Organisms have evolved with biological timekeeping systems to synchronize various biological processes with the cyclic environmental changes caused by earth’s rotation. The biological clock rhythmically regulates many aspects of metabolism, physiology and behaviour in diverse organisms. The clock genes that control the circadian rhythm were first identified in Drosophila and the fly circadian oscillatory mechanisms have mammalian counterparts (Konopka and Benzer, 1971; Sehgal et al., 1994; Allada et al., 1998; Gekakis et al., 1998; Rutila et al., 1998; Kume et al., 1999). The −24 h period of the endogenous clock is maintained by an interlocked transcription translation feedback loop (TTFL) (Hardin et al., 1990). The two basic helix-loop-helix transcription factors; CLOCK (CLK) and CYCLE (CYC) form a heterodimer and binds to the E-box located at the promoter region of the genes period (per) and timeless (tim) in Drosophila (Taylor and Hardin, 2008). PER and TIM form a heterodimer and upon increased accumulation, this heterodimer enters the nucleus and binds to CLK-CYC dimer through a PER-CLK interaction (Lee et al., 1999). This inhibits the transcription of per and tim by repressing the CLK-CYC transcriptional activity (Menet et al., 2010). This TTFL occurs within a span of approximately 24 h and ensures robust circadian oscillation of clock gene transcripts and their proteins.

Zeitgebers are the cyclic environmental time cues capable of entraining the circadian clock, amongst which, light is considered to be the most potent zeitgeber for many organisms (Schlichting and Helfrich-Förster, 2015). Blue light photoreceptor CRYPTOCHROME (CRY), the compound eyes, ocelli and Hofbauer-Buchner (HB) eyelets are involved in the light mediated clock entrainment in Drosophila (Rieger et al., 2003; Veleri et al., 2007; Yoshii et al., 2016). CRY is present in a subset of the clock neurons as well as in the photoreceptor cells and resets the phase of the clock by leading to the degradation of TIM in a light-dependent manner (Ceriani et al., 1999; Busza et al., 2004). The compound eyes, ocelli and HB eyelets express a variety of rhodopsins (RH1-7), each with distinct spectral sensitivity (Sharkey et al., 2020). The clock neurons, particularly the pigment dispersing factor (PDF)-positive ventral lateral neurons (LNvs), which are responsible for the morning peak (M peak) of activity, have dendritic connections projecting to the accessory medullae (AMe) where they communicate directly with the projections from the HB eyelets and indirectly with the compound eyes via the interneurons (Helfrich-Förster et al., 2002; Li et al., 2018). In addition, the interaction between PDF-positive small ventral lateral neurons (sLNvs) and posterior dorsal neurons (DN1ps) plays an important role in governing the morning anticipatory activity under 12 h light: 12 h dark cycles (LD). The presence of either of these neuronal subsets is sufficient to drive morning anticipation (Stoleru et al., 2004; Zhang et al., 2010). The PDF neuropeptide is critical for proper timing of locomotor activity under LD and for maintenance of rhythm under constant darkness (DD) (Renn et al., 1999). In addition to PDF-positive neurons, the dorsal lateral neurons (LNds), which regulate the evening peak (E peak) of activity also have anatomical connections to the AMe (Helfrich-Förster et al., 2007).

Though the clock neuronal circuitry, as well as the clock gene transcript/protein oscillations are well studied, additional regulatory mechanisms which confer robustness to these circadian oscillations are not well understood. The importance of microRNA (miRNA) mediated posttranscriptional regulation in circadian rhythm has been recognized in previous studies (Kadener et al., 2009). miRNAs are endogenous small (19–25 nucleotides long) noncoding RNAs that repress the translation of mRNA or degrade it by forming an imperfect hybrid at the 3′ untranslated region (3′UTR) of the mRNAs (Carthew and Sontheimer, 2009). Recent studies indicate that posttranscriptional regulation by miRNAs has a potential role in maintaining the robustness of the circadian clock as well as in the proper phasing of the rhythm (Herranz and Cohen, 2010; Ebert and Sharp, 2012; Xue and Zhang, 2018). Although several miRNAs are expressed in clock neurons (Kadener et al., 2009), only a few miRNAs among these were examined systematically in Drosophila to elucidate their specific roles in maintaining the circadian rhythm (Kadener et al., 2009; Luo and Sehgal, 2012; Vodala et al., 2012; Chen et al., 2014; Sun et al., 2015; Garaulet et al., 2016; Zhang et al., 2016; Chen and Rosbash, 2017; Cusumano et al., 2018; Nian et al., 2019; Niu et al., 2019; Xia et al., 2020; Nian et al., 2022).

