Vol 461 | 29 October 2009 | doi:10.1038/nature08501

LETTERSA regulatory circuit for piwi by the large Maf genetraffic jam in Drosophila
Kuniaki Saito1, Sachi Inagaki1, Toutai Mituyama2, Yoshinori Kawamura1,3, Yukiteru Ono4, Eri Sakota5,
Hazuki Kotani1, Kiyoshi Asai2,6, Haruhiko Siomi1 & Mikiko C. Siomi1,7

PIWI-interacting RNAs (piRNAs) silence retrotransposons in
Drosophila germ lines by associating with the PIWI proteins
Argonaute 3 (AGO3), Aubergine (Aub) and Piwi1,2. piRNAs in
Drosophila are produced from intergenic repetitive genes andpiRNA clusters by two systems: the primary processing pathway
and the amplification loop1–7. The amplification loop occurs in a
Dicer-independent, PIWI-Slicer-dependent manner3,4,8. However,primary piRNA processing remains elusive. Here we analysed
piRNA processing in a Drosophila ovarian somatic cell line where
Piwi, but not Aub or AGO3, is expressed; thus, only the primarypiRNAs exist. In addition to flamenco, a Piwi-specific piRNA cluster3,
traffic jam (tj)9, a large Maf gene, was determined as a new piRNA
cluster. piRNAs arising from tj correspond to the untranslated
regions of tj messenger RNA and are sense-oriented. piRNA loading
on to Piwi may occur in the cytoplasm. zucchini10, a gene encoding aputative cytoplasmic nuclease, is required for tj-derived piRNA
production. In tj and piwi mutant ovaries, somatic cells fail to intermingle with germ cells and Fasciclin III is overexpressed. Loss of tj
abolishes Piwi expression in gonadal somatic cells. Thus, in gonadal
somatic cells, tj gives rise simultaneously to two different molecules:
the TJ protein, which activates Piwi expression, and piRNAs, which
define the Piwi targets for silencing.
Genetic studies have shown that piwi and aub are essential in germline stem-cell self-renewal and pole-cell formation, respectively11,12.
Mutations introduced into piwi and aub cause de-repression of retrotransposons and a loss of piRNA accumulation in ovaries8. A recent
study has revealed that strong loss-of-function mutations in AGO3 also
increase expression of selfish genetic elements in germ lines5. Thus, thePIWI proteins, with their associated piRNAs, function in retrotransposon silencing. piRNA production in Drosophila ovaries occurs in a
Dicer-independent manner8. A model for piRNA biogenesis—thepiRNA amplification loop3,4—was proposed as a result of deepsequencing and bioinformatic analyses of Drosophila piRNAs. In thismodel, Aub/Piwi and AGO3 reciprocally guide the 59 end formation ofpiRNAs.
Classification of piRNAs according to their origins indicated thatpiRNAs derived from a particular piRNA cluster locus—flamenco(flam)—on the X chromosome are exclusively loaded on to Piwi1,3,
indicating that those piRNAs are produced by a pathway independentof the amplification loop1,2. This pathway is called the primary processing pathway1,2. Recently, two independent groups deduced theexistence of the primary piRNA processing pathway from extensive
bioinformatic analyses of piRNAs in a broad range of piRNA mutants;
these studies reconfirmed that primary piRNAs derived from flam aremost likely loaded directly on to Piwi and not further amplified5,6.

