NonA and CPX Link the Circadian Clockwork to Locomotor Activity in Drosophila

Abstract

Drosophila NonA and its mammalian ortholog NONO are members of the Drosophila behavior and human splicing (DBHS) family. NONO also has a strong circadian connection: it associates with the circadian repressor protein PERIOD (PER) and contributes to circadian timekeeping. Here, we investigate NonA, which is required for proper levels of evening locomotor activity as well as a normal free-running period in Drosophila. NonA is associated with the positive transcription factor CLOCK/CYCLE (CLK/CYC), interacts directly with complexin (cpx) pre-mRNA, and upregulates gene expression, including the gene cpx. Downregulation of cpx expression in circadian neurons phenocopies NonA downregulation, whereas cpx overexpression rescues the nonA RNAi phenotypes, indicating that cpx is an important NonA target gene. As the cpx protein contributes to proper neurotransmitter and neuropeptide release in response to calcium, these results and others indicate that this control is important for the normal circadian regulation of locomotor activity.

Keywords: CLOCK; CPX; Drosophila; NonA; PDF; circadian rhythm; neuropeptide; neurotransmitter; vesicle release.

Figure 1.. NonA Physically Interacts with CLK in Fly Head Extracts.

A. Schematic flow chart showing purification of the CLK protein complex from fly head extracts. CLK protein is pull down efficiently by two-step IP by western blotting. Only ZT14 time point was shown here. Lane 1: input. Lane 2: supernatant after 1st FLAG IP. Lane 3: bead wash. Lane 4: protein elution from FLAG beads. Lane 5: supernatant after 2nd streptavidin IP. B. More NonA peptides were identified in the CLK complex at ZT10 and ZT14. CLK and NonA peptide number detected by mass spectrometry (average of two experiments) (Top). The ratio of NonA to CLK peptide numbers (left bottom). The ratio of NonA to CLK peptide numbers normalized to their molecular weights (right bottom). C. Co-IP of NonA with CLK by western blotting. Note that the maximal interaction of NonA and CLK at ZT14. The immunoprecipitates were assayed by anti-FLAG (CLK) and anti-PER, anti-NonA Western blotting. D. Anti-CLK, anti-NonA, anti-PER ChIP assay at the tim E-box. Note that CLK and NonA have the maximal binding on tim E-box at ZT14. Y-axis represents the IP signals relative to the input signals assayed by Q-PCR at the tim E-box. Averages of three experiments are shown.

Figure 2.. Downregulation of NonA Lengthens Circadian Free-run Period and Inhibits Evening Activity Anticipation.

A. nonA RNAi knockdown by tim-gal4, tubgal80ts or dvpdf-gal4 resulted in modest long period. Flies were entrained for 3–4 d in 12-hrs-light-12-hrs-dark (LD) cycles and released into constant darkness (DD) for at least 5 days at 29°C. τ: free-run period. Average activity actograms under DD1-5 were shown for nonA RNAi/+, tim-gal4, tubgal80ts>NonA RNAi, dvpdf-gal4>NonA RNAi flies (Levine et al 2012). tim-gal4, tubgal80ts>per RNAi as a control for the driver. Note: Knockdown of NonA by tim-gal4 causes lethality of flies. B. Evening activity anticipation is inhibited in the nonA RNAi mutants. Flies were entrained as A. Average activity plots at 4 LD cycles were shown. Evening locomotor activity (anticipation) was dramatically inhibited by the tim-gal4, tub-gal80ts and dvpdf-gal4 drivers (black arrows). The inhibition in the pdf-gal4> nonA RNAi stain is modest probably due to its weaker driver. Evening anticipation index is calculated as the percentage of the total activity 3 hours before light-off relative to total evening activity. Statistical analysis was done by 2-tailed, unpaired Student's t tests. Compared to the control, the evening anticipation indexes in all RNAi strains tested are significantly reduced (***p<0.001). LD cycles light-on (white bar), light-off (gray bar).

Figure 3.. Cpx pre-mRNA is a Bona Fide NonA Target.

