Volume 66, Issue 1, Pages 129-140.e7 (April 2017) LSM12 and ME31B/DDX6 Define Distinct Modes of Posttranscriptional Regulation by ATAXIN-2 Protein Complex in Drosophila Circadian Pacemaker Neurons  Jongbo Lee, Eunseok Yoo, Hoyeon Lee, Keunhee Park, Jin-Hoe Hur, Chunghun Lim  Molecular Cell  Volume 66, Issue 1, Pages 129-140.e7 (April 2017) DOI: 10.1016/j.molcel.2017.03.004 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 66, 129-140.e7DOI: (10.1016/j.molcel.2017.03.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 A Genetic Screen Identifies Lsm12 and me31B Important for Circadian Locomotor Rhythms in Drosophila (A) A schematic diagram of ∼150 circadian pacemaker neurons in each hemisphere of fly brain. Large and small ventral lateral neurons (l-LNv and s-LNv, respectively) express a circadian neuropeptide PDF important for the behavioral rhythms in constant dark (DD). LNd, dorsal LN; 5th s-LNv, PDF-negative LNv; DN, dorsal neuron; LPN, lateral posterior neuron. (B) Lsm12 or me31B RNAi flies show long-period locomotor rhythms or poor rhythmicity, respectively. Each RNAi transgene was overexpressed by TD2 (tim-Gal4 > UAS-DCR2). Circadian periods of the locomotor rhythms in DD were determined by chi-square periodograms (y axis). Power of rhythmicity was calculated by power (P) minus significance (S) values (x axis). Data were averaged from 12–105 male flies per each RNAi transgene. (C) The averaged actograms of TD2 control, Lsm12 RNAi, and me31B RNAi flies for three light:dark (LD) cycles of 12 hr on and 12 hr off followed by seven DD cycles. White colors, L phases; gray colors, D phases. (D) Depletion of LSM12 or ME31B in PDF neurons is sufficient to affect circadian behaviors in DD. Circadian periods (top) or power of rhythmicity (bottom) were measured in individual flies. Data represent mean ± SEM (n = 20–91). PD2, Pdf-Gal4 > UAS-DCR2. ∗∗p < 0.01, ∗∗∗p < 0.001 to heterozygous controls as determined by one-way ANOVA, Tukey’s post hoc test. See also Figure S1 and Table S1. Molecular Cell 2017 66, 129-140.e7DOI: (10.1016/j.molcel.2017.03.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Lsm12 in Circadian Pacemaker Neurons Sustains 24 hr Periodicity in Circadian Behaviors (A) A schematic diagram of genomic deletions in Lsm12 mutant alleles. Darker gray, exons; lighter gray, untranslated regions; arrowheads, transcriptional termination sites. (B and C) A chromosomal deletion Df(1)ED7153 fails to complement Lsm12 mutant alleles. Circadian periods were measured in male flies hemizygous for control (Lsm12Δ2) or Lsm12 deletion alleles (B), or in female trans-heterozygotes with Df(1)ED7153 (C). Data represent mean ± SEM (n = 15–63 male flies; n = 16–41 female virgins). n.s., not significant; ∗p < 0.05, ∗∗∗p < 0.001 to Lsm12Δ2 or heterozygous controls as determined by one-way ANOVA, Tukey’s post hoc test. (D and E) LSM12 overexpression in cry-expressing clock neurons rescues long-period locomotor rhythms in Lsm12 mutants. The averaged actograms per each genotype (n = 23–42) were double-plotted (D). White colors, L phases; gray colors, D phases. Circadian periods were measured in control or Lsm12Δ3 flies with each combination of Gal4 and UAS transgenes (E). Data represent mean ± SEM (n = 23–48). n.s., not significant; ∗∗∗p < 0.001 to cry-Gal4 control in wild-type as determined by one-way ANOVA, Tukey’s post hoc test. See also Figure S2 and Tables S2 and S3. Molecular Cell 2017 66, 129-140.e7DOI: (10.1016/j.molcel.2017.03.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Dampened PER Expression Causes Long-Period Locomotor Rhythms in Lsm12 Mutants (A) Lsm12 mutant flies (Lsm12Δ6) display lower PER levels in circadian pacemaker neurons than control (Lsm12Δ2). PER and TIM protein levels in clock neurons were quantified at the indicated zeitgeber times (ZT; lights on at ZT0, lights off at ZT12) in LD cycles and normalized to the values in control at ZT0 (set as 100%). Data represent mean ± SEM (n = 10–26). Representative confocal images of PDF-expressing s-LNv were shown on the left. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 to Lsm12Δ2 control at each ZT as determined by Student’s t test. (B) A genomic per transgene harboring an ∼13.2 kb wild-type per locus (p{13.2per-HAHis}) rescues long-period locomotor rhythms in Lsm12 mutants. Data represent mean ± SEM (n = 29–63). n.s., not significant; ∗∗∗p < 0.001 as determined by one-way ANOVA, Tukey’s post hoc test. (C) PER overexpression in cry-expressing clock neurons rescues long-period locomotor rhythms in Lsm12 mutants. Data represent mean ± SEM (n = 25–42). (D) Depletion of LSM12 but not ME31B exaggerates the period-lengthening effects of TYF dominant-negative (TYFΔC5). TYFΔC5 was co-expressed with me31B or Lsm12 RNAi transgene in PDF neurons by Mz520-Gal4. Data represent mean ± SEM (n = 21–36). See also Figure S2 and Tables S4 and S5. Molecular Cell 2017 66, 129-140.e7DOI: (10.1016/j.molcel.2017.03.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 ME31B Sustains Rhythmic Outputs from Circadian Pacemaker Neurons via a PER-Independent ATX2 Pathway (A) ME31B depletion does not dampen PER and TIM rhythms in PDF-expressing s-LNv. PER and TIM protein levels were quantified at each time point in LD cycles and normalized to the peak values in TD2 control flies (set as 100%). Data represent mean ± SEM (n = 10–22). ∗p < 0.05 to TD2 control at a given time point as determined by Student’s t test. (B and C) ME31B depletion abolishes daily oscillations in PDF levels at the axonal terminus of s-LNv. Dorsal projections from s-LNv were visualized by confocal imaging of anti-PDF staining at each ZT in LD cycles (B). The area positive for anti-PDF staining and its integrated intensity were measured from the fluorescence signals above a threshold level (C). Data represent mean ± SEM (n = 12–19). n.s., not significant; ∗∗p < 0.01, ∗∗∗p < 0.001 to TD2 control at the same ZT as determined by one-way ANOVA, Tukey’s post hoc test. (D) Modest depletion of ATX2 and ME31B non-additively dampens the rhythm amplitude in circadian behaviors. Atx2 and me31B RNAi transgenes were overexpressed in cry-expressing clock neurons by the relatively weak cry-Gal4. Data represent mean ± SEM (n = 21–69). n.s., not significant; ∗∗∗p < 0.001 to controls for the me31B RNAi transgene as determined by one-way ANOVA, Tukey’s post hoc test. See also Table S6. Molecular Cell 2017 66, 129-140.e7DOI: (10.1016/j.molcel.2017.03.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 LSM12 and ME31B Associate with the ATX2-TYF Complex (A) LSM12 associates with the ATX2-TYF complex in Drosophila S2 cells. Soluble S2 extracts were immunoprecipitated by pre-immune control, anti-ATX2, or anti-TYF antibodies. Purified immunoprecipitation (IP) complexes were resolved by SDS-PAGE and immunoblotted with specific antibodies (left). Input, 4.5% of soluble extracts used in each IP. Asterisk indicates rabbit immunoglobulin. Protein size markers were shown on the right. (B) The translation-activation domain of TYF (TYF-C5) is necessary for the association with ATX2 and LSM12 in circadian pacemaker neurons. FLAG-tagged TYF or TYFΔC5 proteins were expressed in tim-expressing clock neurons of transgenic flies. Head extracts were prepared at each ZT in LD cycles and immunoprecipitated by anti-FLAG antibody. (C) The C-terminal RecA-like domain of ME31B associates with the ATX2-TYF complex. S2 cells were transfected with expression vectors for EGFP or EGFP-ME31B fusion proteins as indicated at the top. Soluble extracts were prepared at 48 hr after transfection and immunoprecipitated by anti-GFP antibody. Arrowhead indicates each EGFP-fusion protein. See also Figure S3. Molecular Cell 2017 66, 129-140.e7DOI: (10.1016/j.molcel.2017.03.