However, a molecular mechanism of the primary processing pathway
remains elusive.
We established a stable cell line of ovarian somatic cells (OSCs)from the parental cell line fGS/OSS, comprising germline stem cells
and sheets of somatic cells (OSS)13. fGS/OSS culture was shown to be
Vasa-positive13, whereas our OSCs are Vasa-negative (Supplementary
Fig. 1a). OSCs express Fasciclin III (FasIII; Supplementary Fig. 1b) andundergo rounds of passage in culture for several months. All these data
support the idea that OSCs contain only mitotically active early follicle(somatic) cells.
Piwi is expressed in somatic gonadal cells3,14,15, whereas Aub and
AGO3 are not expressed in this cell type3,4,16. Western blot analysis
revealed that Piwi, but not Aub and AGO3, was detectable in OSCs
(Supplementary Fig. 1a). As in ovaries, Piwi in OSCs was localized in
the nucleus (Supplementary Fig. 1c). The absence of AGO3 expression in OSCs was further confirmed by polymerase chain reaction
with reverse transcription (RT–PCR; Supplementary Fig. 1d, e).
Piwi in OSCs was detected in a form bound to small RNAs of 24–30
nucleotides (Fig. 1a). The size distribution of these small RNAs wasvery similar to that of gonadal Piwi-associating piRNAs (PiwipiRNAs)3,4,14. In addition, they showed resistance to periodate oxidation and b-elimination treatments, which are hallmarks of 29-Omethyl modification at the 39 end, as in ovarian piRNAs8,17,18 (Supplementary Fig. 1f). Thus, the small RNAs associated with Piwi in
OSCs could be categorized as piRNAs and, importantly, thesepiRNAs are produced in an Aub/AGO3-independent manner.
We examined whether the nuclear localization of Piwi is requiredfor piRNA production and for Piwi loading in OSCs. A mutant of
Piwi (Piwi-DN) in which its putative nuclear localization signals weredeleted, resulting in cytoplasmic localization of the mutant (Fig. 1b),
was loaded with piRNAs in a similar manner to wild-type Piwi
(Fig. 1c). We also observed that a Slicer-deficient Piwi mutant—
Piwi-DDAA, where two aspartic acids (D614 and D685) in the
PIWI domain, which are required for Slicer activity, are altered toalanines—was loaded with piRNAs, similarly to wild-type Piwi
(Fig. 1c). Depletion of endogenous Piwi from OSCs did not affectpiRNA loading on to a double mutant of Piwi (Piwi-DN-DDAA)(Supplementary Fig. 1g). Piwi does not seem to homodimerize invivo (data not shown). These results support a model in which theprimary piRNA processing and the piRNA loading on Piwi may
occur in the cytoplasm in a Piwi-Slicer-independent manner.
Piwi-associating piRNAs in OSCs (OSC piRNAs) are mainly
derived from the antisense strand of retrotransposons (Fig. 2a andSupplementary Fig. 2a), similar to the derivation of ovarian Piwi
piRNAs3,4,14,19. The size distribution of OSC piRNAs is shown in

1

Keio University School of Medicine, Tokyo 160-8582, Japan. 2Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology(AIST), Tokyo 135-0064, Japan. 3Institute of Health Biosciences University of Tokushima, Tokushima 770-8503, Japan. 4Department of Life Sciences, Information and Mathematical
Science Laboratory, Inc., Tokyo 112-0012, Japan. 5Japan Biological Informatics Consortium (JBIC), Tokyo 135-8073, Japan. 6Graduate School of Frontier Sciences, University of Tokyo,
Chiba 277-8583, Japan. 7JST, CREST, Saitama 332-0012, Japan.

1296©2009 Macmillan Publishers Limited. All rights reserved

LETTERS

NATURE | Vol 461 | 29 October 2009b

Anti-Myc

DAPI

Mergeda

54.2%
Piwi-ΔN

1

5

10

15

20

25

5′

Piwi-ΔN
Piwi-DDAA

tj ORF
probe

xx

1,000
0
1,000
5 Mb

Figure 1 | Piwi in OSCs is associated with endogenous small RNAs. a, Piwiassociating small RNAs in OSCs were visualized by 32P-labelling. n.i., nonimmune IgG was used as a negative control. b, Subcellular localization of
Myc-tagged wild-type Piwi, Piwi-DN and Piwi-DDAA in OSCs. Scale bars,
20 mm. DAPI, 49, 6-diamidino-2-phenylindole. c, Myc-tagged wild-type Piwi,
Piwi-DN and Piwi-DDAA expressed in OSCs are bound with piRNAs.
M, molecular mass marker.

tj 3′ UTR
probed6.6 kb
5.0 kb
3.6 kbChr 2L:19,445,598–19,485,883

CG10026

traffic jam

cg10195

2.6 kb
1.9 kb
1.4 kbChr 2L:19,464,129–19,468,157

0.9 kb– rRNA

Supplementary Fig. 2b. Examination of their nucleotide bias indicated that OSC piRNAs mostly have a bias for U as the first nucleotide
in the sequence (1st-U), but no other prominent bias was observed
throughout their entire sequence (Fig. 2b). Exclusion of piRNAs with
1st-U from the piRNA pool did not uncover any obvious bias,
including 10th-A (Supplementary Fig. 2c). piRNA pairings throughthe ten nucleotides from the 59 ends were negligible (Supplementary
Fig. 2d). Thus, Piwi does not self-amplify piRNAs. All these observations correlate well with the data obtained from a recent deepsequencing study that was performed using a Drosophila ovariansomatic sheet (OSS)7.
The origins of OSC piRNAs were examined (Fig. 2c andSupplementary Fig. 3). Unique mapping (see Methods for definition)of OSC piRNAs on the Drosophila genome revealed that flam is themain source (1,365 perfectly matched and 186 one-base mismatchedpiRNAs; Supplementary Figs 3 and 4), as it is for ovarian Piwi
piRNAs3 and OSS piRNAs7. In addition to flam, we found anotherlocus on chromosome 2L that also produces piRNAs uniquely
mapped to the particular region (322 perfectly matched and 29
one-base mismatched piRNAs; Fig. 2c; chromosome 2L, unique).
This locus corresponds to the 39 untranslated region (UTR) of aprotein-coding, single-exon gene, traffic jam9 (tj, Fig. 2d).
TJ is a soma-specific large Maf factor necessary for controlling
gonad morphogenesis in Drosophila9. TJ is the only Drosophila orthologue of the transcriptional factors c-Maf and MafB/Kleisler in vertebrates9. In tj mutant gonads, somatic cells fail to intermingle andproperly develop germ cells. This eventually causes an early blockage
in germ-cell differentiation and no follicle cells are detected in adultovaries of tj mutants9. All of the OSC piRNAs derived from tj weresense-oriented (Fig. 2d), indicating that the tj transcript may serve
not only as the template for TJ synthesis but also as the precursor of
the piRNAs. The tj transcript in OSCs was not dedicated to piRNA
production, because the TJ protein was strongly expressed in OSCs
(Supplementary Fig. 5a). The existence of piRNAs derived from the
39 UTR of tj was further confirmed by northern blot analysis (Supplementary Fig. 5b). tj-derived piRNAs also appeared in ovarian total

