The EMBO Journal (2009) 28, 3820–3831 | & 2009 European Molecular Biology Organization | All Rights Reserved 0261-4189/09
www.embojournal.org

THE

EMBO
JOURNAL

Functional involvement of Tudor and dPRMT5 in
the piRNA processing pathway in Drosophila
germlines
Kazumichi M Nishida1,2, Tomoko N
Okada1, Takeshi Kawamura3, Toutai
Mituyama4, Yoshinori Kawamura1,2,
Sachi Inagaki1, Haidong Huang5,
Dahua Chen5, Tatsuhiko Kodama3,
Haruhiko Siomi1 and Mikiko C Siomi1,6,*
1Department of Molecular Biology, Keio University School of Medicine,Tokyo, Japan, 2Institute of Health Biosciences, University of Tokushima,
Tokushima, Japan, 3Research Center for Advanced Science andTechnology, University of Tokyo, Tokyo, Japan, 4Computational Biology
Research Center (CBRC), National Institute of Advanced Industrial
Science and Technology (AIST), Tokyo, Japan, 5Institute of Zoology
Chinese Academy of Sciences, Beijing, People’s Republic of China and
6
JST, CREST, Saitama, JapanIn Drosophila, the PIWI proteins, Aubergine (Aub), AGO3,
and Piwi are expressed in germlines and function insilencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) andthat dPRMT5/Capsuleen/DART5 is the modifying enzyme.
Here, we show that Tudor (Tud), one of Tud domaincontaining proteins, associates with Aub and AGO3,
specifically through their sDMA modifications and thatthese three proteins form heteromeric complexes. piRNA
precursor-like molecules are detected in these complexes.
The expression levels of Aub and AGO3, along with theirdegree of sDMA modification, were not changed by tud
mutations. However, the population of transposon-derived
piRNAs associated with Aub and AGO3 was altered by tud
mutations, whereas the total amounts of small RNAs on
Aub and AGO3 was increased. Loss of dprmt5 did notchange the stability of Aub, but impaired its association
with Tud and lowered piRNA association with Aub. Thus,in germline cells, piRNAs are quality-controlled by
dPRMT5 that modifies PIWI proteins, in tight association
with Tud.
The EMBO Journal (2009) 28, 3820–3831. doi:10.1038/
emboj.2009.365; Published online 3 December 2009
Subject Categories: RNA
Keywords: Drosophila; piRNA; PIWI; sDMA; Tudor

Introduction
In RNA silencing, small RNAs of 20–30 nucleotides (nt)trigger gene silencing mechanisms by associating with*Corresponding author. Keio University School of Medicine,
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Tel.: þ 81 3 5363 3498; Fax: þ 81 3 5363 3266;
E-mail: siomim@sc.itc.keio.ac.jp
Received: 1 July 2009; accepted: 10 November 2009; published
online: 3 December 2009

