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Gene silencing mechanisms mediated by Aubergine–piRNAcomplexes in Drosophila male gonad
KAZUMICHI M. NISHIDA, KUNIAKI SAITO, TOMOKO MORI, YOSHINORI KAWAMURA,TOMOKO NAGAMI-OKADA, SACHI INAGAKI, HARUHIKO SIOMI, and MIKIKO C. SIOMI
Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan

ABSTRACT
Genetic studies have shown that Aubergine (Aub), one of the Piwi subfamily of Argonautes in Drosophila, is essential for germ
cell formation and maintaining fertility. aub mutations lead to the accumulation of retrotransposons in ovaries and testes, andStellate transcripts in testes. Aub in ovaries associates with a variety of Piwi-interacting RNAs (piRNAs) derived from repetitive
intergenic elements including retrotransposons. Here we found that Aub in testes also associates with various kinds of piRNAs.
Although in ovaries Aub-associated piRNA populations are quite diverse, piRNAs with Aub in testes show a strong bias. The
most abundant piRNAs were those corresponding to antisense transcripts of Suppressor of Stellate [Su(Ste)] genes known to be
involved in Stellate gene silencing. The second most abundant class was made up of those from chromosome X and showedstrong complementarity to vasa transcripts. Immunopurified Aub–piRNA complexes from testes displayed activity in cleaving
target RNA containing sequences complementary to Stellate and vasa transcripts. These results provide the first biochemical
insights into gene silencing mechanisms mediated by Aub and piRNAs in fly testes.
Keywords: Aubergine; piRNA; Stellate; RNA silencing; germlines

INTRODUCTION
RNA silencing is an evolutionarily conserved physiologicalprocess involved in regulating gene expression, defending
against viruses, and maintaining genome stability (Tomari
and Zamore 2005; Zaratiegui et al. 2007). RNA interference(RNAi) (Fire et al. 1998) is the most typical of such processes and causes gene silencing by cleaving (slicing) thetarget mRNAs in a sequence-dependent manner (Tomari
and Zamore 2005). A molecule rendering sequence specificity is a 21–23 nucleotide (nt) long RNA, termed shortinterfering RNA (siRNA) (Tomari and Zamore 2005).
microRNAs (miRNAs) are a large group of endogenous
small RNAs (21–23 nt in length) encoded in the genome ofa variety of organisms (Ambros 2004; Du and Zamore
2005; Kloosterman and Plasterk 2006). miRNA also functions in RNA silencing, as does siRNA, a process throughwhich the expression levels of genes involved in various
developmental and metabolic processes are modulated.
Besides their functional commonality, siRNA and miRNA
Reprint requests to: Mikiko C. Siomi, Institute for Genome Research,
University of Tokushima, Tokushima 770-8503, Japan; e-mail: siomim@
genome.tokushima-u.ac.jp; fax: 81-88-6339451.
Article published online ahead of print. Article and publication date areat http://www.rnajournal.org/cgi/doi/10.1261/rna.744307.

show strong similarities even in maturation events. Bothare processed from a longer precursor by an RNase III
enzyme (Dicer), unwound, and then loaded onto a memberof the Argonaute family of proteins (Carmell et al. 2002;
Parker and Barford 2006). After maturation and association
with Argonautes, they ultimately exert their effects in RNA
silencing as guide molecules to specify the RNA targets tobe silenced.
Members of the Argonaute family are defined by thepresence of the PAZ and PIWI domains (Carmell et al.
2002). In Drosophila, five genes—AGO1, AGO2, AGO3,
Piwi, and Aubergine (Aub)—exist in the genome as members of the Argonaute family. AGO1 and AGO2 are expressed ubiquitously through development, whereas theother three are only expressed in germline cells (Williams
and Rubin 2002). The latter are grouped as the Piwi
subfamily of Argonautes because they show strong sequencesimilarities to each other.
AGO1 and AGO2 in Drosophila function in genesilencing through binding with miRNA and siRNA, respectively (Okamura et al. 2004; Tomari et al. 2004; Miyoshi
et al. 2005). Recent studies have shown that the structure of
a double-stranded small RNA intermediate strongly influences its partitioning between AGO1- and AGO2-RISC(RNA-induced silencing complex) (Forstemann et al. 2007;

RNA (2007), 13:1911–1922. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2007 RNA Society.

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Nishida et al.

