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1 doi: /nature10810 Supplementary Fig. 1: Mutation of the loqs gene leads to shortened lifespan and adult-onset brain degeneration. a. Northern blot of control and loqs f00791 mutant flies. loqs f00791 mutants have reduced processing of mirnas 1,2, including mir-277. Note higher levels of the precursor mirna (arrow head) and 1

2 lower levels of mature mir-277 compared to control. Bottom panel, 2S ribosomal RNA (rrna) loading control. Male flies. Genotypes: control: loqs f00791 /+. loqs mutant: loqs f00791 / loqs f b. loqs f00791 mutant flies (red) have a shortened lifespan compared to control flies (black). Flies were raised at 25 C. Mean ± s.e., n 180 male flies per genotype. Genotypes as in a. c. loqs f00791 mutant flies show late-onset brain degeneration. Top left panel, paraffin section of the fly brain shows that loqs f00791 mutant flies have normal brain morphology at 3d. Note major anatomical structures: CB (central brain), Lo (lobula), LoP (lobula plate), Me (medulla), La (lamina) and Rt (retina). Top right panel, control flies have normal brain morphology at 25d. Bottom panel, aged loqs f00791 mutants (25d) show striking vacuoles in the lamina (arrowheads) and retina (arrows). Male flies. Genotypes as in a. Scale bar: 0.1mm. d. Northern blot of control and loqs f00791 mutant flies. loqs f00791 mutant flies (right lane) have reduced processing of mirnas 1,2, including mir-34. Note higher levels of the mir-34 precursor (arrow head) compared to control at young and older age. Bottom panel, 2S rrna loading control. RNA was from male heads. Genotypes: control: loqs f00791 /+. loqs mutant: loqs f00791 / loqs f e. Western immunoblot showing that E74A protein is dysregulated in loqs f00791 mutant flies, consistent with E74A being a target of mirna function. Protein was from male heads. Genotypes. control. mir-34 -/-: mir-34 null-1 in 5905 homogenous genetic background. loqs mutant is loqs f00791 / loqs f

3 Supplementary Fig. 2: The conserved mirna, mir-34, has an adult-onset expression in the fly brain. a. mirna northern of indicated developmental stages shows that mir-34 has an adult-onset expression. Genotype: iso31. b. mirna northern of indicated tissues shows that mir-34 is enriched in the brain compared to the rest of the fly body (other parts). Note that mir-34 has higher expression in 30d animals compared to 3d animals. Genotypes as in a. c. mirna northern of indicated transgenes for mir-34 expressed by GAL4 drivers, showing that all mir-34 isoforms are generated upon induction. Two different transgenes were made with different lengths of genomic DNA encompassing the mir-34 locus. The red arrow represents the location of mir-34 precursor sequence in each genomic DNA fragment. Note higher levels of precursor mirna (arrow head) due to upregulation of the transgene, and correspondingly higher levels of mature mir-34 compared to control lanes. Bottom panel, 2S rrna loading control. RNA was from male fly heads. Genotypes: control flies gmr-gal4, mir-34 (286bp): gmrgal4/+, UAS-miR-34-I (286bp)/+. heatshock-gal4, mir-34 (936bp): heatshock-gal4/+, UASmiR-34-II (936bp)/+. d. Sequence alignment showing striking conservation of mir-34 orthologues in C. elegans, fly, mouse and human. Fly isoforms are shown; mouse and human have multiple mir-34 genes. 3

4 Supplementary Fig. 3: mir-34 mutant and genomic rescue. a. Map of the mir-34 locus, mir-34 mutant, and mir-34 genomic rescue. The mir-34 region deletion removes mir-277, mir-34 and part of dfmr1, and was generated by site-specific recombination between the two piggybac P-element insertions, PBac(XP)d02752 and PBac(RB)fmr1 e02790 (shown on map). A mir277/dfmr1 transgenic rescue construct was generated to re-introduce the genomic sequences of mir-277 and dfmr1, but not mir-34 (a 9kb genomic DNA fragment containing mir-34 and its endogenous cis-regulatory elements; deleted for mir-277 to retain normal gene dosage levels of mir-277 in rescued animals). b. mir-34 mutant and rescue lines. Selective mir-34 nulls were made by combining the homozygous mir-277/dfmr1 transgenic rescue fragment with the homozygous mir-34 region deletion. In these lines, mir-277 and dfmr1 are intact, and mir-34 is selectively deleted. mir- 34 rescue flies were made by introducing the mir-34 genomic rescue into mir-34 mutant flies. c. mir-34 mutant flies express normal levels of mir-277 and dfmr1. Two independent mir-34 mutant lines mir-34 null-1 and mir-34 null-2, were generated. In these lines, mirna northern (left) and western immunoblot (right) show that mir-34 is selectively deleted, whereas mir-277 and dfmr1 are expressed at normal levels. 3d adult male animals. Genotypes: control: mir-34 -/-: mir-34 null-1 and mir-34 null-2, in 5905 homogenous genetic background. d. mirna northern shows that mir-34 genomic rescue restores mir-34 expression in mir-34 mutant flies. In mir-34 rescue flies, expression of mir-34 also increases with age; mir-34 expression is restored by the genomic insertions to 19.6±0.9%; mean ± s.d. (n = 3 independent experiments) of control. The lack of full rescue with the independent transgenic lines generated suggests a complexity of the mir-34 gene regulatory elements and region. RNA was from male heads. Genotypes are control: mir-34 -/-: mir-34 null-1 in 5905 homogenous genetic background. mir-34 -/-; mir-34 (+): mir-34 genomic rescue in mir-34 null-1 in 5905 homogenous genetic background. 4