3820 The EMBO Journal VOL 28 | NO 24 | 2009

Argonaute proteins, the core factors of the RNA silencing
machinery (Carthew and Sontheimer, 2009; Siomi and Siomi,
2009). Each member of the Argonaute family of proteins
contains two domains, the PAZ and PIWI domains, which
confer two distinct activities to Argonaute: to associate withsmall RNAs and to catalytically interfere with target gene
expression, respectively (Jinek and Doudna, 2009). Small
RNAs guide Argonaute proteins to target transcripts andfollowing this target recognition step, Argonaute proteins
exhibit activities for gene silencing, either transcriptionally,or post-transcriptionally (Moazed, 2009; Siomi and Siomi,
2009). In the latter case, Argonaute proteins function as anendonuclease, or Slicer, to cleave the target transcripts, or asa translation inhibitor (Jinek and Doudna, 2009).
PIWI proteins are a subset of Argonaute proteins, primarily
expressed in germlines (Siomi and Kuramochi-Miyagawa,2009). In Drosophila, PIWI proteins consist of three members,
Piwi, Aubergine (Aub), and AGO3. All PIWI members areassociated with PIWI-interacting RNAs (piRNAs), a subset ofgermline-specific small RNAs that are mainly derived fromtransposons and other intergenic regions and which arefound in clusters throughout the genome (Aravin et al,2007; Siomi and Siomi, 2008). Earlier studies have shownthat each PIWI protein loads a specific set of piRNAs withunique characteristics, such as piRNA origin and nucleotide
and strand bias (Brennecke et al, 2007; Gunawardane et al,2007). These bioinformatic observations led to the proposalof two models for piRNA production, the amplification
loop model and the primary processing pathway (Aravin
et al, 2007; Houwing et al, 2007). Dicer activities, which arenecessary for processing small interfering RNA andmicroRNAs (Kim et al, 2009), are dispensable for piRNA
production (Vagin et al, 2006). This suggests that theprecursors of piRNAs may be single-stranded RNAs. In theamplification loop model, piRNAs induce reciprocal Slicerdependent cleavage of sense and antisense transcripts oftransposons, which is mediated by PIWI proteins. Aubmediated silencing of sense transcripts generates sense
piRNAs, which associate with AGO3 to direct the silencing
of antisense transposon transcripts. The products of Slicer
activity give rise to antisense piRNAs, which in turn bind to
Aub and guide the slicing of sense transposon transcripts
to generate sense piRNAs. Thus, in the amplification loop
model, the interaction of AGO3 and Aub should occur atleast transiently, in vivo. However, how they interact witheach other and how the interaction is regulated remain
unknown. Factors necessary for the primary processing
pathway remain largely unidentified, except that loss of
zucchini function leads to a marked reduction of processed
piRNAs in ovaries and in ovarian somatic cells (Malone et al,
2009; Saito et al, 2009).
Recently, PIWI proteins in various species, including
Xenopus, Drosophila and mice, were shown to have
& 2009 European Molecular Biology Organization

Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et ala post-translational modification, called symmetric dimethylarginine (sDMA), in their N-terminal regions (Kirino et al,
2009; Reuter et al, 2009; Vagin et al, 2009). sDMA is a methylgroup modification that occurs on specific arginine residues
in protein molecules (Bedford and Clarke, 2009). Like othermethyl-group modifications found on arginines, such as
mono methyl-arginine and asymmetric dimethyl-arginine(aDMA), sDMA is known to modify the ability of a protein
to interact with other factors and to modulate its biologicalactivity. A good example is Sm splicing factors (Yong et al,
2004; Neuenkirchen et al, 2008). Nascent Sm proteins, suchas B/B0 , D1, and D3, have been shown to contain sDMAs in
their arginine- and glycine-rich (RG) domains. Further studies
showed that a complex, termed the methylosome and whichcontains protein arginine methyltransferase 5 (PRMT5), a
member of the PRMT family of proteins, catalyses sDMA
modification in eukaryotes. After such dimethylation at specific arginines in the RG domains, Sm proteins associatethough their sDMAs, with a Tudor (Tud) domain-containing
protein, survival motor neuron (SMN) protein. This Sm-SMN
association concomitantly recruits U snRNA. Finally, by thissequential mechanism, U snRNP is efficiently assembled.
Recent studies have shown that the PRMT5-SMN-system
serves as a chaperone system that prevents the mis-assemblyof Sm proteins to non-target RNA and also prevents Sm
protein aggregation (Pellizzoni et al, 2002; Chari et al,2008). Many other Tud-domain proteins have been shownto interact with particular proteins, and this binding requires
methylated arginine residues in the target proteins (Côtéand Richard, 2005). These observations in mammalian cells
led us to infer that PIWI proteins with sDMA modification
could be associated with Tud domain-containing proteins
and that such sDMA-specific interaction may regulatepiRNA association with PIWI proteins in germline cells. In
fact, a fly homolog of PRMT5, dPRMT5 (also known as
DART5, or Capsuleen (Csul)) (Gonsalvez et al, 2006;
Anne et al, 2007) was shown to be responsible for thesDMA modification of PIWI proteins in Drosophila (Kirino
et al, 2009).
In this study, we found that in Drosophila, a Tud domaincontaining protein, Tud, associates with Aub and AGO3 in an
sDMA-dependent manner. The fly homolog of mouse Tud
domain-containing protein-1 (Tdrd1), dTdrd1, and Spindle-E(Spn-E), both of which belong to the fly Tudor domaincontaining protein family, did not show sDMA-dependentinteraction with Aub or AGO3. It seems that Tud, Aub, andAGO3 are able to form various heteromeric complexes indifferent combinations. Of these, particular complexes that
contain both Tud and Aub (and/or AGO3) contained
pre-piRNA-like molecules. The expression levels of Aub andAGO3, along with their degree of sDMA modification, were
not changed by tud mutations. However, the association oftransposon-derived piRNAs with Aub and AGO3 was alteredby the loss of tud, whereas the total amounts of small
RNAs on Aub and AGO3 were increased. Loss of dprmt5
did not change the stability of Aub, but did impair its
association with Tud and lowered piRNA association with
Aub. Taken together, these results suggest that, in germline
cells, piRNAs are quality-controlled by two factors: dPRMT5,
which sDMA-modifies PIWI proteins, and Tud, which associates with PIWI proteins specifically through such sDMA
modification.
& 2009 European Molecular Biology Organization

