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Specific association of Piwi with rasiRNAs derived fromretrotransposon and heterochromatic regions in the Drosophila
genomeKuniaki Saito, Kazumichi M. Nishida, Tomoko Mori, Yoshinori Kawamura, Keita Miyoshi, Tomoko Nagami,
Haruhiko Siomi and Mikiko C. Siomi
Genes & Dev. 2006 20: 2214-2222; originally published online Aug 1, 2006;
Access the most recent version at doi:10.1101/gad.1454806

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Specific association of Piwi with
rasiRNAs derived from retrotransposonand heterochromatic regionsin the Drosophila genome
Kuniaki Saito, Kazumichi M. Nishida, Tomoko Mori, Yoshinori Kawamura, Keita Miyoshi,
Tomoko Nagami, Haruhiko Siomi, and Mikiko C. Siomi1
Institute for Genome Research, University of Tokushima, Tokushima 770-8503, JapanIn Drosophila, Piwi (P-element-induced wimpy testis), which encodes a protein of the Argonaute family, isessential for germ stem cell self-renewal. Piwi has recently been shown to be a nuclear protein involved ingene silencing of retrotransposons and controlling their mobilization in the male germline. However, little is
known about the molecular mechanisms of Piwi-dependent gene silencing. Here we show that endogenous
Piwi immunopurified from ovary specifically associates with small RNAs of 25–29 nucleotides in length.
Piwi-associated small RNAs were identified by cloning and sequencing as repeat-associated small interfering
RNAs (rasiRNAs) derived from repetitive regions, such as retrotransposon and heterochromatic regions, in the
Drosophila genome. Northern blot analyses revealed that in vivo Piwi does not associate with microRNAs(miRNAs) and that guide siRNA was not loaded onto Piwi when siRNA duplex was added to ovary lysate. In
vitro, recombinant Piwi exhibits target RNA cleavage activity. These data together imply that Piwi functions
in nuclear RNA silencing as Slicer by associating specifically with rasiRNAs originating from repetitive
targets.
[Keywords: Piwi; retrotransposon; rasiRNA; Slicer; Drosophila]
Supplemental material is available at http://www.genesdev.org.
Received June 2, 2006; revised version accepted June 26, 2006.

RNA interference (RNAi) is a gene silencing pathway
triggered by double-stranded RNA (dsRNA) (Tomari andZamore 2005). dsRNA is processed by Dicer to short interfering RNA (siRNA) (21–23 nucleotides [nt] in length),
unwound, and loaded onto the RNA-induced silencing
complex (RISC) as a single-stranded guide siRNA (Hammond 2005). RISC then binds the target RNA and cleaves
it at sites completely complementary to the siRNA(Hutvagner 2005). microRNAs (miRNAs) are a large
group of small noncoding RNAs (21–22 nt in length)
functioning in gene silencing by repressing translation of
the target mRNA (Ambros 2004; Du and Zamore 2005).
The expression of miRNA is developmentally regulated,
implying an important role for miRNAs in regulating
gene silencing through development (Ambros 2004; Du
and Zamore 2005). Members of the Argonaute family,
defined by the presence of the PAZ and PIWI domains,play essential roles in gene silencing triggered by small
RNAs such as siRNA and miRNA (Carmell et al. 2002).
1

Corresponding author.
E-MAIL siomim@genome.tokushima-u.ac.jp; FAX 81-88-6339451.
Article published online ahead of print. Article and publication date areonline at http://www.genesdev.org/cgi/doi/10.1101/gad.1454806.

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In Drosophila, there are five genes encoding membersof the Argonaute family, AGO1, AGO2, AGO3, Aubergine, and Piwi (P-element-induced wimpy testis). Amongthese, AGO1 and AGO2, members of the Argonaute subfamily, are expressed ubiquitously through development, whereas the other three, members of the Piwi subfamily, are expressed only in germline cells (Williams
and Rubin 2002). AGO1 and AGO2 in Drosophila function in gene silencing through specific binding with
miRNA and siRNA, respectively (Okamura et al. 2004).
miRNA and siRNA are derived from different sources ofprecursors; miRNA is produced from a hairpin-shaped
precursor (pre-miRNA) by Dicer1/Loquacious (R3D1)(Forstemann et al. 2005; Jiang et al. 2005; Saito et al.
2005), whereas siRNA is produced from long dsRNA by
Dicer2/R2D2 (Liu et al. 2003). siRNA-loaded AGO2
functions in RNAi as a factor directly responsible forcleaving a target completely complementary to siRNA(Miyoshi et al. 2005). AGO2 has recently been shown toalso be involved in RISC formation in RNAi by slicing
the passenger strand of the siRNA duplex (Matranga et
al. 2005; Miyoshi et al. 2005; Leuschner et al. 2006).
miRNA-associated AGO1 is thought to repress translation of mRNA targets without cleaving them; however,

