Circadian rhythms allow organisms to orchestrate behavioral and physiological outputs in anticipation of predictable diurnal changes in the environment. These rhythms are generated by endogenous molecular clocks that entrain to environmental cycles, predominantly light and temperature, and can maintain rhythms when released into constant conditions (free-run). A conserved mechanistic feature of circadian clocks is an auto-regulatory transcriptional feedback loop in which circadian proteins rhythmically regulate their own expression to generate a clock, and also drive a global program of cycling gene expression. Discovery of this clock mechanism was revolutionary and has received well-deserved recognition, but critical aspects of how the clock is sustained remain unclear.

PERIOD (PER) and TIMELESS (TIM) are the auto-regulating elements of the circadian clock in Drosophila (Allada and Chung, 2010; Hardin, 2011; Zheng and Sehgal, 2012). The expression of per and tim is driven by circadian transcription factors CLOCK (CLK) and CYCLE (CYC), and peaks around the early night. Relative to their mRNA peak, accumulation of PER and TIM proteins is delayed by ~6 hr. In the mid-to-late night, PER and TIM are predominantly nuclear, and once in the nucleus, they repress CLK-CYC activity to decrease per and tim expression. Degradation of TIM and then of PER in the morning resets the transcription cycle and restarts the loop. In order to maintain rhythmicity and set the proper pace of the circadian clock, both PER and TIM need to be dynamically regulated on multiple levels. For instance, a stable circadian molecular oscillator requires temporal delays to separate the phases of gene transcription and repression and thereby prevent these from reaching equilibrium (Zheng and Sehgal, 2012).

The overall levels and stability of TIM constitute a critical circadian modality. Although PER is the more important factor for transcriptional regulation, levels and activity of PER depend upon TIM (Price et al., 1995; Dubruille and Emery, 2008). TIM levels are acutely modulated by light, which promotes the degradation of TIM, and thereby PER, during the day and allows the rise in circadian transcription (Suri et al., 1999; Yang and Sehgal, 2001). Subsequently, TIM accumulation is necessary to stabilize PER and promote its nuclear accumulation (Jang et al., 2015). Thus, in the presence of light:dark cycles, light delays the accumulation of TIM-PER and so contributes to the lag in repression. These temporal relationships are largely preserved in constant darkness, and are also entrained by temperature cycles regardless of light cues, although the mechanisms under these conditions are not known.

While regulated protein stability and translation have been directly explored as mechanisms that could contribute to maintenance of the feedback loop (Dembinska et al., 1997; Chen et al., 1998 ;Lim and Allada, 2013; Zhang et al., 2013), and regulation of protein stability is indeed critical (Zheng and Sehgal, 2012), little investigation has focused on a potential role of alternative splicing. To date, the best-studied role for alternative splicing in Drosophila rhythms is in the temperature-dependence of the behavioral siesta (Majercak et al., 1999; Majercak et al., 2004; Collins et al., 2004). Splicing is driven by spliceosomes, dynamic RNA-protein complexes composed of five core small nuclear ribonucleoprotein particles (U1, U2, U4, U5, U6 snRNP) and >150 additional proteins specific for each snRNP complex (Wahl et al., 2009). In this study, we report a circadian role for Pre-mRNA Processing factor 4 (PRP4), a conserved component of the spliceosomal U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex. We identified PRP4 in a screen for novel regulators of the free-running circadian period, and established that PRP4 is necessary in tim+ clock cells to maintain 24 hr period and robust rhythms of the circadian clock. In addition to prp4, downregulation of multiple tri-snRNP components affected circadian period length and rhythmicity, which led us to implicate this entire spliceosomal complex in circadian regulation. Using unbiased RNA-Sequencing, we characterized the splicing events regulated by PRP4 and identified a novel intron retention event in tim. We show that alternative splicing of this intron in tim represents an important mechanism to time the daily accumulation of TIM, in constant darkness following entrainment to light:dark cycles and also in temperature cycles. Together, these findings identify a mechanism contributing to the maintenance of clock function.

In this study, we identify a novel alternative splicing (AS) mechanism that affects the pace of endogenous circadian oscillations and implicates PRP4 and other tri-snRNP components in circadian clock regulation. Importantly, our findings contribute to the understanding of a longstanding question in the circadian field, specifically how negative feedback by clock proteins is delayed in order to permit distinct phases, and therefore oscillations, of transcriptional activation and repression. In Drosophila, PER translation does not appear to be delayed relative to its mRNA production in a daily cycle (Chen et al., 1998), but the protein is initially destabilized through its phosphorylation by the DOUBLETIME (DBT) kinase (Price et al., 1998). TIM stabilizes PER by alleviating this effect of DBT, and so accumulation of TIM, which only occurs after dark as TIM is degraded by light, determines the rise of PER. How this mechanism persists when light is not a cycling cue, for instance in constant darkness or in temperature cycles in constant light, was not known. Alternative splicing of the tim-tiny intron may be critical under these conditions.

