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Biogenesis pathways of piRNAs loaded onto AGO3 in the
Drosophila testis
Akihiro Nagao, Toutai Mituyama, Haidong Huang, et al.
RNA 2010 16: 2503-2515 originally published online October 27, 2010
Access the most recent version at doi:10.1261/rna.2270710

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Biogenesis pathways of piRNAs loaded onto AGO3
in the Drosophila testis
AKIHIRO NAGAO,1,2 TOUTAI MITUYAMA,3 HAIDONG HUANG,4 DAHUA CHEN,4 MIKIKO C. SIOMI,1,5
and HARUHIKO SIOMI1
1

Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan
Institute of Health Biosciences, University of Tokushima, Tokushima 770-8503, Japan
3
Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064,
Japan
4
Institute of Zoology, Chinese Academy of Sciences, Beijing, People’s Republic of China
5
JST, CREST, Saitama, Japan
2

ABSTRACTPIWI-interacting RNAs (piRNAs) silence transposable elements in animal germ cells. In Drosophila ovaries, piRNAs areproduced by two distinct pathways: the ‘‘ping-pong’’ amplification cycle that operates in germ cells and a ping-pongindependent pathway termed the primary pathway that mainly operates in somatic cells. AGO3, one of three PIWI proteinsin flies, is involved in the ping-pong cycle in ovaries. We characterized AGO3-associated piRNAs in fly testes and found that likein ovaries, AGO3 functions in the ping-pong cycle with Aubergine (Aub) for piRNA production from transposon transcripts. In
contrast, most AGO3-associated piRNAs corresponding to Suppressor of Stellate [Su(Ste)] genes are antisense-oriented andbound to Aub. In addition, the vast majority of AGO3-bound piRNAs derived from the AT-chX locus on chromosome X are
antisense-oriented and are also found among Aub-associated piRNAs. The presence of very few sense Su(Ste) and AT-chX
piRNAs suggests that biogenesis of both Su(Ste) and AT-chX piRNAs by a ping-pong mechanism only is highly unlikely.
Nevertheless, the mutual interdependence of AGO3 and Aub for the accumulation of these piRNAs shows that their production
relies on both AGO3 and Aub. Analysis of piRNA pathway mutants revealed that although the requirements for piRNA factors
for Su(Ste)- and AT-chX-piRNA levels mostly overlap and resemble those for the ping-pong mechanism in the ovaries, Armitage(armi ) is not required for the accumulation of AT-chX-1 piRNA. These findings suggest that the impacts of armi mutants on theoperation of the piRNA pathway are variable in germ cells of fly testes.
Keywords: AGO3; Aubergine; piRNA; Drosophila; RNA silencing; germline

INTRODUCTION
Recent studies have shown that eukaryotic cells express alarge number of different small RNAs, 20- to 30-nucleotide(nt) long, which trigger various forms of sequence-specificgene silencing by guiding the Argonaute complex to target
RNAs by base-pairing (Ghildiyal and Zamore 2009; Kimet al. 2009; Siomi and Siomi 2009). This process is referred
to as ‘‘RNA silencing.’’ RNA silencing is an evolutionarily
conserved nucleic acid–based immunity that restrains theexpression of parasitic and pathogenic invaders such as vi-

Reprint requests to: Haruhiko Siomi, Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan; e-mail:awa403@sc.itc.keio.ac.jp; fax: 81-3-5363-3266.
Article published online ahead of print. Article and publication date areat http://www.rnajournal.org/cgi/doi/10.1261/rna.2270710.

ruses and transposable elements (Girard and Hannon 2008;
Siomi and Siomi 2008).
In Drosophila, the endogenous small interfering RNA(endo-siRNA or esiRNA) pathway restrains the expressionof transposable elements in somatic cells, whereas the
PIWI-interacting RNA (piRNA) pathway represses themin germline cells (Ghildiyal and Zamore 2009; Kim et al.
2009; Siomi and Siomi 2009). esiRNAs are produced by
the Dicer2-dependent pathway, indicating that they arederived from double-stranded RNA (dsRNA) precursors(Czech et al. 2008; Ghildiyal et al. 2008; Kawamura et al.
2008; Okamura et al. 2008). Processed esiRNAs are loaded
onto AGO2 to form RNA-induced silencing complexes(RISCs) that silence transposable elements by cleaving theirtranscripts. In contrast, the production of piRNAs is Dicerindependent, indicating that the biogenesis of piRNAs isdistinct from that of esiRNAs and does not involve stable
dsRNA intermediates (Vagin et al. 2006; Houwing et al.

RNA (2010), 16:2503–2515. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2010 RNA Society.

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Nagao et al.

