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Roles for the Yb body components Armitage and Yb in primarypiRNA biogenesis in Drosophila
Kuniaki Saito, Hirotsugu Ishizu, Miharu Komai, et al.
Genes Dev. 2010 24: 2493-2498 originally published online October 21, 2010
Access the most recent version at doi:10.1101/gad.1989510

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RESEARCH COMMUNICATION

Roles for the Yb bodycomponents Armitage and Yb
in primary piRNA biogenesis
in Drosophila
Kuniaki Saito,1,3 Hirotsugu Ishizu,1,3
Miharu Komai,1 Hazuki Kotani,1Yoshinori Kawamura,1 Kazumichi M. Nishida,1Haruhiko Siomi,1,5 and Mikiko C. Siomi1,2,4
1

Keio University School of Medicine, Tokyo 160-8582, Japan;
Core Research for Evolutional Science and Technology(CREST), Japan Science and Technology Agency (JST),
Saitama 332-0012, Japan
2

PIWI-interacting RNAs (piRNAs) protect genome integrity from transposons. In Drosophila ovarian somas,
primary piRNAs are produced and loaded onto Piwi. Here,
we describe roles for the cytoplasmic Yb body components Armitage and Yb in somatic primary piRNA biogenesis. Armitage binds to Piwi and is required for
localizing Piwi into Yb bodies. Without Armitage or Yb,
Piwi is freed from the piRNAs and does not enter the
nucleus. Thus, piRNA loading is required for Piwi nuclear entry. We propose that a functional Piwi–piRNAcomplex is formed and inspected in Yb bodies before its
nuclear entry to exert transposon silencing.
Supplemental material is available at http://www.genesdev.org.
Received September 3, 2010; revised version accepted
September 22, 2010.

In Drosophila, three sets of endogenous small RNAs havebeen identified so far: microRNAs (miRNAs), endogenous
siRNAs (endo-siRNAs/esiRNAs), and PIWI-interacting
RNAs (piRNAs) (Ghildiyal and Zamore 2009; Malone
and Hannon 2009; Siomi and Siomi 2009). Of these,
piRNAs are considered unique because of their germline-specific expression and specific interaction with
germline-specific Argonaute proteins, PIWI proteins (Coxet al. 2000; Harris and Macdonald 2001; Saito et al. 2006;
Brennecke et al. 2007; Gunawardane et al. 2007; Nishida
et al. 2007; Li et al. 2009). The identification of the piRNAs
associated with three PIWI proteins (Aubergine [Aub],
Argonaute 3 [AGO3], and Piwi) has revealed distinct
features of piRNAs associated with each PIWI and has
led to two models for piRNA biogenesis: the primaryprocessing pathway and the amplification loop pathway(Aravin et al. 2007; Klattenhoff and Theurkauf 2008;
Ghildiyal and Zamore 2009). In the amplification loop
model, the Slicer (endonuclease) activity of Aub and[Keywords: Piwi; Armitage; Yb; Zucchini; primary piRNA; Drosophila]
3These authors contributed equally to this work.
Corresponding authors.
4
E-MAIL siomim@sc.itc.keio.ac.jp; FAX 81-3-5363-3266.
5
E-MAIL awa403@sc.itc.keio.ac.jp; FAX 81-3-5363-3266.
Article published online ahead of print. Article and publication date areonline at http://www.genesdev.org/cgi/doi/10.1101/gad.1989510.

AGO3 determines the formation of the 59 end of piRNAs(Brennecke et al. 2007; Gunawardane et al. 2007). Zucchini (Zuc), a putative cytoplasmic nuclease (Pane et al.
2007), is involved in the primary processing pathway (Liet al. 2009; Malone et al. 2009); however, its precise
molecular function remains unclear. Furthermore, thefactors other than zuc required for primary piRNA biogenesis are unknown.
The ovarian somatic cell (OSC) line consists of ovariansomas only (Saito et al. 2009). The expression of Aub and
AGO3 is not detectable in OSCs (Saito et al. 2009)
because both proteins are germ cell-specific. This implies
that the amplification loop does not operate in OSCs
(Saito et al. 2009). However, OSCs express piRNAs andare loaded onto Piwi, indicating that the piRNAs in OSCs
are generated specifically through the primary processing
pathway (Saito et al. 2009). Thus, OSCs are an ideal tool
to elucidate the molecular mechanisms of primarypiRNA processing and Piwi function. Loss of zuc function
drastically reduced the level of primary piRNAs in theovaries (Malone et al. 2009). We recapitulated this in
OSCs: Zuc depletion by RNAi caused a severe reductionin the piRNA level in OSCs (Saito et al. 2009). This result
prompted us to screen for other factors necessary forprimary piRNA production using RNAi in OSCs.