The circadian profile of tim-tiny intron retention (i.e. high during the daytime) is consistent with it delaying the accumulation of TIM protein. Indeed, this mechanism is particularly robust in DD (Figure 6) and also in temperature cycles in constant light conditions (Glaser and Stanewsky, 2005; Yoshii et al., 2005). The retention of tim-tiny peaks at high temperatures, which correlates with low TIM levels (Glaser, F.T., and Stanewsky, R., 2005; Yoshii et al., 2005). We propose a model (Figure 7) whereby the splice choice at the tim-tiny locus modulates the rate of TIM accumulation, which in turn affects the total levels of nuclear PER and TIM. The intron retention of tim-tiny, therefore, likely contributes to a delay between initial tim expression and accumulation of TIM, ensuring stability and robustness of the circadian oscillator. At the same time, the response of this splicing event to temperature promotes flexibility of the clock.

Figure 7

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Traditionally, it has been accepted that AS is driven by a set of auxiliary splicing factors, such as serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs), that act on cis-regulatory splicing modules and recruit U1 and U2 snRNP machinery (Matera and Wang, 2014; Bradley et al., 2015; Han et al., 2011). However, studies from a range of organisms increasingly point to the involvement of the core spliceosomal machinery not only in AS execution but also in splice junction selection (Brooks et al., 2015; Burckin et al., 2005; Clark et al., 2002; Park et al., 2004; Pleiss et al., 2007). Our RNA-seq findings further support the idea that the abundance/stability of at least some components of the core spliceosome has an effect on a select subset of AS events, while constitutive splicing seems to be unperturbed (Supplementary file 3 and 4). A functional network of spliceosomal proteins has been proposed on the basis of their knockdown phenotypes (Papasaikas et al., 2015). According to this model, PRP4 resides within the tri-snRNP regulatory module, which largely coincides with the previously reported physical interactions between the components of the tri-snRNP (Dellaire et al., 2002; Bottner et al., 2005; Schneider et al., 2010). Our findings of similar circadian behavioral effects caused by loss of any of several tri-snRNP components strongly implicate the tri-snRNP complex in circadian regulation (Table 1). It remains to be established how the decreased abundance of tri-snRNP components triggers changes in alternative splicing. We hypothesize that tri-snRNP level/activity is limiting for a subset of AS reactions, likely the ones with weaker splice sites. This hypothesis is supported by two recent studies reporting that (1) Prp4 in fission yeast is necessary to recognize and splice the introns with weak splice sites (Eckert et al., 2016) and (2) decreased availability of mammalian Prpf8 leads to the selective retention of introns that harbor weak 5’ splice sites (Wickramasinghe, V.O., et al., 2015).

We speculate that tri-snRNP components constitute a well-conserved regulatory module for circadian clocks. Conservation of the circadian role of tri-snRNP is suggested by several findings. First, the human homolog of PRP4 was identified as a hit in a genome-wide RNAi screen for regulators of the circadian clock (Zhang et al., 2009). Secondly, SM-like (LSM) proteins that are associated with U6 snRNP were recently shown to regulate circadian rhythmicity in both Arabidopsis and in mammalian cell culture (Perez-Santángelo et al., 2014). Finally, some tri-snRNP components (Prpf8, Prpf31 and SART1) physically associate with mammalian PER2 complexes, further highlighting potential cross-talk between central clock components and the tri-snRNP (Kim et al., 2014).

Although AS has been implicated in the regulation of circadian clocks, it has not been linked to clock function in the manner we report here. In Neurospora crassa, the ratio of alternatively spliced frequency (frq) isoforms determines the robustness of circadian rhythmicity and fine-tunes the period length (Garceau et al., 1997; Liu et al., 1997). In Arabidopsis thaliana, AS regulates the circadian clock by multiple mechanisms, such as the production of new isoforms that competitively inhibit functional clock proteins (Seo et al., 2012) and the modulation of clock RNA levels via the nonsense-mediated decay (NMD) pathway (James et al., 2012; Kwon et al., 2014). In Drosophila, the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) modulates PER levels locally in the pacemaker neurons to regulate circadian rhythms (Beckwith et al., 2017). The key theme that emerges from these studies is that AS acts directly on core clock components to set their levels. Alternatively, splicing mechanisms could regulate diurnal rhythmicity of neuronal excitability, as proposed for splicing of BK channels in the suprachiasmatic nucleus (Shelley et al., 2013).

