LETTER

doi:10.1038/nature11509

Structure and function of Zucchini endoribonucleasein piRNA biogenesis
Hiroshi Nishimasu1*, Hirotsugu Ishizu1,2*, Kuniaki Saito2, Satoshi Fukuhara1, Miharu K. Kamatani2, Luc Bonnefond1{,
Naoki Matsumoto1, Tomohiro Nishizawa1, Keita Nakanaga3, Junken Aoki3, Ryuichiro Ishitani1, Haruhiko Siomi2,
Mikiko C. Siomi1,2,4 & Osamu Nureki1,4

PIWI-interacting RNAs (piRNAs) silence transposons to maintaingenome integrity in animal germ lines1–4. piRNAs are classified as
primary and secondary piRNAs, depending on their biogenesis
machinery5–10. Primary piRNAs are processed from long noncoding RNA precursors transcribed from piRNA clusters in the
genome through the primary processing pathway5,8–10. Although
the existence of a ribonuclease participating in this pathway has
been predicted, its molecular identity remained unknown. Here weshow that Zucchini (Zuc), a mitochondrial phospholipase D (PLD)
superfamily member11, is an endoribonuclease essential for primarypiRNA biogenesis. We solved the crystal structure of Drosophilamelanogaster Zuc (DmZuc) at 1.75 Å resolution. The structure
revealed that DmZuc has a positively charged, narrow catalytic
groove at the dimer interface, which could accommodate a singlestranded, but not a double-stranded, RNA. DmZuc and the mouse
homologue MmZuc (also known as Pld6 and MitoPLD)12–14 showedendoribonuclease activity for single-stranded RNAs in vitro. The
RNA cleavage products bear a 59-monophosphate group, a hallmark
of mature piRNAs. Mutational analyses revealed that the conserved
active-site residues of DmZuc are critical for the ribonuclease activity in vitro, and for piRNA maturation and transposon silencing invivo. We propose a model for piRNA biogenesis in animal germlines, in which the Zuc endoribonuclease has a key role in primarypiRNA maturation.

Zuc is conserved among animals, and loss-of-function mutations in
zuc in flies and mice cause a severe defect in piRNA accumulation in
the germ lines, implying a role for Zuc in piRNA biogenesis8,10,11,13,15,16.
Zuc contains a mitochondrial localization sequence (MLS) at the amino
terminus and an HKD (His-Lys-Asp) motif, a hallmark of PLD superfamily members, in the central region (Supplementary Fig. 1a)12. The
PLD superfamily members have diverse cellular functions17, and Zucshares the highest sequence similarity with the bacterial nucleaseNuc18,19 among the superfamily members (Supplementary Fig. 1b).
Zuc has therefore been considered to be a candidate for the RNase
required for piRNA biogenesis11. However, previous attempts to detect
the nuclease activity in Zuc were unsuccessful12,13.
To obtain structural clues to Zuc’s function, we solved the 1.75-Å
resolution crystal structure of the cytoplasmic region (residues 41–253) of DmZuc, which lacks the N-terminal MLS and a predictedtransmembrane helix (Fig. 1a and Supplementary Fig. 2a–c). The
structure consists of a catalytic domain (residues 49–62 and 89–245)
and a zinc-binding domain (residues 63–88), with the two protomers
in the asymmetric unit forming a dimer. Co-immunoprecipitation
experiments suggested this oligomeric state in vivo (Supplementary
Fig. 2d). The catalytic domain consists of an eight-stranded mixed
b-sheet flanked by a-helices on both sides, and shares structuralsimilarity with the catalytic domains of other PLD superfamily members, such as Nuc19,20 (PDB 1BYR; 26% sequence identity, root mean

73

a
81
Zn2+
α2

α1

b

Zinc-binding
domain

β1
PO42–

β1

Cys 8890°

α2
Cys 83

Cys 63

His 67

β1
α2
Zn2+

49

α1

76

Zinc-binding α1domain68

41

Figure 1 | Crystal structure of DmZuc. a, Overall structure. The two
protomers are coloured green and yellow-green, and the zinc-binding domains
are coloured gold and yellow. The bound zinc ions are shown as grey spheres.
Cys 63, His 67, Cys 83, Cys 88, His 169, Lys 171 and the bound phosphate ion

41

are shown as sticks. Disordered regions are shown as dashed lines. b, The zincbinding domain. The bound zinc ion is shown as a grey sphere, and a simulated
annealing Fo 2 Fc omit electron density map (contoured at 4s) is shown as a
blue mesh.

