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Nature. Author manuscript; available in PMC 2011 Aug 17.
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PMCID: PMC3073513

twenty-four defines a critical translational step in the Drosophila clock


Daily oscillations of gene expression underlie circadian behaviours in multicellular organisms1. While attention has been focused on transcriptional and posttranslational mechanisms13, other posttranscriptional modes have been less clearly delineated. Here we report mutants of a novel Drosophila gene twenty-four (tyf) that display weak behavioural rhythms. Weak rhythms are accompanied by dramatic reductions in the levels of the clock protein PERIOD (PER) as well as more modest effects on TIMELESS (TIM). Nonetheless, PER induction in pacemaker neurons can rescue tyf mutant rhythms. TYF associates with a 5′-cap binding complex, poly(A)-binding protein (PABP) as well as per and tim transcripts. Furthermore, TYF activates reporter expression when tethered to reporter mRNA even in vitro. Taken together, these data suggest that TYF potently activates PER translation in pacemaker neurons to sustain robust rhythms, revealing a novel and important role for translational control in the Drosophila circadian clock.

Transcriptional feedback loops are critical for setting time of eukaryotic circadian clocks. In Drosophila, the Clock (Clk)/cycle (cyc) dimer activates the transcription of period (per), timeless (tim), vrille (vri), Par domain protein 1 (Pdp1), and clockwork orange (cwo) genes, which in turn feed back to inhibit CLK-activated transcription or regulate Clk transcription2. These components are also modified posttranslationally to alter core clock timing23. Regulation at multiple levels is thought to impose temporal delays in feedback allowing sustained oscillations on a circadian time scale.

To discover novel clock components, we performed a genome-wide behavioural screen. Using the KAIST-GenExel Drosophila library, we identified ~4000 EP lines containing P elements bearing the Upstream Activating Sequence (UAS) for the yeast GAL4 transcription factor inserted near transcription start sites. These flies were crossed with transgenic flies expressing GAL4 under the control of the tim promoter (tim-GAL4) to drive downstream gene expression in clock cells4. One EP line identified by a long-period rhythm was the G10872 line that contains an insertion 893 bp upstream of the CG4857 transcription start site (Supplementary Fig. 1a). Sequence analyses of the predicted amino acid sequence for CG4857 did not reveal any apparent functional domains or obvious vertebrate homologues but do reveal conservation with genes from different Drosophila species and other insects (Supplementary Fig. 2). We termed this novel gene twenty-four (tyf).

To characterize the phenotype in flies bearing tyf loss-of-function mutations, we generated a ~2.5 kb deletion by imprecise P element excision (Supplementary Fig. 1a, tyfΔG14151; tyfΔ), deleting amino acids 79–449 of tyf and resulting in a frame-shift and premature termination. In addition, we identified a piggyBac insertion line that shows dramatically reduced levels of tyf transcript (tyfe00614; tyfe) without affecting the transcript levels of adjacent genes (Supplementary Fig. 1b). Wild-type flies display morning and evening peaks under 12 h light- 12 h dark (LD) cycles, anticipate the transitions between light-on and light-off by gradually increasing their activity, and maintain their locomotor rhythm in subsequent constant dark (DD). In tyf mutants, morning anticipation of lights-on was reduced and their rhythm was immediately less robust, resulting in weak but long periods in DD (Fig. 1 and Supplementary Table 1). Precise piggyBac excision in tyfe restored wild-type circadian behaviour (Supplementary Table 1), indicating that the tyf gene disruption is responsible for its circadian phenotype. Analyses in trans-heterozygous females show that tyf alleles are recessive and not complemented by deletions of the CG4857 locus (Supplementary Fig. 3).

Figure 1
Robust behavioural rhythms require tyf

While circadian clocks are evident in multiple tissues, brain clocks are largely responsible for circadian behaviours4. Neuroanatomical studies have established two oscillator models in which distinct groups of clock cells control morning and evening locomotor activity and behavioural rhythms5,6. The neuropeptide gene, Pigment-dispersing factor (Pdf), expressed in ventral lateral neurons (LNvs) has been implicated in driving morning anticipation and resetting evening clocks in the dorsal LNs (LNds)/dorsal neurons (DNs)79. To map the neurons important for tyf effects, we generated tyf-GAL4 lines containing the tyf promoter region (from −3.0 kb to +0.5 kb), and visualized its expression using a UAS-GFP reporter. tyf-GAL4 expression was relatively restricted to a subset of neurons in the adult brain (Supplementary Fig. 4a–f). Anti-PER antibody staining revealed that it is strongly expressed in PDF+ LNvs and weakly in LNds assessed in independent lines (data not shown). In contrast, tyf-GAL4 expression was not detectable in the DNs. Consistent with the idea that tyf-GAL4 reflects endogenous tyf expression, tyf-GAL4 along with a UAS-tyf transgene fully rescues the behavioural phenotypes in tyf mutants (data not shown).