Our present study was aimed at elucidating the role of miRNA-277 (miR-277) in governing the circadian rhythm in Drosophila. Downregulation of miR-277 in the PDF positive neurons advanced the phase of circadian activity-rest rhythm under LD and amplitude under DD. miR-277 expressed in PDF-positive neurons is demonstrated to be important for proper activity distribution around the morning and evening peak under lower light intensities of LD. In addition, the diurnal oscillation of Clk mRNA was found to be dependent on the optimal expression level of miR-277 in the fly head.

Although miRNAs are important regulators of diverse physiological processes, our understanding of their potential roles in maintaining proper clock function is still limited. Recent studies showed that miRNAs are involved in maintaining the phase, amplitude and period of the circadian clock (Kadener et al., 2009; Mehta and Cheng, 2013; Chen et al., 2014; Garaulet et al., 2016; Xue and Zhang, 2018). In the present study, we assessed the role of miR-277 in regulating the circadian time keeping in Drosophila. In order to investigate the role of miR-277 in the clock tissues including the PDF positive clock neurons, miR-277 was downregulated using tim G4 and Pdf G4. Under LD, downregulation of miR-277 in the entire clock neuronal circuit under tim G4 resulted in an increased activity prior to lights-on at ZT23. These flies also exhibited some increased activity after lights-off. Under DD, the robustness of the rhythm was found to be reduced. Downregulation of miR-277 in the PDF-positive neurons, the principal regulators of the M peak, resulted in the advancement of the morning peak phase by about 40 min. The amplitude of the rhythm was reduced in the experimental flies indicating the importance of miR-277 in maintaining robust rhythms under DD. However, we did not find any significant difference in the free-running period of these flies indicating that the phase advancement observed in the experimental flies is not due to a change in the period of the circadian clock. miR-124 KO flies are known to have a similar effect, where the circadian phase is advanced without any change in the circadian period (Zhang et al., 2016). miR-210 KO flies also have an advancement in the phase of the evening peak while having normal FRP, amplitude and PER cyclic expression (Cusumano et al., 2018). Previous studies have shown that at higher temperatures, DN1p neurons promote the morning anticipatory activity without affecting the free-running period because it is temperature-compensated (Pittendrigh, 1954; Lamaze et al., 2017). Similarly, glass ^60j mutants which do not have DN1p are rhythmic under DD but do not exhibit morning anticipatory activity (Helfrich-Förster et al., 2001). The results of our study suggest that the M peak phase advancement observed in flies upon miR-277 downregulation in the PDF neurons is without affecting the speed of the clock. The amplitude of DD rhythm was found to be decreased in the clock/ PDF neurons specific downregulation of miR-277 indicating the importance of miR-277 expression in clock neurons for robust rhythms.

The role of miR-277 in the clock neurons was further investigated by overexpressing miR-277 in the whole clock tissues. This also resulted in a phase advance of the morning peak by a magnitude of approximately 40 min when compared to the controls. In addition, these flies enhanced the activity prior to lights-off. However, when miR-277 was overexpressed in PDF neurons, we found a phase advance in the morning peak of activity without an effect on the activity prior to lights-off. The FRP however was not different for the miR-277 overexpressed flies compared to the controls. This indicates that the overexpression of miR-277 in the PDF neurons might be affecting the PDF signaling from sLNvs to DN1ps and hence altering the activity around M peak without changing the speed of the clock (Zhang et al., 2010). Downregulation as well as upregulation of miR-277 in PDF neurons specific manner producing a similar behavioural phenotype in the phase of morning activity peak is perplexing. Several studies have previously reported similar instances where miRNA downregulation as well as overexpression showing similar phenotype. Downregulation as well as overexpression of miR-276a in the clock neurons is reported to result in decreased rhythmicity (Chen and Rosbash, 2016). Constitutive expression as well as downregulation of miR-277 is known to result in reduced lifespan in flies (Esslinger et al., 2013). This study identified miR-277 as a regulator of branched chain amino acid (BCAA) catabolism and proposed that fine tuning of BCAA catabolism by miR-277 is required for optimal longevity. MicroRNAs are known to fine tune the expression of target genes to physiologically relevant levels (Hobert, 2007; Karres et al., 2007). Hence, we assume that the level of mRNA responsible for phasing of M peak might be fine-tuned to an optimal level by miR-277 and downregulation or overexpression of miR-277 is possibly altering the optimal expression of the target mRNA, leading to phase advancement.