Tomari et al. 2007). siRNA-loaded AGO2 functions in
RNAi as Slicer and is directly responsible for cleaving atarget completely complementary to siRNA (Miyoshi et al.
2005). AGO2 is also involved in RISC formation by slicing
the passenger strand within the siRNA duplex (Matranga
et al. 2005; Miyoshi et al. 2005; Rand et al. 2005; Kim et al.
2006; Leuschner et al. 2006). Although AGO1 is thought tofunction in miRNA-mediated translational repression, like
AGO2 it also possesses Slicer activity (Miyoshi et al. 2005).
Piwi, one of the Piwi subfamily proteins in Drosophila, isan essential factor in germline stem cell (GSC) self-renewal
in both males and females (Cox et al. 1998, 2000; Szakmary
et al. 2005). piwi mutations impact transposon mobility(Sarot et al. 2004; Kalmykova et al. 2005); this most likely
results from the deleterious effects of transposon activation.
Indeed, recent studies have shown that Piwi in ovaries
is associated with repeat-associated siRNAs (rasiRNAs)
derived from a variety of repetitive intergenic elements
such as retrotransposons (Saito et al. 2006; Vagin et al.
2006; Brennecke et al. 2007). Their longer length distinguishes rasiRNAs (24–30 nt) from siRNAs and miRNAs(Aravin et al. 2003). Recombinant Piwi protein producedin Escherichia coli is able to exhibit Slicer activity in vitro(Saito et al. 2006), and Piwi is localized in the nucleus (Coxet al. 2000; Saito et al. 2006; Brennecke et al. 2007). Taken
together, it can be postulated that Piwi functions, at least inpart, like Slicer in silencing repetitive/transposable genes
through its association with rasiRNAs in the nucleus, andthus protects the genome from invasive elements. rasiRNAs
in Drosophila are also called piRNAs (Brennecke et al. 2007;
Zamore 2007); we use the latter term hereinafter.
Genetic studies have revealed that Aub is requiredfor pole cell formation (Harris and Macdonald 2001) andfor activating RNAi during Drosophila oocyte maturation(Kennerdell et al. 2002). Aub is also involved in silencing
retrotransposons in the germline (Vagin et al. 2004, 2006;
Savitsky et al. 2006) and Stellate genes in testis throughthe homologous Suppressor of Stellate [Su(Ste)] repeats on
chromosome Y from which Su(Ste) piRNAs are derived(Aravin et al. 2004; Vagin et al. 2006). Aub is required foraccumulation of Su(Ste) piRNAs (Aravin et al. 2004; Vagin
et al. 2006). Mutations in Aub cause male sterility, which is
directly attributable to the failure in silencing the repetitive
Stellate locus. Aub in ovaries is associated with piRNAs
originating mainly from retrotransposon antisense transcripts (Vagin et al. 2006; Gunawardane et al. 2007). A
large-scale study identifying small RNAs associated with
Aub in ovary has recently been reported (Brennecke et al.
2007). However, questions for which answers have yet to beelucidated include:
1. What are the differences between piRNAs associatedwith Aub and Piwi in male and female gonads?
2. Is Aub in testes physically associated with Su(Ste)piRNAs?
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3. If so, how is the Aub–piRNA complex involved in genesilencing at the molecular level?
Here we closely investigated the profiles of piRNAs associated with Piwi and Aub in ovaries and testes. Although
cellular localizations of Piwi and Aub in ovaries differ fromeach other, they bind to a similar set of piRNAs. In contrast
to this, we found that piRNAs associated with Aub in testes
were rather unique. Of these piRNAs, the most abundant(z46% of the total) were those corresponding to Su(Ste)antisense transcripts. Aub–piRNA complex immunopurified from testes was able to cleave target RNAs containing
sequences of Su(Ste) and Stellate. The second largest class ofpiRNAs associated with Aub in testes contained only two
kinds of sequences, both of which were mapped to tiny,
repetitive regions on chromosome X. Interestingly, thesetwo, termed AT-chX-1 and -2, showed strong complementarity to vasa (vas) mRNA, a germline-specific transcript
involved in oocyte differentiation and cyst development(Lasko and Ashburner 1988; Styhler et al. 1998). Indeed,
the protein levels of VAS in aub mutant testes wereincreased by about twofold compared to those in wild type(wt). The Aub–piRNA complex from testes even showed
activity in cleaving target RNAs containing part vas mRNA.
Immunofluorescence showed that Aub is localized in thecytoplasm, as is AGO2 (Findley et al. 2003); however, 21-nt
siRNA incubated in ovary lysate was only loaded onto
AGO2, not onto Aub. Taken together, we propose thatboth in ovaries and testes, Aub functions in cytoplasmic
RNA silencing only through its association with 24–30-nt
piRNAs. Our studies provide the first biochemical insights
into gene silencing mechanisms mediated by Aub in accord
with piRNAs in fly testes.
RESULTS
Expression of Aub in germline cells
To biochemically elucidate the functional involvementof Aub in the presumptive RNA silencing process in fly
gonads, we produced monoclonal antibodies against Aub.
Western blotting on ovary and S2 cell lysates using the antiAub antibody revealed that Aub is strongly expressed in
ovaries, but little in S2 cells (Fig. 1A). AGO1 and AGO2
expressed in S2 cells (Miyoshi et al. 2005) were not crossreacted by anti-Aub (Fig. 1A). Western blotting on Piwi
and AGO3 recombinant proteins produced in E. coli
confirmed that the anti-Aub cross-reacts to neither of
those (Supplemental Fig. 1A), indicating that the antibody
is specific to Aub. In early embryos, Aub is strongly
expressed, but its protein levels gradually diminish
throughout development (Supplemental Fig. 1B).
To examine the expression patterns of endogenous Aub
in ovaries and embryos, immunofluorescent staining was
carried out. In the germarium, Aub was found in theDownloaded from rnajournal.cshlp.org on January 4, 2017 - Published by Cold Spring Harbor Laboratory Press

Silencing by Aub–piRNAs in fly testes

FIGURE 1. Aub expression. (A) Aub expression is detected in Drosophila ovaries, but not in S2
cells. Western blotting was performed on lysates of fly ovaries and S2 cells using an anti-Aub
antibody produced in our laboratory. Anti-tubulin was utilized as a loading control. (B) Fluorescentimage of a germarium region stained with anti-Aub, which clearly indicates that Aub is localized
in the cytoplasm of germline stem cells (GSC) and germline cyst cells (GCC), but Aub appears not
to be expressed in somatic cells in the germarium. (C) Fluorescent image of Aub in an egg chamberat stage 6. Aub is found both in oocytes (Oo) and nurse cells. In the latter, Aub accumulation at the
peripheral region of the nuclei and in the cytoplasmic particles is observed (nuage). (D) Fluorescentimage of Aub in an egg chamber at stage 10A. Around this stage, Aub accumulation to the posteriorpole (PP) in oocytes is clearly found. (E) Fluorescent image of an embryo (stage 14) stained with
anti-Aub. Pole cells are indicated as Pc. (F) The DIC image of the embryo shown in E and theimmunostaining in E were merged. (G) This immunostaining image clearly shows that Aub islocalized in the cytoplasm of pole cells in the interior of embryos. (H–M) Testes were doublestained with anti-Aub and anti-Piwi antibodies (Saito et al. 2006). Fluorescent images of (H,K) Auband (I,L) Piwi. Merged images are shown in J and M, which clearly indicate that the expressionsof Aub and Piwi are mutually exclusive in testes. Aub expression is restricted to GSC, gonialblast
cells (Gb), and spermatogonia cells (Sg), while Piwi is only expressed in hub (Hub) and supporting somatic cells (SSC). All images shown in this figure represent a single confocal section.