2007). In fly gonads, three distinct PIWI proteins of the
Argonaute family—AGO3, Aubergine (Aub), and Piwi—areexpressed (Williams and Rubin 2002). In fly ovaries, piRNAs
associated with Aub and Piwi are derived mainly from theantisense strand of retrotransposons, while AGO3-associatedpiRNAs arise mainly from the sense strand. Aub- and Piwiassociated piRNAs show a strong preference for uracil (U) at
their 59 ends, while AGO3-associated piRNAs show a preference for adenine (A) at the tenth nucleotide from the 59 end(Brennecke et al. 2007; Gunawardane et al. 2007). The first 10
nt of Aub-associated piRNAs can be complementary to thefirst 10 nt of AGO3-associated piRNAs. These PIWI proteinsretain the endoribonuclease or Slicer activity that allows
them to cleave an RNA substrate across from position 10 of
their bound piRNA (Saito et al. 2006; Gunawardane et al.
2007). These observations suggest a Slicer-dependent selfamplifying loop model, called the ‘‘ping-pong cycle,’’ forpiRNA biogenesis in which sense piRNAs bound to AGO3
cleave long antisense transcripts and guide the formation of
the 59 end of the antisense piRNA bound to Aub, and viceversa (Brennecke et al. 2007; Gunawardane et al. 2007).
Therefore, in this amplification loop, transposons are botha source gene of piRNAs and a target of piRNA-mediatedsilencing. Signatures of this ping-pong cycle are conserved
among metazoans (Aravin et al. 2007; Houwing et al. 2007;
Grimson et al. 2008; Kawaoka et al. 2009).
Both AGO3 and Aub are cytoplasmic proteins expressed
only in germline cells in the ovaries (Harris and Macdonald
2001; Brennecke et al. 2007; Gunawardane et al. 2007;
Nishida et al. 2007). In contrast, Piwi is nuclear and isexpressed in both germline cells and somatic support cells
such as follicle cells in the ovaries (Cox et al. 2000; Megosh
et al. 2006; Saito et al. 2006; Brennecke et al. 2007;
Gunawardane et al. 2007; Nishida et al. 2007). Piwi istherefore spatially separated from AGO3 and Aub at thecell-type and subcellular levels in the ovaries. The pingpong cycle in fly ovaries operates specifically in germ cells
and engages mainly AGO3 and Aub (Brennecke et al. 2007;
Gunawardane et al. 2007; Li et al. 2009; Malone et al. 2009).
piRNAs produced by the ping-pong cycle are often referred
to as secondary piRNAs. Classification of piRNAs according to their origins has indicated that piRNAs derived froma particular piRNA cluster locus, the flamenco locus, areexclusively loaded onto Piwi and not further amplified(Brennecke et al. 2007; Malone et al. 2009; Saito et al.
2009). These piRNAs are overwhelmingly antisense. The
flamenco locus was originally identified as a repressor oftransposon expression in somatic follicle cells (Pelisson et al.
1994), where Piwi, but not AGO3 and Aub, is expressed.
These results indicate that piRNAs from the flamenco locus
are produced by a pathway independent of the ping-pongcycle in ovarian somatic cells. This pathway is called the‘‘primary piRNA pathway’’ (Brennecke et al. 2007; Malone
et al. 2009; Saito et al. 2009; Siomi and KuramochiMiyagawa 2009). It is believed that the primary pathway
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also generates piRNAs that can initiate the ping-pong cyclein the ovarian germ cells (Brennecke et al. 2007; Malone
et al. 2009). Although Aub receives some piRNAs via a
primary biogenesis pathway operating in the ovarian germ
cells, AGO3 contains mostly secondary piRNAs (Brennecke
et al. 2007; Li et al. 2009; Malone et al. 2009).
In Drosophila testes, the X-linked Stellate locus is silenced
by piRNAs derived from antisense transcripts of the homologous Suppressor of Stellate [Su(Ste)] repeats on chromosome Y (Balakireva et al. 1992; Bozzetti et al. 1995;
Aravin et al. 2001, 2004; Vagin et al. 2006). Aub is required
for accumulation of Su(Ste) piRNAs (Aravin et al. 2004;
Vagin et al. 2006). Mutations in Aub result in the formationof Stellate protein crystals in primary spermatocytes, whichcauses male sterility (Bozzetti et al. 1995; Aravin et al. 2001,
2004; Kotelnikov et al. 2009). We previously demonstrated
that, among piRNAs associated with Aub in fly testes, thosederived from Su(Ste) antisense transcripts were the mostabundant (Nishida et al. 2007). The second largest class ofpiRNAs associated with Aub in the testes is derived froma repetitive region on chromosome X, termed AT-chX. Oneof these piRNAs, termed AT-chX-1, shows 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). The
AT-chX-1 piRNA down-regulates the protein levels of VAS(Nishida et al. 2007; Li et al. 2009). piRNAs from the twoloci, Su(Ste) and AT-chX, are not bound to Piwi (Nishida
et al. 2007). Recently, Li et al. (2009) produced Ago3 mutants and demonstrated that Ago3 is required for accumulation of both Su(Ste) and AT-chX piRNAs in fly testes.
However, how piRNAs are produced in fly testes remains
largely unknown.
We sought to determine whether these abundant piRNAs
in fly testes were produced in a ping-pong-dependent manner, as is the case for piRNAs derived from transposons in
ovaries. Here, we analyzed piRNAs associated with AGO3
and Aub immunopurified from fly testes. Our data provide
support for the ping-pong cycle in which transposonderived piRNAs are amplified by AGO3 and Aub in fly
testes. However, a large number of piRNAs with exactly thesame sequences, derived from antisense strands of the twoloci, Su(Ste) and AT-chX, were associated with both AGO3
and Aub. Therefore, biogenesis of these piRNAs througha ping-pong mechanism only is highly unlikely. We examined the accumulation of Su(Ste) and AT-chX piRNAs
in mutant testes defective for nine piRNA pathway proteins.
We found that AGO3, Aub, spindle-E (Spn-E), Krimper(Krimp), Maelstrom (Mael), and VAS are required for the
production of both types of piRNAs. However, the production of Su(Ste) piRNAs, but not AT-chX-1 piRNA,
depends on the RNA helicase Armitage (Armi). Together,
these results suggest that distinct piRNA pathways, withdifferent genetic requirements probably depending on thepiRNA loci, operate in germ cells of fly testes.

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piRNAs bound to AGO3 in fly testes

RESULTS
Expression of AGO3 in fly testes
To biochemically investigate piRNA biogenesis in fly testes, we produced antibodies against AGO3 (Nishida et al.
2009). Western blotting of testis lysates
prepared from yellow white wild-type(WT), trans-heterozygous ago3 mutants (ago3t2/ago3t3), and aub mutants(aubHN2/aubQC42) using the anti-AGO3
antibody revealed that the amount of
AGO3 protein was severely reduced in
aub mutant testes (Fig. 1A). The ago3mRNA levels were also significantly affected in aub mutant testes (Fig. 1A,
lower panel). In contrast, levels of AGO3
protein were not affected in aub mutant
ovaries, suggesting that Aub is required
for stabilizing ago3 mRNA and AGO3
protein in testes. Conversely, levels of
Aub protein were not altered in either
testes or ovaries of the ago3 mutants(Fig. 1A). Immunofluorescent stainingof AGO3 in testes revealed that AGO3
was present in the cytoplasm of germline stem cells (GSC), gonialblasts, andspermatogonia, as was the case for Aub(Fig. 1B). However, AGO3 was below
the level of detection in primary spermatocytes, where Aub is expressed. No
expression of AGO3 or Aub was de- FIGURE 1. AGO3 expression in fly testes. (A) The expression levels of Aub and AGO3 weretected in somatic cells surrounding the analyzed by Western blotting using anti-Aub and anti-AGO3 antibodies. The expression levels
gonialblasts and spermatogonia, or in of AGO3 were severely reduced by loss of Aub expression in testes, while Aub was expressed inago3 testes to the same extent as in wild-type (yw) testes. The expression of ago3 mRNA wasthe hub (Fig. 1B), where the strong ex- reduced to almost 50% of the control level. The expression levels of aub and ago3 mRNAs inpression of Piwi was observed (Cox et al. aub and ago3 mutant testes standardized on aub/+ hetero or ago3/+ hetero line testes (red line)
2000; Saito et al. 2006). Both AGO3 and by qRT-PCR. This result showed that ago3 mRNA was unstable in aub mutants. In contrast,
Aub accumulated in the nuage (Snee and such a severe reduction in AGO3 was not observed in aub mutant ovaries, indicating that therequirement for Aub in stabilizing AGO3 expression differs between the ovaries and the testes.
Macdonald 2004; Brennecke et al. 2007), (B) Fluorescent image of testes stained with anti-AGO3 and anti-Aub. Double-staining of fly
a ring around the cytoplasmic face of testes [wild-type (wt) (top), aub (middle), ago3 (bottom)] with anti-Aub (red) and anti-AGO3
the nuclei in germline cells (Eddy 1975). (green) antibodies. Aub and AGO3 expression was detected in germline stem cells (GSCs),
Recent work has suggested the nuage to gonialblast cells (Gb), spermatogonia (Sg), and spermatocytes (Sc). (White triangle) The hub.
Aub was expressed in primary spermatocytes, where AGO3 expression was below the limit of
be a potential site for RISC-mediated detection. Aub and AGO3 co-localize at the nuage, which is a perinuclear electron-dense
post-transcriptional transposon silenc- structure in GSCs and gonialblasts. Aub localization at the nuage was not altered by loss ofing and piRNA biogenesis (Brennecke ago3 function; however, AGO3 did not accumulate at the nuage in aub mutants, indicating
AGO3 accumulation at the nuage depends on Aub. The AGO3-positive, large foci were
et al. 2007; Lim and Kai 2007; Li et al. thatobserved near the nucleus in gonialblasts.
2009; Malone et al. 2009). In aub mutant testes, no nuage staining of AGO3
was observed. Instead, AGO3-positive
and to localize to the nuage in the testes. Thus, in the testes,
large dots were occasionally observed in the cytoplasm,
the dependence of these PIWI proteins for their stability
which was also the case for aub mutant ovaries (Malone
et al. 2009). However, mutations in ago3 did not disrupt
and localization to the nuage is different from thatobserved in the ovaries, where loss of AGO3 does not
the localization of Aub to the nuage in the testes. These
affect Aub protein levels and localization of AGO3 andresults show that Aub is required for AGO3 to be stabilized
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Nagao et al.

