SUPPLEMENTARY INFORMATION

doi:10.1038/nature11509

Supplementary Table 1 Data collection and refinement statistics
DmZuc WT

DmZuc K171A

SeMet-DmZuc WT(residues 41–253)(residues 41–253)(residues 89–250)

P21

P21

P21

a, b, c (Å)

55.0, 71.2, 56.3

55.9, 70.2, 56.9

35.3, 52.4, 38.8

α, β, γ (°)

90, 107.9, 90

90, 108.8, 90

90, 104.9, 90

Data collection
Space group
Cell dimensions

Peak

Inflection

Wavelength (Å)

1.000

1.000

0.97930.9800Resolution (Å)

50.00–1.75

50.00–2.2

50.00–2.2

50.00–2.5(1.78–1.75)(2.24–2.20)(2.24–2.20)(2.54–2.50)

Rsym

0.064 (0.34)

0.083 (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)

Resolution (Å)

42.8–1.75

45.8–2.2

37.5–2.2

No. reflections

39,874

19,684

6,836

0.187 / 0.230

0.190 / 0.246

0.223 / 0.270

Protein

3,080

2,986

1,173

Ligand/ion

31

10

1

Water

180

96

16

Protein

28.4

40.4

39.0

Ligand/ion

33.6

35.3

24.0

Water

34.3

38.0

30.4

Bond lengths (Å)

0.008

0.005

0.002

Bond angles (º)

1.09

0.95

0.57

Refinement

Rwork / Rfree
No. atoms

B-factors

R.m.s deviations*Highest resolution shell is shown in parentheses. WT, wild-type.

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Predicted transmembrane helixaDmZuc dimer construct(residues 41–253)

Zinc-binding domain

β1
1

Fly
Human
Mouse
Frog
Zebrafish

10

20

30

40

α1

50

60

70

80

90

MVITQIIMKQIRDYPIVSTISIAVSTVLASEVIWKLVQCSRSKREKASRVHEVIIFNELGEICAAVHMRNSSMGSQKPQVSPCCNMHCSL...MGRLS.......WQVAAAAAVGLALTLEALPWVLRWLRSR..RRRPRREAPFFPSQVTCTEALLRAPGAELAELPEGCPCGLPHG.
...MGRSS.......WRLVFAAGAGLALALEALPWLMRWLLAG..RR.PRREVLFFPSQVTCTEALLQAPG..LPPGPSGCPCSLPHS.
...MLLWG.......RWKLAAGLAGLALSLELFYRYMR.......RRKPLREVLFFPVPVTCIEPVLSPMK........QCSCPLPHT.
...MDVFKQMSFKELMKVLGLGTVAFVLGVEWLNWLTRRLRD...SRGPLKEVLFFPSPQVCVEHLFTSHR......SFPCACPLPHGI
α2

β2
100

Fly
Human
Mouse
Frog
Zebrafish

α3

110

β3

120

α4

130

140

β4

150

β5

160

170

RNVAKIVEQIDRAVYSIDLAIYTFTSLFLADSIKRALQRGVIIRIISDGEMVYSKGSQISMLAQLGVPVRVPITTN.LMHNKFCIIDGFESALSRLLRALLAARASLDLCLFAFSSPQLGHAVQLLHQRGVRVRVVTDCDYMALNGSQIGLLRKAGIQVRHDQDPG.YMHHKFAIVDK..
SSLSRLLRALLAARSSLELCLFAFSSPQLGRAVQLLHQRGVRVRVITDCDYMALNGSQIGLLRKAGIQVRHDQDLG.YMHHKFAIVDK..
SAFSRLLVQLLGAQRSLELCVFTFSSPSLARALLILHRRDVRVRVITDNDYMAAPGSQIGPLRSAGVAVRHDQSSG.YMHHKFAVVDG..
TSFSRLLEHLLSARTSLEMCIFSFSNMEMSRAILLLHKRGVVVRVVTDRDYMTITGSQIGALRKAGISVRHEMSSAVHMHHKFALVDG..
α5

β6

180

Fly
Human
Mouse
Frog
Zebrafish

190

α6

200

β7

210

α7

220

β8

230

240

250

RVEEIRLLRKLKFMRPCYSIVISGSVNWTALGLGGNWENCIITADEKLTATFQAEFQRMWRAFAKTEGSQIQLK................
..................RVLITGSLNWTTQAIQNNRENVLITEDDEYVRLFLEEFERIWEQFNPTKYTFFPPKKSHGSCAPPVSRAGGR..................KVLITGSLNWTTQAIQNNRENVLIMEDTEYVRLFLEEFERIWEEFDPTKYSFFP.QKHRGH...........
..................TVVLTGSLNWTVQAFQSNKENILITDDTVIVKAYQKEFERLWEEYDPATYNFFPEKENK.............
..................RKLISGSLNWTLTAVQSNKENVIITEEPELVRPFQQEFLKLWEASDPANHKLQSKNGQIKK...........