cytoplasm of germline stem cells (GSC)
and germline cyst cells (GCC) (Fig. 1B).
There are strong accumulations of Aubat the cytoplasmic side of perinuclear
regions, called the nuage, a germlinespecific body conserved in species(Eddy 1975). In egg chambers at stages
6 and 10A, Aub was clearly detected in
the cytoplasm of both nurse cells andoocytes (Fig. 1C,D). Nuage staining
was observed only in nurse cells, notin oocytes at stage 6 (Fig. 1C), which isconsistent with the notion that nuage isknown to be lost from oocytes (Mahowald and Strassheim 1970). Clear, sharp
accumulation of Aub was observed at
the posterior of oocytes at stage 10A(Fig. 1D). Aub signals were under thedetection limit in somatic cells such as
terminal filament cells, cap cells, andfollicle cells (Fig. 1B,C), where strong
Piwi expression has been observed(Saito et al. 2006). These staining patterns rather resembled those previously
reported with GFP-tagged Aub (GFPAub) overexpressed in fly ovaries(Harris and Macdonald 2001) and withpolyclonal antibodies against Aub(Brennecke et al. 2007). In embryos,
endogenous Aub was clearly accumulated in the cytoplasm of pole cells, as
expected (Fig. 1E–G).
Testis staining with anti-Aub revealed
Aub in GSC, gonialblast, and spermatogonia cells (Fig. 1H,K). However, Aub
expression was undetected in somatic
cells surrounding gonialblast and spermatogonia cells, and in hub (Fig. 1H,K),
where the strong expression of Piwi
was observed (Fig. 1I,L; Cox et al. 2000;
Saito et al. 2006). Hub is a tiny clusterof post-mitotic somatic cells localized at
the apical tip of the testis and functionsin the maintenance of GSC identity andinfluences its behavior (Ohlstein et al.
2004; Snee and Macdonald 2004).
Double-staining of testes with antiAub and anti-Piwi confirmed that Aub
signals were only detected in germ cells,
whereas Piwi signals were restricted
to somatic cells in testes (Fig. 1J,M).
Namely, expression of these two proteins was mutually exclusive in respect
to both cellular localization and celltype.
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Comparison of piRNAs associatedwith Aub and Piwi in fly ovaries
We previously identified small RNAs
associated with Aub immunoprecipitated from ovary lysate with the antiAub antibody (Gunawardane et al.
2007). Silver staining patterns of protein
and RNA components in the immunoprecipitate are shown in Supplemental
Figure 2. Western blotting analyses
on the immunoprecipitate using antiAGO3 and anti-Piwi antibodies confirmed that the Aub complex does notcontain either protein (Supplemental
Fig. 2C). To gain insight into thepiRNAs associated with Aub and Piwi
in ovaries, we reexamined the compositions of those clones, which are listed
in Supplemental Tables 1, 2, and 3.
Northern blotting analyses demonstrated that piRNAs associated with
Aub seemed also to be associated with
Piwi (Fig. 2A–C). A size difference wasclearly observed between piRNAs with
Piwi and Aub (Fig. 2A–C). This agrees
with previous data showing that 32Pend-labeled piRNAs associated with Piwi
in fly ovaries contained molecules migrating slightly slower than those associatedwith Aub on denaturing acrylamide gels
(Brennecke et al. 2007; Gunawardane
et al. 2007). piRNAs associated with
Aub and Piwi most likely contain an
equal amount of monophosphate at the
59-ends (data not shown) and are methylated at their 39-ends (Saito et al.
2007). Thus, piRNAs with Aub might
simply be shorter than those associatedwith Piwi.
We previously reported that miRNAs
were not present in Piwi complex
immunopurified from ovaries and thatsiRNAs pre-programmed in ovary
lysate were not loaded on Piwi (Saito
et al. 2006). We speculated that neither
siRNAs nor miRNAs were loaded onto
Aub, as was the case for Piwi. To
examine this possibility, small RNAs
isolated from Aub complex in ovaries
were probed with an oligo that specifically recognizes miR-310, one of themiRNAs expressed in fly ovaries. A
positive band was detected in just the
AGO1 lane (Fig. 2D). Another oligo
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FIGURE 2. Detection of Aub association with piRNAs. (A) Northern blotting with probes for17.6 and GATE retrotransposons shows that piRNAs derived from the transposable elements
are involved in Aub and Piwi complexes immunopurified from ovary lysate. It should be notedthat the Piwi complex contains piRNAs that are apparently a little longer than those within the
Aub complex. (B) The same set of samples used in A was probed with a roo retrotransposon
sequence. As in A, the fraction of piRNAs with Piwi seems to contain longer molecules
compared with those with Aub. (C) An oligo recognizing #4 roo piRNA, originally found toexist in fly ovaries (Vagin et al. 2006), was used for Northern blotting analysis. The anti-Piwi
lane clearly contains bands migrating slower than those observed in the total and anti-Aublanes (an asterisk shows one such prominent band). (D) microRNAs are not loaded onto Aub
in ovaries. RNA molecules within the AGO1 and Aub complexes immunopurified from ovary
lysate were probed with a DNA oligo to detect miR-310. The band corresponding to miR-310
is only found in the anti-AGO1 lane. It should be noted that no miRNAs were obtained
through the experiment identifying the small RNAs within Aub complex in ovaries, indicatingthat it is most likely that no miRNAs are loaded onto Aub in ovaries. (E) luc siRNA duplex was
first added to ovary lysate. After incubation, AGO2 and Aub were immunopurified from thereaction mixture using specific antibodies. RNA molecules were isolated from the complexes
and run on a denaturing acrylamide gel. In the anti-AGO2 lane, the luc guide siRNA appears,
but not in the anti-Aub and (n.i.) non-immune lanes, indicating that luc guide siRNA is
specifically loaded onto AGO2 in the ovary lysate.