To map the loci of tyf function, we restricted TYF overexpression to the PDF+ LNvs using Pdf-GAL47. This results in a long period similar to the tim-GAL4 driver, while GAL4 inhibition in PDF+ cells by a Pdf-GAL80 transgene5 suppressed the long period phenotype (Supplementary Table 2). Independent UAS-tyf insertions confirmed these results. TYF expression restricted to PDF+ cells was also sufficient to rescue free-running locomotor rhythms in mutants (Supplementary Table 3). In addition, RNAi-mediated knockdown of tyf expression in PDF+ cells phenocopied circadian behaviours in tyf hypomorphic mutants (Supplementary Table 3). These data indicate that tyf expression in the PDF+ pacemaker neurons is necessary and sufficient for robust behavioural rhythms.

To determine tyf effects on the core clock, we analyzed molecular rhythms from head extracts, which largely reflect eye clocks10. We found that cycling expression of PER, TIM, and PDP1 proteins in tyf mutants is comparable to wild type (data not shown). tyf transcript levels were relatively constant in LD and not affected in clock mutants (Supplementary Fig. 4g,h). We then focused on the behaviourally relevant pacemaker neurons. Anti-PDF immunofluorescence revealed no overt defects in the neural projections from PDF+ LNvs of tyf mutants (Supplementary Fig. 5a). Adult-specific TYF expression using a drug-inducible GAL4 was sufficient for behavioural rescue in tyf mutants (Supplementary Table 4 and Supplementary Fig. 5c), further reducing the likelihood that tyf phenotypes are due to developmental defects.

Strikingly, we found that PER protein was barely detectable in LN clock cells of tyf mutants (Fig. 2a). PER cycling was dampened but not absent (Fig. 2b and Supplementary Fig. 6). tyf mutant effects were less severe in the DNs with PER at ~50% of wild-type peak levels. TYF expression in PDF+ neurons rescued PER cycling only in PDF+ clock cells of tyf mutants (Supplementary Fig. 7). Consistent with dramatic PER reductions, PDF levels increased in dorsal projections from the small LNvs of tyf mutants (Supplementary Fig. 5b), as observed in per01 flies11. TIM levels were also reduced in tyf mutants, but to a lesser extent than PER, with peak levels in tyf mutants reduced to ~50% of wild-type (Supplementary Fig. 8a). Such effects may be indirect through PER as we found that TIM reductions were also observed in per01 flies and there was little effect of loss of tyf on TIM in per01 mutants.

Figure 2
tyf is crucial for PER expression in pacemaker neurons

In contrast to strong effects on PER, tyf mutants normally expressed PDP1ε, CWO, and CLK proteins (Fig. 2a and Supplementary Fig. 8b). The oscillating expression of PDP1ε protein as well as tim and Pdp1ε transcripts in mutant flies was comparable to wild-type (Supplementary Fig. 8c,d). We reason that LD cycles, the clock neural network and/or multiple feedback loops may buffer the molecular clock against loss of tyf function.

Given the robust reductions in PER may be responsible for the arrhythmic behaviour, we hypothesized that PER expression via the GAL4/UAS system could rescue tyf locomotor rhythms12. Indeed, we find that PER, but not TIM or CLK, overexpression specifically in PDF+ cells of tyf mutants restored wild-type levels of rhythmicity (Fig. 3, Supplementary Fig. 9, and Supplementary Table 5). These rescue data indicate that neither posttranslational regulation of PER protein by TIM3 nor transcriptional activation of per gene expression by CLK2 would be limiting for normal circadian behaviour in tyf mutants. Moreover, these behavioural data suggest that PER is a major target of TYF in PDF neurons.

