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1 SUPPLEMENTARY INFORMATION doi: /nature11509 Supplementary Table 1 Data collection and refinement statistics Data collection DmZuc WT DmZuc K171A SeMet-DmZuc WT (residues ) (residues ) (residues ) Space group P2 1 P2 1 P2 1 Cell dimensions a, b, c (Å) 55.0, 71.2, , 70.2, , 52.4, 38.8 α, β, γ ( ) 90, 107.9, 90 90, 108.8, 90 90, 104.9, 90 Peak Inflection Wavelength (Å) Resolution (Å) ( ) ( ) ( ) ( ) R sym (0.34) (0.27) 0.14 (0.93) 0.13 (0.90) I/σI 27.5 (2.2) 16.6 (2.6) 22.9 (2.7) 24.5 (3.1) Completeness (%) 97.2 (91.2) 95.0 (87.7) 100 (100) 100 (100) Redundancy 5.8 (3.7) 2.8 (2.2) 8.4 (8.2) 8.3 (8.3) Refinement Resolution (Å) No. reflections 39,874 19,684 6,836 R work / R free / / / No. atoms Protein 3,080 2,986 1,173 Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s deviations Bond lengths (Å) Bond angles (º) *Highest resolution shell is shown in parentheses. WT, wild-type. 1
2 RESEARCH SUPPLEMENTARY INFORMATION a Predicted transmembrane helix DmZuc dimer construct (residues ) Fly MVITQIIMKQIRDYPIVSTISIAVSTV L AS E VIWKLVQCSRSKREKASRVH E V II F NELGEICAAVHMRNSSMGSQKPQVSP C CNM H CSL Human... M GRLS...WQVAAAAAVGLA L TL E ALPWVL R WLRSR..R R R P RR E AP F F P SQVT C T E ALLRAPGAELAELPEG C P C G LP H G. Mouse... M GRSS...WRLVFAAGAGLA L AL E ALPWLM R WLLAG..R R. P RR E V L F F P SQVT C T E ALLQAPG..LPPGPSG C P C S LP H S. Frog... M LLWG...RWKLAAGLAGLA L SL E LFYRYM R...R R K P LR E V L F F P VPVT C I E PVLSPMK...Q C S C P LP H T. Zebrafish... M DVFKQMSFKELMKVLGLGTVAFV L GV E WLNWLT R RLRD...S R G P LK E V L F F P SPQV C V E HLFTSHR...SFP C A C P LP H GI α2 β2 α3 β3 α4 β4 β Fly RNVAKIVEQIDR A VY S ID L AIYT F T S LF L ADSIKRALQ R G V II R IIS D GEMVYSK GSQI SM L AQL G VP VR VPITTN.L MH N KF CII D GFE Human SAL SRLL RA LL A A RA S L D LC L F A F SS PQ L GH A VQL LH Q R G V R V R V V T D C DYM ALN GSQI G L L R K A G IQ VR H DQDPG.Y MH H KF A I V D K.. Mouse SSL SRLL RA LL A A RS S L E LC L F A F SS PQ L GR A VQL LH Q R G V R V R V I T D C DYM ALN GSQI G L L R K A G IQ VR H DQDLG.Y MH H KF A I V D K.. Frog SAF SRLL VQ LL G A QR S L E LC V F T F SS PS L AR A LLI LH R R D V R V R V I T D N DYM AAP GSQI G P L R S A G VA VR H DQSSG.Y MH H KF A V V D G.. Zebrafish TSF SRLL EH LL S A RT S L EM C I F S F S NMEMSR A ILL LH K R G V V V R V V T D R DYM TIT GSQI G A L R K A G IS VR H EMSSAVH MH H KF A L V D G.. α5 β6 α6 β7 α7 β Fly RVEEIRLLRKLKFMRPCYSIV I S GS V NWT ALGLGG N W EN CI I T A D EKLTAT F QA EF Q R M W RAFAKTEGSQIQLK... Human...RVL I T GS L NWT TQ A I Q N N R EN VL I T E D DEY V RL F LE EF E R I W E QFN P TKYTFFPPKKSHGSCAPPVSRAGGR Mouse...KVL I T GS L NWT TQ A I Q N N R EN VL I ME D TEY V RL F LE EF E R I W E EFD P TKYSFFP.QKHRGH... Frog...TVVLT GS L NWT VQ A F Q S N K EN IL I T D D TVI V KAYQK EF E R L W E EYD P ATYNFFPEKENK... Zebrafish...RKL I S GS L NWT LT A V Q S N K EN VI I T EEPEL V RP F QQ EF LKL W E ASD P ANHKLQSKNGQIKK... Fly... Human LLSWHRTCGTSSESQT Mouse... Frog... Zebrafish... b DmZuc MVITQIIMKQIRDYPIVSTISIAVSTVLASEVIWKLVQCSRSKREKASRVHE V IIFNELGEICAAVHMRNSSMGSQKPQVSPCCNMHCSL MmZuc...MGRSSWRLVFAAGAGLALALEALPWLMRWLLAGRRPR...RE V LFFPSQ.VTCTEALLQAP..GLPPGPSGCPCSLPHSE Nuc...VEPSVQ V GYSPEG... α2 β2 α3 β3 α4 β4 β DmZuc RNVAKIVEQIDR A VY S