GENES & DEVELOPMENT 20:2214–2222 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org

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Piwi association with rasiRNA in flies

AGO1 also possesses target RNA cleavage (Slicer) activity, as does AGO2, and is capable of cleaving target
RNAs when their sequences show complete complementarity to miRNA (Okamura et al. 2004; Miyoshi etal. 2005).
Aubergine and Piwi, two of the Piwi subfamily members in flies, were shown to play important roles in germline cell formation. Aubergine is required for pole cell
formation (Harris and Macdonald 2001) and for activating RNAi during Drosophila oocyte maturation (Kennerdell et al. 2002). It has also been shown that Aubergine isinvolved in silencing retrotransposons in the female germline (Vagin et al. 2004) and Stellate genes by the homologous Suppressors of Stellate [Su(Ste)] repeats that
produce small RNAs in testis (Aravin et al. 2004). Piwi isessential in germline stem cell (GSC) self-renewal (Coxet al. 1998, 2000; Szakmary et al. 2005). It has been proposed that niche Piwi maintains GSCs by repressing bagof-marbles expression in GSCs, and the germline Piwi
functions by promoting germ cell division (Szakmary et
al. 2005). One of the piwi mutations, piwi1, causes malesterility due to severe defects in spermatogenesis. piwi1
ovarioles are mostly devoid of germline cells (Lin and
Spradling 1997). Piwi has also recently been shown to be
involved in silencing LTR retrotransposons such as
copia and mdg1 in testis (Kalmykova et al. 2005). Transposition burst of mdg1 was also observed in piwi mutants. However, little is known at the molecular levels
about how Piwi is involved in gene silencing and in GSC
self-renewal.
In this study, we investigated the molecular functionof Piwi in gene silencing. We found that the Piwi complex immunopurified from ovary contained a class of
small RNAs, distinct in their size from miRNAs andsiRNAs. Through cloning and sequencing the Piwi-associated small RNAs, we identified them as repeat-associated siRNAs (rasiRNAs) derived from various kinds ofrepetitive elements in the genome such as the retrotransposon and heterochromatin regions. rasiRNAs are a subset of small RNAs and were originally found in a small
RNA profiling study in fly testis and embryo (Aravin etal. 2003). Since rasiRNAs are cognate to transposable
elements and other repetitive elements in the fly genome, it is suggested that they participate in defining
chromatin structures and regulating transposon activities. We noticed that the Piwi-associated rasiRNAs areoften found in both sense and antisense orientations andthat the 5⬘ ends of the rasiRNAs are predominantly uridine, suggesting that they are processed by an RNase III
domain enzyme. Specificity of Piwi association with rasiRNAs was assessed by Northern blot analyses, whichshowed that Piwi does not associate with miRNAs invivo and that guide siRNA was not loaded onto Piwi
when siRNA duplex was added to ovary lysate. We foundthat Piwi does exhibit Slicer activity, as do AGO1 andAGO2; however, Piwi localizes in the nucleus while
AGO1 and AGO2 are found in the cytoplasm. Taken
together, the results imply that Piwi functions in nuclear
RNA silencing as Slicer by associating specifically with
rasiRNAs that originate from the repetitive targets.

Results
Piwi expression in ovary, testis, and embryo
To investigate the molecular function of Piwi in genesilencing, we produced monoclonal antibodies against
the protein. As an antigen, we used the N-terminal region of Piwi that shows much less similarity to the othermembers of the fly Argonaute family. Western blotting
using the Piwi-specific antibody we raised revealed that
Piwi is strongly expressed in the ovary and in the early
embryo, but later in development the protein levels in
the embryo decrease (Fig. 1A). Piwi was not detected in
Schneider 2 (S2) cell lysate (Fig. 1A). The specificity of
the anti-Piwi antibody is shown in Supplementary Fig.
S1. To examine the expression pattern of Piwi in fly testis, ovary, and embryo, immunofluorescent staining was
carried out (Fig. 1B). In ovarioles, Piwi was found to beclearly accumulated in nuclei of both GSCs and somatic
cells such as terminal filament cells (TFCs), cap cells,
and follicle cells (Fig. 1B, panels II,III, IV). It has previously been shown that piwi mRNA is detected in TFCs
(Cox et al. 1998), and that myc-tagged Piwi protein isfound in both somatic and germline cells in adult ovary(Cox et al. 2000). Relatively low expression was observedin cystoblasts, as in the case of myc-Piwi (Cox et al.
2000; Szakmary et al. 2005). In embryos, Piwi was foundaccumulated in nuclei of pole cells (Supplementary Fig.
S1). In testis, Piwi appeared to be expressed in the hub, atiny cluster of post-mitotic somatic cells localized at theapical tip of the testis (Fig. 1B, panels V,VI; panel VII
shows the location of the hub stained with anti-Drosophila E [DE]-cadherin) and in cyst progenitor cells. The
hub functions in the maintenance of GSC identity andinfluences its behavior (Ohlstein et al. 2004), while Piwi
is required for the asymmetric division of GSCs to produce and maintain a daughter GSC but is not essential
for further differentiation of committed daughter cells(Cox et al. 1998). We found that Piwi seems not to be
expressed in male germline stem cells (GSCs) (Fig. 1B,
panels V,VI). This observation agrees well with the Western data showing that the relative amount of Piwi intestis is quite low (data not shown).