Results
LC-MS/MS analysis of Aub-sDMA modification
To examine which arginines in Aub are sDMA modified, LCMS/MS analysis was performed on Aub immunopurified
from ovaries using anti-Aub antibodies (Gunawardane et al,2007; Nishida et al, 2007). The isolated protein was firsttrypsinized and analysed with LC-MS. A database search
confirmed the protein as Aub with 54% sequence coverage(Figure 1A). The m/z ¼ 501.5413 peak of the MS spectrum(Figure 1B) matched to the N-terminal end of Aub, spanningfrom methionine1 to arginine17 (M1–R17). N-terminal acetylation, oxidation on M1, and three sDMAs on arginines, R11,
R13, and R15 (Figure 1C) were observed with a parent masserror of 0.4 p.p.m. and a Mascot ion score of 38. No otherpeptides corresponding to M1–R17, with or without partial
modifications, were detected.
Tud associates with Aub in an sDMA-dependent manner
To identify proteins that associate with Aub in a mannerdependant on sDMA modification, pull-down assays were
performed using a peptide corresponding to the first 25
amino acids of Aub (the sequence is shown in Figure 2A).
The peptide was synthesized with and without sDMAs according to the LC-MS/MS results (Figure 1). We could notentirely exclude the possibility of an sDMA modification on
R17; therefore, we made the Aub-sDMA peptide with sDMA
on R17. A prominent, B280 kDa protein was shown to be
specifically associated with the sDMA-containing Aub (AubsDMA) peptide (Figure 2A). MS analysis identified thisprotein as Tud, one of the Tudor domain-containing proteins
expressed in ovaries. Mutants lacking Tud and Aub show very
similar phenotypes; for example, both fail to make germplasm, which is a specialized, maternally provided cytoplasm
that is sequestered at the posterior pole of the oocyte during
oogenesis and that contains the determinants of the germline(Snee and Macdonald, 2004; Thomson and Lasko, 2004;
Arkov et al, 2006). Thus, the identification of Tud in thisexperiment as a candidate Aub-binding partner is consistent
with the findings of earlier genetic analyses.
Western blot analysis using anti-Tud antibodies confirmedthat Tud is abundant in the protein pool obtained from the
Aub-sDMA peptide pull-down (Figure 2B). An earlier study
showed that Aub and Tud co-precipitate; however, no evidence was provided for their direct interaction or for therequirement of the sDMA modification (Thomson et al,2008). Another Tud domain-containing protein, Spn-E (alsoknown as Homeless) (Gillespie and Berg, 1995; Ponting,1997), was not detected with either of the Aub peptides,
regardless of sDMA modification (Figure 2B). The specificityof the anti-Spn-E antibodies raised in our laboratory is shownin Supplementary Figure 1A and B. In mice, Tdrd1 associateswith Mili, Miwi, and Miwi2 (PIWI proteins in mice) in an
sDMA-dependent manner throughout spermatogenesis(Reuter et al, 2009; Vagin et al, 2009); however, the closest
homolog of Tdrd1 in flies, dTdrd1 (CG14303), was only
weakly associated with the Aub peptides, independent ofsDMA modification (Supplementary Figure 1C). Thus,
dTdrd1 does not seem to be the functional counterpart ofmouse Tdrd1. Mouse Tdrd6 is a putative orthologue ofDrosophila Tud, based on their peptide sequence similarities(Hosokawa et al, 2007), and Tdrd6 was shown to bind
The EMBO Journal

VOL 28 | NO 24 | 2009 3821

Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et al

01Aub15ulETD # 2945 RT: 61.45 AV: 1 NL: 3.07E5
T: FTMS + p NSI Full ms [400.00-1600.00]
501.7920
R =60700
z =4501.5413
100
R =59405
z =490

Relative abundance

80502.0424
R =59604
z =4

70
60
50
40

502.2932
R =59004
z =4

30
20
501.9154
R =49304
z =?