1
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan. 2Department of Molecular Biology, Keio University School of Medicine, Tokyo
160-8582, Japan. 3Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi 980-8578, Japan. 4Japan Science and Technology Agency (JST), Core Research for Evolutional Science andTechnology (CREST), Saitama 332-0012, Japan. {Present address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR7104 CNRS-UDS, INSERM U964, 1 rue Laurent Fries, BP 10142,
67404 Illkirch Cedex, France.
*These authors contributed equally to this work.

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LETTER RESEARCH
squared deviation 1.8 Å for 145 Ca atoms) (Supplementary Fig. 3a, b).
His 169 and Lys 171 in the HKD motif in the two protomers form anactive site at the dimer interface, and Asp 176 in the motif helps tomaintain the structural integrity of each protomer (Supplementary
Fig. 2e). The crystal structure revealed that a zinc ion is tetrahedrally
coordinated by Cys 63, His 67, Cys 83 and Cys 88 in the zinc-binding
domain, which is mostly disordered, and is absent from the other PLD
superfamily members (Fig. 1b and Supplementary Fig. 4a, b). In the
CHCC motif, only Cys 83 is conserved in animals (Supplementary
Fig. 1a). In the corresponding region of MmZuc, the residues
Cys 49, Glu 51, Cys 66 and Cys 68 are highly conserved among animals
except for flies (Supplementary Fig. 1a), suggesting that these residues
may coordinate a zinc ion in these species.
The active-site groove is composed of conserved residues from thetwo protomers, including Tyr 112, His 169, Lys 171, Ser 204, Asn 206,
Trp 207, Thr 208, Asn 215 and Glu 217, which correspond to Tyr 35,
His 94, Lys 96, Ser 109, Asn 111, Phe 112, Thr 113, Asn 120 and
Glu 122, respectively, in Nuc (Fig. 2). Nuc cleaves substrates througha two-step catalytic mechanism, in which His 94 in one protomer
forms a phosphoenzyme intermediate with a substrate phosphorus
atom, followed by the hydrolysis of this intermediate by a watermolecule, activated by His 94 in the other protomer19,21. Lys 96,
Ser 109, Asn 111 and Glu 122 in Nuc form a hydrogen-bonding
network and participate in catalysis19. The active-site residues are
similarly arranged in DmZuc and Nuc (Fig. 2b, c and Supplementary Fig. 3c), suggesting that Zuc cleaves a substrate phosphodiester
linkage through a similar catalytic mechanism. However, a structuralcomparison revealed a notable difference in their active-site architectures (Fig. 2a and Supplementary Fig. 5a, b). Nuc cleaves single-stranded
and double-stranded nucleic acids in vitro18,22 (Supplementary Fig. 6),
and has a wide, positively charged groove that can readily accommodate
double-stranded nucleic acids19 (Supplementary Fig. 5a). In contrast, theactive-site groove of DmZuc is narrower than that of Nuc, and canapparently accommodate single-stranded, but not double-stranded,
nucleic acids (Fig. 2a and Supplementary Fig. 5b). This structural difference is partly due to the replacements of Gly 64 and Ala 115 in Nuc withthe bulkier Met 141 and Leu 210 residues in DmZuc, respectively(Fig. 2a, b and Supplementary Fig. 3c). A phosphate ion derived froma
Active-site groove

*

*

70°

*

*
*

*

PO42–

*
*

20°

c

b
Thr 208

Ala 115
Asn 120

Asn 215

Thr 113
Trp 207*

Met 141
Tyr 112

Glu 217
Asn 206
His 169

Tyr 112
Met 141

Lys 171
Ser 204 Lys 171*

Asn 206
His 169 Ser 204
Asn 215
Thr 208
Glu 217
Trp 207
Leu 210*

Glu 122

Phe 112

Asn 111
His 94

Tyr 35

Gly 64Gly 64
Tyr 35

Lys 96
Ser 109 Lys 96

Asn 111
His 94 Ser109
Asn 120
Thr 113 Phe 112
Glu 122
Ala 115

Figure 2 | Active site of DmZuc. a, Molecular surface of DmZuc. Conserved
residues are coloured green, and Met 141 and Leu 210 are indicated by red andyellow asterisks, respectively. b, Active site of DmZuc. c, Active site of Nuc(PDB 1BYR).