Chr 2L, unique

300
200
100
0

D614A/D685A
Deleted peptide sequence (1–72)
MADDQGRGRRRPLNEDDSSTSRGSGDGPRVKVFRGSSSGDPRADPRIEASRERGALEEAPRREGGPPERKPW

Chr 2L, all

S2

e

OSC

PIWI

S2

PAZ
Piwi

OSC

Piwi-DDAA

Piwi-ΔN

Piwi

VectorM

IP with anti-Myc

3′

c

3.0%
rRNA
tRNA
Coding gene
RepeatTransposons
No annotation

Piwi-DDAA

20 nt –

20 nt –

0.0

16.8%

30 nt –

30 nt –

0.5

10.3%

40 nt –

40 nt –

3.8%

11.9%

50 nt –

c

1.0

b

Piwi

Anti-Piwin.i.

M

IP from
OSC

Bits

a

5′ UTR

traffic jam

ORF

3′ UTR

Figure 2 | Piwi-associated piRNAs in OSCs. a, The contents of Piwiassociated small RNAs in OSCs perfectly match the Drosophila genome
sequence. b, Examination of nucleotide bias indicates that Piwi piRNAs havemostly uracil at the 59 ends (1st-U). c, Mapping data on chromosome 2L.
Unique mapping data (bottom; shown as ‘unique’) indicate that tj is thesource of OSC piRNAs. y axis shows the read number. d, The locus
containing tj (shown in c) is enlarged. Green bars (bottom) indicate piRNAs
corresponding to tj. Two thick black lines show probes (ORF and 39 UTR) ine. e, Northern blot analysis. Both ORF and 39 UTR probes visualized a single
band of the same length corresponding to the tj transcript in OSCs.

small RNA libraries produced by ref. 20 (Supplementary Fig. 6) andref. 6 (see later).
We assessed whether the transcriptional unit of tj is first divided
into two parts, each with an individual function—one for TJ synthesis and the other for piRNA production—or if one full-length tj
transcriptional unit contains both functions. Northern blot analysis,using two probes corresponding to either the open reading flame(ORF) or the 39 UTR of tj, visualized a single discrete band of thesame length (Fig. 2e), indicating that the latter scenario might be thecase.
piRNAs derived from 39 UTRs (and from slightly extended
regions) of protein-coding genes other than tj were also found(Supplementary Table 1). For example, the 39 UTRs of brat21 andKlp10A22 also produce piRNAs (Supplementary Fig. 7). Interestingly,
they are all derived from the sense strand; thus the parental genes areapparently not the targets for gene silencing by Piwi. The parental
genes are also not repetitive. By contrast, piRNAs derived from retrotransposons or flam in OSCs are mainly antisense-oriented and arethought to arise from much longer, repetitive precursors1.
Mutations in zucchini (zuc), a gene encoding a putative nuclease,cause female sterility10 and a reduction of roo- and flam-derived
piRNAs in ovaries6,10. However, whether or not zuc is expressed ingonadal somatic cells and, if it is expressed, where Zuc accumulates
was unknown. We observed that OSCs express zuc (data not shown)1297©2009 Macmillan Publishers Limited. All rights reserved