Results and Discussion
Armitage is required for primary piRNA biogenesis
and accumulates at distinct cytoplasmic foci in OSCs
To identify the genes required for somatic primary piRNAbiogenesis, we performed RNAi-based screening in OSCs.
The genes screened included armitage (armi), spindle-E(spn-E), and maelstrome (mael) (Supplemental Table S1),
all of which are implicated in piRNA biogenesis (Vagin
et al. 2006; Lim and Kai 2007; Malone et al. 2009).
However, their roles in somatic primary piRNA production remain unknown. Depletion of Armi reduced
the piRNA levels to an extent very similar to that of Piwi
(Fig. 1A) and Zuc depletion (Saito et al. 2009), indicatingthat Armi is necessary for primary piRNA biogenesis in
OSCs. Depletion of Mael and Spn-E showed little or noeffect on piRNA accumulation in OSCs (Fig. 1A). Mutations in both genes have been shown to significantly
reduce the piRNA levels in ovaries (Vagin et al. 2006; Lim
and Kai 2007; Malone et al. 2009). Thus, spn-E and mael
are factors functioning in the amplification loop. Depletion of Dicer1 and Dicer2 had little or no effect on thepiRNA levels (Fig. 1A), confirming that neither protein isnecessary for piRNA production (Vagin et al. 2006).
Armi is the Drosophila ortholog of Arabidopsis Silencing-Defective 3 (SDE3) and mammalian Moloney leukemia virus 10 (MOV10) (Cook et al. 2004). These orthologs
contain a conserved ATP-dependent RNA helicase domain at their C termini (Cook et al. 2004) and have been
implicated in small RNA-mediated gene silencing (Dalmay
et al. 2001; Cook et al. 2004; Tomari et al. 2004; Meister
et al. 2005; Klattenhoff et al. 2007; Haussecker et al.
2008). However, their precise functions remain unknown.
To gain further insight into the function of Armi insomatic primary piRNA processing, we produced a monoclonal antibody against Armi. Western blotting showed

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Saito et al.

nurse cell nuclei (Supplemental Fig. S1C). Thus, Armi
might not be a component of the nuage per se. This
correlates well with the fact that armi mutations barely
affected the ability of the ovaries to amplify endogenous
piRNAs (Malone et al. 2009). In ovarian somas, Armi
accumulated strongly at discrete cytoplasmic foci (Supplemental Fig. S1C), as has been shown previously (Cooket al. 2004). Each somatic cell contained one or several
foci. Interestingly, the Armi-positive foci were often
located near the nucleus in both ovaries and OSCs.
Armi is a novel component of Yb bodies

Figure 1. Armi, a novel component of Yb bodies, is necessary forprimary piRNA production. (A) RNAi-based depletion of Armi, but
not of Spn-E and Mael, caused a severe reduction in the expression
levels of tj-piRNA and Idefix-piRNA in OSCs. (B) OSCs and ovaries
immunostained with anti-Armi and anti-Yb antibodies. Merged
images show that Armi colocalizes with Yb at Yb bodies.