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Silencing by Aub–piRNAs in fly testes

specifically recognizing miR-186 showed the same result(data not shown). This strongly suggested that Aub does
not function together with miRNAs. Indeed, only a trace ofmiRNAs was obtained (nine of a total 925 Drosophila small
RNAs; 0.97%) as Aub-associating small RNAs (Supplemental Table 1). We then assessed if the siRNA duplex of
21 nt in length, a triggering molecule of RNAi mediated by
AGO2, was loaded onto Aub as well as AGO2 upon
addition to ovary lysate. luc guide siRNA was efficiently
loaded onto AGO2 but not onto Aub (Fig. 2E). These datasuggested that the 21 nt siRNA loading machinery composed with Dicer-2 and R2D2 (Liu et al. 2003) was unable
to load the small RNA onto both Piwi (Saito et al. 2006)
and Aub (this study) proteins.
Su(Ste) piRNAs are the most abundant associatedwith Aub in testes
Immunoprecipitation of Aub from testes was also performed using the anti-Aub antibody. As in ovaries, small
RNAs 24–30 nt long were observed (Supplemental Fig. 3).
A small RNA library was constructed, and 458 clones weresequenced (Table 1). The list of piRNAs obtained is shownin Supplemental Table 4. Although piRNAs associated with
Aub in testes also contain those derived from LINE and
LTR-type transposons (Table 1) and show a strong preference for U at their 59-ends (z87%) (Supplemental Fig. 4),
the most abundant class of piRNAs identified was made upof those derived from Su(Ste) antisense transcripts (195 of
425 piRNAs; z46%) (Fig. 3A; Table 1; Supplemental Table
4). Consistent with these findings, previously genetic
studies have shown that both aub and Su(Ste) mutations
cause an accumulation of Stellate mRNA in testes (Bozzetti
et al. 1995, Aravin et al. 2004; Vagin et al. 2006). Su(Ste)piRNAs in a sense orientation were found only once in thisscreening. This bias was apparently not a consequence ofany loading bias to Aub because it has been reported thateven in total RNAs, Su(Ste) piRNAs in sense are hardly
detected in testes (Vagin et al. 2006). piRNAs generated
from Stellate sense transcript were also obtained in thisexperiment (two of 425) (Fig. 3A; Table 1; Supplemental
Table 4). Notably, Su(Ste) piRNAs seemed not evenly
produced from the precursor molecules (Fig. 3A). Particularly, one Su(Ste) piRNA, termed Su(Ste)-4, was soabundant that it appeared 120 times [including closely
related ones such as Su(Ste)-4a shown in Supplemental
Table 4] in all the clones sequenced. The association ofSu(Ste)-4 with Aub in testes was further confirmed by
Northern blotting analysis using a DNA oligo specific forthe small RNA (Fig. 3B).
A second large class of piRNAs associated with Aub intestes was made up of those derived from two tiny regions(A, 274 nt; and B, 173 nt) on chromosome X (Fig. 4A). The
A and B regions are both located between retrotransposons(Fig. 4A). Interestingly, one of these piRNAs, termed AT-

TABLE 1. (A) Profiles of small RNAs in Aub and Piwi complexes
in testes. (B) 195 Su(Ste) piRNAs were found in the Aub complex
from testes, but only one piRNA originated from the Su(Ste)sense transcript
A. Profiles of small RNAs in Aub and Piwi complexes in testes
SpeciesType

Aub in testis

Piwi in testis

D.m.

rRNA
tRNA
miRNA
piRNA
mRNA
snoRNA
snRNA
mito.genome.
7SL
Total

1
0
0
425
9
0
0
0
0
435
23
458

14
1
0
246
80
6
0
0
0
347
62
409

D.m.
OthersTotal

B. 195 Su(Ste) piRNAs were found in the Aub complex from testes,
but only one piRNA originated from the Su(Ste) sense transcript
piRNAsAub in testis

Piwi in testis

LINE
LTR
DNA
Satellite
RepeatSu(Ste)AT-chX
Stellate
Others
Total

31
68
14
1
1
195
104
2
9
425

42
169
3
0
3
4
1
0
24
246

Notably, we found no Su(Ste) piRNAs in the Aub complex fromovaries. 104 AT-chX piRNAs (including both 1 and 2) (Fig. 4) appeared as piRNAs associated with Aub in testes (24.5%). AT-chX-2
was found in just one of these. The appearance of AT-chX in
piRNAs associated with Aub in ovary was just 0.44%.

chX-1, whose sequence appeared 103 times in this screening (z24% of the total piRNAs), showed strong complementarity to vas mRNA (Fig. 4B), a germline-specifictranscript involved in oogenesis (Lasko and Ashburner
1988; Styhler et al. 1998). Northern blotting using an oligo
specific for AT-chX-1 verified its association with Aub intestes (Fig. 4C). AT-chX-2, another piRNA found in thissubgroup, appeared only once in this experiment but also
showed high complementarity to vas mRNA (Fig. 4B).
Western blotting revealed that the protein levels of VAS in
aub /aub mutant fly testes were increased by about
twofold compared with those of wild-type (wt) andaub /+ (Fig. 4D; Supplemental Fig. 5A). Mislocalization
or ectopic expression of VAS was not observed in aub
mutant testes (data not shown). We also found that VAS
expression was not increased in piwi mutant testes (Supplemental Fig. 5B). Although it has been shown that thephosphorylation of VAS in ovaries is altered in mutants
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Nishida et al.