LETTERS

NATURE | Vol 461 | 29 October 2009ac

5′ UTR
traffic jam

3′ UTR
ORF

160

Zuc

Merged

Reads

120

OSC

80
40

Reads

0
30

DAPI

DIC

flam

20
10

Reads

0
30

10
0
30

Reads

sizuc

siEGFPbzuc

20– tj-piR-1

squ

20
10
0
30

Reads

– miR310

armi

20
10
0

Figure 3 | Involvement of zuc in the tj–piRNA production pathway. a, Myctagged Zuc protein was overexpressed in OSCs and its subcellular
localization was determined using an anti-Myc antibody (red). Zucaccumulates in the perinuclear region in the cytoplasm of OSCs. DIC,
differential interference contrast. b, Accumulation of tj-derived piRNAs, but
not of miRNAs, requires zuc in OSCs. c, The read numbers of tj-derived
piRNAs in various piRNA mutants. These data, obtained from the data set ofref. 6, strongly support the idea that zuc is required for tj-piRNA production.

piwi2/piwi2

piwi2/CyO

a

b
Piwi

TJ

TJ

Vasa

Vasa

TJ/Vasacand that Zuc is predominantly localized in the cytoplasm of OSCs,particularly in the perinuclear region (Fig. 3a). We then sought todetermine whether zuc is necessary for tj piRNA production in OSCs.
Depletion of zuc by RNA interference (RNAi) significantly reduced
the expression level of tj piRNAs, but not of microRNAs (Fig. 3b),suggesting that zuc is involved in the tj piRNA production pathway.
This was further supported by analysis of the data set of ref. 6, whichindicated that the read number of tj piRNAs in zuc mutants was much
lower compared with those in other mutants (Fig. 3c).
It was previously reported that tj mutant somatic cells show afailure to intermingle with germ cells in third instar larval ovaries9.
Notably, we noticed that the piwi mutants23 piwi2 and piwi3 showed asimilar phenotype: somatic cells in the ovaries of third instar larvae ofpiwi mutants adhere to each other and exclude Vasa-positive germ
cells (Fig. 4a and Supplementary Fig. 8a). aub mutants did not phenocopy the piwi mutants (Supplementary Fig. 8b). piwi mutants
express the tj transcript (data not shown) and the TJ protein(Fig. 4a and Supplementary Fig. 8a) at approximately wild-type
levels. Thus it is unlikely that tj piRNAs target the parental tj gene.
We next examined the expression of Piwi in larval ovaries of tj
mutants. Vasa and TJ are known to be expressed in germline stem cells(and in their developing cells) and somatic cells, respectively, whereas
Piwi is known to be expressed in both cell types3,11,14. In wild-type larval
ovaries, Vasa- and TJ-positive cells are mutually exclusive, but Piwi isexpressed in both cell populations (Fig. 4b and Supplementary Fig. 9a).
By contrast, in tj mutants, Piwi expression was restricted to Vasapositive cells (Fig. 4b and Supplementary Fig. 9a). These results indicate that TJ is the activator of piwi expression in gonadal somatic cells.
Expression of Piwi in tj mutant testes was also examined. As in
ovaries, tj mutations in testes caused a lack of Piwi expression in
gonadal somatic cells, including hub, cyst progenitor cells and earlycyst cells. In contrast, Piwi signals were clearly seen in these cell typesof wild-type testes (Supplementary Fig. 9b). Interestingly, without
functional TJ, germline stem cells and their developing cells in testes,
which normally show a faint signal for Piwi, highly express Piwi
(Supplementary Fig. 9b). It seems that TJ in testes negatively controls
Control
TJ

Piwi/TJ/Vasa

Relative FasIII mRNA level0 1 2 3 4 5 6

tjeo2/CyO
tjeo2/tjeo2

DAPI

tjeo2/Df(2L)E55
TJ

TJ/Vasa

Piwi/TJ/Vasadtj geneTransposon piRNA cluster

Relative FasIII mRNA level
0
1
2
3

DAPI

Piwi

Primary
processing

TJ
piwi geneOn

piRNAspiwi2/CyO
piwi2/piwi2

Piwi

Loading

Relative FasIII mRNA level
012
3

MergedMergedpiwi3/CyO
piwi3/piwi3

Figure 4 | Phenotypes of tj and piwi mutant ovaries and testes. a, In
control (piwi2/CyO) ovaries, TJ-positive cells and primordial germ cells(PGCs) form a mixed cell population. In piwi2/piwi2 larval ovaries, TJpositive cells do not intermingle with PGCs, but instead form coherent
clusters. TJ and Vasa are shown in green and magenta, respectively. Scalebars, 20 mm. b, In tj mutant (tjeo2/Df(2L)E55) larval ovaries, Piwi (magenta)

Gonadal somatic cell

Silencing
Silencingtarget genes
target genes(e.g. transposons) (e.g. FasIII)

is expressed only in Vasa-positive (blue) cells. TJ is shown in green. Scalebars, 20 mm. c, Quantitative RT–PCR shows that the expression level of
FasIII is upregulated by loss of piwi or tj expression. d, Two functions of tj in
the regulation of the function of piwi in gonadal somatic cells. The firstfunction of tj is to activate piwi expression. The second is to supply piRNAs
for Piwi. Genes targeted by the complex might include FasIII.