a discrete band in both ovary and cultured Schneider2 (S2)cell lysates (Supplemental Fig. S1A), indicating that Armi
expression is not germline-specific. The ;150-kDa protein immunopurified from S2 cells with the anti-Armi
antibody was confirmed to be Armi by mass spectrometry (Supplemental Fig. S1B).
Immunostaining of OSCs and ovaries with the antiArmi antibody confirmed an earlier observation thatArmi is a cytoplasmic protein (Supplemental Fig. S1C;
Cook et al. 2004). The Armi signals were detected in both
somatic and germ cells of ovaries (Supplemental Fig.
S1C), as has been reported previously (Cook et al. 2004).
The somatic signal was considered a background signal
because it did not disappear even in armi homozygous
mutant egg chambers (armi72.1/armi72.1) (Cook et al.
2004). In the present study, the cytoplasmic signal in
OSCs mostly disappeared when Armi was depleted byRNAi (Supplemental Fig. S1D). Thus, we conclude thatArmi is expressed in both somatic and germ cells in
ovaries.
The subcellular localization of Armi in the armi transheterozygous mutants (armi72.1/armi1) appeared very
similar to that in the homozygous mutants (armi72.1/
armi72.1) (Supplemental Fig. S1E; Cook et al. 2004). In
addition, Western blotting revealed a band corresponding
to Armi in the armi ovaries (Supplemental Fig. S1F). By
what mechanisms Armi is expressed in the mutant
somas remains unclear. The simplest explanation is thatthe armi gene uses two distinct genomic elements aspromoters in ovarian somas. In fact, the armi homozygous mutants armi1/armi1 and armi72.1/armi72.1 weakly
express a shorter armi transcript than that expressed in
the wild-type strain (Supplemental Fig. S1G; Cook et al.
2004).
The Armi signal in germ cells was rather weak, andonly a small proportion of Armi accumulated at, or near,
the nuage, an electron-dense structure associated with

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We noted that the immunostaining patterns of the Armipositive foci in the ovaries looked very similar to those ofYb (Szakmary et al. 2009). Genetic studies have shownthat Yb is necessary for the self-renewal of germline stem
cells and somatic stem cells via the hedgehog- and piwimediated niche signaling pathways, respectively (Kingand Lin 1999; King et al. 2001). Yb accumulates strongly
at cytoplasmic foci, which do not overlap with known
cytoplasmic granules such as P bodies and U bodies(Supplemental Fig. S2A; Szakmary et al. 2009). Thus,
the Yb-positive foci were deemed novel structures andtermed Yb bodies (Szakmary et al. 2009). Yb bodies areoften located close to spherical structures highly enrichedin RNAs, suggesting the involvement of Yb in RNA
metabolism (Szakmary et al. 2009). Immunostaining with
antibodies for Yb and Armi clearly indicated that the two
proteins colocalize at Yb bodies in OSCs (Fig. 1B). Such
colocalization was also observed in the follicle cells in
ovaries (Fig. 1B). Thus, Armi is a novel component of Yb
bodies. To determine if Armi localization in Yb bodies
depends on Yb, or vice versa, immunofluorescence was
performed in OSCs in which Yb or Armi was depleted byRNAi. Depletion of Armi in the OSCs did not cause any
notable change in Yb localization in the Yb bodies(Supplemental Fig. S2B). However, depletion of Yb drastically affected Armi localization; Armi became distributed evenly throughout the cytoplasm (Supplemental Fig.
S2B). Thus, Yb and Armi have different requirements fortheir localization at Yb bodies.
Armi and Yb are required for Piwi nuclear localizationand piRNA association
Loss of Yb function caused a severe reduction in the levelof Piwi in the ovaries, implying that Yb controls theexpression of Piwi in ovaries (Szakmary et al. 2009). We
therefore examined how depletion of Yb affects the levelof Piwi in OSCs. Yb depletion did not affect the expression level of Piwi protein (Supplemental Fig. S3A). Yb
might not be necessary for maintaining Piwi stability
once it is expressed. Loss of Armi affected the subcellular
localization of Piwi: Although the majority of Piwi
remained in the nucleus, some were distributed throughout the cytoplasm (Supplemental Fig. S3B). The nuclear
localization of Traffic Jam (TJ), the only large Maftranscriptional factor in Drosophila (Li et al. 2003), which
regulates Piwi expression in gonadal somas (Saito et al.
2009), was unaffected by Armi depletion (Supplemental
Fig. S3B), suggesting that the effect is specific to Piwi.
Piwi depletion in OSCs did not affect Armi localization(data not shown). We speculated that the nuclear signal
for Piwi found in Armi-depleted cells (Supplemental Fig.
S3B) might reflect Piwi that was localized to this cellularDownloaded from genesdev.cshlp.org on November 15, 2010 - Published by Cold Spring Harbor Laboratory Press