the crystallization buffer binds to a positively charged patch near theactive-site groove (Supplementary Fig. 5c), suggesting that this patch
could interact with the phosphate group of nucleic acid substrates.
The molecular surface on the side opposite the active-site groove ispositively charged (Supplementary Fig. 5d). The N termini of the two
protomers are located on the same face, and the N-terminal residues
41–48 of one protomer form a positively charged helix, which would
follow the transmembrane helix (residues 41–48 are disordered in theother protomer). This structural feature supports a previous model inwhich Zuc localizes on the outer mitochondrial membrane12–15. Taken
together, the structural features of DmZuc suggested that Zuc is asingle-stranded nuclease that functions on the mitochondrial surface.
To determine whether DmZuc is a nuclease, purified DmZuc(residues 41–253; hereafter referred to as wild-type (WT) DmZuc) was
incubated with a 42-nucleotide (nt) single-stranded RNA (ssRNA)labelled with 32P at the 59 end. DmZuc cleaved the ssRNA substrate(Fig. 3a). In contrast, the DmZuc mutants H169A, K171A, N206A andN215A failed to cleave the substrate (Fig. 3a), highlighting the importance of the conserved residues for the ssRNase activity. These results also
confirmed that the observed cleavage was not due to contaminants. A
DmZuc D63–88 mutant, lacking the zinc-binding domain, showeddecreased ssRNase activity (Fig. 3a), indicating the importance of thisdomain for the nuclease activity. The crystal structure of a shorter
DmZuc construct (residues 89–250), lacking the b1 strand that contributes to dimerization, was solved as a monomer (Supplementary Fig. 2b).
This shorter construct failed to cleave the ssRNA substrate (Fig. 3a),indicating the requirement of dimerization for the ssRNase activity.
Purified MmZuc (residues 35–221) also showed a ssRNase activity comparable to that of DmZuc (Fig. 3a). Thus, Zuc is an evolutionarily conserved RNase. Unlike Nuc (Supplementary Fig. 6), DmZuc cleavedpoly(U) and circular ssRNA, and also ssDNA (Supplementary Fig. 7a),but not double-stranded RNA (dsRNA) (Fig. 3b). Thus, DmZuc is asingle-strand-specific endonuclease. Although the poly(U) cleavage products showed an even distribution, DmZuc did not always cleave thessRNA substrates at specific nucleotides (Fig. 3b), suggesting that DmZucdoes not have strict sequence specificity. DmZuc showed similar activity
in the presence of magnesium ions or EDTA (Fig. 3c), indicating that, like
Nuc19,22, DmZuc is a metal-independent nuclease. The DmZuc ssRNase
activity was inhibited by NaCl at concentrations of 50 mM or higher(Fig. 3c). Sodium ions may inhibit substrate binding, as observed forT4 DNA ligase23 and the dsRNA-specific RNase Pac1 (ref. 24). We
characterized the 59 end structure of the cleavage products, using the
42-nt ssRNA labelled with 32P at the 39 end. The cleavage products
showed resistance to treatment with 59-phosphate ssRNA-specificexonuclease (Terminator Exonuclease) after treatment with calf intestinal phosphatase (Fig. 3d), suggesting that the cleavage products
contain a 59 monophosphate. The endonuclease activity of DmZuc is
modest in vitro, although it showed dose dependence (Supplementary
Fig. 7b). The DmZuc activity may be stimulated in vivo by unknown
cofactors. Although MmZuc reportedly hydrolysed the mitochondrial
lipid cardiolipin to phosphatidic acid12, the active-site groove of
DmZuc seems too narrow to accommodate such a bulky lipid molecule
as a substrate (Fig. 2a and Supplementary Fig. 5b). Indeed, neither
DmZuc nor MmZuc hydrolysed cardiolipin in vitro (Supplementary
Fig. 8). The reason for this discrepancy is currently unclear. Together,
these results indicated that Zuc is an endonuclease specific for singlestranded nucleic acids.
To explore the biological relevance of the DmZuc ssRNase activity
in transposon silencing, we expressed RNA-mediated interference(RNAi)-resistant WT and mutants of full-length DmZuc (residues
1–253) in DmZuc-depleted ovarian somatic cells (OSCs), and thenmonitored the expression levels of the mdg1 transposon by quantitative PCR with reverse transcription (RT–PCR). Western blotting confirmed the similar expression levels of DmZuc WT and mutants(Fig. 4a). WT DmZuc rescued mdg1 derepression, whereas the
H169A and K171A mutants failed to rescue it (Fig. 4a). The mdg1
8 NO V E M B E R 2 0 1 2 | VO L 4 9 1 | N AT U R E | 2 8 5©2012 Macmillan Publishers Limited. All rights reserved

RESEARCH LETTER
a

b
MmZuc

–
WT
89–253
H169A
K171A
N206A
N215A
Δ63–88

DmZuc

42-nt

40-nt 40-nt 40-nt dsRNA ssRNAS
AS poly(U)circular
– +
– +
– +
– + – + T*

NaCl
(mM)
DmZuc
nt
nt
150
150
100
100

nt
150
100

DmZuc
nt
150
100

50
40
30

50
40
30

50
40
30

20

20

20

10

10

10

– +

5′ end-labelled 42-nt ssRNA

Mg2+ (+)
EDTA (–)

c5′ end-labelled ssRNA

Mg2+ (–)
EDTA (+)

dTer. Exo.

