Author response: Drosophila PSI controls circadian period and the phase of circadian behavior under temperature cycle via tim splicing Article Swipe

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Lauren E. Foley , Jinli Ling , Radhika Joshi , Naveh Evantal , Sebastián Kadener , Patrick Emery · YOU? · · 2019 · Open Access · · DOI: https://doi.org/10.7554/elife.50063.sa2

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Introduction Circadian rhythms are the organism's physiological and behavioral strategies for coping with daily oscillations in environment conditions. Inputs such as light and temperature feed into a molecular clock via anatomical and molecular input pathways and reset it every day. Light is the dominant cue for entraining the molecular clock, but temperature is also a pervasive resetting signal in natural environments. Paradoxically, clocks must be semi-resistant to temperature: they should not hasten in warm summer months or lag in the winter cold (this is called temperature compensation), but they can synchronize to the daily rise and fall of temperature (temperature entrainment) (Pittendrigh, 1960). Not only can temperature entrain the clock, it also has a role in seasonal adaptation by affecting the phase of behavior (see for example Majercak et al., 1999). Molecular circadian clocks in eukaryotes are made up of negative transcriptional feedback loops (Dunlap, 1999). In Drosophila, the transcription factors CLOCK (CLK) and CYCLE (CYC) bind to E-boxes in the promoters of the clock genes period (per) and timeless (tim) and activate their transcription. PER and TIM proteins accumulate in the cytoplasm where they heterodimerize and enter the nucleus to feedback and repress the activity of CLK and CYC and thus downregulate their own transcription (Hardin, 2011). This main loop is strengthened by a scaffolding of interlocked feedback loops involving the transcription factors vrille (vri), PAR domain protein 1 (Pdp1) and clockwork orange (cwo). Post-translational modifications are well-established mechanisms for adjusting the speed and timing of the clock (Tataroglu and Emery, 2015). Increasing evidence indicates that post-transcriptional mechanisms controlling gene expression are also critical for the proper function of circadian clocks in many organisms. In Drosophila, the post-transcriptional regulation of per mRNA has been best studied. per mRNA stability changes as a function of time (So and Rosbash, 1997). In addition, per contains an intron in its 3'UTR (dmpi8) that is alternatively spliced depending on temperature and lighting conditions (Majercak et al., 1999; Majercak et al., 2004). On cold days, the spliced variant is favored, causing an advance in the accumulation of per transcript levels as well as an advance of the evening activity peak. This behavioral shift means that the fly is more active during the day when the temperature would be most tolerable in their natural environment. The temperature sensitivity of dmpi8 is due to the presence of weak non-canonical splice sites. However, the efficiency of the underlying baseline splicing is affected by four single nucleotide polymorphisms (SNPs) in the per 3'UTR that vary in natural populations and form two distinct haplotypes (Low et al., 2012; Cao and Edery, 2017). Also, while this splicing is temperature-sensitive in two Drosophila species that followed human migration, two species that remained in Africa lack temperature sensitivity of dmpi8 splicing, (Low et al., 2008). Furthermore, Zhang et al. (2018) recently demonstrated that the the trans-acting splicing factor B52 enhances dmpi8 splicing efficiency, and this effect is stronger with one of the two haplotypes. per is also 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. This