Piwi in ovary associates with small RNAs distinct
from miRNAs
We next performed immunoprecipitation from ovary lysate using the anti-Piwi antibody. Silver staining of theimmunoprecipitate failed to reveal any prominent bands
copurifying with Piwi under physiological conditions(Fig. 2A), although some weak bands were perceived.
Western blotting confirmed that the discrete band of ∼92
kDa was Piwi itself (Fig. 2B). We then investigated if
immunoprecipitated Piwi associates with any small
RNAs. Total RNAs were isolated from the immunopurified Piwi complex, labeled with 32P-pCp, and separated
on a denaturing acrylamide gel. As a control, the AGO1
complex—an essential factor in miRNA-mediated genesilencing—was also immunopurified from the ovary, andGENES & DEVELOPMENT

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Saito et al.

Figure 1. (A) Piwi expression is detected
in Drosophila ovaries and embryos, but
not in S2 cells. Western blotting was performed on lysates of fly embryo (at earlyand late stages), ovary, and S2 cells using
an antibody against Piwi. Anti-tubulin
was utilized as a loading control. (B) Piwi
expression pattern in ovary and testis. All
the images shown in this figure represent
one confocal section. (Panels II,III,IV) The
images of ovariole staining show that Piwi(red) is found clearly to be accumulated innuclei of both GSCs and somatic cells
such as TFCs, cap cells (CpC), and follicle
cells (FC). Panel I shows the DIC image ofpanel II. (Panel V–VIII) In testis, Piwi (red)is expressed in the hub (shown in green;stained with anti-DE-cadherin) at the apical tip of the testis and in the cyst progenitor cells (CC). Male GSCs are not stainedwith anti-Piwi. Panel VI shows the enlarged image of a part of panel V. Panel VIII
shows a merged image of panels VI andVII.

miRNAs associated with AGO1 were simultaneously visualized. The Piwi-associated small RNAs migrated
slightly slower than miRNAs (Fig. 2C). Compared withthe size markers, Piwi-associated small RNAs were estimated to be 25–29 nt in length. Northern blot analysis
using probes specific for miRNAs (miR-1 and miR-310)
clearly revealed that the Piwi-associated small RNAs are
not miRNAs (Fig. 2D).

Identification of small RNAs associated with Piwi
in ovary
To identify the Piwi-associated small RNAs, we cloned
and sequenced them. Of 800 clones sequenced, 392 hit to
the Drosophila genomic sequences in databases. (Database searches were carried out by BLAST search using

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the databases at the National Center for Biotechnology
Information [NCBI] and at FlyBase.) The compositions of
the small RNAs obtained are listed in Table 1. The largest class of cloned RNAs (84.1%) was rasiRNAs, originally found in a small RNA profiling study in Drosophilamelanogaster embryo and testis (Aravin et al. 2003).
Breakdown products of rRNA, tRNA, mRNA, andsnoRNA were also obtained (6.6%, 0.2%, 4.8%, and
4.1%, respectively). It is noteworthy that no miRNAs
were obtained in this experiment. Among the rasiRNAs(330 clones), ∼40% (130 out of 330) matched to various
kinds of transposable elements, including LTR (long terminal inverted repeat) retrotransposons and LINE (longinterspersed nuclear elements)-like elements (Table 2).
Those LINE-like elements include Het-A and TART,
both known to transpose only to the chromosome ends
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Piwi association with rasiRNA in flies

Figure 2. (A) Protein components of the Piwi complex
immunopurified from ovary lysate are visualized by silver staining. A strong discrete band appearing at ∼92
kDa on the gel is Piwi. h.c. shows where the heavy
chain of the antibodies used migrated on the gel, andn.i. indicates nonimmune antibody used as a negative
control. (B) Western blotting with anti-Piwi on the immunoprecipitates shown in A confirms that the 92-kDa
band is indeed Piwi. (C) Visualization of small RNAs
associated with Piwi in ovary. RNA molecules wereisolated from the Piwi immunopurified complex shown
in A, labeled with 32P-pCp, and run on a 7 M ureacontaining denaturing acrylamide gel. RNAs associatedwith AGO1 in ovary involve molecules ∼21–23 nt inlength (blue lines), which from their size are expected tobe miRNAs. Piwi-associated small RNAs (red lines) migrated slightly slower than miRNAs. The size was estimated at ∼25–29 nt in length. (n.i.) Nonimmune antibody used as a negative control. (D) Northern blot
analyses using specific probes for miR-1 and miR-310
show that Piwi-associated small RNAs do not include
the miRNAs.