0

25

50

100

0

– +

– +

– +

– +

– +

+

CIP – +
nt
150
100

– +

50
40

50
40

30

30

20

20

10

10

5′ end-labelled 42-nt ssRNA

T*, Exonuclease T

DmZuc
–

3′ end-labelled
42-nt ssRNA

Figure 3 | Zuc is an endonuclease for single-stranded nucleic acids.
a, DmZuc and MmZuc, but not monomeric DmZuc or the DmZuc mutants,
cleave a 42-nt ssRNA substrate. b, DmZuc cleaves ssRNAs of various sequences
and circular ssRNA, but not dsRNA. Exonuclease T failed to cleave the circular
ssRNA. S and AS correspond to the sense and antisense strands of dsRNA,

respectively. c, DmZuc endonuclease activity requires no magnesium ions, andis inhibited by NaCl. d, The ssRNA products cleaved by DmZuc have a 59
monophosphate. The cleavage products were resistant to treatment withTerminator Exonuclease (Ter. Exo.) after treatment with calf intestinal
phosphatase (CIP).

expression was higher in OSCs expressing the H169A or K171A
mutant than in control OSCs. The mutants may have dominantnegative effects, although the underlying mechanism is unknown.
The C63A, H67A and D63–88 mutants rescued mdg1 derepression.
The D63–88 mutant had a lower ssRNase activity (about 5% of the WT
activity) (Fig. 3a), which may suffice for transposon silencing. None of
the mutants Y112A, S204A, N206A, W207A, T208A, N215A or
E217A rescued mdg1 derepression (Fig. 4a). These residues are conserved (Supplementary Fig. 1a) and form the active-site groove(Fig. 2b). The N218A mutant rescued mdg1 derepression (Fig. 4a).
Asn 218 is buried within the protein, and thus would not contributeto substrate binding. The H169A, K171A, N206A and N215A mutants
lacked ssRNase activity in vitro (Fig. 3a) and the ability to repress
transposons in vivo (Fig. 4a), indicating that the DmZuc ssRNase
activity is critical for transposon silencing. The expression of WT
DmZuc, but not that of the H169A and K171A mutants, rescued thedefects in Idefix-piRNA maturation in DmZuc-depleted OSCs
(Fig. 4b), suggesting that the DmZuc ssRNase activity is required forprimary piRNA maturation. RT–PCR after immunoprecipitation of
DmZuc from OSCs revealed that DmZuc interacts with the piRNA
precursors in OSCs (Supplementary Fig. 9). Together, these results
indicate that Zuc is an endoribonuclease essential for primarypiRNA processing.
We propose a model for primary piRNA biogenesis in Drosophila
OSCs (Supplementary Fig. 10). Mature piRNAs show a strong bias
for 59 uridine (1-U)5,25. DmZuc cleaved ssRNAs with little sequencespecificity, to produce ssRNA fragments bearing a 59 monophosphate(Fig. 3b, d), a hallmark of mature piRNAs associated with Piwi, suggesting that DmZuc generates the 59 end of mature piRNAs. Theseobservations suggested that DmZuc cleaves piRNA intermediates togenerate the 59 end of mature piRNAs during the processing step, andthat Piwi then preferentially binds 1-U piRNAs during the loading
step. Our findings provide a starting point for explaining the molecularmechanism by which Zuc recognizes genuine piRNA intermediates to
produce mature piRNAs, which in turn should shed light on themechanism of primary piRNA biogenesis in animal germ lines.
b

2

8

2

1

1

1

4

1

0
Rescueplasmid

0

0

1
0

0

nt

Rescueplasmid
piRNA
intermediate?

100
75
50
40
30
Idefix-piRNA
20

Anti-Piwi

Piwi

Anti-Myc

Zuc

Figure 4 | DmZuc endoribonuclease activity is required for transposon
silencing. a, Myc-tagged full-length WT and mutant DmZuc, mutated at ashort interfering RNA (siRNA) recognition site and thus resistant to RNAi,
were expressed in DmZuc-depleted OSCs. The expression levels of Piwi and
DmZuc were examined by western blotting, and the expression levels of the
mdg1 transposon were monitored by quantitative RT–PCR. Results are shownas means 6 s.e.m. (n 5 3). As a control, OSCs were transfected with an empty

siZucControl
WT
H169A
K171A

2

Control
WT
Y112A
M141A

2

Control
WT
S204A
N206A
W207A
T208A
N215A
E217A
N218A

3

Control
WT
C63A
H67A

12

Control
WT
Δ63–883

Control
WT
H169A
K171A

3

Relative mdg1 expression

3

OSC
siEGFPamiR-310vector. b, Northern blotting with a piRNA probe, Idefix-piR1-R, showed that
WT DmZuc, but not H169A or K171A, rescued the defects in piRNAmaturation in DmZuc-depleted OSCs. The expression of WT DmZuc
increased mature piRNAs with a concomitant decrease in piRNA intermediatelike molecules. miR-310, one of the microRNAs in OSCs, was used as aninternal loading control. ‘piRNA intermediate?’ corresponds to ‘piRNAintermediate-like molecules’.

