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Emerging roles for post-transcriptional regulation in circadian clocks

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Chunghun Lim1 & Ravi Allada2 Circadian clocks temporally organize behavior and physiology across the 24-h day. Great progress has been made in understanding the molecular basis of timekeeping, with a focus on transcriptional feedback networks that are post-translationally modulated. Yet emerging evidence indicates an important role for post-transcriptional regulation, from splicing, polyadenylation and mRNA stability to translation and non-coding functions exemplified by microRNAs. This level of regulation affects virtually all aspects of circadian systems, from the core timing mechanism and input pathways that synchronize clocks to the environment and output pathways that control overt rhythmicity. We hypothesize that post-transcriptional control confers on circadian clocks enhanced robustness as well as the ability to adapt to different environments. As much of what is known derives from nonneural cells or tissues, future work will be required to investigate the role of post-transcriptional regulation in neural clocks. Circadian clocks have evolved to adaptively align internal biological processes to daily environmental changes. Circadian rhythms are self-sustaining, persisting even in the absence of external time cues. However, the period of these clocks only approximates 24 h. These self-sustaining clocks are reset by oscillating inputs such as light, temperature or feeding to synchronize with the 24-h environment. Period length is relatively constant over a wide range of physiological temperatures, reflecting the robustness of the timekeeping mechanism. Based on these canonical properties, clock systems have been described in terms of a core clock timekeeping mechanism (the ‘gears’), inputs through which environmental signals reset clocks and outputs (the ‘hands’) that generate overt rhythms, such as sleep-wake cycles. Reviews of the molecular mechanisms of circadian timekeeping have largely focused on oscillating transcriptional feedback networks whose components are post-translationally modified1–3. Heterodimeric transcription factors CLOCK and CYCLE (CLK and CYC) in Drosophila and their mammalian homologs CLOCK and BMAL1 activate transcription from E boxes in promoters of circadian clock genes (Fig. 1a,b). Drosophila PERIOD (PER) and TIMELESS (TIM) and mammalian Periods (Per1, Per2 and Per3) and Cryptochromes (Cry1 and Cry2) then feed back to bind the heterodimeric activators and repress their transcriptional activities. The transcriptional repressor CLOCKWORK ORANGE (CWO) in Drosophila and its mammalian homologs Dec1 and Dec2 also feed back to bind E boxes and suppress transcriptional activation by the CLK-CYC and CLOCK-BMAL1 heterodimers, respectively. In contrast, rhythmic transcription of the heterodimeric activator genes is regulated differently between Drosophila and mammals. Two basic leucine zipper transcription factors, VRILLE and PAR-DOMAIN PROTEIN 1, drive oscillating transcription from 1School

of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea. 2Department of Neurobiology, Northwestern University, Evanston, Illinois, USA. Correspondence should be addressed to C.L. ([email protected]) or R.A. ([email protected]). Received 10 June; accepted 12 September; published online 28 October 2013; doi:10.1038/nn.3543

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the Drosophila Clk promoter through their common binding sites, whereas orphan nuclear receptors REV-ERB and ROR proteins govern rhythmic transcription of the mammalian Arntl (also known as Bmal1) gene. In addition, Drosophila CRY primarily functions as a light sensor to regulate light-dependent stability of TIM protein, whereas mammalian Cryptochromes functionally replace TIM to form a transcriptional repressor complex with PER, inhibiting transcription dependent on CLOCK-BMAL1. Phosphorylation of core clock proteins has been implicated in regulating ubiquitin- and proteasome–dependent stability and/or activity of the clock proteins 1. The feedback network architecture of post-translationally modified circadian transcription factors is conserved in a wide range of species including bacteria, fungi, plants and animals3–5. Both neurons in the central nervous system (central clocks) and somatic cells in peripheral tissues (peripheral clocks) exhibit the molecular rhythms that reflect cell-autonomous circadian clock function. However, cycling gene expression in specific subsets of neurons, called central pacemaker neurons, is crucial for maintaining robust behavioral rhythms. In Drosophila, a network of ~150 interconnected neurons drives increases in locomotor activity in anticipation of lights on (morning peak) and lights off (evening peak) (Fig. 1c,e). Discrete subsets of these neurons, the ventral lateral neuron (LNv)–dorsal neuron 1 (DN1) and dorsal LN (LNd)-DN1, drive morning and evening behavior, respectively6–8. The LNvs express the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) that coordinates molecular oscillations across the network. This pacemaker network is a model for understanding how central brain circuits guide behavior. A similar dual dorsal-ventral oscillator system operates in the suprachiasmatic nucleus in the hypothalamus, the master central circadian pacemaker in mammals, timing sleep-wake and coordinating rhythms in peripheral clocks via humoral factors as well as physiological temperature rhythms9–11 (Fig. 1d,f). In addition to these master pacemakers, circadian clocks are also evident in other neurons, including visual photoreceptors, olfactory sensory neurons, learning and memory circuits as well as glia in Drosophila12. Similarly, molecular clocks are evident in the cortex, hippocampus, striatum, olfactory bulb as well as glia VOLUME 16 | NUMBER 11 | NOVEMBER 2013  nature neuroscience