10
0
501.2

501.4

501.6

501.8

502.0

502.2

502.4

502.5439
R =58304
z =4
502.6

Relative abundance

m /z

100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0

501.98658.84650.32987.84668.85 803.25
924.34400.15562.64321.23 477.96
160.16 217.15

200

400

600

1003.30

689.72895.65774.51 842.97

800

1363.58
1918.18
1306.50
1604.641122.16 1285.71
1685.03 1845.77
1517.92

1000

1200

1400

1600

1800

2000

m /z

Figure 1 LC-MS/MS analysis of Aub modification. (A) Aub peptide identified by database searching. Letters with yellow background aresearch hits. Those with green background indicate amino acids with modifications; of those, M, R, and C indicate methionine with oxidation,arginine with sDMA, and cysteine with carbamidomethylation, respectively. Ac on the first methionine indicates that the amino acid is
N-terminal acetylated. (B) MS spectrum of the Aub peptide, from M1 to R17 (M1–R17; m/z ¼ 501.5413, theoretical value ¼ 501.5415). (C) ETD
MS/MS spectrum of the Aub peptide, M1–R17. Ion masses in the fragmentation well match those of C ions from the Aub peptide, spanningfrom alanine10 to glycine16 (A10–G16) A-2meR-G-2meR-G-2meR-G-R, where ‘2meR’ indicates an arginine that is sDMA modified.
Fragmentation peaks of the region, asparagine2 to isoleucine10 (N2–L9), are not assigned.

Miwi and Mili (Vagin et al, 2009; Vasileva et al, 2009).
However, sDMA-dependency of such interactions remains
undetermined.
dprmt5 deletion mutant flies, csulRM50/Df(2R)Jp7, showeda strong impairment in Aub-sDMA modification (Kirino et al,2009). Using another dprmt5 mutant strain, csule00797/
csule00797, we also verified that dPRMT5 is required for
Aub-sDMA modification, (Figure 2C). In the dprmt5 mutant
3822 The EMBO Journal VOL 28 | NO 24 | 2009

ovaries, the association of Tud with Aub was no longer
detectable (Figure 2C). These data correlate well with theobservation that Tud recognizes the sDMA-modified Aub
peptide as a binding partner (Figure 2A and B).
Immunoprecipitation from wild-type (wt) ovary lysatesusing anti-Tud antibodies confirmed the association of Aub
with Tud (Figure 2D). Like Aub, AGO3 is also sDMA modifiedin ovaries (Kirino et al, 2009). Thus, we also performed& 2009 European Molecular Biology Organization

Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et al

AGO3-2-sDMA peptide

AGO3-2 peptide

Aub peptide

AGO3-2-sDMA peptide

AG3O-2 peptide

AGO3-1-sDMA peptide

Aub-sDMA peptide

AGO3-1 peptide

Aub peptide

Beads only

Input

~280 kDa

Aub-sDMA peptide

Pull-down fromovary lysate

Pull-down from ovary lysate

Beads only

Input

Aub peptide

Aub-sDMA peptide

Pull-down fromovary lysate

Beads only

Aub-sDMA peptide

Aub peptide

MW

Pull-down fromovary lysate

Tudor

220k

Tud

Aub

Spn-E

AGO3

160k
Spn-E

60k
50k

IP from
ovary

AGO3-1-sDMA peptide
MSGRGNLLSLFNKNAGNMGKSISSK
AGO3-2 peptide
TLQHVNISVGRGRARLIDTLKTDDH
Tud

40k

AGO3-2-sDMA peptide
TLQHVNISVGRGRARLIDTLKTDDH

Input

70k

AGO3-1 peptide
MSGRGNLLSLFNKNAGNMGKSISSK

IP from ovary
AntiInput n.i. Aub
yw
dprmt5 – /–yw
dprmt5 – /–yw
dprmt5 – /–80k

Anti-AGO3

100k

n.i.