1298©2009 Macmillan Publishers Limited. All rights reserved

LETTERS

NATURE | Vol 461 | 29 October 2009

piwi expression and indirectly controls expression in germline stem
cells and their developing cells.
This study has uncovered two functions of tj in the regulation ofpiwi’s functions. In gonadal somatic cells, TJ supposedly controls
transcription of various genes. Our study indicates that piwi is highly
likely to be one of the genes under strong TJ control because loss of tj
in gonadal somatic cells abolished Piwi expression. Further support
for the hypothesis that TJ controls piwi expression was provided by
DNA sequences near the putative transcriptional start site of the piwi
gene, which show a weak but significant similarity to the Maf binding
consensus sequence24, and which were bound with TJ in OSCs
(Supplementary Fig. 10a, b). Thus, the first function of tj is to activate
the expression of Piwi in gonadal somatic cells. The second function
is to supply piRNAs for Piwi. Without the supplement of tj piRNAs,
Piwi would lose the activity to target genes that should be silenced by
Piwi and the tj–piRNA complex. A likely target of such silencing is
FasIII because FasIII, a cell adhesion molecule concentrated at the
hub cell junction, is ectopically overexpressed in other somatic cells
in tj larval testes9. Indeed, the FasIII expression level was higher inpiwi mutant testes than in control testes (Fig. 4c). Some of the tj
piRNAs identified in this study showed strong complementarity to
the FasIII primary transcript (Supplementary Fig. 10c). Although all
the tj-derived piRNAs are sense-oriented and thus unlikely to target
the tj mRNA, given the nuclear localization of Piwi, it is conceivablethat the Piwi–piRNA complex could associate with the tj gene.
These findings suggest a novel regulatory circuit where tj mRNAsimultaneously produces two types of functional molecules (Fig. 4d):
TJ protein, which activates expression of Piwi, and piRNAs, which
are loaded on to Piwi to silence specific target genes, such as FasIIIand other, as yet undiscovered, genes.
METHODS SUMMARYThe OSC line was developed from fGS/OSS13. Piwi was immunopurified from
OSCs using an anti-Piwi antibody14. Cloning of small RNAs associated with Piwi
in OSCs was carried out as described25. Genome mapping and annotation was
performed as described in the Methods. Western blotting4, RT–PCR, perioxidation/b-elimination4,17, northern blotting14 and immunostaining26 were performed
as described.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 3 August; accepted 16 September 2009.
Published online 7 October 2009.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank Y. Niki, D. Godt, H. Lin, E. Matunis, S. Kobayashi,
Y. Kitadate, Y. Kageyama and H. Sano for providing reagents. We also thankBloomington and Kyoto Drosophila Stock Center for the supply of Drosophila strains.
We thank K. Yamada, E. Hattori, K. M. Nishida and T. N. Okada for technicalassistance; S. Takahashi and K. Kataoka for discussions and suggestions; and othermembers of the Siomi laboratory for discussions and comments on the manuscript.
We also thank K. Greer and D. McGowan for encouragement. This work was
supported by MEXT grants to H.S. and NEDO (New Energy and Industrial
Technology Development Organization) grants to M.C.S., T.M. and K.A. M.C.S. is
supported by CREST from JST. M.C.S. is Associate Professor of Global COE for
Human Metabolomics Systems Biology by MEXT.
Author Contributions K.S., S.I., Y.K. and H.K. conducted biochemical experiments.
T.M., Y.O., E.S. and K.A. performed bioinformatics. K.S., T.M., H.S. and M.C.S
designed experiments, interpreted data and prepared the manuscript.
Author Information Small RNA sequences have been deposited at the GEO
database under accession number GSE15137. Reprints and permissions
information is available at www.nature.com/reprints. Correspondence andrequests for materials should be addressed to H.S. (awa403@sc.itc.keio.ac.jp) or
M.C.S. (siomim@sc.itc.keio.ac.jp).

1299©2009 Macmillan Publishers Limited. All rights reserveddoi:10.1038/nature08501