5 Supplementary Fig. 4: E74A is a target of mir-34. a. Predicted mir-34 target sites within the 3 UTR of the Eip74EF mrna. Top panel, Eip74EF is located on chromosome 3L, and expresses two major protein isoforms, E74A and E74B. Both 5

6 isoforms share the identical 3 UTR (grey) that contains two putative mir-34 target recognition sites (marked in red). A transposon insertion (p(gt) BG01805 ) in the E74A intronic region affects E74A expression as a hypomorphic mutation. Bottom panel, the mir-34 isoform-c (21mer) is shown in a 3 to 5 orientation, while the homologous sequences from the 3 UTR of the orthologous Eip74EF gene across Drosophila species is shown in a 5 to 3 orientation (alignment from TargetScan 3 ). b. Luciferase reporter assays confirmed that the predicted mir-34 binding sites in the 3 UTR of E74A are functional. Three bases pairs in the seed sequences of the mir-34 sites (red boxes in a) were mutated from GCC to AAA in the E74A 3 UTR. The 3'UTR was co-expressed with mir- 34 in the presence of dsrna for control RNAi (β-gal gene) or Ago1 RNAi (to reveal the dependence on mirna modulation of the 3'UTR). Loss of Ago1 abolished mir-34 regulation of E74A on the wild-type 3'UTR. mir-34 could not silence the mutated 3'UTR. c. The Eip74EF gene isoform-b mrna (E74B) shows low expression in the adult fly. Expression of E74A mrna, however, persists in adults (top panel). RNA from whole pupae during development, and 3d adult male heads. Genotypes: control: mir-34 -/-: mir-34 null-1 in 5905 homogenous genetic background. d. Western immunoblots show that E74A protein is highly expressed in adults <12h old (left panel), but is silenced in older animals. Arrowhead indicates approximate size of E74A protein. Protein was from male fly heads. Genotypes as in b. 6

7 Supplementary Fig. 5: mir-34 contributes to adult-onset silencing of E74A protein. Top panel, western immunoblot for E74A protein. The level of E74A protein is high in heads of newly born adult male flies and undergoes a dramatic reduction within a 24h time window. In mir-34 mutants, the reduction still occurs, but the extent of decrease is damped. Bottom graph, quantification of E74A protein from multiple experiments (the level of the protein in 3d controls was set to 1). In controls, the difference in E74A protein expression is significant between adults <12h old and 1d flies; mir-34 mutants have a defect in adult-onset silencing E74A protein (p<0.05); mean ± s.d., n = 3 independent experiments; signal density normalized to tubulin loading control (**=p < 0.001, one-way analysis of variance, with post test: Tukey's multiple comparison test). Protein was from male heads. Genotypes: control: mir-34 -/-: mir-34 null-1 in 5905 homogenous genetic background. 7

8 Supplementary Fig. 6: E74A mediates key aspects of mir-34 age-associated activities. a. Control flies heterozygous or homozygous for the E74A BG01805 mutation have a normal lifespan. Flies were raised at 29 C. Mean± s.e., for lifespan curve in the left panel, n=167, 157 male flies for control and E74 BG /+, respectively; for lifespan curve in the right panel n=209, 128 male flies for control and E74 BG / E74 BG, respectively. p>0.5, ns (log-rank test). Genotypes: control: 5905, 8

