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. 2013 May 17;340(6134):879-82.
doi: 10.1126/science.1234746.

A role for Drosophila ATX2 in activation of PER translation and circadian behavior

Yong Zhang  1 , Jinli Ling, Chunyan Yuan, Raphaëlle Dubruille, Patrick Emery
Affiliations

A role for Drosophila ATX2 in activation of PER translation and circadian behavior

Yong Zhang et al. Science. .

Abstract

A negative transcriptional feedback loop generates circadian rhythms in Drosophila. PERIOD (PER) is a critical state-variable in this mechanism, and its abundance is tightly regulated. We found that the Drosophila homolog of ATAXIN-2 (ATX2)--an RNA-binding protein implicated in human neurodegenerative diseases--was required for circadian locomotor behavior. ATX2 was necessary for PER accumulation in circadian pacemaker neurons and thus determined period length of circadian behavior. ATX2 was required for the function of TWENTY-FOUR (TYF), a crucial activator of PER translation. ATX2 formed a complex with TYF and promoted its interaction with polyadenylate-binding protein (PABP). Our work uncovers a role for ATX2 in circadian timing and reveals that this protein functions as an activator of PER translation in circadian neurons.

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Figures

Fig. 1
Fig. 1
Requirement of ATX2 in LNvs for normal circadian behavior. (A) Effects of depleting ATX2 in pacemaker neurons on period length of circadian behavior. (Upper) Bar graph showing period length (Y axis), percentage of rhythmic flies and number of flies tested (in the bars). Error bars = SEM. TG4 is short for tim-GAL4, TD2 for tim-GAL4, UAS-dicer2, PD2 for Pdf-GAL4, UAS-dicer2 and PG80 for Pdf-GAL80. Dicer2 increases RNAi effects (15). (Lower) Double-plotted actograms of control and TG4/Atx2RNAi-1 flies showing the last day of the light-dark (LD) cycle and 5 days of DD. (B) (Upper) ATX2 is severely downregulated in circadian neurons expressing Atx2 dsRNA. Fly brains were dissected at Zeitgeber Time (ZT) 0 (ZT0 corresponds to the beginning of the light phase of the LD cycle) and immunostained with anti-ATX2, anti-PER and anti-PDF antibodies. (Lower) Quantification of ATX2 levels in sLNvs, lLNvs, LNds and DN1s. Between 11 to 20 neurons were quantified per data point. *** = P< 0.001 as determined by Tukey’s multiple comparison test after one-way ANOVA, n.s. = not significant at the 0.05 level, in both panel A and B.
Fig. 2
Fig. 2
Regulation of PER accumulation in sLNvs by ATX2. (A) sLNvs of brains from wild-type and Atx2 RNAi flies, dissected at different time points (Circadian Time, CT) during the 4th day of DD and stained with anti-PDF and anti-PER antibodies. (B) Quantification of PER staining in 12–16 sLNvs per data point. (C) PER overexpression corrects the period lengthening induced by Atx2 knock-down. Statistics and abbreviations as in Fig. 1.
Fig. 3
Fig. 3
Functional interaction between Atx2 and tyf. (A) Effects of Atx2 RNAi and tyf mutations on circadian period. Statistics and abbreviations as in Fig. 1. (B) Requirement of ATX2 for TYF to promote per translation. MS2 and TYF-MS2 expression plasmids were cotransfected in Drosophila S2R+ cells with firefly luciferase reporter plasmids (FLUC) controlled by MS2 binding sites, in the presence or absence of the per 3′-UTR. Luciferase activity with MS2 was set to 1. dsRNAs targeting Atx2 were added two days before transfection and strongly reduced ATX2 levels (see insertion). N=3. Error bars = SEM. * = P< 0.05, *** = P<0.001, n.s. = not significant (P>0.05) as determined by Student’s t test with Bonferroni correction. (C) Physical association of ATX2 with TYF. Protein from head extracts of flies expressing (+) or not (−) TYF-V5 were immunoprecipitated (IP) with an antibody to ATX2 (left panel) or V5 (right panel). A serum obtained prior to immunization with ATX2 antigens was used as negative control (pre-imm). * = cross-reacting band. (D) The association between ATX2 and TYF is resistant to RNase A treatment, and thus independent of RNA. (E) Association of ATX2 with per and other transcripts. The RNA enrichment index (Y axis) was calculated by subtracting the relative amount of RNA found “immunoprecipitated” with the pre-immunization control from the relative amount found in the immunoprecipitation with the antibody to ATX2, N=4. (F) ATX2 promotes TYF-PABP interactions. Much less PABP is co-immunoprecipitated with TYF-V5 when ATX2 is downregulated by RNAi.
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