METHODSOSC culture. Culture medium was prepared essentially as previously
described13. Cross and Sang’s M3 (BF) medium was prepared from Shields
and Sang M3 Insect Medium supplemented with 0.6 mg ml21 glutathione,
10% FBS, 10 mU ml21 insulin and 10% fly extract. We collected adult Oregon
R flies and measured their total weight. Flies were treated with 70% ethanol for
5 min, and then washed several times with PBS. Flies were transferred to a mortar
and 10 ml of M3 medium supplemented with 10% FBS (M3/FBS) was added.
Flies were homogenized using a pestle on ice, after which M3/FBS was added tothe homogenate, giving a final concentration of 0.2 g (fly weight) per ml. This
homogenate was centrifuged at 6,000g for 10 min. The supernatant was divided
into 500 ml aliquots and then heat treated at 60 uC for 5 min. This preparation was
spun at 17,000g for 10 min. The supernatant was centrifuged at least twice more
and the clear supernatant then collected. The supernatant (fly extract) was stored
at 230 uC before use. Fly extract was sterilized by filtration and added to theculture medium to a final concentration of 10%. For passaging, OSCs were
washed in PBS and then exposed to a solution of trypsin-EDTA for 1 min at
37 uC. Culture medium was then added to neutralize the trypsin. The cells were
washed by aspiration in the medium and aliquots were pipetted into a 6-wellplate (Falcon) or a 90-mm cell culturing dish (FPI). These were then incubated at
26 uC (the detailed protocol is available on request).
Drosophila strains. yellow white (y w) and Oregon R were used as wild-typestrains. The piwi alleles used were piwi2 and piwi3 (Bloomington stock number
12225) (gift from H. Lin). Df(2L)BSC145 (Bloomington stock number 9505) is a
deficiency line that covers the piwi locus. The tj alleles used were tjeo2 and tjz4735(gift from D. Gobt, S. Kobayashi and H. Sano). Df(2L)E55 (DGRC stock number
106822) is a deficiency line that covers the tj locus. The aub alleles used were
aubHN2 cn bw/CyO and aubQC42 cn bw/CyO. aubHN2 cn bw/CyO and aubQC42cn
bw/CyO were crossed to yield aub heterozygous flies, aubQC42/aubHN2. To recognize piwi2/2, tj2/2 and aubQC42/aubHN2 larvae, w*; L2 Pin1/CyO, P{GAL4Kr.C}DC3 P{UAS-GFP.S65T}DC7 (Bloomington stock number 5194) were usedas balancer chromosomes. All stocks were maintained at 25 uC.
Immunoprecipitation and cloning of small RNAs associated with Piwi in
OSCs. Piwi was immunopurified from OSCs using a specific antibody14.
1 3 108 OSCs were homogenized in a hypotonic buffer (30 mM HEPES,
pH 7.3, 2 mM magnesium acetate, 5 mM dithiothreitol (DTT) and 1 mg ml21
Pefablock SC) to prepare OSC lysate. Piwi and AGO1 were immunopurified
using anti-AGO1 (ref. 27) and anti-Piwi (P3G11) antibodies14 immobilized on
GammaBind beads (GE Healthcare). Just before immunoprecipitation,
potassium acetate and NP-40 were added to the lysates to 150 mM and 0.1%,
respectively. Reaction mixtures were rocked at 4 uC for 2 h and beads were
washed five times with washing buffer (30 mM HEPES, pH 7.3, 150 mM
potassium acetate, 2 mM magnesium acetate, 5 mM DTT, 0.1% NP-40 and
1 mg ml21 Pefablock SC). After immunoprecipitation, total RNAs were isolated
from the immunoprecipitates with phenol–chloroform and precipitated withethanol. RNAs were dephosphorylated with CIP (NEB) and labelled for visualization with 32P-c-ATP using T4 polynucleotide kinase (Takara). Cloning of
small RNAs associated with Piwi in OSCs was carried out as previously
described25. Deep-sequencing was performed on a GS FLX system (Roche).
Processing sequence tags. The adaptor sequences attached to the 59 and 39 endsof every sequence produced from the Roche/454 FLX system were removed.
Ideally, the raw sequence belonged to one of the following canonical patterns:
59-ATCGTCTCGGGATGAAA(N...)TTTCATCCCGAGACGAT-39 and 39-ATT
GACCCGAGTTACAG(N...)TTTCATCCCGAGACGAT-59, where (N...) represents an arbitrary nucleotide sequence for subsequent analyses. The adapters canhave deletions at their 59/39 ends. We used the NCBI bl2seq program (-pBLASTN -g F -W 10 -e 10 -D 1) to detect fragments of the adaptor sequencesin the raw sequence and extracted 21,211 sequences belonging to the canonical/
near-canonical patterns. Sequences containing ambiguous Ns were discarded.
Genome mapping and annotation. We followed a previously described procedure to map and annotate sequences25. 21,089 sequences were mapped to the
Drosophila melanogaster (dm3) genome. The annotation results are shown as apie chart in Fig. 2a. Supplementary Fig. 2a compares the characteristics of small
RNAs associated with ovarian Piwi, Aub, AGO33 and OSC Piwi in terms of
strand bias (left) and cloning frequency (right). The strand bias is computedby comparing the mapping strand of a piRNA with the strand of a coinciding
transposon (112 popular classes are used) of the natural transposable element(FlyBase28) track of the UCSC GenomeBrowser for Functional RNA29. To distinguish a piRNA solely mapped on to a single genomic locus from piRNAs
mapped on to multiple genomic loci, we use a term ‘unique mapping’.
Sequence logo. We used the Web Logo program30 to produce Fig. 2b andSupplementary Fig. 2c. The sequences, ranging from 25 to 27 bases, are aligned
to the 59 end with appended ‘-’ to fill the gaps for sequences shorter than 27 bases.