1995). Another 60% (200 out of 330) corresponded toheterochromatic regions in the genome (Table 2). By
looking at the nucleotide sequences of all the clones(Supplementary Table S1), we noticed that the Piwi-associated rasiRNAs include both the sense and the antisense of a transposable transcript, although sense (+) orientation clones tended to be less abundant than thosewith an antisense (−) orientation (Table 2; Fig. 3A), andthat the first nucleotide at the 5⬘ end was predominantly
U (∼78%) (Fig. 3B). In addition, the Piwi-associated rasiRNAs were cloned by the procedure originally developed for miRNA cloning (Maniataki et al. 2005), indicating that they contain 5⬘ phosphates and 2⬘,3⬘-hydroxyl
termini. These characteristics suggested that the Piwiassociated rasiRNAs are produced from dsRNA precursors by an RNase III domain-containing enzyme. In somecases, such as stalker4, rasiRNAs are only found in anantisense (−) orientation (Table 2; Fig. 3A). However,where the orientation bias of rasiRNA loading onto Piwi
comes from is unknown and requires further investigation.
Specific association of Piwi with rasiRNAs
To confirm that rasiRNAs associated with Piwi indeed
involve both the sense and antisense of a transcript,
Northern blotting analysis was performed. roo rasiRNAwas chosen for examination because it was one of mostabundant clones obtained (11 out of 300) and in bothsense and antisense orientations (Table 2). Both sense
and antisense roo rasiRNAs were detected in the immunopurified Piwi complex as expected (Fig. 4A). Neither was found in the AGO1 complex, indicating that
the Piwi association with rasiRNAs is specific (Fig. 4A).
roo rasiRNA that is derived from the roo antisense transcript (hereinafter, we refer to it as roo anti-rasiRNA)seems more abundant in the Piwi complex (Fig. 4A) than
Table 1. Compostitions of RNAs found in the
Piwi-associated small RNA identification experiment
Type
rRNA
tRNA
mRNA
snoRNA
rasiRNA
Total

Clone number

Percent

26
1
19
16
330
392

6.6
0.2
4.8
4.1
84.1
100

The largest class of cloned RNAs (84.1%) was rasiRNAs, originally found in the small RNA profiling study in D. melanogaster embryo and testis (Aravin et al. 2003). Breakdown productsof rRNA, mRNA, and snoRNA were also obtained (6.6%, 4.8%,
and 4.1%, respectively). It is noteworthy that no miRNAs wereobtained in this experiment.

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Saito et al.

Table 2. Identification of rasiRNAs associated with Piwi inovary
Repeat

Name

Total

+

−

LINE-likeBS
F-element
G-element
HeT-A-element
TART-element
TAHRE
X-element
R1Dm
R2Dm
pilger
strider
Total (LINE)
1731
412
blood
gypsy
springer
HMS-Beagle
Max-element
mdg1
mdg3
opus
roo
Stalkerninja
297
cruisercirce
BEL
TirantMicropia-Dm11
rover
copia
Total (LTR)
HoppelBari-1
Subtotal

2
1
2
5
3
1
2
8
2
5
1
32
4
9
3
4
7
8
4
1
2
4
11
17
2
5
3
3
3
1
1
3
1
96
1
1
130

0
0
0
3
0
0
1
4
2
3
0
13
4
4
2
0
1
2
2
0
1
2
1
0
0
1
1
2
1
1
0
1
0
26
1
1
41

2
1
2
2
3
1
1
4
0
2
1
19
0
5
1
4
6
6
2
1
1
2
10
17
2
4
2
1
2
0
1
2
1
70
0
0
89

LTR

TIR

Heterochromatic repeats
Repeat on chr. 2L
41DE region on chr. 2R
42AB region on chr. 2R2h
3h
Responder
Repeat on chr. 3L
Chr.4
Repeat on chr. X
Unspecified
Subtotal
Total clones

1
1
24
2
12
1
7
1
13
138
200
330

(+) Same orientation with the ORF of the LINE-like and LTR
retrotransponsons.