Our study identifies a novel splicing event in tim that can regulate TIM levels in both cell culture and in flies (Figure 5). How does the splicing affect TIM levels? It is unlikely that the retention of tim-tiny leads to NMD-mediated RNA decrease because the flies with prp4 downregulated do not have reduced tim levels (Figure 4—figure supplement 1). On the contrary, overall tim RNA levels tend to be increased in those flies. Additionally, expression of tim cDNA constructs with constitutively retained tim-tiny (Figure 5) decreases TIM levels without altering tim mRNA levels (data not shown). Therefore, we suggest that it is either the tim mRNA translation step or the stability of the truncated TIM isoform, TIM^tiny, produced by tim-tiny that is sub-optimal. The 267 amino acids at the C-terminus of TIM that are predicted to be lost in TIM^tiny include a putative cytoplasmic localization signal (Saez et al., 2011) as well as a predicted threonine phosphorylation site (Bodenmiller et al., 2007), both of which could significantly change stability and function of TIM. Notably, TIM^tiny is typically not detected in western blots of TIM expression in flies, so it appears that tim-tiny retention serves only to reduce the amount of tim RNA that can effectively produce protein.

While studying the effect of tim-tiny retention in isolation can give us a quick snapshot of its importance, overall splicing of tim is considerably more complex. In parallel to tim-tiny, in this study we examined the splicing profile of a previously reported tim-cold intron (Boothroyd et al., 2007). Cycles of tim-tiny and tim-cold intron retention are similar under light:dark and constant dark conditions (Figure 6), but temperature cycles have different effects on the splicing of these two introns. tim-tiny intron retention increases with hot temperature, whereas tim-cold intron retention increases at the onset of the cold cycle (Figure 6). Also, tim-cold retention does not cycle in temperature cycles in LL, suggesting that it does not contribute to rhythmicity under these conditions. tim-tiny intron is upstream of tim-cold, which means that tim-tiny retention should lead to TIM downregulation regardless of the splicing decision at the tim-cold locus. Following differential splicing at tim-tiny locus, tim-cold could get either retained or spliced, introducing an additional regulatory layer. The interplay of AS at the level of these two introns can produce a range of TIM isoforms, the roles of which remain to be elucidated, particularly with respect to temperature entrainment.

In Drosophila, splicing of D. melanogaster per intron 8 (dmip8), the intron located in the 3’ untranslated region (UTR) of per, is regulated by both light and temperature (Collins et al., 2004; Majercak et al., 2004; Majercak et al., 1999). This splicing mechanism allows flies to delay their evening behavior during long photoperiods and/or high temperatures, and might play a role in seasonal adaptation. On a molecular level, dmip8 retention delays per mRNA and PER protein accumulation. While dmip8 retention was not identified in our initial RNA-Seq data (Supplementary file 3 and 4), our follow up qPCR splicing analysis suggested that PRP4 modestly regulates per splicing (Figure 4—figure supplement 2C). dmip8 retention has a small effect on the free-running period length (~25 hr period) (Cheng et al., 1998; Majercak et al., 1999), but it cannot fully account for the period lengthening phenotype we report for flies with downregulated prp4 (Figure 1A–B; Table 1).

Studies in Arabidopsis suggest that the expression of a number of tri-snRNP components is regulated by the circadian clock (Perez-Santángelo et al., 2013). In Drosophila, recent profiling of mRNA cycling in different neuronal clusters detected prp4 mRNA oscillations in DN1 clock neurons and brr2 cycling in LNvs (Abruzzi et al., 2017). While these RNA-Seq findings have not been verified through other approaches, they lead us to hypothesize that diurnal oscillations in tri-snRNP components drive circadian splicing of tim (Figure 6) and potentially other circadian output genes locally in specific clock neurons. This hypothesis is further strengthened by another recent report (Wang et al., 2018) that suggests heterogeneous alternative splicing profiles for different circadian neuronal groups. In addition to changes in total levels of PRP4, circadian regulation of its kinase activity could contribute to differential splicing of tim over the course of the day. We establish a role for PRP4 in LNvs, the key pacemaker cluster necessary for the maintenance of circadian cycles under constant dark conditions (Figure 1). However, based on our findings that splicing of tim is regulated by temperature and persists in constant light (Figure 6), we speculate that PRP4 also functions in DN1s, clock cells implicated in temperature sensing and entrainment (Yadlapalli et al., 2018; Zhang et al., 2010). In summary, while much of the focus in the circadian field has been on transcriptional or post-translational control, our findings indicate a critical role for alternative splicing, perhaps in a cell-type-specific manner.

Sequencing data have been deposited in GEO under accession code GSE115163.

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Author details

1. Iryna Shakhmantsir

   Chronobiology Program at Penn, Howard Hughes Medical Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2138-0236

2. Soumyashant Nayak

   1. The Institute for Translational Medicine and Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States
   2. Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States

   Contribution

   Software, Formal analysis, Writing—original draft

   Competing interests

   No competing interests declared

3. Gregory R Grant

   1. The Institute for Translational Medicine and Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States
   2. Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States

   Contribution

   Data curation, Software, Formal analysis, Supervision, Methodology, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

4. Amita Sehgal

   1. Chronobiology Program at Penn, Howard Hughes Medical Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States
   2. The Institute for Translational Medicine and Therapeutics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States
   3. Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing—review and editing

   For correspondence

   amita@pennmedicine.upenn.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7354-9641

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