Supplementary Fig. 1 Expression levels of 5 mirnas cycling in PDF cells under LD conditions.
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1 Supplementary Fig. 1 Expression levels of 5 mirnas cycling in PDF cells under LD conditions. RT-qPCR quantification of mirna levels in PDF cells entrained under LD cycles in WT flies (PDF-GAL4;UAS-mCD8::GFP). mirna expression levels are normalized to 2S rrna. 2 biological replicates are concatenated to show cycling. a.u. represents artificial units.
2 Supplementary Fig. 2 Expression levels of mir-92a under LD or DD conditions. The average expression levels of 3 biological replicates under either LD or DD conditions in PDF-GAL4;UAS-mCD8::GFP flies are double plotted and superimposed to show the phase difference.
3 Supplementary Fig. 3 Statistical analysis of maximum axonal crosses quantification. Axonal crosses were quantified with Sholl analysis (Fig. 2A). Maximum axonal crosses indicate the position where maximum amounts of intersections are found between axon branches of a projection and concentric circles in Sholl analysis. N = 14. Error bars represent ±SEM, n.s. indicates non-significant, *P < 0.05, **P < 0.01, one-way ANOVA.
4 Supplementary Fig. 4 Adult-specific manipulation of mir-92a levels changes PDF cell fasciculation status. Immunostaining of PDF cell projections with anti-pdf antibody at ZT2 or ZT14. mir-92aoe indicates PDF-GSG;UAS-mCD8::GFP;UAS-mir-92aOE flies and is compared to its corresponding control PDF-GSG;UAS-mCD8::GFP/+ (with w1118 background). mir-92asp
5 indicates PDF-GSG;UAS-mCD8::GFP;UAS-mir-92aSP flies and is compared to its control, PDF-GSG;UAS-mCD8::GFP;UAS-scramble. Flies were fed on food containing 0.2 mg/ml Mifepristone (Sigma-Aldrich) for 1 week and entrained for at least 3 days under LD cycles prior to the assay. (A) Representative images of PDF cell projections of the indicated genotype at the indicated time. Scale bar equals 25 µm. (B) Quantification with Sholl analysis. Statistics were done on the points with maximum axonal crosses. N = 14. Error bars represent ±SEM, n.s. indicates non-significant, **P < 0.01, one-way ANOVA.
6 Supplementary Fig. 5 GCaMP6 live imaging of PDF neurons may show an altered responsiveness to nicotine in mir-92aoe and mir-92asp flies. Flies expressing GCaMP6 in PDF cells (PDF-GSG;UAS-GCaMP6f) in addition to mir-92a manipulations (UAS-mir-92aOE or UAS-mir- 92aSP) in an adult-specific manner were imaged for fluorescence level changes with 3x10-6 M nicotine perfusion after 30 seconds of baseline recording and the washedout at 60 seconds. Flies were fed on food containing 0.2 mg/ml Mifepristone (Sigma-Aldrich) for 1 week and entrained for at least 3 LD cycles prior to the assay. Measurements for both scramble and mir-92asp were performed between ZT18 22 (when endogenous mir-92a levels in PDF cells are high), and w1118 and mir-92aoe between ZT6 10 (when endogenous mir-92a levels in PDF cells are low). Average fluorescence levels of PDF cell bodies (l-lnvs) are plotted (top panels) and quantified (bottom panel). Each dot represents one brain. Bars represent the mean. Changes are statistically insignificant by two-way ANOVA.
7 M e a n f l u o r e s c e n c e s i g n a l * * * s c r a m b l e m i r a S P w m i r a O E Z T Z T Supplementary Fig. 6 GCaMP6 baseline fluorescence levels in PDF cell projections are affected by manipulation of mir-92a in PDF cells. Flies expressing GCaMP6 in PDF cells (PDF-GAL4;UAS-GCaMP6f) in addition to mir-92a manipulation (UAS-mir-92aOE or UAS-mir-92aSP) were imaged for baseline fluorescence levels. Fluorescence levels in PDF cell projections (s-lnvs) are plotted. Each dot represents an individual measurement. Measurements for both scramble and mir-92asp were performed between ZT18 22 (when endogenous mir-92a levels in PDF cells are high), and w1118 and mir-92aoe between ZT6 10 (when endogenous mir-92a levels in PDF cells are low). Error bars represent ±SD, *P < 0.05, ** P < 0.01, two-tailed t-test.
8 Supplementary Fig. 7 Sleep profiles of mir-92a mutant and control flies. (A) Sleep profiles of female flies entrained under LD cycles. mir-92a levels were manipulated in wake-promoting neurons driven by Dvpdf-GAL4. (B) Sleep profiles from the control experiment with no GAL4 driver. Sleep duration is quantified to the right. N = 32. Error bars represent ±SEM, n.s. represents non-significant, **P < 0.01, **** P < , two-way ANOVA.