Aub to the nuage is mutually interdependent (Li et al. 2009; Malone et al.
2009).
Analysis of piRNAs associatedwith AGO3 and Aub in fly
testes by sequencing
We previously performed a small-scalesequencing study to identify small
RNAs associated with Aub, immunoprecipitated from testis lysate with ananti-Aub antibody (Nishida et al. 2007).
To gain further insight into piRNAbiogenesis in testes, we immunopurified
AGO3 and Aub with specific antibodies
from fly testes and performed a largescale sequencing to comprehensively
examine their associated small RNAs.
Both immunopurified AGO3 and Aubin testes (Fig. 2A) were associated withsmall RNAs 23–28 nt long (Fig. 2B).
To characterize the piRNAs associated with AGO3 and Aub, we generated
70,323 and 108,439 sequencing reads,
respectively, for AGO3- and Aub-associated piRNAs. The list of piRNAs obtained is shown in Table 1 and Supplemental Table 1. piRNAs were mapped
to the Drosophila genome and annotated (Fig. 2C). Fifty-four percent of the
AGO3-associated piRNAs corresponded
to transposons. In sharp contrast, only
7% of the Aub-associated piRNAs corresponded to transposons. The Aubassociated piRNAs corresponding to
FIGURE 2. Analyses of small RNAs associated with AGO3 and Aub in testes. (A) Immutransposons mainly arose from antisense noprecipitation was performed from wild-type fly testes using anti-AGO3 and anti-Aubtranscripts and showed a strong prefer- antibodies. (B) RNA molecules extracted from the immunoprecipitated complexes wereence for U at their 59 ends, while the visualized by 32P-ATP labeling on denaturing acrylamide gel. Small RNAs, 23–28 nt long, were
AGO3-associated transposon piRNAs observed associated with AGO3 and Aub in the testes. (C) Profiles of small RNAs associatedwith AGO3 and Aub in fly testes. The most abundant class of piRNAs associated with Aub waswere mainly derived from sense tran- those derived from the Suppressor of Stellate [Su(Ste)] antisense transcripts. The second most
scripts and showed a strong preference abundant class of piRNAs associated with Aub was made up of those derived from anfor A at the tenth nucleotide from the intergenic repetitive region on the X-chromosome, termed AT-chX. On the other hand, the59 end (Fig. 3A,B). Among transposon- majority of piRNAs associated with AGO3 were derived from transposons and other repetitive
DNA elements (repeats) found in the genome. Su(Ste) piRNAs (z5%) as well as AT-chX
derived piRNAs in Aub, z17% have piRNAs (z10%) were also present in the AGO3 small RNA library.
ping-pong partner piRNAs, while z27%of transposon-derived piRNAs in AGO3
with findings that the testis expression of several retrotranshave ping-pong partner piRNAs (Supplemental Fig. 1).
posons was not significantly affected by aub mutations
Thus, piRNAs corresponding to transposons in the testes(Aravin et al. 2001), and these findings together raise theshow signatures of the ping-pong amplification cycle. Thesepossibility that there exist unknown AGO3/Aub-indepenresults suggest that piRNAs of transposon origin are prodent mechanism(s) controlling transposon silencing induced by the amplification loop in testes as in ovaries.
germline cells in the testes.
Curiously, we found that the expression levels of transposons
Although most transposon-derived piRNA species idenin the testes were only slightly affected by the loss of aub or
tified in the ovaries were sequenced only once (Brenneckeago3 functions (Supplemental Fig. 2). This is in agreement
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piRNAs bound to AGO3 in fly testes

TABLE 1. Top 50 small RNAs in AGO3 complexes in testes
Sequence

Read(AGO3)

Read(Aub)

mismatch

Annotation(category)Annotation(detail)

StrandUGUUUCAUCGUUAGACGGCUCGGGC
AAAACCUGAAGAUGAUCCUGAGUCGCUGCUCUCGAAUGUAUGCCCGAUAUUCUCAUCGUCGUAGAACAAGCCCGAUGUUUCAUCGUUAGACGGCUCGGGAUGGCUCUCUCAGUCGCUUCCCGGGAUGCUCUCGAAUGUAUGCCCGAUAUGUUUCAUCGUUAGACGGCUCGGGCGUGCUCUCGAAUGUAUGCCCGAUAUAUGAGACUACAAACGGAUUGCGGACC
AGCUUCAUCCAAGCGAACUGUGCCCGAACGAGUAAUAUGAGCGCCCAGAGUUGCUCUCGAAUGUAUGCCCGAUAUAC
AAAACCUGAAGAUGAUCCUGAGUCAAAACCUGAAGAUGAUCCUGAGUCGUGUUUCAUCGUUAGACGGCUCGG
AAACCUGAAGAUGAUCCUGAGUCGC
CCCGAAAGCAGACGAGGUAACGUACC
AACUGAAUAAACGAAAUGGAUGACAACGAGUAAUAUGAGCGCCCAGAGUGAAAAAACCUGAAGAUGAUCCUGAGUCGCUUCAAGGUUACCCCAGGAUGCUGU
UACUUGUUCCCCGGAUAGUUUAGUUAC
GUAACUUCGGGAUAAGGAUUGGCUCUACAUUCAUAACCGAGCGACUGUUCAAACCGAAUUAAUCGCGAGGCUGCAAGAUGUUUCAUCGUUAGACGGCUCGGGCGG
AACUGAAUAAACGAAAUGGAUGACGCCGAACGUAGUCUCUCGAGAGCGACAAACCGAUAUGCUAUCUUGGCUGAUGGGAACACCGCGUGUUGUUGGCCUUCUCAUCGUCGUAGAACAAGCCCG
ACCGAAUUAAUCGCGAGGCUGCAACAAGCUAGACUAUGUAUCCCCGGGUAAAAAACCUGAAGAUGAUCCUGAGUCGCACCGAACUUGAGACCGCCCCUGGACGACC
AACUUCGGGAUAAGGAUUGGCUCUUACGACGAGAACGAGAUCAGCGCGACAAACCAAUCCCAACCAAACCCGUCAAUUCAAGGUUACCCCAGGAUGCUGUGCUUUGCAAAAACGAGAGCGAUAGAGCGCCAAUUACAUUAGACGAGACGGCCC
GUGGCUUUGAGAGGCGCCGUAGUUAAGCUUCAUCCAAGCGAACUGUGCCCG
ACCCUUGAUAAACGGUUGACUUUCGUGAGAUAUGCUAGCAGACUGGAGAUCGGCCCGAACCCUUCUGGUGUGUAACUGAAUAAACGAAAUGGAUGACACACCCUUGAUAAACGGUUGACUUUC
UCGGCCCGAACCCUUCUGGUGUGUU