Frequency map. Frequency maps for OSC piRNAs were generated (Fig. 2c andSupplementary Fig. 3) representing the number of piRNAs that are 100%
identical to the genomic sequence in a 5-kb sliding window. piRNAs in theforward strand are rendered in green whereas piRNAs in the reverse strand are
rendered in red.
Surveying ten-base binding partners. We performed a sequence similarity
search using the NCBI BLASTN program among small RNAs associated withovarian Piwi, Aub, AGO33 and OSC Piwi to detect potential binding partners
that bind each other with 10-base full complementarity. We used the sequencesthat were 100% identical to the genomic sequence.
Northern blot, western blot and b-elimination analyses. Northern blot analysis
was carried out essentially as previously described14. Total RNAs of OSCs and S2
cells were isolated using ISOGEN (Wako). Total RNAs from the immunoprecipitates were isolated with phenol–chloroform and precipitated with ethanol.
DNA fragments for detecting tj transcripts (accession number AY325814) werecloned into pBS SK1. The ORF probe corresponded to nucleotides 801–1300
and the 39 UTR probe corresponded to nucleotides 2143–2402. Sequences of theprimers were as follows: tj-ORF-forward, 59-CATGACATGATGTGGCTGAC-39;
tj-ORF-reverse, 59-GATCTGGTCGAGCTGGCG-39; tj-utr-forward, 59-TTTTCAAGAGAAGTGCATTCCC-39; tj-utr-reverse, 59-TATCTCATCTATCTCAATCTCTGTC-39. PCR products were used as templates. DNA probes were synthesizedusing a random primed labelling kit (Takara) in the presence of 32P-dCTP. For
small RNA northern blot analyses, probes used for miR-310, tj-piR-1, tj-piR-2,
klp10a-piR-1 and brat-piR-1 were as follows: miR-310, 59-AAAGGCCGGGA
AGTGTGCAATA-39; tj-piR-1, 59-GGTAATGGGAATGCACTTCTCTTGAA-39;
tj-piR-2, 59-TCTCATCTATCTCAATCTCTGTCGA-39; klp10a-piR-1, 59-GAT
TGCAGTTCGGTTTGCGTGTGA-39; brat-piR-1, 59-TTGTGTCGCGGTTCGG
TTTTGGGT-39. The DNA oligonucleotides were labelled with T4 polynucleotide
kinase in the presence of 32P-c-ATP. Western blot analysis was performed as
described previously4. Ten micrograms of protein from each sample was loadedon gels. Culture supernatants of anti-Piwi hybridoma cells (P3G11)14, a mousemonoclonal antibody for Aub16, culture supernatants of anti-AGO3 hybridoma
cells4, a rabbit polyclonal antibody to Vasa (1:1,000 dilution), a guinea-pig
antibody to TJ (1:1,000 dilution)9 and anti-tubulin (from DSHB) (1:5,000 dilution) were used. Periodate oxidation/b-elimination treatment was performed aspreviously described17.
RT–PCR analyses. Total RNAs of ovaries, S2 cells and OSCs were isolated using
ISOGEN according to the manufacturer’s instructions. Total RNAs were treatedwith DNase to eliminate DNA contamination. Five-hundred nanograms of total
RNA was annealed with an oligo-dT primer. Reverse transcription was performed
using Accuscript High Fidelity Reverse Transcriptase (Stratagene) according to
the manufacturer’s instructions. The resultant cDNAs were amplified using
KOD-plus DNA polymerase and primers for each gene. Sequences of the oligonucleotide primers used are: Dmhen1/Pimet, 59-ATGTTTTCGCACAAGTTTATTTG
CGGG (forward), 59-GCCCAAACACCAGTTCTTGAAC (reverse); AGO3,
59-GCGAGACGAAGTACGGTCAGATAAC (forward), 59-CAATCAAATAAG
CCAATTTGTGAGCG (reverse). For quantitative RT–PCR, total RNA (0.1 mg)
was used to reverse transcribe target sequences using a Transcriptor First strand
cDNA Synthesis Kit (Roche) according to the manufacturer’s instructions. The
resulting cDNAs were amplified with a LightCycler 480 Real-Time PCR
Instrument II (Roche) using the LightCycler 480 SYBR Green I Master (Roche).
The primers used are shown in Supplementary Table 2.
Myc-tagged protein expression and RNAi in OSCs. A short DNA fragment
encoding a Myc-tag was inserted into the KpnI site of pAc5.1/V5-HisA(Invitrogen) to produce pAcM. Full-length piwi and zuc cDNA were inserted
into pAcM to yield Myc–Piwi wild type and Myc–Zuc, respectively. To produce
Myc–Piwi-DN, a partial fragment of the piwi gene (amino acids 73–843) was
amplified and inserted into pAcM. Mutagenesis to yield Myc–Piwi-DDAA and
Myc–Piwi-DN-DDAA was performed with a QuikChange PCR kit using Pfu
Turbo (Stratagene). Primers used are shown in Supplementary Table 2.
Trypsinized OSCs (3 3 106 cells) were suspended in 100 ml of Solution V of
the Cell Line Nucleofector Kit V (Amaxa Biosystems) and mixed with 5 mg ofexpression plasmid or 200 pmol of siRNA duplex. Transfection was conducted inan electroporation cuvette using the Nucleofector instrument (Amaxa
Biosystems). The transfected cells were transferred to fresh OSC medium andincubated at 26 uC. In the case of RNAi, cells were transfected again after 48 h (thedetailed protocol is available on request). The siRNA duplexes used are shown in
Supplementary Table 2.
Immunostaining. OSCs were fixed with 4% formaldehyde in PBS for 15 min andtreated with 0.1% Triton X-100 in PBS for 15 min. After blocking with 2% BSA in
PBS, they were incubated with the primary antibody for 1 h. Immunostaining oflarval ovaries was performed essentially as previously described26. Briefly, third
larval or adult ovaries were dissected manually in PBS. The samples were fixed in
4% paraformaldehyde in PBS for 20 min and treated with methanol and detergent.