120k

Tudor
Aub

(Da)

Aub

Aub peptide
MNLPPNPVIARGRGRGRKPNNVEAN
Aub-sDMA peptide
MNLPPNPVIARGRGRGRKPNNVEAN

Aub-sDMA*

Anti-Tud

n.i.

Input

IP from
ovary

Tud
Aub
AGO3

Figure 2 Tud is specifically associated with sDMA-modified Aub.
(A) Pull-down assays from ovary lysates were performed using Aub
peptides corresponding to methionine1 to asparagine25 (M1–N25).
The sequences of the peptides are shown beneath the figure. ‘Aub
peptide’ and ‘Aub-sDMA peptide’ were made without and with
sDMA modification, respectively. Rs in red indicates Rs that weresDMA modified. A prominent protein of B280 kDa associatedspecifically with the Aub-sDMA peptide. MS analysis revealed thatthe protein corresponds to Tud. (B) Western blot analysis with antiTud antibodies of the protein pools associated with Aub peptides in(A) confirmed that the B280 kDa band is Tud. Spn-E was notdetected in either protein pool. (C) Immunoprecipitation experiments were performed from ovary lysates of wt and dprmt5
mutants using anti-Aub antibodies. Non-immune IgG (n.i.) wasused as a negative control. The immunopurified complexes werethen probed with anti-Tud, anti-Aub, and SYM11, an antibody
specifically recognizing sDMA-modified proteins. Tud was observed
only in the Aub complex obtained from wt ovaries. Aub in dprmt5
mutants was not detected with SYM11, as has been reported earlier(Kirino et al, 2009). The signals marked with an asterisk in ‘Input’
lanes were background. (D) The anti-Tud immunopurified complexes from wt ovary lysates were probed with anti-Tud, anti-Aub,and anti-AGO3 antibodies. Both Aub and AGO3 were detected in
the complexes, suggesting the sDMA-dependent association of Tud
with AGO3.

western blot analysis on the anti-Tud immunoprecipitateswith anti-AGO3 antibodies and AGO3 was also detected in
the complexes (Figure 2D). We inferred from these data that
Tud might also recognize AGO3 in an sDMA-dependent
manner.
& 2009 European Molecular Biology Organization

AGO3

Figure 3 The sDMA-dependent association of Tud with AGO3.
(A) Two AGO3 peptides, AGO3-1 and AGO3-2, which correspondto methionine1 to lysine25 (M1–K25) and to threonine58 to histidine82 (T58–H82), respectively, were synthesized with and without
sDMA modification and pull-down assays were performed fromovary lysates as in Figure 2A. Rs in red indicates those Rs that weresDMA modified. The protein pools obtained were probed with antiTud and anti-Spn-E antibodies. Signals for Tud, but not for Spn-E,
were detected in pools pulled-down with Aub-sDMA and AGO3-2sDMA, but not with AGO3-1-sDMA peptides, indicating that the
AGO3 region, T58–H82, is the binding domain for Tud. (B) Proteinsbound with Aub, Aub-sDMA, AGO3-2, and AGO3-2-sDMA peptides
were probed with anti-Aub and anti-AGO3 antibodies. Both proteins
were detected specifically with the sDMA peptides. These results
suggest that Tud is able to simultaneously associate with Aub and
AGO3 to form a heteromeric complex. (C) The anti-AGO3 immunopurified complexes from wt ovary lysates were probed with antiTud, anti-Aub, and anti-AGO3 antibodies.

Tud also associates with AGO3 in an sDMA-dependent
mannerTo determine whether Tud indeed associates with AGO3 in an
sDMA-dependent manner, we produced AGO3 peptides,
AGO3-1 and AGO3-2, which correspond to the far N-terminal
end and to the region from threonine58 to histidine82 (T58–
H82) of AGO3, respectively. Both peptides were synthesizedwith and without sDMAs (Figure 3A) according to the resultsof the LC-MS/MS analysis, which revealed that R4, R68, andR70 of AGO3 have sDMAs (Supplementary Figure 2). The
possibility that R72 is also sDMA modified was not entirely
clarified; thus, AGO3-2 peptide was synthesized with the R72sDMA modification (Figure 3A). Pull-down experiments fromovary lysates revealed that Tud was able to associate with
AGO3, through the T58–H82 peptide only, in an sDMAdependent manner (Figure 3A). Spn-E was again undetectedwith all the peptides used (Figure 3A). We also confirmedthat the AGO3-2-sDMA peptide even without R72-sDMA
interacted with Tud (Supplementary Figure 3A). To examine
whether Tud, when associated with Aub (or AGO3), coassociated with AGO3 (or Aub), the protein pools pulled
down with the peptides were probed with anti-Aub and
The EMBO Journal