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Figure 1  Molecular and neural bases of circadian clocks and behaviors. (a,b) Canonical transcriptional feedback networks important for circadian clocks in Drosophila (a) and mammals (b). Stars indicate sites of documented post-transcriptional control. (c) Drosophila locomotor activity under light-dark conditions. Zeitgeber time 0 (ZT0) indicates time of lights on, and ZT12 is the time of lights off. Orange and blue arrows indicate anticipation of morning and evening, respectively. White and black bars indicate locomotor activities in light and dark phases, respectively. Error bars, s.e.m. (n = 54). (d) Circadian wheel running behavior in mice under light-dark (LD) and dark-dark or constant dark (DD) conditions. (e) Schematic of circadian pacemaker neuron clusters in adult fly brain (adapted with permission from ref. 8). l-LNv, large ventral lateral neuron; s-LNv, small LNv; LNd, dorsal LN; DN, dorsal neuron; LPN, lateral posterior neuron; Ca, calyces of the mushroom bodies (MB); CC, central complex; PI/PL, pars intercerebralis/lateralis; Oc, ocelli; AL, antennal lobe; aMe, accessory medulla (Me); La, lamina; R1–8, photoreceptor cells of the compound eye (Ey). (f) Neuroanatomical structure of the circadian pacemaker suprachiasmatic nuclei (SCN) in mammals (reproduced with permission from ref. 11). The SCN are bilaterally located on both sides of the third ventricle (dashed lines) and above the optic chiasm. The SCN are functionally divided into dorsal and ventral regions in part marked by neuropeptides, arginine vasopressin (red in dorsal region of left image) and vasoactive intestinal peptides (red in ventral region of right image), respectively, reflecting a coupled oscillator network. Transgenic expression of CLOCK∆19-HA in secretogranin-positive cells is visualized in green in both images.

in mammals10. How different neuronal oscillators are specialized to conduct their unique functions remains unclear. Although there has been a focus on transcriptional and posttranslational mechanisms in circadian rhythms, a role for intervening post-transcriptional mechanisms has been rapidly emerging13–15. Multiple features are regulated including splicing, polyadenylation, translation and microRNA (miRNA)-mediated gene silencing. Moreover, post-transcriptional regulation impacts multiple aspects of clock function from inputs to core clocks to output pathways. In this Review, we will summarize these findings, emphasizing neural clocks in Drosophila and mammals. Much of what we know about post-transcriptional regulation comes from studies of peripheral nonneural tissues and even of fungi and plants, because of their abundance and accessibility. For example, the majority of mRNAs that are rhythmically expressed in mouse liver are not regulated by circadian transcription16, indicating that post-transcriptional mechanisms drive the observed oscillations in mRNA and protein levels. Yet given the similarity in neural and nonneural mechanisms, we believe that these findings are broadly applicable to the nervous system. Nonetheless, we will also discuss aspects that are unique to clock neurons. Finally, we speculate on why circadian clocks have evolved post-transcriptional regulation in the molecular-clock network. Transcriptional termination Transcriptional termination is a newly identified target for negative feedback regulation in the clock (Fig. 2a). In addition to its well-known transcriptional repressor function, mammalian PER rhythmically associates with the RNA helicases DEAD-box polypeptide 5 (DDX5) and DEAH-box protein 9 (DHX9), two components of the transcriptional termination complex, specifically binding clock gene transcripts such as Per1 and Cry2 mRNAs in a circadian nature neuroscience  VOLUME 16 | NUMBER 11 | NOVEMBER 2013