Armi and Yb in primary piRNA biogenesis

compartment before Armi depletion. Therefore, we knocked
down Armi in OSCs and subsequently expressed myctagged Piwi (Saito et al. 2009). Under such conditions, thecytoplasmic distribution of Piwi was clearer: myc-Piwi
was detected almost exclusively in the cytoplasm (Fig.
2A). myc-Piwi behaved similarly when Yb was depleted
instead of Armi (Fig. 2A). We also found that Yb depletioncaused a severe reduction (;25% of the control signal) in
piRNA levels in OSCs, as did Armi depletion (Supplemental Fig. S3C). When Armi or Yb was depleted, mycPiwi was loaded with far fewer piRNAs than undernormal conditions (Fig. 2B). We propose a model in which
Armi and Yb act as novel primary piRNA factors, both ofwhich have indirect effects on piRNA loading and nuclear
localization of Piwi. The Piwi PAZ mutant (myc-PiwiPAZmt), where the tyrosine residues required for piRNAloading are mutated to alanines (Y327A and Y328A), also
accumulated in the cytoplasm (Supplemental Fig. S3D).
Thus, in OSCs, piRNA loading is required for the nuclear
localization of Piwi.
Loading of primary piRNAs onto Piwi occurs
in Yb bodies
Silver staining of proteins coimmunoprecipitated with
Armi from OSCs revealed three proteins (p120, p90, andp37) bound to Armi (Fig. 3A). Mass spectrometry identified p120 and p90 as Yb and Piwi, respectively, while p37

Figure 3. The Armi complex contains not only Piwi and Yb, butalso piR-ILs. (A) Silver staining of protein components in the
immunoprecipitated complex with anti-Armi antibody from OSCs.
Three proteins—p120, p90, and p37—coimmunoprecipitated with
Armi. (B) The complex immunoprecipitated with anti-Armi antibody contains Piwi and Yb but not AGO1 and AGO2. Yb was notdetected in the complex. In addition, Piwi was not detected in thecomplex immunoprecipitated by anti-Yb. The input lane contains
1% of lysates used for immunoprecipitation. (C) The Armi complex
immunopurified from OSCs contains piR-ILs. RNA molecules isolated from the complexes immunoprecipitated with anti-Piwi andanti-Armi antibodies were probed with a DNA oligonucleotide
recognizing tj-piRNA. Note that the Piwi complex was diluted
1:40 prior to RNA isolation. (D, top panel) Depletion of Yb resulted
in the disappearance of piR-ILs from the Armi complex. (Bottom
panels) Western blotting showed that the association between Piwi
and Armi was maintained under these conditions.

Figure 2. Depletion of Armi and Yb causes Piwi to be mislocalized
to the cytoplasm. (A) After depletion of Armi and Yb, myc-tagged
Piwi was expressed in OSCs by transfection. myc-Piwi accumulated
predominantly in the cytoplasm, suggesting that the nuclear signals
observed in Supplemental Figure S3B (green) reflect endogenous Piwi
that was localized to the nucleus prior to Armi and Yb depletion. (B)32
P-labeling of RNAs showed that, when Armi or Yb was depleted,myc-Piwi was loaded with few or no piRNAs. (Left panel) Note thatthe complex immunoprecipitated from control cells was diluted 1:8
prior to RNA isolation. The amount of Piwi in the immunoprecipitated complex from control cells was eightfold higher than thatfrom Armi RNAi cells (data not shown).

remains unidentified. Western blotting of the immunoprecipitates confirmed that Yb and Piwi exist in the Armicomplex (Fig. 3B). Neither AGO1 nor AGO2 was detectedin the complex (Fig. 3B), indicating that the association
between Piwi and Armi is specific. Reciprocal experiments in which the complexes immunoprecipitated with
anti-Piwi or anti-Yb antibody were probed for Armi, Piwi,
and Yb showed that both Piwi and Yb interacted with
Armi (Fig. 3B). However, Yb was not detected in anti-Piwi
immunoprecipitates, and Piwi was not detected in antiYb immunoprecipitates (Fig. 3B). Thus, the interaction
between Yb and Piwi seems to occur only through Armi.
Neither AGO1 nor AGO2 was detected in the anti-Piwi
and anti-Yb immunoprecipitates (Supplemental Fig. S3E).
We speculated that the existence of Yb in the Armi–
Piwi complexes might affect the RNA content of thecomplexes. To address this, RNAs isolated from bothcomplexes were probed with a DNA oligonucleotide