Figure 3
PER induction rescues tyf mutant rhythms

We next examined at what regulatory step PER expression in tyf mutants is compromised. We observed that PER protein, but not per RNA, levels were reduced in brain extracts (data not shown). While consistent with posttranscriptional regulation, we cannot exclude the possibility that this result could arise from the masking effect of low level non-cycling per RNA in non-circadian tissues. tyf effects were not evident on a CLK-activated per promoter-GAL4 transgene (Supplementary Fig. 10) but were evident on a per transgene lacking its natural promoter13 (Supplementary Fig. 11a), indicating that the per promoter is not necessary nor sufficient for tyf effects. tyf effects were also still observed on a per(Δ)-HAHis transgene that lacks the DOUBLETIME (DBT)-binding domain, thus reducing DBT-mediated PER degradation14. We then examined PER in flies in which we rescued tyf by PER overexpression. Interestingly, neither PER levels driven by constitutive Pdf-GAL4 nor oscillations were affected in tyf mutants (Supplementary Fig. 11b), indicating that tyf is not required for posttranslational regulation of PER. The lack of most per 5′ and 3′ UTRs in the UAS-per transgene15 and/or per overexpression itself may compensate for loss of tyf. Taken together, our observations suggest that tyf posttranscriptionally regulates per expression.

We next asked whether TYF associates with posttranscriptional regulatory factors, circadian clock components and/or specific RNA targets using immunoprecipitation of an epitope-tagged TYF (TYF-V5). We confirmed that GAL4/UAS-driven TYF-V5 rescues behavioural rhythms in tyf mutants (Fig. 3 and Supplementary Table 5) and that TYF-V5 expression is cytoplasmic at all times of day (Supplementary Fig. 4i). TYF-V5 driven by tim-GAL4 was immunoprecipitated from head extracts and then TYF-associating proteins were probed with different antibodies. TYF did not associate with either PER or TIM (Fig. 4a). However, poly(A)-binding protein (PABP) was specifically co-immunoprecipitated with TYF at two times-of-day in a RNase-insensitive manner (Fig. 4a, data not shown). In vitro assays demonstrated that PABP interacts with the N-terminal region of TYF (Supplementary Fig. 12a; TYF-N, amino acids 1–1167).

Figure 4
TYF specifically associates with the 5′ cap-binding complex, PABP, and target gene transcripts

It has been proposed that PABP stimulates translation in part by binding to both 5′ cap-associating translation initiation factor eIF4G and the poly(A) tail in mRNAs, thereby facilitating mRNA circularization16. Therefore, we also examined a possible association of TYF with a cap-binding complex using 5′ cap (7-methylguanosine, m7-GTP) affinity beads and S2 cell extracts expressing epitope-tagged TYF. We found that eIF4E, a translation initiation factor directly recognizing the 5′ cap structure, PABP, and TYF were efficiently and specifically pulled down by m7-GTP affinity beads in a RNase-insensitive manner (Fig. 4b and Supplementary Fig. 12b). Addition of soluble m7-GTP but not GTP inhibited their cap association, validating the specificity. Interestingly, the N-terminal portion of TYF, capable of in vitro PABP binding (Supplementary Fig. 12a), exhibits strongly reduced affinity for the 5′ cap, suggesting that PABP may not solely mediate TYF association with the 5′ cap. Our results link TYF to RNA-binding proteins involved in translation.

We further checked whether TYF associates with clock gene transcripts. RNA in TYF-V5 immunoprecipitation was analyzed by real-time RT-PCR (Fig. 4d). To quantify TYF-specific transcript enrichment, we normalized RNA levels in TYF immunoprecipitation to input RNA (enrichment fold of TYF IP) and then subtracted the signal from a background control immunoprecipitation (PDFR-V5) also normalized to input. We find that TYF pulls down significant amounts of per and tim RNAs relative to the amounts of Pdp1 at the times of their peak expression levels (ZT15, p<0.029). Moreover, per RNA levels in TYF immunoprecipitation were higher at their peak times than trough times (i.e., ZT15 v. ZT3) even after normalizing for input levels (p<0.026). As Pdp1 has comparable input levels to per and tim (data not shown), the low Pdp1 signal is unlikely to be explained by low input levels. However, differential anatomic distributions could contribute to these results17. We also did not detect significant TYF-specific signal for Clk and cyc RNAs in immunoprecipitated RNA, although low input levels could explain the lack of detectable association (data not shown). We could not identify any RNA recognition motif in TYF, suggesting that RNA binding intermediaries, such as eIF4E and PABP, may mediate its association with RNA.

To address whether TYF is incorporated into translating ribonucleocomplexes, we fractionated S2 cell extracts expressing TYF-3xHA by sucrose density gradient in the absence or presence of EDTA to dissociate ribosomes (Supplementary Fig. 13). Sedimentation profiles demonstrated that ribosomal protein P0 and PABP were present in polysomal fractions in an EDTA-sensitive manner. By contrast, a minor fraction of TYF co-sedimented with polysomes and exhibited a modest shift by EDTA. This pattern is similar to that of eIF4E consistent with their association (Fig. 4b,c). Taken together, the association of TYF with eIF4E, PABP, and per RNA suggests a direct role in PER protein synthesis, possibly at a translation initiation step.