9 E74 BG /+: E74A BG01805 /+ in 5905 homogenous genetic background. E74 BG / E74 BG : E74A BG01805 /E74A BG01805 in 5905 homogenous genetic background. b. Western immunoblot shows that, at 29 C, the E74A protein is aberrantly upregulated in the adult by a transgene driven a temperature sensitive promoter. Bottom panel: tubulin loading control. Protein was from 3d male fly heads. Genotype: control: hs-e74a: heatshock- E74A. c-d. Flies with aberrant expression of the E74A protein in the adult show age-associated defects. Flies of this genotype show (c) enhanced brain degeneration and (d) shortened lifespan, compared to control flies. Flies were raised at 18 C, to prevent expression of E74A from the heatshock promoter, then lifespan analysis was performed at 29 C, which upregulated E74A protein levels (see b). The number of vacuoles in hs-e74a brains at 20d was significantly higher than in control brains (15.3± 4.1 vs 2.3±0.7; p =0.0372, two-tailed t-test; mean vacuoles ± s.e.m., n=3). Lifespan: Mean ± s.e., n>=200 flies per genotype, p< (log-rank test). Male flies. Genotypes as in b. 9

10 Supplementary Fig.7: mir-34 mutant flies show Hsp70/Hsc70 positive accumulated material. Left panel, a representative cryosection of a control fly brain (30d), with anatomical structures noted. CB (central brain), Lo (lobula), LoP (lobula plate), Me (medulla), La (lamina) and Rt (retina). Inset box is the region of brain shown at higher magnification to the right. Middle column, antibody staining for Hsp70/Hsc70 (immunostaining indicated with arrowheads); controls (top panel) showed few sporadic inclusions (4.1±0.7, three experiments, 5-6 brains each). In contrast, striking inclusions were seen in mir-34 mutants (middle panel) (22±2, p<0.0001; one-tailed t-test). mir-34 rescue flies had fewer inclusions (bottom panel). Right column, nuclear staining by DAPI indicates that this brain region is highly enriched for cell bodies. Male flies. Genotypes. control: mir-34 -/-: mir-34 null-1 in 5905 homogenous genetic background. mir-34 -/-; mir-34 (+): mir-34 genomic rescue in mir-34 null-1 in 5905 homogenous genetic background. Scale bar: 0.1mm (left panel), 0.02 mm (right panels). 10

11 Supplementary Fig. 8: Upregulation of mir-34 suppresses polyq-induced neural degeneration. a. Schematic timeline illustrating effects of adult-onset expression of SCA3trQ78 which induces age-dependent neurodegeneration. Expression of Ataxin-3 protein with an expanded polyq domain of 78Q (SCA3trQ78) confers severe neurodegeneration in Drosophila. To assess polyq toxicity in adult neurons, SCA3trQ78 is induced by the rh1-gal4 line, which directs expression to photoreceptor neurons (PR) 1-6, initiating at terminal stages of retinal formation. In the adult, SCA3trQ78 protein progressively accumulates in an aggregated form, accompanied by the reduction of the SDS-soluble form of the protein. By 21d, adult flies show massive neural degeneration. b, c. Upregulation of mir-34 increases solubility of SCA3trQ78 protein. Top panel, in heads of flies expressing SCA3trQ78 alone, polyq protein progressively accumulates into SDS-insoluble aggregates from 1d to 7d, seen as high molecular weight protein smears. Middle panel, this is accompanied by reduction in SDS-soluble polyq protein, which is quantitated in c (mean ± s.e.m., soluble protein relative to tubulin loading control, n=3 experiments; p<0.05; one-tailed t- test). These changes were confirmed with immunohistochemistry on the nuclear inclusions, shown in Figure 4g. Bottom panel, tubulin loading control. Male flies. Genotypes: SCA3trQ78: w + ; rh1-gal4, UAS-SCA3trQ78/+. SCA3trQ78; mir34: w + ; rh-1-gal4, UAS-SCA3trQ78/UASmiR-34-I (286bp). d. Upregulation of mir-34 has no effect on expression of the UAS-SCA3trQ78 transgene (ns, two-tailed t-test). Male flies. Genotypes as in b. 11

12 Supplementary Table 1: mir-34 genomic transgene rescues defects due to loss of mir-34. control mir-34 -/- mir-34 -/-; mir-34 (+) p value (rescue) Lifespan median (day) maximal (day) < Locomotion 1 (% that fail to climb) 4.83 ± ± ± 12 <0.001 Thermostress 1 (% dead) 16.8 ± ± ± 16 <0.01 Brain vacuolization 2 medulla 1.5 ± ± ± 3 <0.01 other regions 7 ± ± ± 3.6 < d old animals 2. 30d old animals Restoring mir-34 to mir-34 deletion flies mitigates defects in lifespan, locomotion, thermo tolerance behaviors, and brain morphology. 12

13 References for Supplementary Figures: 1 Jiang, F. et al. Dicer-1 and R3D1-L catalyze microrna maturation in Drosophila. Genes Dev 19, , (2005). 2 Forstemann, K. et al. Normal microrna maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol 3, e236, (2005). 3 Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P. & Burge, C. B. Prediction of mammalian microrna targets. Cell 115, , (2003). 13