2 8 6 | N AT U R E | VO L 4 9 1 | 8 NO V E M B E R 2 0 1 2©2012 Macmillan Publishers Limited. All rights reserved

LETTER RESEARCHMETHODS SUMMARY
DmZuc and MmZuc were overexpressed in Escherichia coli as glutathione
S-transferase (GST)-tagged proteins, and purified by glutathione-Sepharose,
Resource S and Superdex 200 chromatography, as described26. Crystals wereobtained at 20 uC by the hanging-drop vapour diffusion method. X-ray diffraction
data were collected on beamline BL32XU at SPring-8 (Hyogo, Japan). The crystal
structure of DmZuc (residues 41–253) was determined by molecular replacement using the structure of monomeric DmZuc (residues 89–250), which was
solved by the multiwavelength anomalous dispersion (MAD) method using a selenomethionine (SeMet)-labelled protein. Nuclease assays were performed in buffer
containing 25 mM HEPES-KOH pH 7.4, 2.5 mM EDTA and 5 mM dithiothreitol.
Rescue experiments were performed essentially as described previously15.
Full Methods and any associated references are available in the online version of
the paper.
Received 29 May; accepted 14 August 2012.
Published online 14 October 2012.
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3. Pillai, R. S. & Chuma, S. piRNAs and their involvement in male germline
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5. Brennecke, J. et al. Discrete small RNA-generating loci as master regulators oftransposon activity in Drosophila. Cell 128, 1089–1103 (2007).
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11. Pane, A., Wehr, K. & Schüpbach, T. zucchini and squash encode two putative
nucleases required for rasiRNA production in the Drosophila germline. Dev. Cell 12,
851–862 (2007).
12. Choi, S. Y. et al. A common lipid links Mfn-mediated mitochondrial fusion and
SNARE-regulated exocytosis. Nature Cell Biol. 8, 1255–1262 (2006).
13. Watanabe, T. et al. MITOPLD is a mitochondrial protein essential for nuage
formation and piRNA biogenesis in the mouse germline. Dev. Cell 20, 364–375(2011).
14. Huang, H. et al. piRNA-associated germline nuage formation and spermatogenesis
require MitoPLD profusogenic mitochondrial-surface lipid signaling. Dev. Cell 20,
376–387 (2011).
15. Saito, K. et al. Roles for the Yb body components Armitage and Yb in primarypiRNA biogenesis in Drosophila. Genes Dev. 24, 2493–2498 (2010).
16. Olivieri, D., Sykora, M. M., Sachidanandam, R., Mechtler, K. & Brennecke, J. An in vivo
RNAi assay identifies major genetic and cellular requirements for primary piRNAbiogenesis in Drosophila. EMBO J. 29, 3301–3317 (2010).
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17. Ponting, C. P. & Kerr, I. D. A novel family of phospholipase D homologues that
includes phospholipid synthases and putative endonucleases: Identification of
duplicated repeats and potential active site residues. Protein Sci. 5, 914–922(1996).
18. Pohlman, R. F. et al. Genetic and biochemical analysis of an endonuclease encodedby the IncN plasmid pKM101. Nucleic Acids Res. 21, 4867–4872 (1993).
19. Stuckey, J. A. & Dixon, J. E. Crystal structure of a phospholipase D family member.
Nature Struct. Biol. 6, 278–284 (1999).
20. Leiros, I., Secundo, F., Zambonelli, C., Servi, S. & Hough, E. The first crystal structureof a phospholipase D. Structure 8, 655–667 (2000).
21. Gottlin, E. B., Rudolph, A. E., Zhao, Y., Matthews, H. R. & Dixon, J. E. Catalytic
mechanism of the phospholipase D superfamily proceeds via a covalent
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22. Zhao, Y., Studkey, J. A., Lohse, D. L. & Dixon, J. E. Expression, characterization, andcrystallization of a member of the novel phospholipase D family ofphosphodiesterases. Protein Sci. 6, 2655–2658 (1997).
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ribonuclease of Schizosaccharomyces pombe. Nucleic Acids Res. 24, 2377–2386(1996).
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crystallographic analysis of Zucchini from Drosophila melanogaster. Acta
Crystallogr. F. (in the press).
Supplementary Information is available in the online version of the paper.
Acknowledgements We thank A. Kurabayashi, H. Kotani and Y. Tamaki for technicalassistance; other members of the Siomi laboratory for discussions and comments on
the manuscript; the beamline staff at BL32XU of SPring-8 for assistance in datacollection; and T. Suzuki and H. Suga for technical suggestions and comments. This
work was supported by a grant from the Japan Society for the Promotion of Science(JSPS) through its ‘Funding Program for World-Leading Innovative R&D on Scienceand Technology (FIRST) program’ to O.N., by the Core Research for Evolutional Scienceand Technology (CREST) program ‘The Creation of Basic Medical Technologies toClarify and Control the Mechanisms Underlying Chronic Inflammation’ of the Japan
Science and Technology Agency (JST) to O.N. and M.C.S., and by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) of Japan to H.N., H.I., H.S., M.C.S. and O.N.. K.S. is supported by the
JSPS.
Author Contributions H.N., H.I., K.S., H.S., M.C.S. and O.N. conceived and designed theexperiments and wrote the manuscript. H.N., S.F., L.B., N.M., T.N. and R.I. performed thestructural analyses. H.I., K.S. and M.K.K. performed biochemical and biologicalanalyses. K.N. and J.A. performed PLD activity assays. All authors discussed the data
and the manuscript. M.C.S. and O.N. supervised all the work.
Author Information The atomic coordinates and structure factors are deposited in theProtein Data Bank under accession numbers 4GEL (DmZuc WT dimer), 4GEM (DmZucK171A dimer) and 4GEN (DmZuc WT monomer). The authors declare no competing
financial interests. Reprints and permissions information is available atwww.nature.com/reprints. Readers are welcome to comment on the online version of
the paper. Correspondence and requests for materials should be addressed to O.N.
(nureki@biochem.s.u-tokyo.ac.jp) or M.C.S. (siomim@biochem.s.u-tokyo.ac.jp).