time–dependent manner17. It has been suggested that the PER complex interferes with transcriptional termination by inhibiting the activity of another helicase, senataxin (SETX), which is important for termination. Evidence includes circadian time–dependent accumulation of elongating RNA polymerase II and SETX at 3′ termination sites of target RNAs. Inefficient transcriptional termination causes repression of transcription reinitiation, which reveals a negative feedback mechanism by the RNA-binding PER complex. Future studies will be needed to determine whether this mechanism is operative in pacemaker neurons and/or in other organisms. Alternative splicing Alternative splicing is an effective way to generate multiple isoforms with distinct structure and function from a primary transcript (Fig. 2a). It may mediate signal-dependent and/or tissue-specific regulation of gene expression. The application of RNA sequencing (RNA-seq) and exon arrays has identified widespread clock control of alternative splicing in both the Drosophila brain18 as well as the mouse liver19. Mutation in a Drosophila homolog of Arabidopsis thaliana PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5) causes defects in circadian behavioral rhythms and alternative splicing, emphasizing the evolutionary importance of this post-transcriptional strategy in clock regulation20. As PRMT5 regulates hundreds of alternative splicing events, it links circadian clocks to alternative splicing more globally. Gene-specific thermosensitive splicing has been implicated in temperature-dependent regulation of circadian clocks among poikilotherms including Drosophila (Fig. 2b). Although the duration of a circadian period is thermostable, the circadian phase of adult ­locomotor rhythms is not. Evening activity begins earlier at cool temperatures and later at warm temperatures. Much of these effects can be explained 1545

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by thermosensitive splicing of the Drosophila per gene. Alternative splicing of a per 3′ terminal intron in the untranslated region (dmpi8) is clock-regulated but is also stimulated by lower temperature and shorter photoperiod21–23. Temperature-dependent splicing efficiency from several suboptimal splice sites in dmpi8 may account for these effects24. Modulation of dmpi8 splicing explains shifts in evening behavioral phase between cold days with short photoperiods and hot days with long photoperiods. Given that evening clock neurons LNd-DN1 govern the timing of evening activity peaks6,7, it is likely that regulated dmpi8 splicing occurs in these behaviorally relevant neurons. Although the specific factors regulating this splicing are not clear, a mutation in the splicing component tuII (also known as dprp43) has been shown to lengthen the circadian period25. In the equatorial drosophilids D. yakuba and D. santomea, whose environments are not subject to large seasonal changes in day length or temperature, temperature does not impact the phase of the evening peak. Notably, splicing of per 3′ terminal introns is also not affected by temperature in these species because of changes in per splice site sequences24. These results clearly exemplify the molecular evolution of circadian clock–related adaptive behaviors by alternative splicing. Thermosensitive splicing is also used in fungal clocks26–28, which indicates that alternative splicing is an evolutionarily conserved mechanism by which the clock adapts to different thermal environments. Translation Circadian regulation of global translation. Broad circadian clock– mediated control of translation has been demonstrated in the mouse liver. 20% of soluble proteins in the mouse liver show rhythmic changes in their abundance yet half derive from stably expressed mRNAs29. At least 2% of genes exhibit rhythmic association of their transcripts with polyribosomes independent of any circadian changes in total mRNA levels, an indication of clock-mediated control of translation30. 70% of these rhythmic polysome-associated genes have a peak phase around midnight (Zeitgeber time (ZT)16–18; where lights are turned on and off at ZT0 and ZT12, respectively), consistent with data from genome-wide profiling of circadian poly(A) tail lengths31 (see below). Evidence for rhythmic control of ribosome biogenesis as well as expression of general translation initiation factors has been reported in both Drosophila32 and mammals30. Rhythmic activation of signaling pathways that regulate translation initiation33, target of rapamycin (TOR) pathway in particular, results in circadian timedependent phosphorylation of translation factors in suprachiasmatic nucleus (SCN) clock neurons as well as peripheral clock tissues30,34. In fact, the TOR pathway may also have an important role in the SCN in light-mediated entrainment of locomotor rhythms35. The TOR pathway activates cap-dependent translation, i.e., that depends on the 7-methylguanosine cap present at the 5′ end of mRNA. TOR activation results in release of an inhibitory eukaryotic translation initiation 1546