may enable more efficient translation by promoting circularization of the transcript. Interestingly, this mechanism appears to be required only in the circadian pacemaker neurons. Non-canonical translation initiation has also been implicated in the control of PER translation (Bradley et al., 2012). Regulation of PER protein translation has also been studied in mammals, with RBM4 being a critical regulator of mPER1 expression (Kojima et al., 2007). In flies however, the homolog of RBM4, LARK, regulates the translation of DBT, a PER kinase (Huang et al., 2014). miRNAs have emerged as important critical regulators of circadian rhythms in Drosophila and mammals, affecting the circadian pacemaker itself, as well as input and output pathways controlling rhythmic behavioral and physiological processes (Tataroglu and Emery, 2015; Lim and Allada, 2013b). RNA-associated proteins (RAPs) include proteins that either bind directly or indirectly to RNAs. 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. Thus, 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. Results An RNAi screen for RNA-associated proteins controlling circadian behavioral rhythms Under constant darkness conditions (DD) flies have an intrinsic period length of about 24 hr. 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: RAP Screen Dataset). We avoided many, but not all, genes with broad effects on gene expression, such as those encoding essential splicing or translation factors. When possible, we used at least two non-overlapping RNAi lines from the TRiP and VDRC collections. RNAi lines were crossed to two different GAL4 drivers: tim-GAL4 (Kaneko et al., 2000) and Pdf-GAL4 (Renn et al., 1999) each combined with a UAS-dicer-2 transgene to enhance the strength of the knockdown (Dietzl et al., 2007). These combinations will be abbreviated as TD2 and PD2, respectively. tim-GAL4 drives expression in all cells with circadian rhythms in the brain and body (Kaneko et al., 2000), while Pdf-GAL4 drives expression in a small subset of clock neurons in the brain: the PDF-positive small (s) and large (l) LNvs (Renn et al., 1999). Among them, the sLNvs are critical pacemaker neurons that drive circadian behavior in DD (Renn et al., 1999; Stoleru et al., 2005). In the initial round of screening, we tested the behavior of 4–8 males for each RNAi line crossed to both TD2 and PD2 (occasionally, fewer males were tested if a cross produced little progeny). We also crossed some RNAi lines to w1118 (+) flies (most were lines selected for retest, see below). We noticed that RNAi/+ control flies for the TRiP collection were 0.3 hr shorter than those of the VDRC collection (Figure 1A). Furthermore, the mean period from all RNAi lines crossed to either PD2 or TD2 was significantly shorter for the TRiP collection than for the VDRC collection (Figure 1A) (0.2 hr, TD2 crosses; 0.5 hr, PD2 crosses). We also found that many of the VDRC KK lines that resulted in long period phenotypes when crossed to both drivers contained insertions in the 40D locus (VDRC annotation), although this effect was stronger with PD2 than TD2. It has been shown that this landing site is in the 5'UTR of tiptop (tio) and can lead to non-specific effects in combination with some GAL4 drivers, likely due to misexpression of tio (Vissers et al., 2016; Green et al., 2014). Indeed, when we crossed a control line that contains a UAS insertion at 40D (40D-UAS) to PD2, the progeny also had a ca. 0.6 hr longer period relative to the PD2 control (Figure 1B). Thus, in order to determine a cutoff for candidates to further investigate, we analyzed the data obtained in our screen from the TRiP, VDRC, and the 40D KK VDRC lines independently (Figure 1C). These data are represented in