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 LSM12 Is a TYF-Specific Molecular Adaptor in the ATX2 Complex to Support TYF-Dependent Translation (A) Head extracts from control (Lsm12Δ2) and Lsm12 mutant flies (Lsm12Δ6) were immunoprecipitated with pre-immune control or anti-ATX2 antibodies. Purified IP complexes were resolved by SDS-PAGE and immunoblotted with specific antibodies (left). Input, 2.5% of soluble extracts used in each IP. (B) LSM12 depletion disrupts the association of ATX2 with the translation-activation domain of TYF (TYF-C5). FLAG-tagged TYF-C5 or TYFΔC5 proteins were expressed in EGFP (control)- or LSM12-depleted S2 cells. Their soluble extracts were immunoprecipitated by anti-FLAG antibody. (C) Depletion of ATX2 or LSM12 abolishes the 5′-cap association of TYF. Soluble extracts were prepared from dsRNA-treated S2 cells and incubated with m7-GTP affinity beads to purify the cap-binding protein complexes. EIF4E and TUBULIN proteins were positive and negative controls, respectively. Input, 4.5% of soluble extracts used in each pull down. (D) LSM12 depletion suppresses translational activation by RNA-tethered TYF. Firefly luciferase (FLUC) RNA reporter containing MS2-binding sites, Renilla luciferase (RLUC) RNA reporter, and MS2 fusion proteins (MS2 or TYF-MS2) were co-expressed in EGFP- or LSM12-depleted S2 cells. Specific depletion of endogenous LSM12 protein was confirmed by immunoblotting (top). Dual luciferase reporter assays were performed at 48 hr after transfection. Activation fold was calculated by normalizing to the FLUC/RLUC value in EGFP-depleted cells expressing MS2. Data represent mean ± SEM (n = 5). n.s., not significant; ∗∗∗p < 0.001 as determined by one-way ANOVA, Tukey’s post hoc test. See also Figures S4 and S5. Molecular Cell 2017 66, 129-140.e7DOI: (10.1016/j.molcel.2017.03.004) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 ATX2 Associates with NOT1 in a ME31B-Dependent Manner and Supports NOT1-Mediated Gene Silencing (A) NOT1 depletion does not dampen PER and TIM rhythms in PDF-expressing s-LNv. PER and TIM protein levels were quantified at each time point in LD cycles and normalized to the peak values in Pdf-Gal4 control flies (set as 100%). Data represent mean ± SEM (n = 16–23). ∗∗p < 0.01 to Pdf-Gal4 control at a given time point as determined by Student’s t test. (B) NOT1 depletion abolishes daily oscillations in PDF levels at the axonal terminus of s-LNv. The area positive for anti-PDF staining and its integrated intensity were measured from the fluorescence signals above a threshold level. Data represent mean ± SEM (n = 18–23). n.s., not significant; ∗∗∗p < 0.001 as determined by one-way ANOVA, Tukey’s post hoc test. (C) NOT1 depletion and the heterozygosity of hypomorphic Atx2 mutation (Atx2[0]/+) non-additively dampens the rhythm amplitude in circadian behaviors. Data represent mean ± SEM (n = 28–39). n.s., not significant; ∗∗∗p < 0.001 to Pdf-Gal4 controls as determined by one-way ANOVA, Tukey’s post hoc test. (D) ME31B depletion disrupts the association of NOT1 with the ATX2 complex. FLAG-tagged ATX2-N proteins were expressed in dsRNA-treated S2 cells as indicated at the top. Soluble extracts were immunoprecipitated by anti-FLAG antibody. Purified IP complexes were resolved by SDS-PAGE and immunoblotted with specific antibodies (left). Input, 4.5% of soluble extracts used in each IP. (E) Depletion of ATX2 or ME31B de-represses gene silencing by RNA-tethered NOT1 silencing domain (SD). FLUC RNA reporters containing λN-binding sites (boxB), RLUC RNA reporter, and λN fusion proteins (λN or λN-NOT1 SD) were co-expressed in dsRNA-treated S2 cells as indicated at the top. Dual luciferase reporter assays were performed at 48 hr after transfection. Repression fold per dsRNA was calculated by inversely normalizing to the FLUC/RLUC value in λN-expressing S2 cells. Data represent mean ± SEM (n = 3). HhR, hammerhead ribozyme; HSL, histone stem-loop at 3′ end of the FLUC reporter. n.s., not significant; ∗∗p < 0.01, ∗∗∗p < 0.001 to dsEGFP control as determined by one-way ANOVA, Tukey’s post hoc test. See also Figure S6 and Table S7. Molecular Cell 2017 66, 129-140.e7DOI: (10.1016/j.molcel.2017.03.004) Copyright © 2017 Elsevier Inc. Terms and Conditions