VOL 28 | NO 24 | 2009 3823

Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et alanti-AGO3 antibodies. Although the signals were not sostrong, Aub and AGO3 were both specifically detected with
the Aub-sDMA and AGO3-2-sDMA peptides (Figure 3B). In
addition, the anti-AGO3 immunoprecipitates contained both
Tud and Aub (Figure 3C). Linear density gradient experiments also showed that Tud, Aub, and AGO3 co-sedimented
even in relatively heavy fractions (Supplementary Figure 3B).
These results suggest that Tud might, to some extent, simultaneously associate with Aub and AGO3 and form a heteromeric complex. However, even in tud mutant ovaries Aub
was able to interact with AGO3 (Supplementary Figure 3C).
Although we cannot exclude the possibility that loss of tud
could somehow promote association of Aub with AGO3, we
speculated that Aub and AGO3 might not constantly require
Tud as their bridging molecule. Taken together, we concluded
that Tud, Aub, and AGO3 are likely to form multiple, heterogeneous complexes in different combinations in vivo.

3824 The EMBO Journal VOL 28 | NO 24 | 2009

Anti-Aub x 1/10

Anti-Aub x 1/5

Anti-Aub x 1/2.5

Anti-Tud x 1

n.i. x 1

Input

Tud
Aub

100 ntPre-piRNA-like

200 ntAnti-Aub x 1/5

n.i. x 1

Anti-Aub x 1/20

Anti-Aub x 1/10

Anti-Aub x 1/5

Anti-Tud x 1

n.i. x 1
400 ntAnti-Tud x 1

IP from ovary

IP from ovary

Pre-piRNA-like

Tud function in the piRNA processing pathway
The observations that Tud associates with Aub and AGO3 in
an sDMA-dependent manner (this study) and that the
PRMT5-SMN-system serves as a chaperone system that promotes proper assembly of U snRNP (Pellizzoni et al, 2002;
Yong et al, 2004; Chari et al, 2008; Neuenkirchen et al, 2008)
raised the possibility that the sDMA-dependent association of
Tud with Aub and AGO3 might promote association ofpiRNAs with the PIWI proteins. We first compared theamounts of piRNAs in anti-Aub and anti-Tud immunoprecipitates. When approximately equal amounts of antibodies
and ovary lysates were used, the anti-Aub immunoprecipitates contained a greater abundance of Aub compared withthe anti-Tud immunoprecipitates (data not shown); thus, toequalize Aub quantities, the anti-Aub immunoprecipitates
were sequentially diluted before western blot analysis.
Quantified results showed that a 1 in 5 dilution equalized
the amount of Aub in the anti-Aub and anti-Tud immunoprecipitates (Figure 4A). We then performed northern blot
analysis to visualize the roo piRNAs associated with bothcomplexes. Far fewer roo piRNAs were found to be associatedwith Aub residing in the anti-Tud immunoprecipitates compared with Aub in the anti-Aub immunoprecipitates (which
had been diluted 1:5 before analysis) (Figure 4B). Thissuggested that Aub, in a form associated with Tud, is not
significantly loaded with mature piRNAs. We also noticed
that the anti-Tud immunoprecipitates (but not Aub immunoprecipitates) contained a smeary band, spanning B80 to1000 nt, which was positively recognized by roo piRNAprobes (Figure 4B). The simplest explanation for theseobservations is that the smear corresponds to roo piRNA
precursors. RT–PCR, using primers recognizing roo transcripts in total RNAs isolated from the anti-Tud immunoprecipitates, showed that part of the roo antisense transcript(B500 nt), which supposedly gives rise to multiple roo
piRNAs, exists in the complex (Supplementary Figure 4A).
Therefore, antisense roo piRNA precursor-like molecules
were confirmed in the Tud complex. Interestingly, probes in
the opposite orientation, which should recognize sense roo
piRNAs, also identified the smeary band and also somedistinct bands within the smeary band (Figure 4B). Sense
roo piRNAs were barely detected in the anti-Aub immunoprecipitates, as expected (Figure 4B). RT–PCR using primersthat only detect the roo sense transcript showed that the

