morphology

“…The RNAi technique has evolved to be a powerful approach to perform genome-wide or targeted reverse genetic screens to identify genes involved in various cellular pathways ( Sharma and Rao 2009 ; Mohr 2014 ; Chen and Xu 2016 ; Agrawal and Hardin 2016 ; Vissers et al 2016 ). For instance, such screens have identified novel genes involved in Drosophila nervous system development ( Koizumi et al 2007 ) and wound closure ( Lesch et al 2010 ).…”

SUR-8 interacts with PP1-87B to stabilize PERIOD and regulate circadian rhythms in Drosophila

“…We also noticed PP1-96a knockdown showed weak circadian defects: One dsRNA line (BL40906) exhibited slightly lengthened period in tim-GAL4 and Pdf-GAL4 ; the other non-overlapping line (BL42641) only exhibited reduced rhythmicity in tim-GAL4 , not in Pdf-GAL4 . A recent study also found that PP1-96a knockdown lengthened circadian period in tim-GAL4 and Pdf-GAL4 [60]. Although we cannot exclude SUR-8 might also affect PER through PP1-96A since PP1-87B and PP1-96A are in a complex, PP1-87B is likely the main subunit that interacts with SUR-8.…”