observed twofold changes in VAS abundance do not reflect aneffect on its phosphorylation. In contrast, in ovaries, where
little AT-chX-1 and -2 was found in the Aub complex (three of689), we did not observe such an alteration of VAS levels (Fig.
4D) as reported (Wilson et al. 1996). Taken together, it issuggested that vas expression in testes might possibly be
down-regulated by the Aub complex containing AT-chX-1
and -2 through RNA silencing mechanisms (see below).
Piwi and piRNA association in testes
We also carried out a parallel examination of small RNAs
residing within the Piwi complex in testes (Table 1;
Supplemental Fig. 3; Supplemental Table 5). We detected
only a few Su(Ste) piRNAs (four of 246 piRNAs) and no
AT-chX-1 and -2 as Piwi-associating small RNAs (Table 1;
Supplemental Table 5), which suggested that Piwi hardly
contributes to the repression of Stellate and vas gene
expression. It is, however, worth noting that the loss of
Piwi in testes increases the production of Su(Ste) piRNAs(Vagin et al. 2006). The majority of the small RNAs
associated with Piwi in testes were piRNAs derived fromtransposable elements and other repetitive sequences in the
genome, as observed in ovaries (Saito et al. 2006). Interestingly, pieces of protein-coding sense mRNAs wereconspicuous in this screening (80 of 347 small RNAs
present in the Piwi complex in testes) (Table 1; Supplemental Table 5) as has been reported in the small RNA
profiling study done for MIWI, a Piwi homolog expressedin mouse (Girard et al. 2006). However, the significance of
the association of Piwi/MIWI in testes with small RNAs
originating from sense mRNAs remains unclear.
Immunoprecipitated Aub–piRNA complexes fromovaries and testes show Slicer activity
FIGURE 3. Analyses of Stellate and Su(Ste) piRNAs associated with
Aub in testes. (A) The nucleotide sequence of Stellate pre-mRNA(accession number: X15899) (Balakireva et al. 1992) was aligned withthat of Su(Ste) (accession number: Z11734) (Balakireva et al. 1992).
(+1) The transcription start site, (***) the translation start site, and(+++) the stop codon of the Stellate transcript. (Boxes in yellow)
Nucleotides unmatched between Su(Ste) and Stellate sequences.
(Shadowed areas with gray boxes) The intron sequences. Stellate
and Su(Ste) piRNAs identified as being in the Aub complex are
underlined in blue and red, respectively. (A thick red underline)
Su(Ste)-4 piRNA, the most abundantly found through the experiment. (A black dot line) A particular region in Su(Ste) from which
various piRNAs (29 of 425 piRNAs) were originated (Supplemental
Table 4). Note that all the Su(Ste) piRNAs obtained in this experimentcontained sequences of Su(Ste) antisense transcript. Stellate piRNAs
were from the sense mRNA. (B) The Northern blot confirms that the
Aub complex immunopurified from testes contains Su(Ste)-4 piRNA (*).

defective in piRNA production and/or function (Klattenhoff
et al. 2007), we found no change in the electrophoretic
mobility of VAS in aub mutant and wt testes (Fig. 4D;
Supplemental Fig. 5A; data not shown), suggesting that the
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Previously, we demonstrated that Drosophila AGO1 andAGO2 exhibit Slicer activity in vitro (Miyoshi et al. 2005).
Moreover, we recently showed that recombinant Piwi, Aub,and AGO3 produced in E. coli also exhibit Slicer activity(Saito et al. 2006; Gunawardane et al. 2007). We thenassessed if the Aub–piRNA complexes immunopurified
from ovaries and testes could, indeed, show the target
RNA cleavage activity in vitro. A target RNA containing asequence perfectly complementary to #4 roo piRNA, one ofthe piRNAs residing in the Aub complex in ovaries (Fig.
2C), was produced and used in the assays. As shown in
Figure 5A, the #4 roo target RNA was efficiently cleavedwith Aub complex immunopurified from ovaries, indicating that endogenous Aub residing in fly gonads also has
Slicer activity. Similar experiments were carried out using
Aub complex immunopurified from testes. An RNA target
containing a sequence completely matching Su(Ste)-4piRNA [Su(Ste)-4 target] or part of the Stellate sensemRNA (Stellate target), which should be recognized by

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the Aub–Su(Ste)-4 complex, was efficiently cleaved, as
expected (Fig. 5B,C). These results are consistent with data
demonstrating that mutations in aub cause an increase inboth Stellate mRNA and sense Su(Ste) RNA (Schmidt et al.
1999; Aravin et al. 2001; Vagin et al. 2006). Under the same
conditions, Aub complex obtained from ovaries, where
Su(Ste) transcripts are presumably unexpressed, did notcleave the Su(Ste)-4 target (Fig. 5B). In fact, Su(Ste)-4piRNA did not appear at all in the experiment identifying
small RNAs in Aub complex in ovaries (Supplemental
Table 1). Although the activity was relatively weak, a target
RNA containing a sequence of vas mRNA showing complementarity to AT-chX-1 was also cleaved by Aub complex
immunopurified from testes (Fig. 5D). These results imply
that vas is a candidate for in vivo target genes for Aub–piRNA complexes in testes.
DISCUSSIONFIGURE 4. AT-chX-1 shows strong complementarity to vas mRNA.
(A) The length of Chromosome X is 22,422,827 nt (accession number:
AE014298). The A and B regions (shown in purple) where AT-chX-1
and -2 are coded span from 21,765,976 to 21,766,249 (274 nt) andfrom 21,773,609 to 21,773,781 (173 nt), respectively. The distance of
the A and B regions is 7360 nt. AT-chX-1 in both regions corresponds
to the sequences from 21,766,034 to 21,766,058 and from 21,773,667
to 21,773,691, respectively. AT-chX-2 in the A and B regions is from21,766,122 to 21,766,147 and 21,773,775 to 21,773,780, respectively.
The A and B regions are flanked by baggins{}2584 and baggins{}
2585, and baggins{}2427 and R1{}2611, respectively. CG17686 andCG14621 are protein-coding genes located close to the A and B
regions. (B) Schematic drawing of the vas mRNA. Nucleotide
sequences of a partial vas mRNA (black) (173 nt; a region showing
strong complementarity to the B region in A [z81% identity] isshown) (accession number: NM165103), AT-chX-1 (red), and ATchX-2 (blue) are indicated. Lowercase letters show nucleotides
mismatched between vas mRNA and AT-chX-1 or AT-chX-2. Alongwith AT-chX-1, AT-chX-2 is also found in the Aub-associated small
RNA identification experiment, but appeared just once. In contrast,
AT-chX-1 appeared 103 times in 425 piRNA clones (z24%). (C)Association of AT-chX-1 with Aub was confirmed by Northernblotting. The probe used was a DNA oligo containing a sequence
complementary to AT-chX-1. (D) The protein levels of VAS are
increased in the aub mutant fly testes. Ovary and testis lysates of wildtype (wt), aub /+ and aub /aub were each probed with anti-Aub,anti-VAS, and anti-tubulin. In testes, VAS protein levels in aub /aub
are about twofold compared with those in aub /+ and wt, whereas
they are not changed in the ovary lysates with or without Aub. This
figure shows representative data from three individual experiments.
Anti-tubulin was used for normalization. (wt) yellow white; (aub /+)aubHN2/+; (aub /aub ) aubHN2/aubQC42.