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Saito et al.

recognizing a piRNA derived from the tj transcript (tjpiRNA) (Fig. 3C). The anti-Piwi immunoprecipitate wasdiluted 1:40 before RNA isolation to equalize the amountof Piwi in both complexes (Supplemental Fig. S3F).
Unlike the anti-Piwi immunoprecipitates—in which astrong, specific signal for tj-piRNA was observed—theanti-Armi immunoprecipitates showed a smear spanning
25–70 nucleotides (nt) (Fig. 3C), presumably containing
piRNA intermediate-like molecules (piR-ILs) of various
lengths. The smear was also detected with a probe for theidefix transposon (data not shown), suggesting that transposon–piR-ILs may also exist in the complex.
To determine whether the Armi–Piwi–Yb complex
contains piR-ILs, we isolated small RNAs from the complex and constructed a small RNA library. Sequencing andbioinformatics analysis (using 116,680 ‘‘perfect match to
the genome’’ clones of 184,692 total reads) showed thatthe library included small RNAs that are annotated asprotein-coding genes, rRNAs, transposons, and others (Supplemental Fig. S4A). Small RNAs annotated to miRNAs
and tRNAs were negligible (0% and 0.04%, respectively),suggesting that the Armi–Piwi–Yb complex selects small
RNAs to bind. A number of small RNAs were annotatedas actin 5c (act5c) mRNA (453 clones). However, Northern blotting did not detect these in the Armi–Piwi–Yb
complex (Supplemental Fig. S4B). Small RNAs corresponding to the tj ORF, which does not presumably
contribute to the generation of piRNAs (Saito et al. 2009),
were also negative (Supplemental Fig. S4B). We assume
that small RNAs annotated as act5c mRNA and tj ORF arelikely background.
Clones that mapped uniquely to the piRNA locus flam
were identified, and their distribution on the locus wascompared with that of flam-derived piRNAs (Supplemental Fig. S5A). Interestingly, a number of the flam-derived
piR-ILs overlapped with mature flam-piRNAs (497 of
1203; 41.3%). The details of the overlaps in the ;500-nt
region proximal to the dip1 gene are shown in Supplemental Figure S5B. The majority of the 59 ends of the piRILs, as well as their 39 ends, was not perfectly matchedwith those of mature piRNAs. piR-ILs annotated as tj aresummarized in Supplemental Figure S6A. The 59 ends ofpiR-ILs mapped to transposons were not U-rich (Supplemental Fig. S6B), unlike those of mature transposon–piRNAs. These results suggest that a population of RNAs
residing in the Armi–Piwi–Yb complex correspond topiRNA intermediates. However, it is also possible thatthese RNAs in the complex may correspond to the sideproducts of primary piRNA processing.
We then repeated the Northern blotting experiment
after Yb depletion and found that the anti-Armi immunoprecipitates no longer contained piR-ILs (Fig. 3D). Yb
depletion inhibited Armi accumulation at Yb bodies(Supplemental Fig. S2B), but the interaction between
Armi and Piwi was maintained, as in naı̈ve OSCs (Fig.
3D). Thus, it is likely that Yb is required for localizing theArmi–Piwi complex to Yb bodies, and that this molecular
dynamic is required for the assembly of piR-ILs into thecomplex.
Zuc is required for piRNA maturation
Primary piRNA accumulation in OSCs requires zuc (Saito
et al. 2009). To gain further insight into the function of zucin the processing pathway, OSCs were stained with antiYb and anti-Piwi antibodies under conditions in which