9 Supplementary Fig. 8 mir-92a expression levels in dopaminergic neurons of flies are not affected by light pulses. RT-qPCR quantification of mir-92a levels in dopaminergic neurons. Flies were entrained for at least 3 days under LD cycles before exposure to a 10-min light pulse at either ZT15 or ZT21. Dopaminergic neurons were sorted at either ZT16 or ZT22 (50 min after the light pulse). No LP indicates no exposure to light, and LP indicates light pulse exposure. N = 3. Error bars represent ±SEM, n.s. represents non-significant, two-tailed t-test.
10 Supplementary Fig. 9 Sleep and circadian effects from manipulation of mir-92a levels in PDF cells. (A) The left panel shows a scatter plot of rhythmicity index, and the right panel shows a box plot of period lengths. PDF-GAL4 was crossed to w1118, UAS-mir-92aOE, UASscramble and UAS-mir-92aSP for comparison. Each dot indicates one fly. N = 16. Error bars represent ±SD, n.s. represents non-significant, one-way ANOVA. (B) Sleep profiles of female flies of the same genotypes as in (A). Quantification of sleep duration is to the right. N = 16. Error bars represent ±SEM, n.s. represents nonsignificant, *P < 0.05, **P < 0.01, **** P < , two-way ANOVA.
11 Supplementary Fig. 10 Scheme of S2 cell reporter assay. (A) Three plasmids were co-transfected into S2 cells. Ub-GAL4 and UAS-mir-92a cotransfection enabled expression of mir-92a in the cells, and psicheck2-3 UTR served as a reporter for the suppression assay.
12 (B) Plasmid map of psicheck2. The 3 UTR of sirt2 was inserted downstream of renilla. WT sirt2 3 UTR shows base-paring with mir-92a, and the binding site in Mut. sirt2 is mutated (brown). The plasmid map is adapted from Promega psicheck -2 Vector.
13 Supplementary Fig. 11 Quantification of sirt2 RNAi efficiency. Control (#36303, Bloomington stock center, WT background line for transgene injection) and two RNAi lines (RNAi-1: #32482; RNAi-2: #31363) were crossed to Tubulin-GAL4 drivers for expression in whole flies. Total RNA was extracted from fly heads and sirt2 mrna levels were quantified with RT-qPCR with primers listed in Supplementary Table 2. N = 3. Error bars represent ±SEM, ***P < 0.001, one-way ANOVA.
14 Supplementary Fig. 12 Quantification of PDF cell projection axonal crosses of the indicated genotypes. Quantification with Sholl analysis was done as described above. All flies also express the PDF- GAL4 driver. N = 14. Error bars represent ±SEM. n.s. represents non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA.
15 Supplementary Fig. 13 Adult-specific sirt2 knockdown alters the PDF cell fasciculation phenotype. Immunostaining of PDF cell projections with anti-pdf antibody at ZT2 or ZT14. The control is PDF-GSG;UAS-mCD8::GFP/+ (with #36303 genetic background). Sirt2 RNAi-1/2 is PDF- GSG;UAS-mCD8::GFP;UAS-sirt2 RNAi-1/2. Flies were fed on food containing 0.2 mg/ml Mifepristone (Sigma-Aldrich) for 1 week and entrained for at least 3 LD cycles prior to the assay. (A) Representative images of PDF cell projections of the indicated genotype at the indicated time. Scale bar equals 25 µm.
16 (B) Quantification was done with Sholl analysis. Statistics were done at the points with maximum axonal crosses. N = 14. Error bars represent ±SEM, n.s. indicates nonsignificant, ***P < 0.001, one-way ANOVA.
17 F l u o r e s c e n c e l e v e l s * * * * * b a s e l i n e l e v e l s m a x i m u m l e v e l s c o n t r o l s i r t 2 R N A i - 1 s i r t 2 R N A i - 2 Supplementary Fig. 14 GCaMP6 live imaging of PDF neurons shows reduced responsiveness to nicotine with adult-specific sirt2 knockdown. Flies expressing GCaMP6 in PDF cells (PDF-GSG;UAS-GCaMP6f) in addition to RNAi against sirt2 were imaged for fluorescence level changes with 3x10-6 M nicotine perfusion after 30 seconds of baseline recording and then a wash-out at 60 seconds. Flies were fed on food containing 0.2 mg/ml Mifepristone (Sigma-Aldrich) for 1 week and entrained for at least 3 LD cycles prior to the assay. Measurements were performed between ZT2-6. Average fluorescence levels in PDF cell bodies (l-lnvs) are quantified. Each dot represents one brain. Bars represent mean. *P < 0.05, ****P < , two-way ANOVA.
18 Supplementary Fig. 15 sirt2 knockdown in dopaminergic neurons increases sleep duration. (A) sirt2 was knocked-down in dopaminergic neurons using sirt2 RNAi-2 in combination with TH-GAL4/UAS-dicer2 for higher knockdown efficiency. Sleep duration is quantified to the right. N = 16. Error bars represent ±SEM, n.s. represents non-significant, ****P < , two-way ANOVA. (B) A control experiment with no GAL4 driver. Sleep duration was quantified to the right. N = 16. Error bars represent ±SEM, n.s. represents non-significant, two-way ANOVA.