The present study together with our previous findings(Saito et al. 2006; Gunawardane et al. 2007) clearly
demonstrated that both Piwi and Aub, the Drosophila
Argonaute members specifically expressed in gonads, areassociated with a variety of piRNAs in both ovaries andtestes.
Although Piwi and Aub both associate with piRNAs, we
also see clear differences between them in terms of whichcells in fly gonads are expressed and where in the cells they
are localized. By detecting endogenous Piwi and Aub using
the specific antibodies we produced, it was found that all
germ cells including GSC in ovaries simultaneously express
both proteins, but their cellular localization is clearly
distinct from each other; Piwi is found just in the nucleus(Saito et al. 2006) and Aub in the cytoplasm (Fig. 1). In
somatic cells such as cap cells, however, only Piwi seems to
be expressed (Fig. 1; Saito et al. 2006). In testes, we foundthat Piwi is only expressed in somatic cells, but not in GSC
and gonialblast/spermatogonia (Saito et al. 2006), where wefound Aub is specifically expressed (Fig. 1). This suggeststhat at least in testes, Piwi and Aub function in different
RNA silencing pathways. Even in ovaries, it has been shownthat the loss of Piwi or Aub causes specific defects; forexample, piwi1 ovarioles were mostly devoid of germline
cells (Lin and Spradling 1997), Aub was shown to berequired in the germline for the production of functional
oocytes (Harris and Macdonald 2001), and aub mutations
caused de-repression of retrotransposons (Vagin et al.
2004, 2006). Thus it is clear that Aub does not sufficiently
compensate for the loss of Piwi function and vice versa.
This is even though the two proteins are coexpressed in thesame germ cells and many piRNAs are shared by bothproteins.
Although many studies have suggested connections
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Nishida et al.

FIGURE 5. The Aub-piRNA complexes show Slicer activity. (A) In
vitro target RNA cleavage assay using a target containing a sequence
complementary to #4 roo piRNA (Vagin et al. 2006) (#4 roo target).
#4 roo target was radiolabeled with 32P at the 59-end. The Aub
complex immunopurified from ovaries was incubated with #4 rootarget, and after the reaction RNAs were prepared and analyzed on agel. The Aub complex shows its ability to cleave the target, whereas thecontrol (n.i.) shows no such activity. (B) In vitro target RNA cleavage
assay was carried out using the Aub complex immunopurified fromtestes and ovaries. An RNA target, Su(Ste)-4 target, which has asequence completely complementary to Su(Ste)-4 piRNA was produced and used. An expected cleavage product (*) is observed in the
Aub complex from testes, but not in that from ovaries, indicating that
the Aub complex from ovaries does not have the ability to cleave theparticular target RNA. (C) An RNA target that has a partial sequenceof Stellate mRNA was used in the cleavage assay. The Aub complex
used was immunopurified from testes. (D) An RNA target that has apartial sequence of vas mRNA was also cleaved with the Aub complex
from testes.

Su(Ste) transcripts in aub mutants (Aravin et al. 2001;
Vagin et al. 2006), this study is the first to show that Aub intestes is physically associated with Su(Ste) piRNAs and that
the Aub–piRNA complex from testes is able to cleave
Stellate mRNA. The Aub–piRNA complex was also able
to cleave a target RNA, showing complete complementarity
to one of the Su(Ste) piRNAs, Su(Ste)-4, demonstratingthat the Aub–piRNA complex can act in RNA silencing
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RNA, Vol. 13, No. 11

both on the locus piRNA is derived from and loci that show
similarity to the piRNA. We found that two piRNAs,
AT-chX-1 and -2, originating from tiny repetitive regions
located between retrotransposons on chromosome X,
showed considerable complementarity to vas mRNA.
Cleavage on the target RNA sequence in the RNAi pathway
has been shown to occur between the +10 and +11
positions of the guide siRNA (the 59-end of the guidesiRNA is assigned as +1) (Elbashir et al. 2001; Schwarz et al.
2004). Base-pairings between siRNA and the target RNA at
the +10 and +11 positions is required for the cleavage. If
these roles were also applicable to piRNAs, we speculated
that AT-chX-1 (and -2) would possibly guide cleavage ofvas mRNA. Indeed, the vas target was cleaved by the Aub
complex purified from testes (Fig. 5D), although thecleavage efficiency was relatively low compared with thatfor Stellate mRNA. In agreement with this observation, thelevels of vas mRNA were moderately increased by loss of
Aub expression, whereas those of Stellate mRNA wereseverely diminished under the same conditions (Supplemental Fig. 5C). The Aub–piRNA complex may also repress
the translation of vas mRNAs, just as Argonaute–miRNA
complexes repress their mRNAs in various circumstances(Ambros 2004; Du and Zamore 2005; Kloosterman and
Plasterk 2006). It has been reported that spindle-E (spnE)
mutations cause piRNAs not to be accumulated in gonads,
as in the case of aub mutations (Savitsky et al. 2006; Vagin
et al. 2006). It should be noted that similar effects on
derepression of vas gene expression were also observed intestes of spnE mutants (data not shown), in which theamounts of AT-chX-1 are markedly reduced (data notshown). As well, we found that vas overexpression (EGFPVAS transgenic flies) (Sano et al. 2002) results in oversized
apexes in testes, where spermatogonial cells and spermatocytes are located (Supplemental Fig. 5D). Interestingly, aub
mutants also showed similarly oversized apexes in thetestes. These results indicate a connection between suchphenotypes and increased levels of VAS proteins. At present, we do not know what are the biological consequencesof vas gene silencing in testis, but our data support a modelin which vas gene expression is regulated in male germ cells
by Aub in concert with the piRNAs.
Notably, >70% of Aub-associated piRNAs in testes were
either Su(Ste) piRNAs or AT-chXs. In contrast, Piwiassociated piRNAs in testes are mostly derived from thesepiRNAs. At present, we do not know where this strong bias
comes from. Piwi and AGO2 are not required for Stellatesilencing (Vagin et al. 2006). This study showed thatsiRNAs and miRNAs were not loaded onto Aub (Fig.
2D,E). These findings support the view that a distinct
pathway exists for loading of piRNAs onto Aub in testes.
This strong bias could simply be due to a lack of Piwi
expression in primary spermatocytes where Su(Ste) isexpressed (Aravin et al. 2004). Given the model of piRNAbiogenesis (Brennecke et al. 2007; Gunawardane et al.