IDLAIYT F TSLFLADSIKRALQ RGV IIRIIS D..GEMVYSKGSQISMLAQL G VPV R VPITTNLM H N K FC I I D GF MmZuc SSLSRLLRALLA A RS S LELCLFA F SSPQLGRAVQLLHQ RGV RVRVIT D..CDYMALNGSQIGLLRKA G IQV R HDQDLGYM H H K FA I V D.. Nuc SARVLVLSAIDS A KT S IRMMAYS F TAPDIMKALVAAKK RGV DVKIVI D ERGNTGRASIAAMNYIANS G IPL R TDSNFPIQ H D K VI I V D.. β2 α2 β3 α3 β4 β5 α5 β6 α6 β7 α7 β DmZuc ERVEEIRLLRKLKFMRPCYSIVIS GS V N W T ALGLGG N W EN CIITAD.EKLTAT F QAEFQRM W RAFAKTEG S QIQLK... MmZuc...KKVLIT GS L N W T TQAIQN N R EN VLIMED.TEYVRL F LEEFERI W EEFDPTKY S FFPQKHRGH Nuc...NVTVET GS F N F T KAAETK N S EN AVVIWNMPKLAES F LEHWQDR W NQGRDYRS S Y... β6 α4 β7 α5 β8 Supplementary Figure 1. Multiple amino acid sequence alignment. a, Multiple sequence alignment of Zuc proteins from different animal species. The secondary structure of DmZuc is indicated above the sequences. b, Multiple sequence alignment of DmZuc, MmZuc, and Salmonella typhimurium Nuc. The secondary structures of DmZuc and Nuc are indicated above and below the sequences, respectively. Strictly conserved residues are highlighted in red boxes (coloured green in Fig. 2a), and highly conserved residues are indicated by red letters (coloured pale green in Fig. 2a). The HKD motif is indicated by red triangles. The zinc-binding residues in DmZuc are indicated by grey triangles, and the CEC motif is indicated by white triangles. The active-site residues are indicated by green triangles. 2
3 SUPPLEMENTARY INFORMATION RESEARCH a b d input IP (FLAG) α2 α2 N (89) Zuc-FLAG + Myc-EGFP Zuc-FLAG + Zuc-Myc Zuc-FLAG + Myc-EGFP Zuc-FLAG + Zuc-Myc anti-myc anti-flag α2 DmZuc WT (residues ) Monomer DmZuc WT (residues ) Dimer c e α2 α2 α3 α5 α4 Val104 β4 Tyr105 Ser106 β3 α5 β2 Asp176 β6 β5 β7 α2 β8 α7 DmZuc K171A (residues ) Dimer Supplementary Figure 2. Crystal structures of DmZuc. a c, Overall structures of the wild-type (WT) dimer (residues ) (a), the WT monomer (residues ) (b), and the K171A mutant dimer (residues ) (c). in the WT and Ala171 in the K171A mutant are indicated by red asterisks. Disordered regions in the zinc-binding and catalytic s are indicated by dashed lines. In the WT dimer, residues and in one protomer, and residues 41 48, and in the other are disordered. In the K171A dimer, residues 41 43, and in one protomer, and residues 41 48, and in the other are disordered. In the WT monomer, residues and are disordered. The active-site loops of the WT monomer and the K171A dimer have conformations different from that of the WT dimer. d, Self-interaction of DmZuc in OSCs. OSCs were co-transfected with an expression vector encoding C-terminal FLAG-tagged DmZuc and either an expression vector encoding N-terminal Myc-tagged enhanced green fluorescent protein (EGFP) or an expression vector encoding C-terminal Myc-tagged DmZuc. Immunoprecipitation (IP) using an anti-flag M2 antibody, followed by western blotting using anti-myc and anti-flag antibodies, revealed that DmZuc-FLAG interacts with DmZuc-Myc but not Myc-EGFP. e, Asp176 in the HKD motif helps to maintain structural integrity. Dashed lines indicate hydrogen bonds between the side chain of Asp176 and the main-chain amide groups of Val104, Tyr105 and Ser