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Zuc was depleted by RNAi (Supplemental Fig. S7A). A
fraction of Piwi now accumulated in the cytoplasm,
particularly at Yb bodies (Fig. 4A). The cellular localization of Yb and Armi was not affected by Zuc depletion(Fig. 4A; Supplemental Fig. S7B). In OSCs where Zuc wasdepleted by RNAi, myc-Piwi was localized exclusively inthe cytoplasm and strongly accumulated at Yb bodies(Fig. 4A). Immunoprecipitation and Western blotting
showed that Zuc was not necessary for Armi to associatewith Piwi and Yb (Fig. 4B); instead, more Piwi wasassociated with Armi when Zuc was depleted. Zuc depletion slightly decreased the amount of piR-ILs within
the Armi–Piwi–Yb complex (;70% compared with thecontrol sample) (Fig. 4C); however, the amount of maturelike piRNAs loaded onto myc-Piwi was much lower(;18%) than that in control cells (Supplemental Fig.
S7C). We considered these as ‘‘mature-like’’ piRNAs
because they were ;1 nt longer than the mature piRNAs(Fig. 4C). piR-ILs in the complex also appeared to beslightly longer than those in control (Fig. 4C). Thus, thezuc gene is most likely a primary piRNA factor that plays
an important role in producing piR-ILs and maturepiRNAs in OSCs.
We showed previously that Zuc is a cytoplasmic protein (Saito et al. 2009). In that study, the N terminus of
Zuc was tagged with a myc-peptide. Zuc was reported
previously to contain a mitochondrial localization signal(MLS) at the N terminus (Choi et al. 2006). Hence, themyc-peptide at the Zuc N terminus probably interferedwith the mitochondrial localization and led to artefactual
results. Thus, we re-examined this localization by tagging
the C terminus with a myc peptide. This revealed that Zuc
is localized in mitochondria (Supplemental Fig. S7D). Yb
bodies have been reported to be usually associated withmitochondria (Szakmary et al. 2009). Therefore, we assessed whether Zuc signals were adjacent to Yb signals.
Coimmunostaining with anti-myc and anti-Yb antibodies

Figure 4. Zuc functions in the primary piRNA processing pathway
in OSCs. (A, top panels) Depletion of Zuc caused Piwi (green) to belocalized to Yb bodies. Yb localization (red) was not affected by Zucdepletion. (Bottom panels) myc-Piwi (red) was expressed by transfection after Zuc was depleted in OSCs by RNAi. (B) Depletion of
Zuc did not affect the association of Armi with Piwi and Yb. Under
such conditions, Piwi was slightly more abundant in the Armi–
Piwi–Yb complex. (C) Depletion of Zuc did not affect piRNAintermediates in the Armi complex.

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Armi and Yb in primary piRNA biogenesis

showed that Zuc localizes in close proximity to Yb bodies(Supplemental Fig. S7E, left). We also showed that Yb
bodies were also adjacent to mitochondria (Supplemental
Fig. S7E, right). These findings further support the ideathat Zuc is a primary piRNA factor in processing piR-ILs
and mature piRNAs in OSCs. However, the exact molecular functions of Armi, Yb, and Zuc in the somatic
primary piRNA production remain to be elucidated.
piR-ILs do not contain a phosphate group at the 59 end
but do contain a 29,39-cyclic phosphate at the 39 end
piR-ILs were undetectable by Northern blotting in which
the RNAs were cross-linked to a nylon membrane by1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)(Supplemental Fig. S8A). After phosphorylation with
ATP and T4 polynucleotide kinase (PNK), piR-ILs became
detectable by the EDC method (Supplemental Fig. S8A;
Pall and Hamilton 2008). As expected, mature piRNAs
were also detectable by this method, even without
external PNK phosphorylation (Supplemental Fig. S8A).
These findings suggest that piR-ILs are free from phosphate groups at both ends. piR-ILs were barely polyadenylated unless they were pretreated with PNK underacidic conditions (Supplemental Fig. S8B). Together, theseresults suggest that piR-ILs contain a 29,39-cyclic phosphate at their 39 end. It has been shown previously thatmature piRNAs loaded onto Piwi contain a phosphategroup at their 59 end. Thus, the piR-ILs that we detectedin this study (Fig. 3C) are apparently not yet in a form that
can associate directly with Piwi, although they did
associate with the Armi–Piwi–Yb complex (Fig. 3D).
Although it remains to be elucidated how piR-ILs areproduced from the primary transcripts of transposons
and others, including the tj gene, such characteristics ofpiR-ILs would help resolve this issue.
Nuclear localization of Piwi is required
for transposon silencing
Piwi is required for the silencing of transposons in gonads(Ghildiyal and Zamore 2009; Malone and Hannon 2009;
Siomi and Siomi 2009). In fact, Piwi depletion in OSCs
caused derepression of transposons, as with Armi, Yb, andZuc depletion (Supplemental Fig. S9A–C). Under conditions where endogenous Piwi was depleted, expression of
myc-Piwi-r, which was designed to be RNAi-insensitive,
rescued transposon silencing (Supplemental Fig. S9D).
However, myc-Piwi-DN (Saito et al. 2009), which lacks 72
amino acids at the N terminus of Piwi and thus does not
localize to the nucleus, did not rescue transposon silencing (Supplemental Fig. S9D), although it does associatewith piRNAs to the same extent as does the wild-type
Piwi (Saito et al. 2009). myc-Piwi-DN13, which lacks 13
amino acids at the N terminus, behaved similarly (datanot shown). On the other hand, myc-Piwi-DDAA-r, a
Slicer mutant of Piwi, could bind to mature piRNAs in
OSCs, as does the wild-type Piwi (Saito et al. 2009), andrescued transposon silencing (Supplemental Fig. S9E).
These results might suggest that Piwi must be localized
in the nucleus to silence the transposable elements, andthat Piwi Slicer activity is unnecessary for its function.
We assume that this system has evolved to prevent
nascent Piwi, not loaded with piRNAs, from being
imported into the nucleus. In other words, only thefunctional Piwi–piRNA complex (piRISC) formed at Yb