Previous reports suggest that artificially overexpressing miRNAs may cause non-physiological effects (Selbach et al., 2008). Some studies discuss the possibility that overexpression of miRNA could cause downregulation of physiologically non relevant target mRNAs or mRNAs that have weaker affinity binding sites (Schertel et al., 2012). Hence it is possible that the functional target might be different during downregulation and overexpression of the miRNA as the miRNA-target interaction dynamics is dependent on their relative expression levels. Hence the phase advancement observed in downregulation and overexpression of miR-277 might be mediated by two distinct target mRNAs acting on the same or different pathway of M peak phase regulation in the PDF neurons. We also observe that robustness of the rhythm is reduced upon downregulation of miR-277 in the clock neurons but not upon overexpression. This also might be indicative of the fact that two distinct mRNAs are being targeted during downregulation and overexpression.

Since light is the only zeitgeber in an experiment set up with constant temperature and humidity settings, the altered phase under LD condition can be assumed to be the result of changes in the light sensory pathways or due to changes in the core clock components. Blue light photoreceptor CRY and other external photoreceptors are thought to be important in light mediated entrainment (Stanewsky et al., 1998; Helfrich-Förster et al., 2002; Rieger et al., 2003). We assessed the role of miR-277 in governing the phase of behavioural rhythm under various light intensities. Downregulation of miR-277 in PDF-positive neurons altered the activity around the morning peak under LD with various light intensities ranging from 10 to 1000 lux indicating that miR-277 expressed in PDF-positive neurons mediate the proper activity distribution around the morning peak under various light intensities. Whereas we found a difference in the activity around the E peak only at lower light intensities. This might be because the morning oscillator is capable of interacting with the evening oscillator via the PDF neuropeptide and modulates the timing of the evening behaviour (Cusumano et al., 2009). Eventhough both LNds as well as DN1ps are capable of setting the phase of the evening activity, under low light intensities, the morning oscillator enslaves the LNd evening oscillator and this interaction diminishes with increasing light intensity (Chatterjee et al., 2018). miR-277 downregulation in the PDF neuron affected the activity around the evening peak only at lower light intensity and this effect is diminished under high light intensity (1000 lux). Hence, we speculate that miR-277 present in the PDF neurons might be influencing the phasing of the rhythm by controlling the LNv-LNd interaction, possibly via PDF signalling.

The difference in locomotor activity response during light-dark or dark-light transitions under various light intensities of LD might be due to the difference in the entrainment pathways under different light intensities. norpA encoding Phospholipase-C (PLC), which acts downstream of rhodopsin (RH) is critical for entrainment under low light intensities (Zuker, 1996). CRY and 4 Rhodopsin expressing photoreceptors (R1-R6 expressing RH1, yellow R8 inner photoreceptor expressing RH6, pale R7 photoreceptor expressing RH3 and yellow R7 photoreceptor expressing RH4) of compound eyes are also known to be involved in entrainment at low intensities (Stanewsky et al., 1998; Saint-Charles et al., 2016). Entrainment at higher intensities is known to be mediated by HB eyelets possibly with the help of RH6 in a norpA-independent pathway (Schlichting et al., 2019).