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RESEARCH LETTERMETHODS
26

Protein preparation. Protein samples were prepared as described . In brief, thegene encoding DmZuc (residues 41–253) was cloned between the SacI and XhoI
sites of the pCold–GST vector27. The protein was expressed at 20 uC in E. coli
Rosetta 2 (DE3) (Novagen) and purified using glutathione-Sepharose (GEHealthcare). The protein was treated with Turbo3C protease (Nacalai Tesque)to remove the His6–GST tag, and was further purified by NiNTA (Qiagen),Resource S (GE Healthcare) and Superdex 200 gel filtration (GE Healthcare).
The DmZuc mutants were prepared using a PCR-based method, and thesequences were verified by DNA sequencing. The DmZuc mutants and MmZuc(residues 35–221) were prepared using a protocol similar to that used for WT
DmZuc. DmZuc (residues 89–253) was expressed at 20 uC in E. coli Rosetta 2(DE3) and purified using glutathione-Sepharose. The protein was treated with
Turbo3C protease and was further purified by Superdex 75 gel filtration (GEHealthcare). SeMet-labelled DmZuc (residues 89–250) was expressed in E. coli
B834 (DE3) (Novagen) and purified using a protocol similar to that used for thenative protein.
Crystallization. During our expression trials, we found that DmZuc (residues 89–250) and the K171A mutant of DmZuc (residues 41–253) were more highly
expressed than WT DmZuc (residues 41–253) in E. coli. Thus, we first solved
the crystal structure of DmZuc (residues 89–250) by the MAD method, using a
SeMet-labelled crystal. We next screened and optimized the crystallization
conditions using the K171A mutant of DmZuc (residues 41–253). We thencrystallized WT and K171A DmZuc under similar crystallization conditions,and solved the crystal structures by molecular replacement, using the structureof DmZuc (residues 89–250) as a search model.
Crystallization was performed at 20 uC by the vapour diffusion methods.
Crystals of SeMet-labelled DmZuc (residues 89–250) were obtained by mixing
0.4 ml of protein solution (3 mg ml21 DmZuc, 10 mM Tris-HCl pH 8.0, 150 mM
NaCl, 1 mM dithiothreitol (DTT)) and 0.4 ml of reservoir solution (50 mM MES
pH 6.0, 8% polyethylene glycol 400, 100 mM KCl, 10 mM MgCl2, 3% trehalose).
Crystals of WT and K171A DmZuc (residues 41–253) were obtained by mixing
1 ml of protein solution (3–5 mg ml21 DmZuc, 10 mM Tris-HCl pH 8.0, 150 mM
NaCl, 1 mM DTT) and 1 ml of reservoir solution (16% polyethylene glycol 3350,
200 mM KH2PO4).
Crystallography. X-ray diffraction data were collected at 100 K on beamline
BL32XU at SPring-8 (Hyogo, Japan). The crystals were cryoprotected in reservoir
solution supplemented with 30% ethylene glycol. Diffraction data were processedusing HKL2000 (HKL Research Inc.). The structure of DmZuc (residues 89–250)
was determined by the MAD method, using the 2.2-Å resolution data from the
SeMet-labelled crystal. Four Se atoms were located using SHELXD28, and theinitial phases were calculated using SHARP29, followed by automated model building using RESOLVE30. The model was further built manually using COOT31 andrefined using PHENIX32. The structures of WT and K171A DmZuc (residues 41–253) were solved by molecular replacement with MOLREP33, using the structure of
DmZuc (residues 89–250) as a search model. Data collection and refinement
statistics are provided in Supplementary Table 1. Structural figures were preparedusing CueMol (http://www.cuemol.org).
Nuclease assay. The RNA substrates used for nuclease assays were: 59-AUU
UAAUCAAGCUUAUCGAUACCGUCGACCUCGAGGGGGGGC-39 (42-nt),
59-GGUCUGAUUUCGAUCUGGUUCCCUGGAACAAAAGUGGCAG-39 (40-nt
sense strand), 59-CUGCCACUUUUGUUCCAGGGAACCAGAUCGAAAUCAGACC-39 (40-nt antisense strand), and poly(U) (40-nt). ssRNAs were synthesized(Sigma) and labelled at their 59 ends using [c-32P]ATP and T4 polynucleotide
kinase (New England Biolabs). For 39 end labelling, RNAs were labelled at their 39
ends using 59-[32P]pCp and T4 RNA ligase 1 (New England Biolabs). To makecircular ssRNA, 59 end-labelled 42-nt RNAs were circularized by intramolecular
ligation using T4 RNA ligase 1. The labelled RNAs were purified by electrophoresison 12% denaturing polyacrylamide gels. To make dsRNA, 59 end-labelled 40-nt
sense strand RNA and non-labelled 40-nt antisense strand RNA were mixed at amolar ratio of 1:2 (sense:antisense) in annealing buffer (10 mM Tris-HCl pH 7.5,
50 mM NaCl, 1 mM EDTA), heated at 95 uC for 15 min, and then cooled slowly
to 30 uC.
32
P-end-labelled RNA substrate (104 c.p.m.) was incubated with purified
recombinant DmZuc (1.62 mM) in buffer A (25 mM HEPES-KOH pH 7.4,
2.5 mM EDTA, 5 mM DTT) for 1 h at 26 uC (37 uC for MmZuc). The reaction
was terminated by treatment with proteinase K, followed by extraction withphenol–chloroform and precipitation with ethanol. The products were resolved
on 20% denaturing polyacrylamide gels. The effects of NaCl on the nucleaseactivity were tested using the 42-nt ssRNA substrate in buffer B (25 mM
HEPES-KOH pH 7.4, 5 mM magnesium acetate, 5 mM DTT, 0–100 mM NaCl).
Terminator 59-phosphate-dependent exonuclease was used in accordance withthe manufacturer’s instructions (Epicentre). The DNA substrate used for ssDNase