2642
2229
2069
1670
875
857
729
693
693
606
479
441
374
366
357
308
283
274
255
241
241
238
234
216
188
181
175
165
164
158
152
151
147
147
143
139
131
124
123
121
113
111
109
104
103
102
102
101
98
96

13,615119
1743,207
3013
3
4
91
8
0
0
0
2
24
24
134
16
0
0
0
17
80
0
42
0
0
0
0
41
0
2
6212
0
0
41
0
35
0
0
19
0
3
0
0
0
0
2
0
0
1

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

AT-chX
Transposon
Transposon
Su(Ste)AT-chX
ncRNA
Transposon
AT-chX
Transposon
No annotation
Transposon
Transposon
Transposon
Transposon
Transposon
AT-chX
Transposon
Transposon
Transposon
Transposon
Transposon
RepeatncRNA
ncRNA
Transposon
Transposon
AT-chX
Transposon
Transposon
TransposonncRNA
Su(Ste)Transposon
Transposon
Transposon
Su(Ste)ncRNATransposon
Transposon
RepeatTransposon
Transposon
No annotation
Transposon
Transposon
Transposon
Transposon
Transposon
Transposon
Transposon

AT-chX-B
HelenaR1-element
Su(Ste)AT-chX-B
rRNA
R1-element
AT-chX-B
R1-element

Antisense
Sense
Antisense
Antisense
Antisense
Sense
Antisense
Antisense
Antisense

R1-element
opus
R1-element
HelenaHelenaAT-chX-B
HelenaF-element
invader4
opus
Helena
trf
rRNA
rRNA
GATE
mdg3
AT-chX-B
invader4
invader1Doc
5SrRNA
Su(Ste)mdg3
DocHelenaSu(Ste)rRNA
froggerG2
trf
micropia
opus

Antisense
Sense
Antisense
Sense
Sense
Antisense
Sense
Sense
Sense
Sense
Sense

R1-element
Rt1b
G2
baggins
invader4
Rt1bbaggins

Sense
Sense
Sense
Sense
Antisense
Sense
Sense
Sense
Sense
Antisense
Sense
Sense
Sense
Antisense
Antisense
Antisense
Sense
Sense
Sense
Antisense
Sense
Sense
Sense
Sense
Sense
Sense

Note that a large number of piRNAs with exactly the same sequences are present in both AGO3- and Aub-associated complexes.

et al. 2007; Li et al. 2009; Malone et al. 2009), we noted thatpiRNAs derived from some transposons including Helena,
Opus, and Mgd3 elements in the testes, were sequenced
multiple times (Table 1; Supplemental Table 1). For example, a piRNA derived from a sense strand of Helena wassequenced 4187 times in the AGO3 library and 314 times in

the Aub library. These piRNAs with multiple reads tended
to be derived from the sense strands of transposons (Fig.
3A). We found no antisense piRNAs that paired with these
multiple-read sense piRNAs with signatures of an amplification cycle, namely, 1U/10A partners with a 10-nt,
59-overlap.
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FIGURE 3. Bioinformatics analysis of piRNAs in testes. (A) The heat map (left panel) indicates the strand bias of cloned piRNAs with respect tocanonical transposon sequences. Transposons are grouped into long terminal repeat (LTR), long interspersed nuclear (LINE), and inverted repeat(IR) elements. The color intensities indicate the degree of strand bias: (green) sense; (red) antisense; (yellow) unbiased. Aub-associated piRNAs
mainly arose from antisense transcripts of transposons, and AGO3-associated piRNAs were mainly derived from sense transcripts. The cloning
frequencies of individual transposons are also indicated as a heat map (right panel). (B) The basic composition of piRNAs from transposons
indicates that Aub-associated piRNAs show a strong preference for U at their 59 ends. On the other hand, AGO3-associated piRNAs containpredominantly A at the tenth nucleotide from the 59 end. piRNAs corresponding to the Su(Ste) and AT-chX loci were not included in this analysis.

Su(Ste) piRNAs
The most abundant class of Aub-associated piRNAs identified comprised those derived from Su(Ste) antisense tran2508

RNA, Vol. 16, No. 12

scripts (70.4%) (Figs. 2C, 4; Supplemental Table 2). Of all
the Su(Ste) piRNAs associated with Aub, z65% comprised
only one piRNA, termed Su(Ste)-4 piRNA (Nishida et al.
2007). If up to two mismatches were permitted, z87% ofDownloaded from rnajournal.cshlp.org on November 19, 2010 - Published by Cold Spring Harbor Laboratory Press