4 RESEARCH SUPPLEMENTARY INFORMATION a Nuc 90 b DmZuc / Nuc 90 c Asn215 Thr208 Trp207 Trp207 Glu217 Asn206 Tyr112 Glu217 Asn206 Tyr112 Met141 Asn215 Thr208 Ser204 Met141 Tyr112 Met141 Tyr112 Ser204 Asn206 Asn206 Ser204 Glu217 Leu210 Ser204 Thr208 Thr208 Asn215 Met141 Trp207 Asn215 Glu217Leu210 Trp207 Supplementary Figure 3. Structural comparison of DmZuc and Nuc. a, Overall structure of Salmonella typhimurium Nuc (PDB: 1BYR). His94 and Lys96 are shown as sticks. b, Superposition of the overall structures of DmZuc (coloured) and Nuc (PDB: 1BYR) (grey). c, Superposition of the active sites of DmZuc (coloured) and Nuc (PDB: 1BYR) (grey) (stereoview). Conserved residues are shown as sticks. 4 W W W. N A T U R E. C O M / N A T U R E
5 SUPPLEMENTARY INFORMATION RESEARCH a 73 b 73 Cys63 His67 81 Cys83 Cys83 81 His Cys88 Cys63 Cys88 Supplementary Figure 4.. a, A zinc ion is coordinated by Cys63, His67, Cys83, and Cys88 in the zinc-binding. The bound zinc ion is shown as a grey sphere, and an anomalous difference Fourier map at 2.3-Å resolution is shown as a magenta mesh (contoured at 10σ). b, Superposition of the zinc-binding s in the two protomers. The two zinc-binding s are oriented similarly, although the disordered regions differ somewhat (residues in one protomer and residues in the other), because of the different crystal packing environments. 5
6 RESEARCH SUPPLEMENTARY INFORMATION a Nuc Active-site groove b DmZuc Active-site groove 70 2 PO 4 20 c d Active-site groove Lys145 Gly146 PO 4 2 α4 Thr115 Gln148 Outer mitochondrial membrane Supplementary Figure 5. Active-site groove. a, Electrostatic surface potential of Nuc (PDB: 1BYR). b, Electrostatic surface potential of DmZuc. The active sites are indicated by yellow dashed lines in a and b. c, Phosphate-binding site. Hydrogen bonds are indicated by yellow dashed lines, and simulated annealing F O F C omit electron density map is shown as a blue mesh (contoured at 4σ). The phosphate ion interacts with the side chain of Lys145 and the main-chain amide group of Gly146, and is also surrounded by Thr115 and Gln148. Thr115, Gly146, and Gln148 are conserved in Zuc proteins (Thr115 in Drosophila is replaced by a serine in other animals). d, Model of DmZuc localization on the outer mitochondrial membrane. Electrostatic surface potential suggests that DmZuc is anchored on the mitochondrial surface through its N-terminal transmembrane helices, with the positively charged surface facing the outer mitochondrial membrane. N N 6
7 SUPPLEMENTARY INFORMATION RESEARCH ssrna dsrna Substrate Nuc (µm) (nt) Supplementary Figure 6. Nuc cleaves both ssrna and dsrna. The 42-nt ssrna and 40-nt dsrna were incubated with Nuc ( µm) in buffer (25 mm HEPES-KOH, ph 7.4, 2.5 mm EDTA, and 5 mm DTT) at 26ºC for 1 h, and were then resolved on a 20% denaturing polyacrylamide gel. 7
8 RESEARCH SUPPLEMENTARY INFORMATION a DmZuc (µm) b DmZuc (µm) (nt) (nt) nt ssdna nt ssrna Supplementary Figure 7. Characterization of DmZuc nuclease activity in vitro. a, DmZuc cleaves ssdna. The 70-nt ssdna labelled with 32 P at the 5' end was incubated with WT DmZuc (0.60 µm) for 1 h at 26ºC, and was then resolved on a 20% denaturing polyacrylamide gel. b, DmZuc cleaves ssrna in a dose-dependent manner. The 42-nt ssrna labelled with 32 P at the 5' end was incubated with WT DmZuc ( µm) for 1 h at 26ºC, and was then resolved on a 20% denaturing polyacrylamide gel. 8