assays was 59-AATTGGTACGTAGCGCTTGATCTAGAGGGGTTTGCCAAT
AGCAATCCGCACGTTCCTCGTGCTCGACAAT-39.
PLD assay. PLD from Actinomadura sp. no. 362 was purchased from Meito
Sangyo Inc. (Tokyo Japan). Tetraoleoyl cardiolipin (CL) (0.1 mmol; Avanti Polar
Lipids) and dipalmitoyl phosphatidylcholine (PC) (0.5 mmol; Avanti Polar Lipids)
were dried under nitrogen gas and resuspended in 10 mM HEPES pH 7.5 (0.6 ml)
by sonication. The enzyme reaction was performed at 26 uC (DmZuc) or 37 uC(MmZuc and Actinomadura PLD) for 2 h with 10 ml of lipid solution, 38 ml of
assay buffer (50 mM HEPES pH 7.5, 80 mM KCl, 3 mM MgCl2, 2 mM CaCl2,
1 mM DTT) and purified recombinant DmZuc (0.4 mg), MmZuc (0.4 mg) or
Actinomadura PLD (0.3 U). The reaction was stopped by adding 450 ml ofmethanol containing an internal standard (1 mM diheptadecanoyl PC) andcentrifuged at 21,500g. The sample solution (5 ml) was injected into liquid chromatography (LC) and analysed by tandem mass spectrometry (MS/MS). In brief,
lipids were separated by Nanospace LC (Shiseido) with a silica column (5 mm,
2.0 mm 3 150 mm; Shiseido), using a gradient of solvent A (5 mM ammonium
formate in water) and solvent B (acetonitrile), and then analysed by MS/MS usinga Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific).
Lipids were monitored in negative-ion mode and quantified by multiple reaction
monitoring (MRM). Tetraoleoyl CL, dioleoyl phosphatidic acid (PA), dipalmitoyl
PA and dipalmitoyl PC were monitored with the MRM transition of m/z
1,456.1 . 281.2, m/z 699.4 . 281.2, m/z 759.5 . 255.2 and m/z 778.6 . 255.2,
respectively. The ratio between analyte and internal standard peak area was used
for quantification. Calibration curves (0.5–5,000 ng) and LC retention times foreach compound were established using synthetic standards.
Rescue and dimerization assays in OSCs. The RNAi-resistant WT and mutants of
DmZuc were prepared by a PCR-based method, using a pAc-Zuc-Myc vector15 as the
template. Transfection was performed essentially as described previously10. In brief,trypsinized OSCs (5 3 106 cells) were transfected with siRNA duplex (200 pmol),transferred to fresh OSC medium and incubated at 26 uC. Two days after transfection, OSCs (3 3 106 cells) were again transfected with siRNA duplex (200 pmol) andplasmid vector (5 mg). The cells were again incubated at 26 uC for 2 days, and thentotal RNAs were purified using ISOGEN reagent (Nippon Gene). Total RNA (0.5 mg)
was used to reverse transcribe target sequences using oligo(dT) primers. The resulting
cDNAs were analysed by quantitative RT–PCR using a LightCycler real-time PCRsystem (Roche Diagnostics) and SYBR Premix Ex Taq (Takara). Relative steady-statemRNA levels were determined from the threshold cycle for amplification. Ribosomal