To investigate tyf effects on its associating RNAs, we performed a RNA-tethering assay in cultured S2 cells18. TYF fused to the RNA-binding bacteriophage MS2 coat protein is tethered to a luciferase reporter RNA containing MS2-binding sites (Fig. 5a). TYF activity is monitored by assaying luciferase activity. TYF-MS2, compared to MS2 alone, enhanced luciferase expression in a MS2-binding sites-dependent manner (Fig. 5b). The C-terminal region of TYF (TYF-C, amino acids 1161–1911) was necessary and sufficient for the activation (Fig. 5b and Supplementary Fig. 14a–c). TYF-MS2 activation was augmented if the per or tim 3′ UTR is fused to the reporter gene but not the cyc 3′ UTR (Fig. 5b and Supplementary Fig. 14d). TYF without the MS2 domain could not activate reporter containing both per 5′ and 3′ UTRs consistent with a requirement for other RNA binding proteins (Supplementary Fig. 14h). Notably, we found that a transgenic fusion between the per coding region and luciferase lacking the per 3′ UTR (XLG-luc)19 was also reduced in tyf mutants indicating that the per 3′ UTR is not necessary for tyf effects in vivo (data not shown). In contrast to reporter activity, reporter RNA levels and its nuclear/cytoplasmic distribution were comparable between MS2 and TYF-MS2 transfected cells (Supplementary Fig. 14e,f). Moreover, analytical centrifugation through a sucrose cushion revealed that more reporter transcripts associate with high-density ribosomes in the presence of TYF-MS2 (Supplementary Fig. 14g, p<0.027), further supporting a role in translational control.

Figure 5
TYF activates reporter expression when tethered to its RNA

To more directly test the translational activation function of TYF, we reconstituted this tethering system in in vitro translation assays. A C-terminal TYF region (TYFc3, amino acids 1373–1911) fused to MS2, which robustly activated MS2 reporter expression in transfected cells (data not shown), was bacterially expressed, purified and incubated with in vitro transcribed MS2 reporter RNAs and translation-competent S2 cell extracts. TYFc3-MS2 activated translation from a m7-G capped and poly(A)-tailed reporter RNA modestly (Fig. 5c, p<0.001) and a non-polyadenylated RNA even more strongly (2.9×, p<0.001). TYFc3-MS2 effects are evident without affecting reporter RNA levels (data not shown). Moreover, this TYF region is not sufficient to bind PABP in vitro (Supplementary Fig. 12a), suggesting that these effects are not mediated by PABP recruitment. These data clearly demonstrate a translation activation function of TYF in vitro, supporting a possible role in translation of poorly adenylated transcripts.

Relative to studies of transcriptional and posttranslational regulation, little is known about other posttranscriptional/translational mechanisms of core clock regulation in different organisms3,20. While a number of RNA-binding proteins are either rhythmically expressed1 or important for behavioural rhythms2124, direct links between specific transacting factors, specific clock gene transcripts, and in vivo core clock function have yet to be clearly established, especially in metazoans. Indeed, a number of studies have indicated a role for posttranscriptional regulation in modulating per expression10,2528. Here we demonstrate with multiple lines of evidence that TYF activates PER translation to sustain behavioural rhythms, revealing a novel and important role for translational control in the Drosophila circadian clock. We observe robust tyf effects on PER and lesser effects on TIM yet no detectable reduction of other core clock components we assayed. This observation suggests that impairing tyf-dependent translation is not critical for the expression of most clock components. Importantly, transgenic induction of PER, but not other clock components including TIM, can rescue tyf mutant phenotypes.

TYF function is especially important in pacemaker neurons. Both tyf and per expression in the PDF+ LNv is sufficient to strongly rescue behavioural rhythms in tyf mutants. Moreover, tyf effects on PER are, by far, most evident in these pacemaker neurons. The brain pacemaker neurons are also among the few clock cells, which demonstrate robust free-running molecular rhythms in constant dark8,9. Thus, TYF-mediated translational control may be a specialization of networked pacemakers in the brain crucial for sustaining free-running rhythmicity.

Our data also strongly support the model that TYF acts at the level of translational control. TYF associates with per and tim RNAs, as well as translational regulatory components such as the eIF4E-containing 5′ cap binding complex, and PABP, the latter of which are insensitive to RNase treatment. In addition, TYF tethered to a reporter RNA via MS2 can activate reporter expression without altering RNA levels in transfected cells and importantly, in cell extracts providing exogenous RNA templates and purified TYF-MS2.