IP from ovary

400 nt200 nt
100 nt

50 nt

50 nt

roo piRNA
20 nt

20 nt
Antisense

Sense

Figure 4 Mature piRNAs and piRNA precursor-like molecules contained in the Tud–Aub complex. (A) The immunoprecipitatedcomplexes from ovary lysates using anti-Tud and anti-Aub antibodies were probed with anti-Tud and anti-Aub antibodies. Prior to thewestern blot analyses, the anti-Aub immunoprecipitate was diluted
1:2.5, 1:5, and 1:10. It should be noted that the B1:5 dilution of the
Aub complex equalized the amounts of Aub in the anti-Tud andanti-Aub immunoprecipitated complexes. (B) RNA molecules isolated from the anti-Tud and anti-Aub immunoprecipitated complexes were probed with 32P-labelled DNA oligos that recognize
roo piRNAs (antisense and sense on left and right panels, respectively). Although B1:5 dilution of the Aub complex equalized theamounts of Aub in both complexes, lower levels of roo piRNAs weredetected in the anti-Tud immunoprecipitate (left panel), indicatingthat Aub, whereas bound to Tud, is associated with low levels of
piRNAs. piRNA precursor-like signals (indicated as pre-piRNA-like)were observed in the anti-Tud immunoprecipitates with both sense
and antisense probes, but sense roo piRNAs were not detected in
Tud- or Aub-immunoprecipitated complexes (right). We speculatethat the association of Tud with Aub and AGO3, through sDMA
modification, recruits piRNA precursors to the complex, in amechanism analogous to the recruitment of U snRNA into the
SMN-Sm protein complex.

anti-Tud immunoprecipitates contain sense roo piRNA precursor-like molecules (Supplementary Figure 4B). These
results suggest that the Tud complex is able to hold piRNA
precursor-like molecules in both sense and antisense orientations, but most likely as single-stranded forms. A mechanism
analogous to U snRNP assembly may occur for piRNA precursor recruitment to the heteromeric Tud/Aub/AGO3 complexes, previously assembled through Tud recognition ofsDMA-Aub and sDMA-AGO3. Tud may dissociate from thecomplex after piRNA processing and association with PIWI
proteins because Aub, in a form associated with Tud, contains
only a low level of mature piRNAs.
& 2009 European Molecular Biology Organization

Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et al

Tud provides quality-control of piRNA association
with Aub and AGO3
We then asked whether loss of tud affects piRNA loading onto
Aub and AGO3. Western blot analysis of tud mutant and wt
ovary lysates revealed that the expression levels of Aub andAGO3, along with their degree of sDMA modification, were
not changed by mutations in tud (Figure 5A). 32P-labelled
piRNAs in tud and wt ovaries showed that tud mutations
approximately doubled and quadrupled the amounts ofpiRNAs loaded onto Aub and AGO3, respectively(Figure 5B). Northern blot analyses, however, in tud mutants,
roo#4 piRNA, a piRNA derived from roo transcripts, wasloaded onto Aub much less efficiently compared with thatin wt ovaries (Figure 6A). A very similar result was obtained
when probes recognizing piRNAs derived from Het-A transposon transcripts in the antisense orientation were used
(Figure 6A). In contrast, an alkaline-treated, single-stranded
RNA probe that detects I-element-derived piRNAs showedthat a slightly larger, but still negligible amount of I-element
piRNAs was loaded onto Aub in tud mutants (Figure 6B). A
DM297-derived piRNA behaved in the same way (Figure 6B).
Interestingly, some other piRNAs, derived from R1Dm andDMRT1B, were more abundantly associated with Aub in thetud mutants (Figure 6C), suggesting that loss of tud function
might affect the population of piRNAs that associate with
Aub. To comprehensively examine changes to the piRNA
population due to mutations in the tud gene, deep sequencing
was performed for small RNAs associated with Aub in tud
ovaries and in wt controls. Strand bias was not significantly