protein 49 was used as an internal control. The expression levels of Piwi and DmZuc
were analysed by western blotting using culture supernatants of anti-Piwi hybridoma
cells (P4D2; 1:1 dilution)25 and a mouse monoclonal antibody 9E10 against the Myc
tag (1:1,000 dilution; Sigma), respectively.
Carboxy-terminal Flag-tagged DmZuc (pAc-Zuc-Flag) was prepared, by inserting oligonucleotides encoding a 3 3 Flag tag between the XhoI and BamHI sites ofpAc-Zuc-Myc (ref. 15). OSCs (5 3 106 cells) were co-transfected with pAc-ZucFlag and either pAc-Zuc-Myc or pAcM–EGFP. Two days after transfection, theOSCs were homogenized in lysis buffer (30 mM HEPES pH 7.3, 150 mM
potassium acetate, 2 mM magnesium acetate, 5 mM DTT, 0.1% Nonidet P40) to
prepare OSC lysate. Zuc-Flag was immunopurified using an anti-Flag M2
antibody (Sigma) immobilized on GammaBind beads (GE Healthcare). The
mixtures were rocked at 4 uC for 2 h and the beads were washed five times withlysis buffer. After immunoprecipitation, proteins were resolved by SDS–PAGE
and then detected by western blotting using anti-Flag M2 and anti-Myc antibodies.
RNA immunoprecipitation. C-terminal Myc-tagged, full-length DmZuc wasexpressed in OSCs by transfection. Two days after transfection, the OSCs were
homogenized in lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA,
1 mM DTT, 2 mg ml21 pepstatin, 2 mg ml21 leupeptin, 0.5% aprotinin, 20 U ml21
RNasin Plus (Promega), 1% Triton X-100) to prepare OSC lysate. Zuc-Myc wasimmunopurified using a monoclonal anti-Myc antibody (9E10) immobilized on
Dynabeads Protein G (Invitrogen). The mixtures were rocked at 4 uC for 2 h andthe beads were washed five times with lysis buffer. Total RNAs were isolated fromthe immunoprecipitates with phenol–chloroform and were precipitated withethanol. Total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) and a strand-specific RT primer for a fragment of theflamenco transcript. PCR was performed using Ex Taq DNA polymerase(Takara) and primers for a fragment of the flamenco transcript. The primer
sequences were as follows: flamenco forward, 59-AACGTATGCTCAGTCCAGTGAA-39; flamenco RT and reverse, 59-AAAACCTTCTAGCTTGCCCTCT-39.
27.
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Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).
de La Fortelle, E. & Bricogne, G. SHARP: a maximum likelihood heavy-atomparameter refinement program for the MIR and MAD methods. Methods
Enzymol. 276, 472–494 (1997).

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Adams, P. D. et al. PHENIX: building new software for automated crystallographic
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©2012 Macmillan Publishers Limited. All rights reserved