bodies could be transported to the nucleus. At present, themechanisms of this control system remain unclear. In the
nongonadal somatic S2 cell line, where the expression ofpiRNAs is undetectable, transfected Piwi is localized to the
nucleus (data not shown), indicating that ‘‘empty’’ Piwi canbe transported to the nucleus. It seems that the machineries necessary for the nuclear transport of Piwi mightrecognize different features of Piwi in different cell types.
How is piRNA-free Piwi restrained in the cytoplasm in
OSCs? One possibility is that some unknown protein
binds the N-terminal end of Piwi, where its NLS (nuclear
localization signal) resides, and interferes with the nuclear import machinery’s ability to recognize Piwi as
a cargo. The nuclear localization inhibitory factors may
be retained on Piwi until a functional Piwi–piRNAcomplex is formed at Yb bodies. Once the complex isformed, a conformational change in Piwi would be induced, which would release the regulatory factors andreveal the Piwi NLS for recognition by the nuclear importmachinery. It would be very interesting to determine theproteins that are associated with Piwi in OSCs underconditions of Armi or Zuc depletion, thus identifying theprotein factors that restrain Piwi in the cytoplasm until it
is loaded with mature piRNAs at Yb bodies.
Materials and methods
Production of the anti-Armi antibody and Western blotting
A 200-amino-acid fragment of the N terminus of Armi fused withglutathione-S-transferase was used as the antigen to immunize mice. An
anti-Armi monoclonal antibody was produced essentially as described
previously (Saito et al. 2006). Western blotting was performed as described
previously (Saito et al. 2006). Anti-tubulin was obtained from the Developmental Studies Hybridoma Bank and was used at 1:1000 dilution.
Immunofluorescence; immunoprecipitation; analysis of piRNAintermediate chemical structures, Drosophila strains, myc-Piwi expression, and RNAi in OSCs; cloning of small RNAs and processing sequencetags; genome mapping, annotation, and frequency mapping; and Northernblotting are described in the Supplemental Material.

Acknowledgments
We are grateful to H. Lin and D. Godt for antibodies, W. Theurkauf(University of Massachusetts) for armi mutant lines, and I. Sagawa formass spectrometry. We thank Y. Ono and T. Yamada for bioinformatics
analyses. We also thank T. Okada and S. Inagaki for their technicalassistance, and other members of the Siomi laboratory for discussions andcomments on the manuscript. This work was supported by MEXT
(Ministry of Education, Culture, Sports, Science, and Technology of Japan)
grants to K.S., H.S., and M.C.S. H.I, Y.K., and M.K.N. are supported by the
JSPS (Japan Society for the Promotion of Science). M.C.S. is supported by
CREST from the JST.

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