piRNAs bound to AGO3 in fly testes

z52% corresponded to Su(Ste)-4 piRNA(z67% if allowing up to two mismatches) and z85% were antisense-oriented (Supplemental Fig. 3). The majority of AGO3-associated Su(Ste) piRNA
species were antisense-oriented and werealso found among the Aub-associated
Su(Ste) piRNAs (Supplemental Table 2).
These results indicate that Su(Ste) piRNAs
are mostly produced from antisense precursor molecules and that a large numberof Su(Ste) piRNAs, mostly Su(Ste)-4 piRNAand its variants, with exactly the same sequences, are loaded onto both AGO3 andAub.
Among the AGO3-associated Su(Ste)piRNAs, z15% (584 out of 3214 reads)
were sense-oriented (Fig. 4). To examine
whether Su(Ste) piRNAs are produced ina ping-pong-dependent manner, we analyzed all the Su(Ste) piRNAs bound to
AGO3 and Aub to determine whether
the first 10 nt of Su(Ste) piRNAs in thesense orientation were complementary
to the first 10 nt of Su(Ste) piRNAs in
the antisense orientation (Fig. 4). We
found that only four Aub- and 11 AGO3associated sense Su(Ste) piRNAs have 78
FIGURE 4. Classification of Su(Ste) and AT-chX piRNAs loaded onto AGO3 and Aub. Aub- Aub- and 38 AGO3-associated antisense
associated Su(Ste) and AT-chX piRNAs were overwhelmingly antisense. A proportion of Su(Ste) piRNA pairs showing signatures
Su(Ste) and AT-chX piRNAs associated with AGO3 were sense-oriented. The classification ofSu(Ste) and AT-chX piRNA sequences loaded onto both AGO3 and Aub, with the read of an amplification cycle with 1U/10A
numbers, is also listed below. The vast majority of Su(Ste) piRNAs loaded onto both AGO3 partners with a 10-nt, 59-overlap (Supand Aub comprised those derived from Su(Ste) antisense transcripts, including the most plemental Table 3). Although this sugabundant Su(Ste)-4 piRNA. Half of Su(Ste) piRNA sequences loaded onto AGO3 alone (AGO3 gests that the ping-pong cycle may still
Uniq.) comprised those derived from Su(Ste) sense transcripts and only a portion of AT-chX
piRNAs associated with AGO3 (AGO3 Uniq.) were sense-oriented. In contrast, Su(Ste) piRNA operate for the production of Su(Ste)sequences loaded onto Aub alone (Aub Uniq.) were all antisense. AT-chX piRNA sequences piRNAs, albeit rarely, we were unable toloaded onto both AGO3 and Aub (Common) comprised almost exclusively those derived from find sense Su(Ste) piRNAs that paired
AT-chX antisense transcripts, including the most abundant AT-chX-1 piRNAs.
with Su(Ste)-4 piRNA or its variants. We
also failed to detect by Northern blotting
the Aub-associated Su(Ste) piRNAs (58,055 out of 66,734
sense piRNAs that pair with Su(Ste)-4 piRNA (Supplemental
reads) mapped to the Su(Ste)-4 piRNA (Supplemental
Fig. 5A). Taken together, these results suggest that theTable 2). The over-representation of the Su(Ste)-4 piRNA
production of most of the Su(Ste) piRNAs only throughin the Aub library is consistent with our previous resultsa ping-pong pathway is unlikely.
obtained by analyzing a small Aub library that was independently produced and sequenced by a different method
AT-chX piRNAs(Nishida et al. 2007). We also confirmed by Northern blot
analysis that Su(Ste)-4 is a highly abundant piRNA in the
A second large class of piRNAs associated with Aub in thetestis (Supplemental Fig. 5A). This suggests that Su(Ste)testes was made up of those derived from a short repeatedpiRNAs are not evenly produced from the precursor moleregion, termed AT-chX, on chromosome X (Nishida et al.
cules, but they are produced from only very few hotspots2007). One of these piRNAs, termed AT-chX-1 (Nishida et al.
(Supplemental Fig. 3). Su(Ste) piRNAs in the sense orien2007), showed strong complementarity to vas mRNA andtation corresponded to only 31 reads out of 66,734 reads
suppresses the expression of VAS protein (Nishida et al.
among all the Aub-associated Su(Ste) piRNAs in this screen2007). We analyzed AGO3- and Aub-associated AT-chX
ing (Fig. 4). Among the AGO3-associated piRNAs in testes,
piRNAs and found that 10% and 20% of AGO3- and Aubz6% corresponded to Su(Ste) piRNAs (Fig. 2C), in whichassociated piRNAs, respectively, were derived from thewww.rnajournal.org

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Nagao et al.

AT-chX locus (Fig. 2C). These piRNAs
were overwhelmingly antisense: Only 32
out of 5240 reads for AGO3-associatedAT-chX piRNAs, and only one (one outof 19,227 reads) for Aub-associated ATchX piRNAs were sense-oriented (Fig. 4;
Supplemental Fig. 4). Only one pair(AT-chX-71 and AT-chX-71 sense) withping-pong signatures was found among
all these AT-chX piRNAs (Supplemental
Fig. 4). Ninety-three percent of AGO3associated AT-chX piRNA species werealso found among the Aub-associatedAT-chX piRNAs (Fig. 4). If two mismatches were permitted, z91% of all
Aub-associated AT-chX piRNAs, andz93% of all AGO3-associated AT-chX
piRNAs, were mapped to AT-chX-1 andits variants (Supplemental Table 4).
However, we failed to find sense piRNAs
that pair with AT-chX-1 piRNA among
Aub- and AGO3-associated AT-chX
piRNAs. We confirmed by Northern blot
analysis that the AT-chX-1 piRNA is avery abundant piRNA in the testis (Supplemental Fig. 5A). We also confirmed
by Northern blotting the expression of
AT-chX-71 piRNA that pairs with ATchX-71 sense piRNA. However, we failed
to detect AT-chX-71 sense piRNA andsense piRNAs that pair with AT-chX-1
piRNA (Supplemental Fig. 5A). Together,
these results suggest that the productionof piRNAs from the AT-chX locus, particularly for the AT-chX-1 piRNA, througha ping-pong mechanism only is highly
unlikely.
Mutational analysis to define thegenetic requirements for Su(Ste)and AT-chX piRNA productionin testes

FIGURE 5. Genetic requirements for production of Su(Ste) and At-chX piRNAs. (A)Accumulation of Su(Ste)-4 and AT-chX-1 piRNAs in ago3 and aub mutant testes was analyzed
by Northern blotting. The probe used was a DNA oligodeoxynucleotide containing a sequence
complementary to Su(Ste)-4 and AT-chX-1. As expected, Su(Ste)-4 piRNA was not detected intestes from males lacking the Su(Ste) loci (6Ste, Ycry). In ago3 mutant testes, Su(Ste)-4 and ATchX-1 piRNAs were detected only weakly, although in aub testes neither piRNA was detected.
(B) Staining patterns of fly wild-type (yw), aub, ago3, and cry mutant testes with anti-Stellate
antibody (upper panel). As expected, in the cry mutant (6Ste, Ycry) males, which havea deletion of the Su(Ste) locus, needle-shaped Stellate aggregation (crystals) were formed inprimary spermatocytes. Stellate protein crystals were also observed in primary spermatocytes of
aub testes, while ago3 testes contained far fewer crystals. The expression level of stellate mRNA
was analyzed by qRT-PCR. The expression level of stellate mRNA in aub and ago3 mutant
testes by qRT-PCR standardized to testes (red line) from wild type (yw) or each hetero fly line
(aub/+, ago3/+) (lower panel). Loss of aub caused a dramatic increase in stellate mRNA
expression, whereas loss of ago3 caused a slight increase in stellate mRNA expression. (C)Northern analysis of the Su(Ste)-4 and AT-chX-1 piRNAs in testes mutant for piRNA pathway
genes. It should be noted that loss of function of spn-E and krimp resulted in a shift in theoverall size of the population of AT-chX-1 piRNAs toward that of endo-siRNAs and miRNAs.