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Figure 2  Nuclear post-transcriptional control of circadian clocks. (a) Nuclear post-transcriptional steps starting with 5′ capping, transcriptional termination, nuclear polyadenylation, alternative splicing and nuclear export. Examples of clockrelevant control at each step are listed in red. (b) Temperature-sensitive alternative splicing of an intron (dmpi8) within 3′ UTR of Drosophila per gene and diurnal plots of locomotor activitiy under light-dark conditions at the respective temperatures. Zeitgeber time 0 (ZT0) indicates time of lights on, and ZT12 is the time of lights off. Error bars, s.e.m. (n = 61 (left); n = 48 (right)).

factor 4E-binding protein-1 (4EBP1) from the 5′ cap-binding translation initiation factor eIF4E. TOR activation also facilitates S6 rib­ osomal protein phosphorylation, thereby enhancing cap-dependent translation. Clock control may operate to synchronize translation and ribosome biogenesis to a specific time window when energy and nutrients are sufficient to drive these energy-consuming processes (for example, the middle of the night for nocturnal mice)30. Translational control in the core clock. Translational control has also been observed as a key player in the core clock (Fig. 3a). Studies in both Neurospora and cyanobacterial clocks indicate that codon bias and thus translation efficiency of clock gene transcripts has an important role in clock function and adaptation to different environments36,37. Some of the most potent neuronal effects of translational regulators on circadian clock mRNA translation have been observed in Drosophila. From an unbiased genetic screen for clock regulators, Drosophila gene twenty-four (tyf) was identified as a critical activator of PER translation38 (Fig. 3b). However, TYF had no canonical functional domains, raising questions as to its precise molecular mechanism of action. Mass spectrometry analysis revealed that TYF interacts with the Drosophila homolog of the product of the human neurodegenerationassociated gene Ataxin-2 (ATX2)39. It has been further demonstrated that ATX2 is an important player in TYF-dependent translation of PER39,40 (Fig. 3b). Atx2 impairment and tyf mutants display poorly rhythmic circadian behavior with lengthened period as well as strongly dampened PER cycling, particularly in central pacemaker neurons. PER in peripheral clocks, including in retinal photoreceptor neurons as evidenced by assessments in Drosophila heads, appears to be largely spared. Atx2 and tyf phenotypes could be rescued by PER induction in these cases, indicating that PER is the major target of ATX2 and TYF in circadian clock control. Although both TYF and ATX2 associate with per mRNA as well as other clock gene mRNAs, ATX2 requires its interaction with the poly(A)-binding protein (PABP) for RNA binding whereas TYF does not. Depletion of ATX2 also causes dissociation of the TYF-PABP complex and strongly attenuates translational activation by RNA-tethered TYF in cultured cells, suggesting that ATX2 coordinates a TYF-PABP complex to activate PER translation. Why TYF/ATX2 is apparently dispensable for PER translation in peripheral clocks yet critical in central pacemaker neurons remains a mystery. Nonetheless, these studies have collectively identified a neuron-specific translational control system important for behavior. In mammals, translational control may be important for synchronizing peripheral clocks to master neural circadian pacemakers. It has been shown that daily body temperature rhythms in the physiological ranges are sufficient to synchronize peripheral clocks in homeotherms41. A candidate mediator of this rhythm is the cold-inducible RNA-binding protein (CIRP)42 (Fig. 3c). Rhythms in the physiological temperature range (34–38 °C) can drive rhythmic expression of VOLUME 16 | NUMBER 11 | NOVEMBER 2013  nature neuroscience

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Figure 3  Cytoplasmic translational control of circadian clocks. (a) Cytoplasmic translational activation and repression by RNA-binding proteins and miRNAs. RBP indicates regulatory RNA-binding protein, where circles indicate translational activators and hexagons repressors. RISC indicates RNA-induced silencing complex. Examples of clockrelevant control at each step are provided in red. (b) Translational coactivator complex of TYF and ATX2 in activating Drosophila PER translation. ATX2 facilitates the formation of a ribonucleoprotein complex containing TYF, poly(A)-binding protein (PABP) and per RNA competent for high-amplitude PER translation. (c) High-amplitude CLOCK translation driven by cold-inducible RNA binding protein (CIRP) in mammalian clocks. Body temperature cycles generate daily oscillations of Cirp RNA and CIRP protein levels. Subsequently CIRP targets Clock RNA and stimulates its nuclear export and translation.