two overlaid histograms that show period distributions: one for the TD2 crosses (blue) and one for the PD2 crosses (magenta). We chose a cutoff of two standard deviations (SD) from the mean period length for each RNAi line set. RNAi lines were selected for repeat if knockdown resulted in period lengths above or below the 2-SD cutoff. We also chose to repeat a subset of lines that did not pass the cutoff but were of interest and showed period lengthening or shortening, as well as lines that were highly arrhythmic in constant darkness (DD) or had an abnormal pattern of behavior in a light-dark cycle (LD). After a total of three independent experiments, we ended up with 43 candidates (Table 1) that passed the period length cutoffs determined by the initial screen; 31 showed a long period phenotype, while 12 had a short period. One line showed a short period phenotype with PD2 but was long with TD2 (although just below the 2-SD cutoff). Although loss of rhythmicity was also observed in many lines (Supplementary file 1), we decided to focus the present screen on period alterations to increase the probability of identifying proteins that regulate the circadian molecular pacemaker. Indeed, a change in the period length of circadian behavior is most likely caused by a defect in the molecular pacemaker of circadian neurons, while an increase in arrhythmicity can also originate from disruption of output pathways, abnormal development of the neuronal circuits underlying circadian behavioral rhythms, or cell death in the circadian neural network, for example. Figure 1 Download asset Open asset An RNAi screen of RNA associated proteins identifies long and short period hits. (A–B) Background effect of TRiP and VDRC collections on circadian period length. Circadian period length (hrs) is plotted on the y axis. RNAi collection and genotypes are labeled. Error bars represent SEM. (A) Left group (black bars): Patterned bars are the average of period lengths of a subset of RNAi lines in the screen crossed to w1118 (TRiP/+ N = 17 crosses, VDRC/+ N = 46 crosses, 40D KK VDRC/+ N = 20 crosses). Solid bar is the w1118 control (N = 20 crosses). Middle group (blue bars): Patterned bars are the average of period lengths of all RNAi lines in the screen crossed to tim-GAL4, UAS-Dicer2 (TD2) (TRiP/TD2 N = 151 crosses, VDRC/TD2 N = 340 crosses, 40D KK VDRC/TD2 N = 61 crosses). Solid bar is the TD2/+ control (N = 35 crosses). Right group (magenta bars): Patterned bars are the average of period lengths of all RNAi lines in the screen crossed to Pdf-GAL4, UAS-Dicer2 (PD2) (TRiP/PD2 N = 176 crosses, VDRC/PD2 N = 448 crosses, 40D KK VDRC/PD2 N = 69 crosses). Solid bar is the PD2/+ control (N = 36 crosses). One-way ANOVA followed by Tukey's multiple comparison test: *p<0.05, ***p<0.001, ****p<0.0001. Note that the overall period lengthening, relative to wild-type (w1118), when RNAi lines are crossed to TD2 or PD2 is a background effect of our drivers (see main text), while the period differences between the TRiP (shorter) and VDRC (longer) collections is most likely a background effect of the RNAi lines themselves. There is also a lengthening effect of the 40D insertion site in the VDRC KK collection that cannot be explained by a background effect, as it is not present in the RNAi controls (Left panel). Instead the lengthening was only observed when these lines were crossed to our drivers. A modest effect was seen with TD2 (middle panel) and a larger effect was seen with PD2 (right panel). (B) The period lengthening effect of the VDRC 40D KK lines is likely due to overexpression of tio, as we observed lengthening when a control line that lacks a RNAi transgene, but still has a UAS insertion in the 40D (40D-UAS) locus was crossed to PD2. N = 32 flies per genotype, ****p<0.0001, Unpaired Student's t-test. (C) Histogram of period lengths obtained in the initial round of screening. Number of lines per bin is on the y axis. Binned period length (hrs) is on the x axis. Bin size is 0.1 hr. TD2 crosses are in blue and PD2 crosses are in magenta. Dashed lines indicate our cutoff of 2 standard deviations from the mean. Number of crosses that fell above or below the cutoff is indicated. Top panel: TRiP lines. 