It is known that both AGO3 and Aub are
required to produce or stabilize Su(Ste)piRNAs (Vagin et al. 2006; Li et al.
2009), suggesting mutual interdependence of the PIWI proteins for the biogenesis of Su(Ste) piRNAs. It is also known
that AGO3 is required to produce or stabilize AT-chX
piRNAs (Li et al. 2009). We used Northern hybridization
to examine Su(Ste)-4 and AT-chX-1 piRNA production inago3 and aub mutant testes (Fig. 5A). Neither Su(Ste)-4 nor
AT-chX-1 piRNAs were detected in aub mutant testes. A
marked reduction in both piRNA species was observed inago3 mutant testes. However, production of these piRNAs
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was not affected in piwi mutant testes, consistent with thefact that Piwi in the testes is expressed mostly in somatic
cells, where AGO3 and Aub are not expressed (Nishida et al.
2007). These results suggest that both Su(Ste)-4 and AT-chX-1
piRNAs are produced in germ cells by a mechanism requiring both AGO3 and Aub, even though these piRNAs
have few or no signatures of the ping-pong cycle.
Previous genetic studies have shown that both ago3
and aub mutations cause the formation of Stellate crystals

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piRNAs bound to AGO3 in fly testes

in the testes (Fig. 5B; Bozzetti et al. 1995; Aravin et al. 2004;
Vagin et al. 2006; Li et al. 2009). We noted that, as reported(Li et al. 2009), Stellate protein crystals form in primary
spermatocytes in ago3 testes but not as abundantly as in
aub testes. We examined the accumulation of StellatemRNA in these mutant testes and found that the level of
Stellate mRNA was only slightly increased in ago3 testes,
compared with that in aub testes (Fig. 5B). This may be
accounted for by the low expression level of AGO3 protein
in testes. The amount of AGO3 in testes was approximately
10 times lower than that of Aub in the testes (Supplemental
Fig. 6). In addition, residual Su(Ste) piRNAs in ago3 mutant
testes may still be loaded onto Aub to form an RISC thatsilences Stellate mRNA, which may account for the weakerphenotype observed in ago3 mutant testes (Fig. 5A,B).
Mutations in a large number of genes affect piRNA
production in Drosophila (Vagin et al. 2006; Chen et al.
2007; Lim and Kai 2007; Nishida et al. 2007; Pane et al.
2007; Klattenhoff and Theurkauf 2008; Li et al. 2009;
Malone et al. 2009). To understand their involvement in
the production of Su(Ste)-4 and AT-chX-1 piRNAs, we
examined the molecular phenotypes of a series of eightmutants, including putative helicases, nucleases, and Tudor-domain proteins. We used Northern hybridization to
examine Su(Ste)-4 and AT-chX-1 piRNA production inthese mutant testes. As shown in Figure 5C, both Su(Ste)-4
and AT-chX-1 piRNAs were strongly reduced in spn-E andkrimp mutant testes. spn-E encodes a member of the DExH
family of adenosine triphosphatases (ATPases) with a Tudor
domain (Gillespie and Berg 1995), and mutations in thisgene are known to impair Stellate silencing by eliminating
Su(Ste) piRNAs (Aravin et al. 2004; Vagin et al. 2006).
krimp encodes a Tudor-domain protein (Barbosa et al. 2007;
Lim and Kai 2007). Both genes are critical for silencingof transposons and the accumulation of piRNAs in the
Drosophila germline (Vagin et al. 2006; Lim and Kai 2007).
Normal accumulation of both Su(Ste)-4 and AT-chX-1
piRNAs also requires mael and vas. mael was identified asa genetic loss-of-function mutant, whose germline cells
exhibited incorrect posterior localization of several transcripts (Clegg et al. 1997; Lim and Kai 2007). Mael has an
MHG box and a domain homologous to DnaQ-H 39-to-59
exonucleases (Zhang et al. 2008). vas is a germline-specificgene that encodes a DEAD-box RNA helicase involved inoogenesis (Lasko and Ashburner 1988; Styhler et al. 1998).
zucchini (zuc) and squash (squ) were identified in a screen
for female sterile mutations and cause dorso–ventral patterning defects (Schupbach and Wieschaus 1991; Pane et al.
2007). Both encode proteins with homology with nucleases. Mutations in squ slightly reduced the accumulationof Su(Ste)-4 piRNA and AT-chX-1 piRNA. However, theaccumulation of neither piRNA was significantly affected in
zuc mutant testes.
Su(Ste)-4 piRNA was almost absent from armitage (armi)
mutant testes, consistent with previous results (Vagin et al.

2006). Mutations in armi disrupt translational repression
and localization of oskar mRNA and block RNA interference (RNAi) in Drosophila oocytes (Cook et al. 2004;
Tomari et al. 2004). armi encodes a homolog of Arabidopsis SDE-3, an RNA helicase, which plays a role in posttranscriptional gene silencing triggered by transgenes andsome viruses (Dalmay et al. 2001). The mammalian Armi
homolog Mov10 also plays a role in siRNA-directed RNAiin cultured human cells (Meister et al. 2005). However,loss of Armi function had little impacts on AT-chX-1
piRNA levels.
We probed other piRNAs [Su(Ste)-6 and Su(Ste)-pair4]
from the Su(Ste) locus on the Northern blots and foundthat the genetic requirements for their accumulation arealmost identical to those for the Su(Ste)-4 piRNA (Supplemental Fig. 5B,C). We also probed another piRNA (ATchX-71) from the AT-chX locus on the Northern blots andfound that, in contrast with AT-chX-1 piRNA, the accumulation of this piRNA appears dependent on armi, althoughmodest, as compared with piRNAs from the Su(Ste) locus(Supplemental Fig. 5B,C).
Taken together, these results indicate that although production of both Su(Ste)-4 and AT-chX-1 piRNAs requires
several common genes, including ago3, aub, spn-E, krimp,
and vas, which is similar to the requirements for the pingpong mechanism in the ovaries (Malone et al. 2009), theimpacts of armi mutants on the operation of the piRNA
pathway are variable in germ cells of fly testes.
DISCUSSION
The present study demonstrated that piRNAs in the germ
cells of fly testes are produced not only by a ping-pongcycle but probably also by other mechanisms whose genetic
requirements vary depending on the piRNA loci.
A surprisingly large portion of Aub-associated piRNAs in
testes comprised only two piRNA species: Su(Ste)-4 andAT-chX-1. These piRNAs also comprise a large proportionof AGO3-associated piRNAs in testes. These piRNAs are
not bound to Piwi that is expressed only in somatic cells intestes (Nishida et al. 2007). Therefore, these very abundantpiRNAs are produced only in germ cells of the testes. In
germ cells of the ovaries, AGO3 and Aub mostly engage inthe ping-pong cycle to amplify piRNAs derived fromtransposons (Li et al. 2009; Malone et al. 2009). Althoughtransposon-derived piRNAs that associate with AGO3 and
Aub in testes show signatures of the ping-pong cycle,
Su(Ste) and AT-chX piRNAs show very few signatures ofthe ping-pong cycle. The ping-pong cycle requires initiators
to amplify piRNAs. piRNA populations maternally inherited by germline transmission serve as important initiatorsof the ping-pong cycle to amplify piRNAs in the ovaries(Blumenstiel and Hartl 2005; Brennecke et al. 2008).
However, few or no Su(Ste) piRNAs or AT-chX piRNAs
are produced in the ovaries (Nishida et al. 2007). They arewww.rnajournal.org