CIRP protein. Depletion of CIRP proteins in cultured mammalian cells reduced oscillating amplitudes of clock gene and reporter expression. To identify CIRP targets, cross-linking and immunoprecipitation followed by RNA sequencing (CLIP-seq) of CIRP-associated transcripts was performed. In addition, a comparison of gene expression profiles in control versus CIRP-depleted cells revealed several clock gene targets. Upon depletion of CIRP, one such target, Clock, exhibited only modest reductions in RNA levels but profound reductions in protein levels, consistent with a possible role of CIRP in CLOCK translation. Molecular phenotypes in CIRP-depleted cells largely mimicked those in CLOCK-depleted cells. Moreover, circadian rhythms in CIRP-depleted cells were partially rescued by overexpression of CLOCK, suggesting that depletion of CIRP reduced CLOCK translation as a limiting factor for molecular rhythms. Thus, the functional relationship of CIRP to CLOCK in mammals is analogous to that of TYF-ATX2 to PER in Drosophila (Fig. 3b,c). Cap-independent translation in the core clock. Translation can also be initiated via modes independent of the 5′ cap. A role for this capindependent translation has been observed in both mammalian and Drosophila circadian clocks. In flies, depletion of atypical translation factor NAT caused period lengthening in behavioral rhythms and reduced the amount of PER in central pacemaker neurons43. It has been proposed that cooperative interaction between 5′ and 3′ untranslated regions (UTRs) of Drosophila per facilitates NAT1-dependent PER translation when cap-dependent translation is suppressed by Torin, a TOR kinase inhibitor43. Heterogenous nuclear ribonucleoproteins (hnRNPs) have been shown to be involved in translation and degradation of clock and clock-related transcripts through the association with target UTRs in cultured mammalian cells. SYNCRIP binds and activates internal ribosome entry site (IRES)-dependent translation (hence, nature neuroscience  VOLUME 16 | NUMBER 11 | NOVEMBER 2013