the roo rasiRNA that was derived from the roo transcriptin the sense orientation. This was determined from thefact that the signal of roo anti-rasiRNA in the anti-Piwi
lane is about threefold stronger than that of roo antioligo (10 fmol) (Fig. 4A, left panel), whereas the signal ofroo rasiRNA is a little less (∼85%) compared with that of

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Figure 3. Identification of small RNAs associated with Piwi inovary. (A) The regions where roo, 412, and stalker4 rasiRNAs
are identified as Piwi-associated rasiRNAs corresponding to thetranscripts are shown by arrowheads. rasiRNAs in sense andantisense orientations are indicated in white and black, respectively. (B) The first nucleotide of the rasiRNA associated with
Piwi is predominantly U (1st). This tendency was not found tobe obvious in the second and last nucleotides of the rasiRNAs.

roo sense oligo (10 fmol) (Fig. 4A, right panel). This observation correlated well with the fact that we obtained
less roo rasiRNA than roo anti-rasiRNA in the cloning
experiment (Table 2). The Piwi–rasiRNA association
was further verified by Northern blotting on total RNAs
isolated from Piwi and AGO1 immunopurified complexes in ovary. rasiRNAs originating from copia and
TART retrotransposon transcripts, and one of the repetitive elements located in the centromeric heterochromatin region, Responder (Supplementary Table S1; Cabot etal. 1993), were detected only in Piwi but not in AGO1
complexes (Fig. 4B), demonstrating that the association
with rasiRNAs is specific to Piwi. Whether Piwi wasable to associate with siRNA was also investigated. luc
siRNA duplex was first incubated in ovary lysate. Piwi
and AGO2 complexes were then immunopurified fromthe lysate using specific antibodies to each protein.
Northern blotting revealed that guide siRNA was specifically loaded onto AGO2 but not onto Piwi (Fig. 4C).
These data, together with those shown in Figure 2D,
strongly indicate the high specificity of Piwi association
with rasiRNAs. It is suggested that a complex for loading
rasiRNAs specifically onto Piwi exists; as the Dicer1/
Loquacious (R3D1) and Dicer2/R2D2 complexes load, after processing, mature miRNA and guide siRNA onto
AGO1 and AGO2, respectively.

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Piwi association with rasiRNA in flies

cleave target RNA completely complementary tomiRNA (Miyoshi et al. 2005). In this study, we produced
full-length GST-Piwi and examined the activity in an invitro target RNA cleavage assay. Interestingly, we noticed that in the alignment of the Piwi domains of Piwi,
AGO1, and AGO2, the D–D–H (Asp–Asp–His) motif,
shown to be essential for Slicer activity of human Ago2
(Rivas et al. 2005), was not well conserved, and that thethird residue was replaced with Lys (Supplementary Fig.
S2). Thus, we suspected that Piwi may not exhibit Slicer
activity. However, we observed that GST-Piwi was able
to cleave luc target RNA when it was preincubated withluc guide siRNA (21 nt; luc 21) as well as GST-AGO1(Fig. 5A). In this assay, it should be noted that we usedsingle-stranded luc guide siRNA, in contrast to thesiRNA duplex used in Figure 4C. GST-Piwi bound withanother single-stranded siRNA, luc 30, which contains
an additional 9 nt at the 3⬘ end of luc 21, also cleaved theluc target (data not shown). By searching for peptide sequence similarity over species, we noticed that the
D–D–K triad is conserved in a member of the Argonautes(EAL29401) in Drosophila pseudoobscura and one of thePiwi domain-containing proteins (EAR93067) in Tetrahymena thermophila.

Figure 4. Specificities of the Piwi–rasiRNA association. (A)RNAs associated with Piwi in ovary were isolated and probedwith sense and antisense roo transcripts internally labeled with
32
P in vitro. The left panel shows that Piwi associates with roo
anti-rasiRNAs originating from roo antisense transcript. The
right panel shows the Piwi association with roo rasiRNAs thatis derived from the roo transcript in sense orientation. roo sense
and antisense DNA oligos (10 fmol each) were also loaded ongels to show the probe specificities. roo anti-rasiRNA signal in
the anti-Piwi lane was several-fold stronger than that of roo
antisense oligo (left panel), whereas roo rasiRNA signal in theanti-Piwi lane (right) is a little less (∼85%) compared with thatof the roo sense oligo, indicating that Piwi likely associates
preferentially with roo anti-rasiRNA. Note that the AGO1 immunopurified complex (both panels) does not contain roo
rasiRNAs, showing the specificity of the Piwi–rasiRNA association. (B) Blots containing RNAs associated with Piwi and
AGO1 in ovary were probed with copia and TART transcripts
and one of repetitive elements, Responder. Small RNAs corresponding to copia and TART rasiRNAs are detected only inthe anti-Piwi lanes, indicating that the association with bothrasiRNAs is specific for Piwi. (C) siRNA preprogrammed inovary lysate was loaded onto AGO2, but not onto Piwi. Ovarylysate was prepared first and luc siRNA duplex was added to thelysate. After incubation, AGO2 and Piwi were immunopurified
from the mixture using the specific antibodies. RNA molecules
were then isolated from the complexes and probed with luc
siRNA passenger strand to visualize the luc guide siRNA. In theanti-AGO2 lane, the luc guide siRNA strongly appeared, but notin the anti-Piwi and n.i. (nonimmune) lanes, indicating that luc
guide siRNA was specifically loaded onto AGO2.