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Nagao et al.

therefore not maternally passed on to the offspring toserve as inputs to the ping-pong cycle. Thus, these piRNAs
cannot rely on maternally deposited piRNA populations
to initiate the production in testes. In other words, theDrosophila testis must have evolved these sets of piRNA
pathway genes to produce Su(Ste) and AT-chX piRNAs. In
ovaries, the primary piRNAs, whose production is mostly
dependent on Piwi, have been proposed to be important
initiators of the ping-pong cycle (Brennecke et al. 2007;
Malone et al. 2009). Although Piwi is coexpressed with
AGO3 and Aub in germ cells of the ovaries, its expressionin testes is largely restricted to somatic cells where AGO3
and Aub are not expressed. Indeed, production of Su(Ste)-4
and AT-chX-1 piRNAs is Piwi-independent. Together, thesefindings suggest that production of Su(Ste)-4 and AT-chX-1
piRNAs through a ping-pong mechanism only is highly
unlikely. However, it is still formally possible that, assuggested in Li et al. (2009), very few sense piRNAs might
be sufficient to guide the processing of a large number ofantisense piRNAs in a ping-pong manner.
How are these very abundant piRNAs produced in germ
cells of the testes? Genetic analysis has revealed that in fly
ovaries, ago3, aub, krimp, spn-E, and vas are required for thenormal production of the ping-pong-dependent piRNAs
derived from transposons in germ cells (Li et al. 2009;
Malone et al. 2009). However, only piwi and zuc mutations
specifically decrease levels of piRNAs produced by the primary pathway in ovarian somatic cells (Malone et al. 2009;
Saito et al. 2009). armi plays an important role in the pingpong mechanism (Malone et al. 2009). We demonstrated
that in testes, ago3, aub, krimp, spn-E, and vas are required
for the production of both Su(Ste)-4 and AT-chX-1 piRNAs
in germ cells. This resembles the genetic requirements ofthe ping-pong cycle in the ovaries (Malone et al. 2009).
However, these piRNAs in testes do not require piwi for
their production. In addition, the AT-chX-1 piRNA accumulation does not require armi. These results therefore
suggest that the Su(Ste)-4 and AT-chX-1 piRNA pathways
rely on different sets of genes.
The production of both Su(Ste)-4 and AT-chX-1 piRNAs
is dependent on both AGO3 and Aub. How does suchinterdependence between the two proteins occur? Mutations in aub severely impair the expression of AGO3 protein in testes. Thus, neither AGO3 nor Aub is sufficiently
abundant for the loading of piRNAs in aub mutant testes.
It is conceivable that, without the PIWI proteins, piRNAs
are degraded. Supporting this is the observation that increased levels of Argonaute proteins in mammalian cells correlate with increased levels of mature miRNAs (Diederichsand Haber 2007). This effect depends on direct bindingof the Argonaute proteins to the miRNA, suggesting that
Argonaute proteins are limiting and serve to stabilize
miRNAs. In sharp contrast, in ago3 mutant testes, thelevels of Aub are not altered, but piRNA accumulation ismarkedly reduced. As Su(Ste)-4 and AT-chX-1 piRNAs are
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mainly bound to Aub, it is difficult to explain how Aub
could rely on AGO3 to stably produce these piRNAs in thetestes. Because the accumulation of piRNA precursor- orintermediate-like molecules is not observed in ago3 and aub
mutant testes (Fig. 5A), depletion of AGO3 and Aub may
not substantially affect the processing of piRNA precursors. A plausible, although unsatisfying, model is that bothproteins are required to form protein complexes that promote effective loading of piRNAs onto both AGO3 andAub. Spn-E and Krimp are essential for the production ofSu(Ste) and AT-chX piRNAs, and both proteins contain
Tudor domains that recognize and bind to symmetric
dimethyl-arginine residues (sDMAs) on proteins (Côté andRichard 2005; Bedford and Clarke 2009). Recently it has
been shown that PIWI proteins in fly ovaries contain sDMAs(Kirino et al. 2009; Nishida et al. 2009; Siomi et al. 2010) andthat Tudor-domain-containing proteins such as Tudor interact with PIWI proteins specifically through their sDMA
modifications (Nishida et al. 2009). In this context, it is
tempting to speculate that AGO3 and Aub in fly testes may
also contain sDMAs, which are required for the formation offunctional complexes with Tudor-domain-containing proteins including spn-E and Krimp in the piRNA pathway.
This model also implies that complexes containing AGO3
with sDMAs and complexes containing Aub with sDMAs are
independently required for the piRNA pathway.
Our findings suggest that multiple pathways for piRNAbiogenesis may exist in fly testes. More comprehensive bioinformatics, biochemical and genetic characterization of
AGO3, Aub, and Piwi and their associated piRNAs in the
Drosophila testis, should shed light on the molecular pathways of piRNA production.
MATERIALS AND METHODS
Drosophila strains
The yellow white (yw) strain was used as the wild type. The strains
bearing aub mutations, aubHN2 cn bw/CyO and aubQC42 cn bw/
CyO, and ago3 mutations, ago3t2/TM6B Tb and ago3t3/TM6B
Tb (Li et al. 2009), were provided by P. Zamore (University of
Massachusetts, USA). The P[6Ste;Ste-lacZ]; Ycry Bs strains were
provided by A. Aravin (Cold Spring Harbor Laboratory, NY,
USA). Other mutant alleles and allelic combinations used in thiswork were armi1/TM3 Sb Ser and armi72.1/TM3 Sb P[hs-hid],
PBac{WH}krimpf06583/CyO, piwi2/CyO (a kind gift from H. Lin,
Yale Stem Cell Center), maelM391/TM3 Sb (a kind gift from T. Kai,
Temasek Lifesciences Laboratory, Singapore) and Df(3L)BSC554/
TM6C Sb, spn-E100.37,e/TM3,Sb,P{w+,hs-hid}, squPP32 cn bw /CyO andDf(2L)ED1109/SM6a, tud1 bw sp/CyO l(2)DTS5131, vasPH165/CyO(a kind gift from S. Kobayashi, NIBB, Okazaki), and Df(2L)A267 b cn
bw/CyO Adh, zucRS49 cn bw/CyO and Df(2L)Exel6031.