19 Supplementary Fig. 16 A sirt2 amorphic mutant strain shows PDF projection abnormalities. Immunostaining of PDF cell projections with anti-pdf antibody at ZT2 or ZT14. w1118 WT was used to compare with the sirt2 mutant flies. Flies were entrained for at least 3 LD cycles prior to the assay. (A) Representative images of PDF cell projections of the indicated genotypes at the indicated times. Scale bar equals 25 µm. (B) The defasciculation index (DI) was calculated according to 31. High DI indicates more defasciculation and lower DI indicates nire fasciculation. Each dot represents one projection. Bars indicate the mean. n.s. indicates non-significant, *P < 0.05, two-way ANOVA. (C) Axonal crosses were quantified with Sholl analysis as described above. (D) Statistics were done at the points with maximum axonal crosses. N = Error bars represent ±SEM, n.s. indicates non-significant, **P < 0.01, ****P < , two-way ANOVA.
20 Supplementary Fig. 17 sirt2 amorphic flies show increased sleep duration in LD as well as enhanced arrhythmicity in DD. (A) Sleep profiles of female sirt2 amorphic flies compared to w1118 flies under LD conditions. Quantification is to the right. N = 32. Error bars represent ±SEM, n.s. represents non-significant, ****P < , two-way ANOVA. (B) Actograms of male sirt2 amorphic flies compared to w1118 flies during 3 days of LD cycles followed by 8 days of DD cycles. The actograms show the average activity of 32 flies. The white background indicates lights-on, and the grey background indicates lightsoff. The rhythmicity index was calculated for individual flies. N = 32. Error bars represent ±SD, ****P < , two-tailed t-test.
21 Supplementary Fig. 18 Validation of mir-92a expression in PDF cells using traditional stem-loop RT-qPCR. Extracted RNA from sorted PDF cells was quantified. Shown to the left are PDF cell mir-92a levels at ZT6 and ZT18 from flies entrained under LD cycles. Shown to the right are PDF cell mir-92a levels from flies exposed to a 10-min light pulse at either ZT15 or ZT21 (same paradigm as Fig. 4A). ZT16/22 indicates mir-92a expression in controls with no exposure to light pulses. LP16/22 indicates mir-92a expression in flies with light pulse exposure. N = 2 biological replicates. Bar graphs show the average values of the replicates.
22 Supplementary Table 1 TRAP values of mir-92a target candidates with decreased IP/IN levels in mir-92a overexpression flies and increased levels in mir-92asp flies GENE NAMES MIR- 92AOE/W1 118 (IP/IN)/(IP/ IN)-1 MIR- 92AOE/W1 118 (IP/IN)/(IP/ IN)-2 AVERAGE MIR- 92AOE/W1 118 (IP/IN)/(IP /IN) MIR- 92ASP/SCRA MBLE (IP/IN)/(IP/IN )-1 MIR- 92ASP/SCRA MBLE (IP/IN)/(IP/IN )-2 AVERAGE MIR- 92ASP/SCRA MBLE (IP/IN)/(IP/IN ) CG CG CG CG CG REGUCAL CIN CG CG CG L(3)73AH KAT RAB ARR DER CG CG SIRT CG HIL CG CG ONECUT LOLA CG PDK ELAV CG POR
23 Supplementary Table 2 Primer list sirt2 3'UTR fwd sirt2 3'UTR rv sirt2 mut 3'UTR fwd sirt2 mut 3'UTR rv Forward primer: Small RNA PCR Primer 2 Reverse primer RT primer 3' linker (linker1) GCAGTAATTCTAGGCGATCGCACCCTAAGATTAGTTAGTAACATCCGT AGTTAATTTGTAGTTG AATGAAAATAAAGATATTTTATTGCGGCCAGCTTAGCCAGCAATGCC TGCGCTT CCGTAGTTAATTTGTAGTTGAATTcacgttaTTGTTTACATGTGGATTAC GTAATCCACATGTAAACAAtaacgtgAATTCAACTACAAATTAACTACGG AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTATTGA TGGTGCCTACAG ATTGATGGTGCCTACAG rappctgtaggcaccatcaat/ddc/ 5 'linker GUUCAGAGUUCUACAGUCCGACGAUC dme-mir- 92a-3p fwd pr 2S fwd pr dme-mir- 184 fwd pr dme-mir- 999 fwd pr dme-mir- 981 fwd pr dme-mir- 210 fwd pr dme-mir- 276a fwd pr sirt2 fwd sirt2 rv ACACTCCAGCTGGGcattgcacttgtccc ACACTCCAGCTGGGtacaaccctcaacca ACACTCCAGCTGGGtggacggagaactga ACACTCCAGCTGGGtgttaactgtaagac ACACTCCAGCTGGGttcgttgtcgacgaa ACACTCCAGCTGGGttgtgcgtgtgacag ACACTCCAGCTGGGtaggaacttcatacc TTACCATGGGCGACATCGAA GTCGTCACTGGACGAGGAAT
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supplementary information
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0.9 5 H M L E R -C tr l in T w is t1 C M
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