A. Flow chart for manually sorting GFP-labeled dvpdf cells from fly brains. Flies were entrained for 4 LD cycles at 29°C and collected at ZT14 and ZT23. Brains were dissociated and GFP-labeled dvpdf (M- and E-) cells were collected according to Nagoshi et al 2012. RNAseq libraries were prepared according to Abruzzi et al 2017. RNAseq showed that about 70% nonA mRNA is reduced in the M- and E-cells of dvpdf>nonA RNAi line (red oval and the right graph). RNA libraries were 3’ end biased. The blue oval validates the over-expression of nonA dsRNAi replicon in the neurons. B. NonA TRIBE analysis was done from fly brains collected at ZT14. An inducible pan-neuronal driver was used to drive the expression of UAS-nonA-ADAR due to the toxicity of the fusion protein. 4 controls and 3 experiments were done as follows: 1 and 2: elev-gs with RU486; 3, 4: UAS-nonA-ADAR with RU486; 5,6,7: elev-gs>UAS-nonA-ADAR with RU486. Nascent RNAs were extracted from 30 brains and libraries were prepared and analyzed according to McMahon et al 2016. Total edit sites and edits per million mapped reads were show the left. The high confidence (HC) edit was defined as those with at least 20 reads and 10% editing in 2/3 of three experiment group (5,6,7). Negative control edit sites are those present in any of the 4 control groups with at least 20 reads and 10% editing. 1001 genes were identified as HC NonA TRIBE targets within which 639 genes have at least two editing sites. C. The quantification of the location of NonA TRIBE edits according to (McMahon et al., 2016) in nascent RNAs show that NonA preferably binds introns. There is a modest increase of editing percentage (18%) when NonA-ADAR is expressed in the neurons. D. The overlap of CLK direct targets from fly heads and transcripts down-regulated by nonA RNAi in M- and E-cells and NonA TRIBE targets as in Fig. 3C. We intersected transcripts downregulated in the NonA RNAi mutant and CLK direct targets (Abruzzi et al 2011) and found there is only 32 genes shared by these two groups (top graph). There are 14 genes overlapping within CLK targets, NonA RNAi targets and NonA TRIBE targets (bottom graph). E. Intersection of NonA TRIBE targets and RNAi targets suggests that NonA preferably activates gene expression in circadian neurons. There is about 20% percent of NonA TRIBE targets whose transcripts are also downregulated in the nonA RNAi mutant. Only 3% of TRIBE top100 targets whose transcripts are upregulated by the RNAi.

Figure 4.. CPX is a Strong NonA TRIBE Target and Required for Evening Activity Anticipation.

A. RNA-seq analysis shows cpx mRNA is reduced by about 2 folds in the M- and E-cells of dvpdf>nonA RNAi line, Y axis: the FPKM reads from one reprehensive replica in the driver control and RNAi samples (left). Anti-CLK ChIP-seq suggests that CLK binds at the cpx gene locus probably with the aid of NonA and cpx RNA (right). B. CPX is a NonA TRIBE target. Top graph shows comparable cpx mRNA expression levels in the control and nonA-ADAR strains. Middle graph shows there is a dramatic increase in edit sites at the cpx locus in the nonA-ADAR strain. Each solid bar indicates an editing event. Bottom graph showed the total editing sites on the cpx transcripts as in B. C: Downregulation of CPX by the tim-gal4, tub-gal80^ts or dvpdf-gal4 drivers inhibits evening activity anticipation. Experiments and behavior analysis were performed as in Figure 2. D. Down-regulation of CPX by the tim-gal4, tub-gal80^ts driver lengthens the free-run period whereas CPX knockdown in the dvpdf cells has no period effect.

Figure 5.. PDF Signaling is Required for the NonA/CPX-Mediated Evening Locomotor Activity.

A. PDF signaling inhibits overall activity under constant darkness. Overexpression of t-PDF (inhibitory) or knockdown of PDF receptor(excitatory) showed opposite effects on overall locomotor activity. B Quantification of average mean activities as in Fig. 5A. C. Either CPX over-expression or PDF knockdown rescues the evening activity anticipation defect in the nonA RNAi mutant. Experiments were performed as in Fig. 2. Average activity actograms under 4 LD cycles were shown. Arrow shows the evening locomotor anticipation. D. Quantification of Fig. 5B. Note that that cpx overexpression or pdf RNAi knockdowns significantly increases evening anticipation index and therefore rescues the evening defect of the nonA RNAi mutant. Compared to the control, the evening anticipation index in all RNAi strains tested is significantly reduced (*** p<0.001).

Figure 6.. Circadian Neuronal Activity in the nonA RNAi, cpx RNAi and pdf RNAi Mutants Assayed by a Tric-LUC Reporter.

A. Evening neuronal activity is reduced in the nonA or cpx RNAi mutants whereas PDF downregulation increases evening neuronal activity. Flies were entrained under 3-4 LD cycles at 29°C and luciferase activity was recorded in a topcounter monitor. Data was analyzed by Matlab software as described in the method. Y-axis: LUC activity. X-axis: days under LD. B. Quantification of evening and morning peak neuronal activity from the control and RNAi mutants. 3 LD cycles of peak activity were averaged and were plot for the control and RNAi strains Note that nonA RNAi knockdown significantly inhibited evening neuronal activity compared to the driver control. The cpx RNAi mutant exhibited reduced morning and evening neuronal activity relative to the driver control whereas PDF knockdown increases the activity (*P<0.05, **P<0.01). C. PDF signals at the PDF cells dorsal projections are reduced in the nonA and cpx RNAi driven by the dvpdf-gal4, tubgal80ts driver. The RNAi and control lines were entrained for 4 LD cycles at 29°C and PDF immunostaining were performed according to Menegazzi et al 2013. scale bar: 20 μm. Statistical analysis was done by one-way Anova with post-hoc Turkey HSD. * p<0.05, ** p<0.01. D. Model of how NonA and CPX contribute to circadian evening locomotor activity. NonA and CPX may coordinate the PDF-dependent and PDF-independent mechanisms to regulate circadian evening activity.