cap-independent) from 5′ UTRs in Per1 and the arylalkylamine N-acetyltransferase gene, which encodes an enzyme important for pineal melatonin rhythms44,45. IRES-dependent translation of Nr1d1 (also known as Rev-erbA) is enhanced by SYNCRIP and polypyrimidine tract–binding protein (PTBP1, also known as hnRNP I)46. SYNCRIP also stimulates Per3 translation and mRNA degradation by associating with Per3 5′ and 3′ UTRs47. In contrast, PTBP1 and HNRNPD have been shown to mediate rhythmic decay of Per2 and Cry1 mRNAs, respectively, through binding to their 3′ UTRs48,49. Translational control in circadian output pathways. Two RNAbinding proteins with translation functions in both fly and mouse circadian clocks are the Fragile X mental retardation protein (FMRP) and Lark. In both fly and mouse, FMRP functions to affect clock output. FMRP is a well-known RNA-binding protein, mutations in which result in the most commonly identified genetic cause of autism spectrum disorders. Consistent with abnormal sleep and rhythms in patients with Fragile X syndrome50,51, mutations in Drosophila and mouse homologs of human FMRP disrupt circadian behavior52–55. In Drosophila, these phenotypes are accompanied by changes in pacemaker neuron morphology52,54. Loss of FMRP does not affect core clock gene expression in pacemaker neurons, suggesting its role in circadian output pathways52–56. FMRP represses target gene translation possibly via ribosomal stalling57 and/or miRNA pathways58 (Fig. 3a). Future studies should address which FMRP targets are important for circadian behavior. Circadian clocks also gate eclosion (i.e., emergence of flies from pupal cases) with a peak in the morning. The RNA-binding protein gene Lark was identified from a screen for mutants with advancedphase eclosion59. Amounts of LARK proteins oscillate in both flies and mammals60,61. Depletion of LARK in circadian pacemaker neurons also dampens locomotor rhythms62. Oscillations in amounts of PER are unaffected, indicating that LARK acts on output pathways. LARK contains two RNA recognition motifs (RRMs) and a retroviral-type zinc finger (RTZF). Many LARK-associating RNAs have been identified from fly brain extracts, including Eip74EF, which may mediate lark effects on eclosion63. In contrast, mammalian LARK activates PER1 translation by associating with the Per1 3′ UTR, suggesting a role in the core clock61. Depletion of LARK proteins in mammalian cells, however, modestly affects circadian period61. Nonetheless, in Drosophila, both LARK and FMRP are RNA-binding proteins that function post-transcriptionally in discrete neural circuits to regulate clock-controlled behaviors. microRNAs microRNA function in the core clock. High-throughput gene expression profiling has been used to identify rhythmically expressed miRNAs in fly heads64,65, mouse retina66, mouse liver67,68, rat pineal gland69 and Arabidopsis70, among others. Impairment of the microRNA biogenesis pathway in clock cells disrupts circadian locomotor activity rhythms in Drosophila71, indicating a functional role for miRNAs (Fig. 3a). MiRNAs contribute to core clock function in Drosophila. Inhibition of miRNA biogenesis in pacemaker neurons elevated steady-state levels of several primary miRNAs, including the conserved bantam miRNA, revealing their expression relevant to circadian clock neurons71. In addition, RNA immunoprecipitation with Argonaute 1 (AGO1), a key component of the miRNA-induced silencing complex (mRISC), uncovered AGO1-associating clock mRNAs including Clk. The Clk 3′ UTR contains bantam-binding sites targeted by bantam-containing mRISC. Consequently, overexpression of 1547

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bantam in pacemaker neurons lengthens period, likely because of the bantam-mediated suppression of Clk expression, whereas loss of the bantam-binding sites in the Clk 3′ UTR impairs rescue of Clk mutant behavioral rhythms. Thus, bantam-Clk is a miRNA-target pair important in neurons regulating circadian behavioral rhythms. To identify miRNAs involved in mammalian circadian clock function, CLOCK-targeted (by chromatin immunoprecipitation) and CREB-regulated miRNAs were identified72. CREB, which has a pivotal role in mediating light resetting in the SCN, targets miR132, whereas CLOCK targets miR-219-1. Both of these miRNAs show daily oscillations in the SCN72. miR-219-1 inhibition resulted in period lengthening of wheel-running rhythms, possibly by altering cell excitability; miR-132 functions in the photic entrainment pathway. In addition to these miRNAs, cell-based studies implicate miR-192 and miR-194 for regulation of the mammalian Per family73 as well as miR-494 and miR-142-3p for Bmal1 (ref. 74). miRNA function in clock output. Drosophila miR-279 is another miRNA highly expressed in clock cells71. miR-279 mutant flies show weak rhythms in constant darkness with no apparent effects on PER oscillations in canonical pacemaker neurons, consistent with an output function75. miR-279, along with circadian clock genes and the clock neuropeptide PDF, regulates cycling expression of one of the components of the JAK-STAT pathway, STAT92E. These effects are indirect, mediated by miR-279 regulation of outstretched, also known as unpaired (upd), which encodes a secreted activating ligand for JAK-STAT. UPD is likely secreted from the dorsal neurons, other newly identified PER-expressing neurons or perhaps even non-clock neurons to drive rhythmic expression of STAT92E in downstream neurons. Taken together, these data reveal a novel molecular output pathway of miRNA–JAK-STAT signaling in neural circuits regulating circadian behavior. miRNAs also function in clock-driven metabolic and immune functions in Drosophila. Small RNA-seq revealed rhythmic expression of a cluster of miR959-964 in head fat body, which is involved in lipid storage and immunity. Apparently, rhythmic feeding drives these miRNAs rhythms65. The miR959-964 cluster regulates genes important for metabolic and immune function, suppresses foraging and immune responses and alters feeding rhythms, with only modest effects on locomotor activity rhythms. Thus, this miRNA cluster is principally involved in clock outputs linking nutrient state to feeding, metabolism and immunity. In mice, miR-122 is a liver-specific miRNA implicated in circadian physiology and metabolism76. Rhythmic pre-miR-122 transcription is driven by REV-ERBα, a key component of the mammalian core clock, although the mature miR-122 does not itself cycle, apparently because of its long half-life. Nonetheless, miR-122 regulates hundreds of mRNAs, many of which are clock-regulated and are involved in lipid metabolism including the circadian deadenylase encoded by the gene noc (see below). Daily miR-152 and miR-494 levels in mouse serum exhibit bimodal peaks, suggesting that miRNAs may act as humoral factors to reset peripheral clocks74. Cytoplasmic polyadenylation The length of the poly(A) tail is an important determinant for translational efficiency and mRNA stability (Fig. 4). One of the earliest examples of daily regulation of poly(A) tail lengths was reported for vasopressin, a key rhythmic neuropeptide in the SCN77. Differing display of genes rhythmically expressed in photoreceptors of Xenopus laevis retina initially revealed nocturnin (noc), which encodes a rhythmically regulated deadenylase78. Subsequent work demonstrated 1548