Piwi exhibits Slicer activity
Previously, using GST-tagged recombinant full-length
AGO1 (GST-AGO1), we showed that AGO1 is able to

Discussion
For several years, it has been thought that Piwi is involved in gene silencing of LTR retrotransposons since(1) Piwi is one of the members of the fly Argonaute family of proteins, and (2) piwi mutations cause higher expression levels of retrotransposon transcripts in vivo.
Links between protein factors involved in RNAi and the

Figure 5. Target RNA cleavage assay with GST-Piwi and GSTAGO1. Recombinant full-length Piwi and AGO1 fused to GST
were produced in and purified from Escherichia coli. GST wasused as a control. As guide siRNA, luc 21 (luc guide siRNA, 21
nt; Miyoshi et al. 2005) and bantam miRNA (21 nt; Miyoshi etal. 2005) were used. GST fusion proteins were first incubatedwith guide siRNA, and then the luc target labeled at the 5⬘ end(Miyoshi et al. 2005) was added to the reaction mixture. Afterincubation, resultant RNAs were loaded on a denaturing acrylamide gel. The cleaved product was observed not only with
GST-AGO1, but also with GST-Piwi. GST-AGO1 bound withbantam did not cleave the target, as expected.

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Saito et al.

silencing of endogenous transposable elements have also
been made in other species like Caenorhabditis elegans(Ketting et al. 1999; Tabara et al. 1999) and Chlamydomonas reinhardtii (Wu-Scharf et al. 2000). However, evidence at molecular levels to support such a Piwi function has not hitherto been reported. This study is thefirst to show that Piwi is specifically associated with
rasiRNAs in vivo and that Piwi has Slicer activity.
Our data suggest a novel, third kind of gene silencing
pathway in Drosophila, following two distinct gene silencing pathways mediated by AGO1-miRNAs and
AGO2-siRNAs (Okamura et al. 2004). The existence ofrasiRNAs in Drosophila has been demonstrated previously through investigation of small RNA expression
profiling in testis and embryo (Aravin et al. 2003). In thatstudy, it was mentioned that rasiRNAs show somecharacteristics that suggest involvement of an RNase III
domain enzyme in rasiRNA processing (Aravin et al.
2003). In this study, we observed that Piwi-associatedrasiRNAs also show the same characteristics as thosefound in the exhaustive study of Aravin et al. (2003).
Which protein factor(s) then produces rasiRNAs? Dicer1,
Dicer2, and Drosha were not detected in Piwi complex
immunopurified from ovary (data not shown). Although
it may be due to the epitope hindering by Piwi and processing factor association, it is possible that a yet-to-beunidentified protein(s) other than Dicer1, Dicer2, andDrosha might be the crutial factor(s) for the rasiRNAproducing process.
We found that in fly testis, Piwi seems not to be expressed in GSCs, but its expression was clearly observedin the hub, the somatic cell cluster that functions tomaintain GSC identity and influence its behavior. It is
known that piwi1 causes male infertility due to severe
defects in spermatogenesis; thus, the necessity of thePiwi function in the hub for such processes is quite apparent. Although it also will be important to determine
if Piwi is associated with rasiRNAs in the testis, ourfindings have set a new stage for understanding how Piwi
functions in the formation and maturation of GSCs inboth ovary and testis.
Materials and methods
Western blot analysis
Two hundred amino acids at the N terminus of Piwi fused with
GST were used as the antigen to immunize mice. Anti-Piwi
monoclonal antibodies were produced essentially as described
previously (Ishizuka et al. 2002). Western blotting was performed as described previously (Miyoshi et al. 2005). Ten micrograms of proteins from each sample were loaded on gels (Fig.
1A). Anti-tubulin was from the Developmental Studies Hybridoma Bank (1:1000 dilution).
Immunofluorescence
Testes and ovaries were dissected manually from adult flies in
1× PBS. Embryos were collected and dechorionated. Immunostainings were performed under standard procedures. Culture
supernatants of anti-Piwi hybridoma cells were used without
dilution. A rat monoclonal antibody to DE-cadherin (DCAD2)