Fly................
Human
LLSWHRTCGTSSESQT
Mouse................
Frog................
Zebrafish . . . . . . . . . . . . . . . .

Zinc-binding domain

b

β1
1

10

20

30

40

α1

50

60

70

90

80

DmZuc M V I T Q I I M K Q I R D Y P I V S T I S I A V S T V L A S E V I W K L V Q C S R S K R E K A S R V H E V I I F N E L G E I C A A V H M R N S S M G S Q K P Q V S P C C N M H C S L
MmZuc . . . . . . M G R S S W R L V F A A G A G L A L A L E A L P W L M R W L L A G R R P R . . . . . . . R E V L F F P S Q . V T C T E A L L Q A P . . G L P P G P S G C P C S L P H S E
Nuc..............................................VEPSVQVGYSPEG...............................
β1

α2

β2
100

α3

110

120

β3
130

α4
140

β4

150

160

β5
170

DmZuc R N V A K I V E Q I D R A V Y S I D L A I Y T F T S L F L A D S I K R A L Q R G V I I R I I S D . . G E M V Y S K G S Q I S M L A Q L G V P V R V P I T T N L M H N K F C I I D G F
MmZuc S S L S R L L R A L L A A R S S L E L C L F A F S S P Q L G R A V Q L L H Q R G V R V R V I T D . . C D Y M A L N G S Q I G L L R K A G I Q V R H D Q D L G Y M H H K F A I V D . .
Nuc
SARVLVLSAIDSAKTSIRMMAYSFTAPDIMKALVAAKKRGVDVKIVIDERGNTGRASIAAMNYIANSGIPLRTDSNFPIQHDKVIIVD..
α1

β2

α5
180

α2

β6
190

200

α6
210

β3

α3

β7
220

β4

α7
230

β5

β8
240

250

DmZuc E R V E E I R L L R K L K F M R P C Y S I V I S G S V N W T A L G L G G N W E N C I I T A D . E K L T A T F Q A E F Q R M W R A F A K T E G S Q I Q L K . . . .
MmZuc . . . . . . . . . . . . . . . . . . K K V L I T G S L N W T T Q A I Q N N R E N V L I M E D . T E Y V R L F L E E F E R I W E E F D P T K Y S F F P Q K H R G H..................NVTVETGSFNFTKAAETKNSENAVVIWNMPKLAESFLEHWQDRWNQGRDYRSSY........
Nuc
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.

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d
input

IP
(FLAG)

Zn2+
α2

α2

N (89)

β1

204–219∗∗∗

Zuc-FLAG + Zuc-Mycb

Zuc-FLAG + Myc-EGFP

Zinc-binding
domainZuc-FLAG + Zuc-Myc

α1

Zuc-FLAG + Myc-EGFP

aanti-Myc
anti-FLAG

162–171
β1

DmZuc WT (residues 89–250)
Monomer

α2

Zn2+
Zinc-binding
domainα1

DmZuc WT (residues 41–253)
Dimer

c

α1

Zinc-binding
domain

e

α1

Zn2+

Zn2+
α2

α2
β1

α3

α4

∗

α5

Val104∗

β1

Tyr105
Ser106

Asp176

β2
β4

β1
α2

Zn2+
Zinc-binding
domain

β3

β5

β6

β7

Lys171
His169
Lys171

α5β8
α7His169α1DmZuc K171A (residues 41–253)
Dimer

Supplementary Figure 2. Crystal structures of DmZuc.
a–c, Overall structures of the wild-type (WT) dimer (residues 41–253) (a), the WT monomer (residues 89–250)(b), and the K171A mutant dimer (residues 41–253) (c). Lys171 in the WT and Ala171 in the K171A mutant are
indicated by red asterisks. Disordered regions in the zinc-binding and catalytic domains are indicated by dashedlines. In the WT dimer, residues 74–80 and 246–253 in one protomer, and residues 41–48, 69–75 and 246–253 in
the other are disordered. In the K171A dimer, residues 41–43, 75–81 and 245–253 in one protomer, and residues
41–48, 68–77 and 246–253 in the other are disordered. In the WT monomer, residues 209–213 and 242–250 are
disordered. The active-site loops of the WT monomer and the K171A dimer have conformations different from thatof 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 andanti-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 chainof Asp176 and the main-chain amide groups of Val104, Tyr105 and Ser106.
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a