How might TYF control translation of its target RNAs? We observe specific effects on PER and TIM yet not on other clock components while we find that TYF interacts with translation components such as the eIF4E-containing cap binding complex and PABP. We hypothesize that RNA-binding translational repressors associate with newly transcribed per RNA, temporarily postpone translation and thus, delay feedback PER repression on its own transcription (Supplementary Fig. 15). Such a delay could contribute to the observed lag between protein and RNA particularly in pacemaker neurons, although posttranslational mechanisms may also contribute at least in the eyes29. TYF, which does not have a known RNA recognition motif, could then be recruited to target transcripts by these translational repressors, releasing them to stimulate initiation of per translation. We have not been able to biochemically or genetically link TYF to RNA-binding proteins FMR, LARK, or the translation regulator Thor/4E-BP that have been shown to contribute to circadian clock function21,22,24. Nonetheless, TYF association with eIF4E and their similar polysome profiles implicates TYF as a novel translation initiation factor. In addition, TYF effects may be more evident on poorly adenylated transcripts based on our in vitro data (Fig. 5c). Of note, the fly homolog of the clock-regulated deadenylase nocturnin30 has been shown to be important in DNs for circadian light responses but neither an LN function nor an RNA target has been described24. Nevertheless, unique features of TYF-regulated transcripts may mediate the highly selective TYF effects on clock components in vivo.

Posttranscriptional regulation on per RNA has been considered to be modulatory to clock function. The identification of critical role for TYF highlights an important role for PER translation in the Drosophila neural clockwork. It will be of interest to determine if proteins functionally analogous to TYF serve similarly important and specific functions in the mammalian clock.



Total RNA from adult fly heads was isolated using Trizol reagent and reverse-transcribed using Superscript III according to the manufacturer’s instructions (Invitrogen). tyf cDNA was PCR-amplified by Platinum Pfx polymerase (Invitrogen) with the appropriate primer sets and inserted into pUAST vector for regular germ-line transformation and into its modified version with attB site and C-terminal V5-tag for site-specific germ-line transformation.

Fly stocks

All flies were reared with standard cornmeal-yeast-agar medium at 25°C under LD (12-h light/12-h dark) cycles. EP lines G10872 and G14151 were obtained from KAIST-GenExel Drosophila library. To generate a tyf deletion line (tyfΔ), P-element excision lines were established from the G14151 line and molecularly characterized by genomic DNA-PCR with appropriate primer sets. Df(1)HC244, Df(1)rb23, and UAS-mCD8-GFP lines were obtained from Bloomington Drosophila stock center. UAS-tyfRNAi line was obtained from National Institute of Genetics (Japan). Several independent germ-line transformants were established from w1118 embryos injected with UAS-tyf transgenic construct (BestGene Inc.).

Supplementary Material

supplement 1


We thank Issac Edery, Jeffrey Hall, Haig Keshishian, Michael Rosbash, Francois Rouyer, Amita Sehgal, Bloomington Drosophila stock center, Harvard Exelixis Drosophila stock collection, KAIST-GenExel Drosophila library, and National Institute of Genetics for fly strains; Paul Hardin, Elisa Izaurralde, Akira Nakamura, Michael Rosbash, and Nahum Sonenberg for antibodies; Jens Lykke-Andersen for plasmids; Kent E. Duncan for helpful suggestions on in vitro translation assays. This work was supported by grants from the Brain Research Center of the 21st Century Frontier Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology, the Republic of Korea (J.C.) and from the National Institutes of Health (R01NS059042, R01NS052903, R01MH067870; R.A.)


Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.

Supplementary Information accompanies the paper on www.nature.com/nature.

Author Contributions R.A. and J.C. conceived the study; R.A., C.L., and J.C. designed the experiments; C.L. (under supervision of R.A.) and J.L (under supervision of J.C.) jointly completed Figs. 1 and and2,2, Supplementary Figs. 1, 4, 8, and 14, Supplementary Tables 2 and 3; J.L., S.M.P. and S.K.J. performed and analyzed the experiments in Supplementary Fig. 13; J.L., C.C., and J.K. performed the genome-wide behavioural screen; C.C. performed GST pull-down studies in Supplementary Fig. 12a; V.L.K. performed PDF quantification analysis in Supplementary Fig. 5b; C.L. performed and analyzed experiments in all remaining Figs, Supplementary Figs and Tables; C.L. and R.A. wrote the manuscript.

Author Information Reprints and permissions information is available at www.nature.com/reprints.

The authors declare no competing financial interests.


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