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GENES & DEVELOPMENT(1:20 dilution) was a gift from S. Hayashi (Kobe-RIKEN CDB).
Alexa-488 anti-mouse and FITC-conjugated anti-rat IgGs (Jackson ImmunoResearch) were used as the secondary antibodies.
All images were collected using a Zeiss LSM510 laser-scanningmicroscope.
Immunoprecipitation from ovary lysate
Immunoprecipitation was performed essentially as described
previously (Miyoshi et al. 2005). Immunoprecipitation buffer
contained 30 mM HEPES-KOH (pH 7.3), 150 mM KOAc, 2 mM
MgOAc, 5 mM DTT, 0.1% NP40, 2 µg/mL Pepstatin, 2 µg/mL
Leupeptin, and 0.5% Aprotinin. About 300 ovaries were used
per immunoprecipitation. Silver staining of the protein components in the immunoprecipitates was performed using SilverQuest (Invitrogen).
Visualization of small RNAs associated with Piwi and AGO1
After immunoprecipitation, total RNAs were isolated from theimmunopurified complexes with phenol:chloroform and precipitated with ethanol. RNAs were labeled with 32P-pCp with
T4 RNA ligase (TaKaRa) in a cold room overnight. After thereaction, RNAs were extracted with phenol:chloroform and precipitated with ethanol. RNAs labeled with pCp were loaded on
a 7 M urea-containing denaturing 12% polyacrylamide gel andrun. Signals were visualized using BAS-2500 (Fuji).
Small RNA cloning and sequence analysis
Small RNA cloning was carried out essentially as described(Maniataki et al. 2005). The sequences of the adapters were 3⬘
adapter, 5⬘-pUUUaaccgcatccttctctmdT-3⬘ and 5⬘ adapter, 5⬘-tactaatacgactcactAAA-3⬘ (p, phosphate; capital letters, RNA; small
letters, DNA; mdT, 3⬘ amino-modified deoxythymidine). Firststrand cDNA synthesis was performed with Stratascript RT and
RT primer (5⬘-ACTAGCTGGAATTCAAGGATGCGGTTAA
AG-3⬘). For PCR, an RT primer and PCR primer (5⬘-CAGCCAACGGAATTCATACGACTCACTAAA-3⬘) were used after 5⬘end phosphorylation with T4 polynucleotide kinase (TAKARA).
KOD plus (TOYOBO) was used as a polymerase. PCR products
were cloned into the EcoRV site of pBS SK+ vector followed bysequencing. RNA sequences between 5⬘ and 3⬘ adapters wereanalyzed by performing searches in annotated genomic databases (NCBI [http://www.ncbi.nlm.nih.gov/blast] and FlyBase[http://flybase.bio.indiana.edu/blast]) to determine whether thecloned RNAs mapped to the genome. To avoid confusion, weonly accepted sequences that perfectly matched the databases
and categorized sequences that did not match to the D. melanogaster genome as “others.”
Northern blot analysis
Total RNA was isolated from 50 ovaries from flies using Isogen(Invitrogen) according to the manufacturer’s instructions. Fivemicrograms of total RNA were resolved by electrophoresis.
Northern blotting for miRNA (miR-1 and miR-310) and luc
siRNA was performed as reported previously (Miyoshi et al.
2005; Saito et al. 2005). The following probes were used for
detection: 5⬘-CTCCATACTTCTTTACATTCCA-3⬘ for miR-1,
and 5⬘-AAAGGCCGGGAAGTGTGCAATA-3⬘ for miR-310.
Gene fragments for detecting rasiRNAs were cloned into pBS
SK+ vector using primers 5⬘-TTTGGTCAATCGAGCCTTTAATCGACTC-3⬘ and 5⬘-CACAATTGCTGATGTATGGCCTCA
AG-3⬘ corresponding to GenBank sequence AY180917 for roo;
5⬘-AAGCAAGAAAGTTATGAACGCG-3⬘ and 5⬘-CCACAGA

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Piwi association with rasiRNA in flies