0 lines crossed to TD2 and 2 lines crossed to PD2 gave rise to short periods and were selected for repeats. four lines crossed to TD2 and 10 lines crossed to PD2 gave rise to long periods and were selected for repeats. Middle panel: VDRC lines. eight lines crossed to TD2 and 5 lines crossed to PD2 gave rise to short periods and were selected for repeats. 12 lines crossed to TD2 and 20 lines crossed to PD2 gave rise to long periods and were selected for repeats. Bottom panel: VDRC 40D KK lines. one line crossed to TD2 and 1 line crossed to PD2 gave rise to short periods and were selected for repeats. two lines crossed to TD2 and 3 lines crossed to PD2 gave rise to long periods and were selected for repeats. Figure 1—source data 1 40D insertion control – behavior data. https://cdn.elifesciences.org/articles/50063/elife-50063-fig1-data1-v2.xlsx Download elife-50063-fig1-data1-v2.xlsx Figure 1—source data 2 Figure statistics – Figure 1. https://cdn.elifesciences.org/articles/50063/elife-50063-fig1-data2-v2.xlsx Download elife-50063-fig1-data2-v2.xlsx Table 1 Circadian behavior in DD of screen candidates GeneRNAi LineDrivern% of Rhythmic FliesPeriod Average ±SEMPower Average ±SEMAtx-1GD11345TD2247526 ± 0.161.5 ± 4.1PD2177626.4 ± 0.150.7 ± 5.6KK108861TD2247925.7 ± 0.149.1 ± 4.7PD2237426.2 ± 0.161.8 ± 4.5barcGD9921PD2207526.5 ± 0.246.9 ± 5.6KK101606**TD268325.3 ± 0.555.4 ± 12.7PD2167527 ± 0.443.9 ± 5.1bsfJF01529TD2248825.8 ± 0.168.4 ± 4.6PD2246725.7 ± 0.147.6 ± 4.1CG16941GD9241PD280HMS00157PD224423.428.3KK102272PD280CG32364HMS03012PD2248825.7 ± 0.158.9 ± 3CG42458KK106121TD2233526.5 ± 0.238.3 ± 4.9PD2228226.2 ± 0.171 ± 4.1CG4849KK101580TD210PD2246327.3 ± 0.248.8 ± 4.1CG5808KK102720*TD2237027.4 ± 0.145.3 ± 5.1PD2245428.5 ± 0.634.8 ± 2.7CG6227GD11867TD210PD2166326.7 ± 0.251.4 ± 7KK108174TD240PD2203024.2 ± 0.430.9 ± 3.5CG7903KK103182*TD224823.626.3PD2247526.4 ± 0.249.1 ± 3.7CG8273GD13870TD2248325.9 ± 0.147.3 ± 4.6PD21410025.4 ± 0.151.2 ± 4.8KK102147TD2245825.5 ± 0.141.1 ± 5PD22310025.7 ± 0.164.3 ± 3.9CG8636GD13992PD2125026.9 ± 0.236 ± 6.4KK110954TD210PD2196326.3 ± 0.351.4 ± 5.6CG9609HMS01000PD2244626.3 ± 0.246.1 ± 6.5KK109846TD2237825.3 ± 0.148.5 ± 4.2PD2239126.3 ± 0.156.4 ± 3.9Cnot4JF03203TD2232623.7 ± 0.139.8 ± 6PD2317723.9 ± 0.151.1 ± 3.2KK101997TD2324723.9 ± 0.137.3 ± 2.9PD2279325 ± 0.148 ± 4.1Dcp2KK101790TD2226426 ± 0.149.7 ± 5.3PD2249225.9 ± 0.162.5 ± 4.1eIF1KK109232*PD224423.268.9eIF3lKK102071TD2242126 ± 0.228.9 ± 2.4PD22310025.7 ± 0.162.5 ± 3.9Hrb98DEHMS00342PD2229125.8 ± 0.160.2 ± 4.1l(1)G0007GD8110PD2246326.3 ± 0.242.4 ± 3.7KK102874TD2241726.9 ± 0.432.6 ± 5.5PD2234826.7 ± 0.248 ± 6.1LSm7GD7971PD2223628 ± 0.443.5 ± 5.6ncmGD7819PD280KK100829*PD2193223.3 ± 0.134.4 ± 5.6Nelf-AKK101005TD2246326.4 ± 0.152.9 ± 4.4PD2237424.8 ± 0.159.4 ± 4.5Not1GD9640PD223422.643.6KK100090PD2103023.8 ± 0.339.4 ± 4.7Not3GD4068PD280KK102144PD2211423.6 ± 0.130.8 ± 2.1Patr-1KK104961*TD2233026.3 ± 0.233.6 ± 3PD2246327.1 ± 0.238.3 ± 3.6Pcf11HMS00406PD28132420.1KK100722PD2242123.3 ± 0.135.4 ± 5pcmGD10926TD2166325.7 ± 0.136.6 ± 4.1PD2205526.3 ± 0.240.4 ± 3.8KK108511TD2242125.7 ± 0.240.7 ± 7.8PD2241727.7 ± 0.632.9 ± 6.1PsiGD14067TD2487923.7 ± 0.0749.6 ± 3.0PD2328424.2 ± 0.153.3 ± 4.1HMS00140TD22410024 ± 0.161.8 ± 4.2PD2208524.5 ± 0.152.9 ± 5.6JF01476TD2249224 ± 0.164.7 ± 