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Silencing by Aub–piRNAs in fly testes

2007), identification of AGO3-associated piRNAs in testes
will shed light on the mechanism of the preferential
association of Su(Ste) piRNAs and AT-chXs with Aub.
In this study, we fortuitously encountered a type of
piRNA that originated from non-annotated, intergenic
regions and was likely involved in triggering the silencing of protein-coding mRNA targets in collaboration with
Aub. More comprehensive biochemical characterization ofpiRNAs associated with Aub, Piwi, and/or AGO3 in Drosophila should shed light on the biological processes involvedin the production of the gametes necessary for developmentof new individuals and thus species perpetuation.
MATERIALS AND METHODS
Drosophila strains
The y w strain was used as a wild type. The strains bearing aub mutations, aubHN2 and aubQC42, were provided by P. Zamore (University of Massachusetts). Females of aubHN2 and males of aubQC42
were crossed to yield aub heterozygote flies, aubQC42/aubHN2.

Western blot analysis
Two hundred amino acids at the N terminus of Aub fused with
GST were used as the antigen to immunize mice. Anti-Aub
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 ofproteins from each sample were loaded on gels (Figs. 1A, 4D).
Anti-tubulin was from the Developmental Studies Hybridoma
Bank (1:1000 dilution). Anti-VAS was provided by A. Nakamura(Kobe-RIKEN CDB) and used at 1:2500 dilution.

Immunofluorescence
Testes and ovaries were dissected manually from adult flies in 13
PBS. Embryos were collected and dechorionated. Immunostaining
was performed following standard procedures. Anti-Aub was
purified from culture supernatants of hybridoma cells using
Thiophilic-Superflow Resion (BD Biosciences) and directly
labeled using a HiLyte Fluor 555 Labeling Kit-NH2 (Dojindo
Molecular Technologies). Culture supernatants of anti-Piwi
hybridoma cells were used without dilution. Alexa 488-conjugatedanti-mouse IgG (Molecular Probe) was used as the secondary
antibody to detect the anti-Piwi antibody. All images werecollected using a Zeiss LSM510 laser scanning microscope.

Small RNA cloning and sequence analysis
Cloning of small RNAs associated with Aub in ovaries and testes
and Piwi in testes was carried out essentially as described (Saito
et al. 2006) with minor modifications. Immunoprecipitation was
performed essentially as described previously (Miyoshi et al.
2005). Immunoprecipitation buffer contained 30 mM HEPESKOH (pH 7.3), 150 mM KOAc, 2 mM MgOAc, 5 mM DTT,
0.1% NP-40, 2 mg/mL Pepstatin, 2 mg/mL Leupeptin, and 0.5%
Aprotinin. About 300 ovaries or about 1000 testes were used per
immunoprecipitation. After immunoprecipitation, total RNAs

were isolated from the immunopurified complexes with phenol:
chloroform and precipitated with ethanol. RNAs were dephosphorylated with CIP (NEB) and labeled with [g-32P]ATP with T4
polynucleotide kinase (TaKaRa) for visualization. For cloning of
small RNAs isolated from Aub complex in ovaries, we usedadapters and primers (i.e., RT and PCR primers) as describedby Saito et al. (2006). Adapters (MI-59 Linker and MI-39 Linker)
and primers used for small RNAs associated with Aub and Piwi intestes were included in the DynaExpress miRNA Cloning Kit(BioDynamics Laboratory). The sequences of each oligo were as
follows:
MI-39 Linker: 59-pCTGTAACTCGGGTCAATddC-39 (DNA);
MI-59 Linker: 59-AUCGUCUCGGGAUGAAA-39(RNA);
39 RT primer: 59-ATTGACCCGAGTTACAG-39 (DNA);59 Primer: 59-ATCGTCTCGGGATGAAA-39 (DNA).
First-strand cDNA synthesis was performed with Stratascript RT(Stratagene) or Reverse Transcriptase (BioDynamics Laboratory).
KOD plus (TOYOBO) or Ex Taq polymerase (TaKaRa) was usedas the polymerase. PCR products were cloned into the EcoRV siteof the pBS SK+ vector (for small RNAs associated with Aub in
ovaries) or pTAC-1 (for small RNAs associated with Aub or Piwi
in testes) (BioDynamics Laboratory) and sequenced. RNA sequences between 59 and 39 adapters were analyzed by undertaking
searches in annotated genomic databases (UCSC: http://genome.
ucsc.edu/cgi-bin/hgBlat; NCBI: http://www.ncbi.nlm.nih.gov/
blast/; and FlyBase: http://flybase.bio.indiana.edu/blast/) to determine whether the cloned RNAs map to the genome. We only used
the best matches up to a maximum of two differences (mismatch,insertion, or deletion) for each small RNA sequence. For repeat
annotation, we used results from the RepeatMasker program(UCSC BLAT), NCBI BLAST, or FlyBase BLAST.