oscillating expression of noc homologs in different tissues and species, including Drosophila pacemaker neurons79–81. In Drosophila, knockdown of noc partially restores rhythmic behaviors in constant light, suggesting that noc is involved in light-response behaviors80. In mice, disruption of the noc gene does not affect free-running locomotor rhythms or clock gene expression, suggesting its role in circadian output pathways82. In fact, noc mutant mice show resistance to obesity induced by high-fat diet in contrast to the obese phenotype in Clock mutant mice83, suggesting that cycling expression of noc links circadian clocks to a specific metabolic pathway distinct from those regulated by Clock. Taken together, rhythmic polyadenylation appears to be a major link between the core clock and output pathways including those controlling metabolism. The question of global clock control of polyadenylation state has been addressed most completely in the mouse liver31. The majority of mRNAs displaying rhythmic polyadenylation state also exhibit evidence of rhythmic transcription (as indicated by rhythmic pre-mRNA expression) suggesting co-transcriptional nuclear polyadenylation84 may be largely responsible. The more intriguing set of transcripts (~40) are those with constant levels of pre-mRNA and mRNA but rhythms in polyadenylaton state, suggesting cytoplasmic regulation of polyadenylation85 (Fig. 4). Indeed, genes implicated in cytoplasmic polyadenylation such as genes encoding cytoplasmic polyadenylation element binding proteins (CPEBs), the poly(A) polymerase Gld2 and the poly(A)-specific RNase PARN, exhibit mRNA oscillations in phase with these rhythms of polyadenylation. Several PABPs, poly(A) polymerases and deadenylases also show rhythmic abundance in their steady-state mRNA levels. Rhythmic peaks in poly(A) correlated with rhythmic abundance in corresponding proteins even in the absence of steady-state mRNA rhythms. This possibly explains the temporal coincidence of major peak phases (around ZT18) in mRNA rhythms of polyadenylation and polysome association in mouse liver transcriptomes30,31 (Fig. 4). mRNA degradation Rhythmic control of mRNA half-life can be inferred by comparing estimates of transcription rates (for example, pre-mRNA or nascent RNA levels) with steady-state mRNA levels. Rhythms in mRNA abundance in the absence of rhythms in newly synthesized pre-mRNA are prima facie evidence for clock controlled post-transcriptional regulation, potentially via control of mRNA half-life (Fig. 4). In both Drosophila heads and mouse liver, a substantial fraction of cycling mRNAs showed poor or low-amplitude oscillations in nascent RNA or pre-mRNA, suggesting a major post-transcriptional contribution to the cycling transcriptome16,86,87. The underlying mechanisms for

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Figure 4  Cytoplasmic post-transcriptional control of circadian transcripts. Circadian oscillations in poly(A)-tail length can be generated by rhythmic activities of cytoplasmic adenylases (top) and deadenylases (bottom), which subsequently lead to rhythmic association with translating polyribosomes and mRNA degradation, respectively. Examples of clock-relevant control at each step are listed in red.