4.9PD2249224.3 ± 0.153.2 ± 4KK101882TD2357723.6 ± 0.0661.9 ± 3.7PD2478924.7 ± 0.0656.3 ± 3.4RgaGD9741TD2242126.2 ± 0.132.8 ± 3.2PD2223625.4 ± 0.236.1 ± 4.7RpS3GD4577PD2145726.4 ± 0.248.9 ± 5.9JF01410PD2245025.6 ± 0.234.9 ± 2.3KK109080PD283826 ± 1.334.5 ± 6.3Rrp6GD12195PD2101024.527.2KK100590PD2211023.643.2sbrHMS02414TD2138526.8 ± 0.248.7 ± 5.3PD22110024.9 ± 0.157.2 ± 4.6Set1GD4398TD2209025.8 ± 0.152.1 ± 4.2PD2137725.3 ± 0.142.1 ± 5.5HMS01837TD2237825.6 ± 0.147.9 ± 3.6PD2249224.8 ± 0.150 ± 3.8SmBGD11620PD2136926.2 ± 0.152.1 ± 8HM05097PD2245825.6 ± 0.145.2 ± 4.4KK102021PD2210025.667.1SmEGD13663PD2245825.7 ± 0.337.3 ± 3.3HMS00074PD2810024.5 ± 0.155.1 ± 7.4KK101450PD2156726.5±51.3 ± 7.8SmFJF02276PD2247525.8 ± 0.146.3 ± 3.9KK107814PD2215727.3 ± 0.345.4 ± 4.2smgGD15460PD2245826.5 ± 0.239 ± 3.5Smg5KK102117TD2235223.7 ± 0.138.9 ± 3.7PD2247923.9 ± 0.158.5 ± 4.3SmnJF02057TD236724.225.9PD2245425.7 ± 0.147.2 ± 3.6KK106152TD2246725.3 ± 0.139.7 ± 3.5PD2249626.3 ± 0.248.7 ± 2.7snRNP-U1-CGD11660PD2118225.7 ± 0.156.5 ± 6.1HMS00137PD2249225.8 ± 0.155.9 ± 4.1SpxGD11072PD2146426.5 ± 0.256.1 ± 7.4KK108243TD2410024 ± 0.247.5 ± 10.2PD2197926.9 ± 0.356.4 ± 5Srp54kGD1542PD250KK100462PD2241723.7 ± 0.431.3 ± 6Zn72DGD11579TD2288926.3 ± 0.146.1 ± 4.6PD2228226.4 ± 0.159.4 ± 6.9KK100696TD2267326.8 ± 0.157 ± 3.6PD2248326 ± 0.157 ± 4.5 *Line contains insertion at 40D. ** Unknown if line contains insertion at 40D. Among the 43 candidate genes (Tables 1 and 2), we noticed a high proportion of genes involved or presumed to be involved in splicing (17), including five suspected or known to impact alternative splicing. Perhaps not surprisingly, several genes involved in snRNP assembly were identified in our screen. Their downregulation caused long period phenotypes. We also noticed the presence of four members of the CCR4-NOT complex, which can potentially regulate different steps of mRNA metabolism, including deadenylation, and thus mediate translational repression. Their downregulation mostly caused short period phenotypes and tended to result in high levels of arrhythmicity. Rga downregulation, however, resulted in a long period phenotype, suggesting multiple functions for the CCR4-NOT complex in the regulation of circadian rhythms. Interestingly, two genes implicated in mRNA decapping triggered by deadenylation, were also identified, with long periods observed when these genes were downregulated. Moreover, POP2, a CCR4-NOT component, was recently shown to regulate tim mRNA and protein levels (Grima et al., 2019). gene in our was also recently found to impact circadian behavior et al., 2019). This our screen. Table 2 or known functions of screen candidates function on information from et al., mRNA snRNP mRNA stability, mRNA mRNA mRNA RNA RNA translational protein regulation of of RNA mRNA mRNA RNA mRNA RNA mRNA decapping of mRNA RNA small RNA translation alternative mRNA mRNA mRNA RNA mRNA RNA RNA RNA CCR4-NOT CCR4-NOT mRNA decapping of RNA transcription mRNA mRNA mRNA mRNA CCR4-NOT RNA of mRNA RNA from mRNA RNA contains an RNA RNA snRNP snRNP RNA mRNA mRNA snRNP RNA site mRNA splicing, alternative mRNA mRNA protein targeting to RNA RNA binding of Psi shortens the period of behavioral rhythms A candidate to from our screen was the alternative splicing regulator PSI et al., et al., of Psi with both TD2 and PD2 crossed to two non-overlapping RNAi lines from the VDRC collection and caused a period shortening, to the TD2/+ and PD2/+ controls (Figure Table which the flies to be to the GAL4 drivers in the TD2 and PD2 combination a dominant ca. hr period lengthening (Figure to et al., et al., 1999; Zhang and Emery, 2013; Zhang et al., the RNAi lines did not period on their own (Figure Table While most were performed at we noticed that at TD2/+ control had a period of ca. 24 hr (Figure We could thus flies to both RNAi/+ and TD2/+ control at that temperature. The period of the flies was significantly shorter than both controls (Figure lines from the TRiP collection and also caused period when crossed to TD2 (Table Interestingly, only the that the is important for the control of circadian period (Figure four RNA lines caused a phenotype and only two of (Figure we are that the period was not caused by Moreover, both the and lines have been shown to downregulate Psi et al., and we by that the RNAi line which gave the period phenotype with significantly Psi mRNA levels in (Figure This line was selected for most of the below as it gave the period Figure 2 with 2 see all Download asset Open asset level of Psi the circadian behavior period length and circadian (A) of Psi and of the long and short used in this from et al., of Psi shortens the behavioral period. (B) the average during 3 in and 5 in Left panel: TD2/+ Right panel: Note the short period of Psi knockdown = Circadian period length (hrs) is plotted on the y axis. are on the x axis. Error bars represent SEM. Solid bar is w1118 and bars are bars are Psi knockdown with two non-overlapping RNAi and *p<0.05, ***p<0.001, ****p<0.0001, ANOVA followed by Tukey's multiple comparison (C) multiple comparison and (C) in all circadian Left Note that at the flies are shorter than their RNAi/+ the dominant period lengthening caused by TD2 in circadian pacemaker neurons. in circadian In and only the controls are they are the controls which the flies to be to of the dominant period lengthening caused by PD2 and TD2. of Psi the behavioral period and Left Circadian period length (hrs) is plotted on the y axis. Error bars represent SEM. Right of flies that remained rhythmic in DD is plotted on the y axis. are on the x axis. Not *p<0.05, ****p<0.0001, ANOVA followed by Tukey's multiple comparison of Psi in all circadian the circadian period and the of rhythmic of Psi in circadian pacemaker neurons caused a but period lengthening to the control which is the comparison of the dominant period lengthening caused by was to but not to of Psi in circadian the circadian period and Figure data 1 Psi downregulation and overexpression – behavior data. Download Figure data 2 Figure statistics – Figure Download Table 3 PSI circadian behavior ±SEMPower of Rhythmic downregulation and overexpression at ± ± ± 0.0749.6 ± ± 0.0661.9 ± ± ± ± ± ± 0.0656.3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± downregulation at ± ± ± ± ± ± ± ± ± ± downregulation at ± ± ± ± ± ± ± ± ± ± of effect on circadian ± ± ± ± ± ± ± ± The phenotype caused by Psi downregulation was more with TD2 than with PD2 (Figure Table This was the sLNvs targeted by PD2 determine circadian behavior period in DD et al., et al., 1999). This could if PD2 is less efficient at Psi in sLNvs than or if the short period phenotype is not caused by downregulation of Psi in the To between these two we used combined with TD2 to GAL4 activity in the LNvs et al., while RNAi expression in all circadian this we also observed a period to but the period was not as as with TD2 (Figure

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Author response: Drosophila PSI controls circadian period and the phase of circadian behavior under temperature cycle via tim splicing

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Lauren E. Foley, Jinli Ling, Radhika Joshi, Naveh Evantal, Sebastián Kadener, Patrick Emery

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Circadian rhythm, Period (music), Drosophila (subgenus), RNA splicing, Circadian clock, Biology, Cell biology, Neuroscience, Genetics, Physics, Gene, RNA, Acoustics

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