CATCTGAGTGTAC-3⬘, corresponding to GenBank sequence
X04456 for copia; 5⬘-CGCCTGTGCAGCAGCTATTTAA-3⬘
and 5⬘-CAAATAAAGGATCTGGCAATATGGTAT-3⬘, corresponding to GenBank sequence AY600955 for TART; 5⬘-TCTG
GAGATTCTGATCAACTGG-3⬘ and 5⬘-AGGTGTCTTCTTT
TCGCCTGG-3⬘ corresponding to GenBank sequence M21213
for the Responder element. PCR products carrying T7 or T3
promoters containing the cloned fragments of retrotransposon
or Responder sequences were used as transcription templates.
RNA probes were synthesized in vitro using MaxiScript T7 andT3 kits (Ambion) in the presence of 32P-UTP. The transcribed
RNAs were extracted with phenol:chloroform and precipitatedwith ethanol, then partially hydrolyzed by incubating at 60°C in
a solution containing 40 mM NaHCO3 and 60 mM Na2CO3.
Hybridization and washing were performed as indicated previously (Saito et al. 2005). To test the specificity for roo rasiRNA
hybridization, 10 fmol of roo sense (5⬘-TCCTTTAAGCATCTTACAGCTAAAGG-3⬘) and antisense (5⬘-TTTAGCTGTAAGATGCTTAAAGGAGCT-3⬘) DNA oligos were loaded on gels.
siRNA loading assay
Three hundred ovaries were homogenized in a hypotonic buffer(30 mM HEPES at pH 7.4, 2 mM MgOAc, 5 mM DTT, and 1
mg/mL Pefablock SC) to prepare ovary lysate. luc siRNA duplex(Miyoshi et al. 2005) was incubated in the lysate for 1 h at 26°C.
Piwi and AGO2 complexes were then immunopurified from the
mixture using anti-AGO2 and anti-PIWI antibodies. Sodium
chloride was added to the lysates to 800 mM just before immunoprecipitation was begun. Reaction mixtures were rocked for 1
h at 4°C and the beads washed extensively with a washingbuffer (30 mM HEPES at pH 7.4, 800 mM NaCl, 2 mM MgOAc,
5 mM DTT, and 1 mg/mL Pefablock SC). RNAs were isolated
from the immunoprecipitates and separated on a denaturing
polyacrylamide gel. Northern blotting was performed for detecting luc guide siRNA as described previously (Miyoshi et al.
2005).
Production of recombinant proteins and target RNA
cleavage assay
To obtain cDNA encoding PIWI, poly(A)+ RNAs were purified
from ovaries and RT–PCR was carried out using a ProSTAR
Ultra-HF RT–PCR system (Stratagene). To produce a GST-PIWI
fusion protein, full-length PIWI cDNA was subcloned into a
pGEX-5X-1 expression vector (GE Healthcare Bioscience). GSTtagged protein was expressed in BL21 (DE3) and purified according to the manufacturer’s instructions (GE Healthcare Bioscience). Preparation of cap-labeled luc target RNA (luc180) andthe cleavage assay were carried out essentially as described (Miyoshi et al. 2005). In brief, 1 µg of GST fusion proteins waspreincubated with luc siRNA 21mer (5⬘-UCGAAGUAUUCCGCGUACGUG-3⬘) in a cleavage reaction buffer (25 mM HEPES
at pH 7.4, 50 mM KOAc, 5 mM MgOAc, 5 mM DTT, 0.1 U/µL
RNasin and 0.1 µg/µL yeast RNA) for 1.5 h at 26°C. Then 5000
cpm of luc target RNA (luc180) was added and incubated foranother 1.5 h at 26°C. The cleavage products of the reactions
were analyzed by electrophoresis on 6% denaturing polyacrylamide gels.
Accession numbers
The GenBank accession numbers for the transposons and chromosome sequences used in small RNA mapping and orientation
determination are as follows: BS(X77571), F-element (AY183919),
G-element (X06950), HeT-A (AJ635224 and U06920), TART(U14101 and AY600955), TAHRE (AJ542581), X-element(AF237761), R1Dm (X51968), R2Dm (X51967), pilger(AJ278684), strider (AF456125), 1731 (X07656), 412 (X04132),
blood (AY180916), gypsy (M12927 and Z31368), springer(AF364549), H. M. S. Beagle (AF365402 and AY183918), Maxelement (AJ487856), mdg1 (X59545), mdg3 (X95908), opus
(AY180918), roo (AY180917), stalker (AF420242), ninja(AF420242), 297 (X03431), cruiser (AF364550), circe (AJ132547
and X98424), BEL (U23420), Tirant (AY928610), MicropiaDm11 (X14037), rover (AF492764), Hoppel (AF533772), copia(X04456).

Acknowledgments
We thank members of the Siomi laboratory for discussions andcomments on the manuscript. We also thank S. Hayashi for
anti-DE-cadherin, and T. Suzuki and H. Kose for discussions
and technical advice. K.S. is a postdoctoral fellow of the 21st
century COE Program from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (MEXT), and K.M is a
research fellow supported by the Japan Society for the Promotion of Science (JSPS). This work was supported in part by grants
to M.C.S and H.S from MEXT and the New Energy and Industrial Technology Development Organization (NEDO).

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