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this global control remain unknown but could overlap with those controlling polyadenylation and translation (see above). Time-dependence of mRNA stability of clock genes has been described in different species. Drosophila per mRNAs have higher stability during their accumulating phase and exhibit rhythmic abundance even in the absence of circadian transcription, possibly indicating rhythmic degradation of per mRNAs88. As mentioned above, PTBP1 promotes Per2 mRNA degradation via its 3′ UTR in mammalian clocks. Rhythmic abundance in Per2 mRNAs is in antiphase with that in cytoplasmic PTBP1 protein levels. Consequently, the stability of Per2 mRNAs is higher during their accumulation phase than degradation phase48. A similar observation has been reported for HNRNPD–mediated decay of Cry1 mRNA49 and SYNCRIP mediated decay of Per3 mRNA47. Results from circadian clocks in plants also support light- and time-dependent mRNA stability of core clock and circadian output genes89–93. Conclusion Post-transcriptional mechanisms impact circadian rhythms over a wide range of levels, tissues and organisms. What advantages do posttranscriptional mechanisms specially confer to circadian systems? One likely role is to produce a delay in synthesis of a transcriptional repressor, thus delaying negative transcriptional feedback and enabling sustained transcriptional oscillations. Activation of per translation by TYF and ATX2 may have this role in the fly central pacemaker neurons, one of only a few circadian clock cell types with sustained free-running molecular oscillations. An additional layer of post-transcriptional mechanisms can also confer robustness to circadian clocks, allowing them to adaptively time key processes under challenging environmental conditions. Transcriptional rhythms will lose amplitude or even be lost if the processed mRNA is highly stable. In these situations, additional post-transcriptional rhythms may sustain these rhythms to produce a robust, high-amplitude oscillator. A post-transcriptional mechanism may also allow for global alignment of the control of the transcriptome driving rhythmic outputs to the availability of nutrients and/or substrates for protein synthesis, for example. Taken together, the prevalence of post-transcriptional mechanisms clearly indicates that they provide organisms an adaptive advantage. Although considerable progress has been made in elucidating posttranscriptional mechanisms in circadian rhythms, there remain many questions and challenges. We have described a wide span of clockcontrolled mechanisms, yet several have yet to be well-described, such as the role of the plethora of noncoding RNAs, such as rhythmically expressed long noncoding RNAs in the pineal gland94 as well as 5′ capping and decapping, nuclear export, RNA editing, RNA processing at various cytoplasmic bodies, axonal transport of RNAs and local protein synthesis95,96. A number of studies have defined global posttranscriptional control mechanisms in the mouse liver or the fly head. Future work will be needed to see whether these mechanisms are evident in central pacemaker neurons and other brain regions exhibiting circadian function. The application of novel techniques to isolate translating RNAs from targeted brain regions such as translating ribosome affinity purification97–99 will be needed to define neuronal post-transcriptional mechanisms. A future challenge will be to take studies of global control and link them to the function of specific factors. Coupling of RNA immunoprecipitation using specific RNAbinding proteins with RNA sequencing (‘RIP-seq’) or CLIP-seq100 should define the factor-specific mechanisms in these post-transcriptional clock networks. Although studies in plants, fungi and flies have resulted in the identification of a number of post-transcriptional clock components such as TYF and ATX2, it remains to be seen whether these findings will be conserved in mammals. It will also be interesting nature neuroscience  VOLUME 16 | NUMBER 11 | NOVEMBER 2013

to see whether similar post-transcriptional mechanisms are employed in clock-controlled circuits and behaviors such as sleep. Acknowledgments We thank V. Kilman for helpful comments on the manuscript and M. Flourakis for providing an image in Figure 1d. This work was supported by the year of 2013 Research Fund (project no. 1.130009) and the 2013 Creativity & Innovation Research Fund (project no. 1.130036) of UNIST (Ulsan National Institute of Science and Technology) (C.L.) and by US National Institutes of Health NINDS (R01NS059042) and NIMH (R01MH 092273), and Defense Advanced Projects Research Agency (DARPA; D12AP00023) (R.A). R.A.’s effort is in part sponsored by DARPA, and the content of the information does not necessarily reflect the position or the policy of the Government and no official endorsement should be inferred. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

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