Western blotting
The anti-Aub monoclonal antibody (Nishida et al. 2007) was used
at 1:1000 dilution, and the anti-AGO3 monoclonal antibody

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piRNAs bound to AGO3 in fly testes(Gunawardane et al. 2007) was used at 1:500 dilution. The antitubulin antibody was obtained from the Developmental Studies
Hybridoma Bank and was used at 1:2000 dilution. Western
blotting was performed as described previously (Miyoshi et al.
2005), with 20–40 mg of sample protein loaded on the gels.

Immunofluorescence
Testes and ovaries were dissected manually from adult flies in 13
phosphate-buffered saline (PBS). 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). AntiAGO3 antibody (mouse polyclonal) was generated by using 289
amino acids at the N terminus of AGO3. Anti-AGO3 antibody
was used at 1:5000 dilution. Alexa 488–conjugated anti-mouse
IgG (Molecular Probes) was used as the secondary antibody todetect anti-AGO3 antibody. For Stellate staining, anti-Stellate
antibody (a gift from P. Zamore) was used at 1:1000 dilution.
Alexa 488–conjugated anti-rabbit IgG (Molecular Probes) wasused as the secondary antibody to detect the anti-Stellate antibody. All images were collected using a Zeiss LSM510 laser scanning microscope.

First-strand cDNA synthesis was performed with Stratascript RT(Stratagene) or Reverse Transcriptase (BioDynamics Laboratory).
KOD plus (TOYOBO) or ExTaq polymerase (TaKaRa) was usedas the polymerase. PCR products were cloned into the EcoRV siteof the pBluescript SK+ vector and sequenced. Sequencing was
performed on a GS FLX system (Roche), which produced 63,282
raw reads from the Aub library and 80,882 raw reads from the
AGO3 library. We used a custom Perl script to remove 59- and39-primer sequences found in the raw sequence data, and disjoined
fused sequences that have multiple adapter sequences, which produce 108,439 short reads from the Aub library and 70,323 shortreads from the AGO3 library. We mapped the resulting small RNA
sequences to the genome sequence of Drosophila melanogaster (Apr.
2006 Assembly a.k.a dm3) by SeqMap (Jiang and Wong 2008) with
two admissible mismatches, which resulted in 94,845 mapped sequences (87.5%) in the Aub library and 49,840 mapped sequences(71%) in the AGO3 library. The mapped sequences were annotatedby comparing their genomic positions to track for repetitive elements (RepeatMasker, FlyBase natural transposable elements, andTandem Repeat Finder), coding genes (FlyBase genes), non-coding
genes (FlyBase non-coding genes), or the Functional RNA Database(Mituyama et al. 2008). Small RNA sequences were deposited in theGene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) under theaccession number SRP003750.

Immunoprecipitation
Immunoprecipitation was performed using anti-Aub and antiAGO3 (polyclonal) antibodies immobilized on Dynabeads Protein
G (Invitrogen). Immunoprecipitation buffer contained 50 mM
Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM dithiothreitol (DTT),
0.1% SDS, 1% Triton-X, 0.5% sodium deoxycholate, 2 mg/mL
pepstatin, 2 mg/mL leupeptin, and 0.5% aprotinin. Approximately
1500 testes were used per immunoprecipitation. The reaction
mixtures were rocked for 1 h at 4°C, the bead fractions were
washed five times with binding buffer, and the protein pools
bound to the beads were eluted with SDS-PAGE sample buffer.
After heating for 5 min to 95°C, protein samples were run on
SDS-PAGE gels and processed for Western blot analysis.

Small RNA cloning and sequence analysis
The cloning of small RNAs associated with Aub and AGO3 in thetestes was carried out essentially as described (Saito et al. 2006)
with minor modifications. After immunoprecipitation, total RNAs
were isolated from the immunopurified complexes with phenol:chloroform and precipitated with ethanol. RNAs were dephosphorylated with calf intestinal phosphatase (CIP; NEB) andlabeled with [g-32P]ATP with T4 polynucleotide kinase (TaKaRa)
for visualization. For cloning of small RNAs isolated from the
Aub and AGO3 complexes in the testes, we used adapters andprimers [namely, the reverse transcription (RT) and PCR primers]
as described in Saito et al. (2006). The adapters (MI-59 Linker andMI-39 Linker) and primers used for small RNAs associated with
Aub and Piwi in the testes were included in the DynaExpress
miRNA Cloning Kit (BioDynamics Laboratory). The sequences ofeach oligonucleotide 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)Northern blotting
Total RNA from fly testes was isolated using ISOGEN (Nippon
Gene). Northern blotting was performed as reported previously(Saito et al. 2006) with minor modifications. Five micrograms of
total RNA from each sample was separated on 12% acrylamidedenaturing gels and transferred onto Hybond-N membrane(Amersham Pharmacia) in distilled water and 1-ethyl-3 [3dimethylaminopropyl]carbodiimide hydrochloride cross-linking
reactions for 2 h at 60°C to enhance detection of small RNA
molecules (Pall and Hamilton 2008). After cross-linking, hybridization was performed at 42°C in 0.2 M sodium phosphate (pH 7.2),
7% SDS, and 1 mM EDTA with end-labeled antisense oligodeoxynucleotide, and washed at 42°C in 23 saline sodium citrate and0.1% SDS. The probes used for detecting Su(Ste)-4 piRNAs, ATchX-1 piRNAs, and U6 snRNAs are listed in Supplemental Table 5.

RT-PCR
Quantitative RT-PCR (qRT-PCR) was performed as reported
previously (Saito et al. 2009). One microgram of total RNA fromeach sample was used to reverse-transcribe target sequences usinga Transcriptor First Strand cDNA Synthesis Kit (Roche) according
to the manufacturer’s instructions. The resulting cDNAs were
amplified with a LightCycler 480 SYBR Green I Master (Roche).
The primers used are shown in Supplemental Table 5.

SUPPLEMENTAL MATERIAL
Supplemental material can be found at http://www.rnajournal.org.

ACKNOWLEDGMENTS
We thank P. Zamore, A. Aravin, H. Lin, S. Kobayashi, and T. Kai
for fly stocks and antibodies. We also thank S. Inagaki andwww.rnajournal.org

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Nagao et al.

H. Ishizu for technical advice, and Y. Iyoda for collecting fly testes.
We thank members of the Siomi laboratory for discussion andcomments on the manuscript. This work was supported by the
Ministry of Education, Culture, Sports, Science and Technology ofJapan (MEXT) grants to H.S. M.C.S. is supported by CREST(Core Research for Evolutional Science and Technology) from JST(Japan Science and Technology Agency) and the New Energy andIndustrial Technology Development Organization (NEDO).
Received May 17, 2010; accepted September 16, 2010.

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