Nuc

90°

b

Zinc-binding
domainDmZuc / Nuc90°

Zinc-binding
domainc
Asn215

Thr208
Trp207

Trp207

Glu217
Asn206

Tyr112

Glu217
Asn206

Tyr112

His169
Met141

Asn215

Thr208

Lys171
Lys171
Ser204

Met141

Tyr112

His169
Met141
Tyr112

Lys171
Lys171
Ser204
Asn206

Asn206
His169

Ser204

His169Glu217 Leu210

Ser204
Thr208Thr208Asn215

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 theactive sites of DmZuc (coloured) and Nuc (PDB: 1BYR) (grey) (stereoview). Conserved residues are shown as
sticks.

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73

a

b

Zinc-binding
domain81

Zinc-binding
domain

73

81

His67
Zn2+
Cys63

68

Cys83

76

Cys83

Cys88

Cys63
Cys88

His67
Zn2+

Supplementary Figure 4. Zinc-binding domain.
a, A zinc ion is coordinated by Cys63, His67, Cys83, and Cys88 in the zinc-binding domain. The bound zinc ion isshown 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 domains in the two protomers. The two zinc-binding
domains are oriented similarly, although the disordered regions differ somewhat (residues 74–80 in one protomer
and residues 69–75 in the other), because of the different crystal packing environments.

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Nuc
Active-site groove

a

70°

20°

DmZuc
Active-site groove

b

PO42−

70°

20°

c

d

Active-site groove

Lys145

His169PO42−

Gly146

α4

Lys171
Thr115
Gln148Outer mitochondrial
membraneNN

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 FO–FC 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 alsosurrounded by Thr115 and Gln148. Thr115, Gly146, and Gln148 are conserved in Zuc proteins (Thr115 inDrosophila 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 throughits N-terminal transmembrane helices, with the positively charged surface facing the outer mitochondrial
membrane.

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2.33

Substrate
1.17

0.58

0.29

0.15

0

2.33

dsRNA
1.17

0.58

0.29

0.15

0

ssRNA

Nuc (µM)

(nt)
150
100
50
40
30
20

10

Supplementary Figure 6. Nuc cleaves both ssRNA and dsRNA.
The 42-nt ssRNA and 40-nt dsRNA were incubated with Nuc (0.15–2.33 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 polyacrylamidegel.

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b

(nt)

(nt)

150
100

150

50
40
30

70-nt ssDNA

1.62

0.81

0.41

0.20

0.10

DmZuc (µM)

0

0.60

DmZuc (µM)

0

a

100

50
40

42-nt ssRNA

30
20
20

10

10

Supplementary Figure 7. Characterization of DmZuc nuclease activity in vitro.
a, DmZuc cleaves ssDNA. The 70-nt ssDNA labelled with 32P at the 5' end was incubated with WT DmZuc (0.60µM) for 1 h at 26ºC, and was then resolved on a 20% denaturing polyacrylamide gel. b, DmZuc cleaves ssRNA ina dose-dependent manner. The 42-nt ssRNA labelled with 32 P at the 5' end was incubated with WT DmZuc(0.10–1.62 µM) for 1 h at 26ºC, and was then resolved on a 20% denaturing polyacrylamide gel.

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PC cleavage assay

CL cleavage assay

Lipid concentration (µM)

3.0

No enzyme

2.0

Actinomadura PLD
DmZuc
1.0

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, DmZucand MmZuc hydrolysed neither dipalmitoyl PC nor tetradioleoyl CL.

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Strand-specific RT-PCR

Western blotting

160

anti-Myc

n.i.

total RNAIP
anti--Myc

n.i.

(kDa)

input

IP

flamenco(chrX:21510622..21510771)

120
100
80
70
60
50
40

30

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.

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Nucleus

Cytoplasm

piRNA primary precursor
piRNA intermediate
piRNA cluster
Transposon
Yb body
Silencing

Armi

Piwi

Piwi

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) andTudor domain-containing RNA helicase Yb localize in Yb bodies, cytoplasmic perinuclear non-membranousorganelles, which are often located near mitochondria15,33. Armi and Yb form a complex that contains piRNAintermediates with 5'-hydroxyl and 3'-cyclicphosphate ends15. Nascent, piRNA-free Piwi transiently localizes at Yb
bodies, and interacts with the Armi–Yb complex15. Zuc is anchored on the mitochondria surface, with the catalytic
site facing the cytosol12–15, 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.

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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, 613–627 (2009).

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