Northern blot analysis
Total RNA either from fly ovaries/testes or immunoprecipitatedcomplexes was isolated using ISOGEN (Nippon Gene). Fivemicrograms of total RNA were used in Figures 2, C and D, 3B,
and 4C. Northern blotting was performed as reported previously(Saito et al. 2006). Probes used for roo piRNA and miR-310 wereas described previously (Saito et al. 2006). Probes used for
detecting #4 roo piRNA, Su(Ste)-4 and AT-chX-1 were as follows:
For #4 roo piRNA: 59-TCGACTCAGTGGCACAATAAAT-39;
for Su(Ste)-4: 59-TCGGGCTTGTTCTACGACGATGAGA-39;
for AT-chX-1: 59-GCCCGAGCCGTCTAACGATGAAACA-39.
DNA fragments for detecting piRNAs that originated from 17.6
(accession number: XD1472) and GATE (accession number:
AJ010298) retrotransposons were obtained by PCR on the
Drosophila melanogaster genomic DNA and cloned into a pBS
SK+ vector. The primers used are as follows:
For 17.6: 59-ATTCTTGTAAACAAATCTTA-39 and 59-CAGCCTCTCACAAATTCAAT-39;
for GATE: 59-TCGAGCAGGGGCACTTCTCAGTC-39 and 59-AC
TATGGCATGCGCGTCGCCA-39.
PCR was again performed using primers for the T7 and T3
promoter sequences on each construct, and the PCR products
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Nishida et al.

were used as templates for in vitro transcription using MAXIscript
T7 and T3 kits (Ambion) in the presence of 32P-UTP. RNAstranscribed were extracted with phenol:chloroform, precipitatedwith ethanol, and partially hydrolyzed as described previously(Saito et al. 2006).

siRNA loading assay
Three hundred ovaries were homogenized in a hypotonic buffer(30 mM HEPES-KOH at pH 7.4, 2 mM MgOAc, 5 mM DTT, and1 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.
Of the luc siRNA duplex, only guide siRNA was labeled with[g-32P]ATP for visualization. Aub and AGO2 complexes werethen immunopurified from the mixture using the specific antibodies. Sodium chloride was added to the lysates to 800 mM justbefore immunoprecipitation was started. Reaction mixtures wererocked for 1 h at 4°C, and the beads were washed extensively witha washing buffer (30 mM HEPES-KOH at pH 7.4, 800 mM NaCl,
2 mM MgOAc, 5 mM DTT, 1 mg/mL Pefablock SC). RNAs wereisolated from the immunoprecipitates, separated on a denaturing
polyacrylamide gel, and visualized on BAS-2500 (Fuji).

In vitro target RNA cleavage assay
Target RNA cleavage assays using immunoprecipitates were
performed in a reaction buffer containing 30 mM HEPES-KOH(pH 7.4), 100 mM KOAc, 2 mM MgOAc, 5 mM DTT, 10 mM
creatine phosphate, 0.5 mM ATP, 30 mg/mL creatine kinase(Roche), 0.1 U/mL RNasin (Promega), and 0.5 mg of yeast RNA(Ambion). After reaction for 3 h at 26°C, RNAs were isolated
from the whole reaction mixtures, run on gels, and visualized on
the BAS 2500. To make a target harboring a sequence completely
complementary to miR-310, a short DNA fragment produced byannealing a set of oligo DNAs (59-AGCTTAAAGGCCGGGAAGTGTGCAATAA-39 and 59-AGCTTTATTGCACACTTCCCGGC
CTTTA-39) was inserted in a pBS SKII+ vector digested with
HindIII (pBS310). To make a target harboring a sequence
completely complementary to #4 roo piRNA (#4 roo piRNAtarget), a short DNA fragment produced by annealing a set ofoligo DNAs (59-GATCCATAGTCGACTCAGTGGCACAATAAATAAAGGC-39 and 59-GGCCGCCTTTATTTATTGTGCCACTGAGTCGACTATG-39) was inserted in a pBS310 vector digested with
BamHI and NotI. To make a target harboring a sequence
completely complementary to Su(Ste)-4 piRNA [Su(Ste)-4 target],a short DNA fragment produced by annealing a set of oligo DNAs
(59- GATCTCGGGCTTGTTCTACGACGATGAGA-39 and 59GGCCTCTCATCGTCGTAGAACAAGCCCGA-39) was inserted
in a pBS310 vector digested with BamHI and NotI. To make atarget harboring a partial sequence of Stellate sense mRNA
(Stellate target), a short DNA fragment produced by annealing a
set of oligo DNAs (59-AATTCTCTGGCTTGTTGTACGGCGATG
AAAG-39 and 59-GATCCTTTCATCGCCGTACAACAAGCCAGA
G-39) was inserted into a pBS SK+ vector digested with BamHI
and EcoRI. To make a target harboring a partial sequence of vas
mRNA (vas target; as a target for AT-chX-1), a short DNA
fragment produced by annealing a set of oligo DNAs (59-TTC
CTCCCGAGCCGTCCAACGATGCAATTGAGA-39 and 59-TCT
CAATTGCATCGTTGGACGGCTCGGGAGGAA-39) was inserted
in a pBS SKII+ vector that was digested with BamHI and NotI and

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blunted. PCR was again performed using primers for the T7 andT3 promoter sequences, and the PCR products were then usedas templates for in vitro transcription using a MEGAscript T7
kit (Ambion). The resultant RNAs were radiolabeled at the 59-G
cap by guanylyltransferase (Ambion) as previously described(Okamura et al. 2004) or with [g-32P]ATP using T4 polynucleotide kinase and gel-purified. vasa target (Fig. 5D) was labeled with
pCp at the 39-end and gel-purified before use.

Accession numbers
The accession numbers are: Aub-associated piRNAs in ovary,
AB296387-AB297014; Aub-associated piRNAs in testis,AB297015-AB297190; and Piwi-associated piRNAs in testis,AB297191-AB297373.

SUPPLEMENTAL DATA
Supplemental materials are available upon request by sending
an e-mail message containing the keyword ‘‘Aub-supplement’’ tosiomim@genome.tokushima-u.ac.jp.

ACKNOWLEDGMENTS
We thank P. Zamore for providing the aub mutant stocks
aubHN2 and aubQC42 and A. Nakamura for the anti-VAS antibody and technical advice. We also thank H. Kose, T. Suzuki,
and V. Vagin for technical advice. We thank members of theSiomi laboratory for discussion and comments on the manuscript. 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). This study 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).
Received July 19, 2007; accepted August 15, 2007.

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Gene silencing mechanisms mediated by Aubergine−piRNAcomplexes in Drosophila male gonad
Kazumichi M. Nishida, Kuniaki Saito, Tomoko Mori, et al.
RNA 2007 13: 1911-1922 originally published online September 13, 2007
Access the most recent version at doi:10.1261/rna.744307

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