©2009 Macmillan Publishers Limited. All rights reserveddoi:10.1038/nature08501

The samples were washed three times (20 min each) in PBTx (PBS containing 0.1%
Triton X-100) and blocked in PBTxb (PBTx containing 5% bovine serum (Sigma))for 1.5 h. Subsequently, samples were incubated with primary antibodies in PBTxb
for 16 h at 4 uC. After washing three times (20 min each) in PBTx, samples wereincubated with secondary antibodies in PBTxb for 16 h at 4 uC and rinsed threetimes (20 min each) in PBTx. Culture supernatants of anti-Piwi hybridoma cells(P4D2, 1:1 dilution)14, a mouse monoclonal antibody to Myc-tag (1:1,000 dilution,
Sigma), a guinea-pig antibody to TJ (1:2,000 dilution)9, a rabbit polyclonal
antibody to Vasa (1:1,000 dilution) and a mouse antibody to FasIII (1:50 dilution)
were used for primary antibodies. Alexa-488-conjugated anti-mouse IgG (Molecular Probes), Cy3-conjugated anti-mouse IgG (Sigma), Alexa-546-conjugatedanti-rabbit IgG (Molecular Probes), Alexa-633-conjugated anti-rabbit IgG(Molecular Probes), Alexa-546-conjugated anti-guinea-pig IgG (Molecular
Probes) and Alexa-488-conjugated anti-guinea-pig IgG (Molecular Probes) wereused as the secondary antibodies. DNA was stained with DAPI. All images werecollected using a confocal microscope (Zeiss LSM5 EXCITER).
ChIP assay. ChIP assays were performed using the EZ ChIP Chromatin
Immunoprecipitation Kit (Upstate). Briefly, 2 3 107 cells were crosslinked with

1% formaldehyde for 10 min at room temperature and sheared to produce
crosslinked DNA (,200–1,000 bp in length). The DNA–protein complexes were
immunoprecipitated overnight with either non-immune IgG antibody or anti-TJ
antibody. After reversing crosslinking at 65 uC for 12–16 h, DNA was recovered.
Short DNA fragments corresponding to piwi gene were amplified by a LightCycler
480 Real-Time PCR Instrument II using SYBR Premix Ex Taq (Takara). The
primers used are listed in Supplementary Table 2.
27. Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H. & Siomi, M. C. Slicer function of
Drosophila Argonautes and its involvement in RISC formation. Genes Dev. 19,
2837–2848 (2005).
28. Wilson, R. J., Goodman, J. L., Strelets, V. B. & FlyBase Consortium. FlyBase:
integration and improvements to query tools. Nucleic Acids Res. 36, D588–D593(2008).
29. Mituyama, T. et al. The Functional RNA Database 3.0: databases to support
mining and annotation of functional RNAs. Nucleic Acids Res. 37, D89–D92(2009).
30. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo, a sequence logo
generator. Genome Res. 14, 1188–1190 (2004).

©2009 Macmillan Publishers Limited. All rights reserved