Misregulation of miR-277 in the PDF positive neurons did not alter the phase of cry transcript diurnal oscillation, however we found a significant increase in the cry mRNA level at ZT06 in these flies compared to the controls. We further analysed the impact of miR-277 expressed in PDF positive neurons on the central oscillator core clock genes such as per, tim and Clk. When miR-277 was downregulated in PDF positive neurons, we did not find any significant difference in the tim and per transcript oscillation. Nonetheless, downregulation of miR-277 in the PDF neurons abolished Clk mRNA oscillation in the fly head. Such an effect on the Clk oscillation upon downregulation of the miRNA in a very small subset of clock neurons suggests the possibility of miR-277 expressed in the PDF neurons affecting the clock machinery in the visual photoreceptors. Clock as well as clock regulated genes are expressed in the different cell types of Drosophila visual system and circadian neuropeptides such as PDF and Ion transport peptide (ITP) are shown to regulate circadian rhythms in the expression of these genes (Damulewicz et al., 2013; Górska-Andrzejak et al., 2013; Damulewicz et al., 2015). Large LNvs have arborizations extending to the distal medulla and PDF receptors are expressed in the fenestrated glia of the outer retina, suggesting potential PDF signalling to the photoreceptor cells (Im and Taghert, 2010).

When we downregulated miR-277 in PDF positive neurons, Clk mRNA oscillation was abolished but it didn’t affect the per and tim oscillation. Although the Clk mRNA exhibits circadian oscillation in wild type flies, the CLK protein level is not known to exhibit any daily oscillation (Bae et al., 1998; Houl et al., 2006). The ClkARK flies with Clk mRNA expression under the control of per or tim promotor exhibited normal per and tim oscillations and had no effects on the circadian regulation of locomotor activity of the flies except the extended morning activity (Kim et al., 2002). In addition, simulation studies showed that fixing the concentration of the CLK to a constant level or to half of its peak value does not affect the circadian oscillation of per or PER (Smolen et al., 2001; Smolen et al., 2004). Taken together these studies support the observation that Clk mRNA oscillation can be abolished without necessarily impacting the per or tim oscillations.

In order to identify whether Clk is under post-transcriptional regulation of miR-277, we looked at data obtained from previous in vivo study of miRNA regulated transcripts (Xia et al., 2020). The CLEAR-CLIP data does not reveal any interaction between Clk and miR-277. This was further confirmed by in silico analyses. These bioinformatic tools uses various criteria such as complementarity between the seed region of the miRNA and seed match region in the 3′UTR of the mRNA, the free energy change of the hybridisation, evolutionary conservation of the miRNA-mRNA pair, etc., to predict the targets of a miRNA. We have used PicTar, TargetScanFly7.2 and Diana Tools for target prediction. Clk or any of the transcripts in the primary TTFL did not come up in the analyses using these softwares. Hence it is likely that miR-277 does not directly target the Clk and it regulates the Clk oscillation via a mediator. Since Clk gene expression is regulated by PDP1 and VRILLE (VRI) (Cyran et al., 2003), we also checked whether these mRNAs are targets of miR-277. Both CLEAR-CLIP and Diana Tools software suggest pdp1ε as a potential target, suggesting a possible mechanism by which miR-277 might be affecting Clk transcript oscillation.

Studies using the Clk ^Jrk mutants have shown that this mutation abolishes the lights-on locomotor activity response and these flies fail to anticipate light to dark transition under LD. ClkSV40 flies with deleted 3′UTR site exhibit defects in the development of the PDF neurons with unequal number of PDF neurons in both the hemispheres (Lerner et al., 2015). These studies along with our results point to the importance of miR-277 mediated proper Clk expression in the PDF neurons which in turn may govern the clock behavioral output. Although Clk mRNA level was measured in the adult fly head, the Pdf G4 driven miR-277 downregulation was not adult stage specific. Hence we cannot tease apart the developmental and adult stage specific roles of miR-277 in the PDF neurons.

In summary, our study shows a previously unknown role of miR-277 in governing the phase of circadian locomotor activity rest rhythm in Drosophila. The results of our study suggest that miR-277 expressed in the PDF neurons controls circadian rhythms in flies probably by fine tuning the Clk diurnal oscillations.