9 SUPPLEMENTARY INFORMATION RESEARCH PC cleavage assay CL cleavage assay 3.0 Lipid concentration (µm) No enzyme Actinomadura PLD DmZuc MmZuc 0 16:0/16:0 PC 16:0/16:0 PA 18:1 X 4 CL 18:1/18:1 PA Substrate Product Substrate Product Supplementary Figure 8. DmZuc and MmZuc displayed no PLD activity. Purified DmZuc, MmZuc, or Actinomadura PLD (positive control) was incubated with dipalmitoyl (16:0/16:0) PC or tetradioleoyl (18:1 X 4) CL, and then phospholipid products were analyzed by LC/MS/MS. Actinomadura PLD failed to hydrolyse tetradioleoyl CL, but hydrolysed dipalmitoyl PC to produce dipalmitoyl PA. In contrast, DmZuc and MmZuc hydrolysed neither dipalmitoyl PC nor tetradioleoyl CL. 9
10 RESEARCH SUPPLEMENTARY INFORMATION Western blotting Strand-specific RT-PCR IP IP (kda) input n.i. anti--myc total RNA n.i. anti-myc flamenco (chrx: ) Zuc-Myc 20 IB: anti-myc Supplementary Figure 9. DmZuc associates with pirna precursors in OSCs. C-terminal Myc-tagged, full-length DmZuc was expressed in OSCs by transfection. RT-PCR was performed on RNAs isolated from the material immunoprecipitated from the DmZuc-Myc expressing cells, using an anti-myc antibody. Western blotting confirmed that DmZuc was efficiently immunoprecipitated from the cells (left panel). RT-PCR indicated that DmZuc-Myc associates with a fragment of the flamenco transcript (right panel). A control experiment using non-immune IgG (n.i.) indicated that the interaction between DmZuc and the flamenco pirna precursor is specific. 10
11 SUPPLEMENTARY INFORMATION RESEARCH Nucleus Cytoplasm pirna primary precursor pirna intermediate pirna cluster Transposon Silencing Piwi Armi Piwi Yb body Yb Processing Zuc Piwi Piwi Mitochondrion Supplementary Figure 10. Model of primary pirna biogenesis in Drosophila OSCs. Long, single-stranded pirna precursors are transcribed from pirna clusters in the genome, and are then partly processed into pirna intermediates through an unknown mechanism. The RNA helicase Armitage (Armi) and Tudor -containing RNA helicase Yb localize in Yb bodies, cytoplasmic perinuclear non-membranous organelles, which are often located near mitochondria 15,33. Armi and Yb form a complex that contains pirna intermediates with 5'-hydroxyl and 3'-cyclicphosphate ends 15. Nascent, pirna-free Piwi transiently localizes at Yb bodies, and interacts with the Armi Yb complex 15. Zuc is anchored on the mitochondria surface, with the catalytic site facing the cytosol 12 15, and processes pirna intermediates into pirna fragments, which are loaded onto Piwi. In this topological environment, Zuc gains access to pirna intermediates, which are held in the Armi Yb complex. In this model, Zuc endoribonuclease plays a critical role in the formation of the 5' end of mature pirnas. An enzyme participating in the formation of the 3' end remains to be identified. 11
12 RESEARCH SUPPLEMENTARY INFORMATION Supplementary reference 33. Szakmary, A., Reedy, M., Qi, H. & Lin, H. The Yb protein defines a novel organelle and regulates male germline stem cell self-renewal in Drosophila melanogaster. J. Cell Biol. 185, (2009). 12
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