affected (Supplementary Figure 5A); however, as inferredfrom northern blotting results, we noticed that the populationof transposon-derived piRNAs was altered by loss of tud(Supplementary Figure 5B). The degree of occupancy ofsmall RNAs corresponding to other RNAs, such as rRNA
and tRNA, were not changed by the loss of tud function(Supplementary Figure 5C). In this regard, Tud function does
not simply mimic that of Tdrd1 in mice. In mice, loss of tdrd1
caused association of aberrant piRNAs with PIWI proteins,which originated largely from rRNA and/or tRNA (Reuter
et al, 2009). A piRNA that was previously recognized to beloaded onto AGO3 (termed minisatellite#1) (Gunawardane
et al, 2007) was detected at about equal levels in AGO3
complexes in tud and wt ovaries (Figure 6D), although far
fewer piRNAs were detected to be associated with AGO3 in wt
ovaries (Figure 5B). These results indicate that loss of tud
function significantly affects the population of piRNAs loaded
onto Aub and AGO3. Thus, Tud provides quality-control of
PIWI-piRNAs by specifically associating with Aub and AGO3,
through dPRMT5-mediated sDMA modification.
sDMA modification of Aub affects PIWI-piRNA loading
We then asked whether piRNA loading onto Aub is also
affected by sDMA modification. We found that loss of
dprmt5 does not affect the stability of Aub (Figure 2C),
although this is in contrast to earlier findings (Kirino et al,2009). We could, however, confirm that Aub is no longer
dimethylated in dprmt5 mutants and thus did not co-immunoprecipitate with Tud (Figure 2C). 32P-labelling of small

IP from ovaryyw

tud – /–

tud – /–

AntiAGO3

n.i.
yw

yw

tud – /–

Input

tud – /–

yw

tud – /–

n.i.
yw

tud – /–

yw

Input

IP from ovary

AntiAub

Tud

Tud

Aub

AGO3

Aub-sDMA

AGO3-sDMA

IP from ovary
Antin.i. Aub

IP from ovaryyw
tud – /–
yw
tud – /–

yw
tud – /–
yw
tud – /–

Antin.i. AGO3

60 nt
60 nt
40 nt

40 nt

30 nt

30 nt
piRNA

piRNA
20 nt

20 nt

Figure 5 Effects of tud mutations on piRNA loading onto Aub and AGO3 in ovaries. (A) The anti-Aub (left) and anti-AGO3 (right)
immunoprecipitates from wt (y w) and tud mutant ovaries were probed with anti-Tud, anti-Aub, andi-AGO3, and SYM11 (bottom panels).
tud mutations did not change the stability or the sDMA modification of Aub or AGO3. (B) Small RNAs associated with Aub (left) and AGO3
(right) in wt and tud mutants were visualized by 32P-labelling. Immunoprecipitation of approximately equal amounts of Aub (left) and AGO3
(right) were checked by western blot analysis, as in (A). The total amounts of small RNAs associated with Aub and AGO3 in tud mutant ovaries
were about two-fold and four-fold greater, respectively, compared with that in wt ovaries (this calculation was done with three individual setsof experimental data).
& 2009 European Molecular Biology Organization

The EMBO Journal

VOL 28 | NO 24 | 2009 3825

Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et al

IP from ovaryyw

tud – /–

AntiAubn.i.
tud – /–

tud – /–

yw

tud – /–yw

yw

tud – /–

n.i.

tud – /–

yw

Total
RNA

AntiAubyw

IP from ovary
Total
RNA

400 ntpiRNA
precursor-like

100 nt-

50 nt30 nt-

50 nt--R1DM piRNA
20 nt-

30 nt- roo#4 piRNA
20 nt- U6 snRNA
IP from ovary

yw

tud

yw

tud

AntiAub
tud – /–

n.i.

– /–

yw

tud – /–

yw

tud – /–

yw

tud – /–

yw

n.i.

Total
RNA

AntiAub– /–IP from ovary
Total
RNA

50 nt30 nt50 nt--DMRT1B piRNA
20 nt-

30 nt-HeT-A piRNA
20 nt-

IP from ovary

tud

– /–

AntiAGO3ywn.i.
tud – /–

tud – /–

yw

tud – /–

yw

tud – /–

yw

tud – /–

yw

n.i.

Total
RNA

AntiAubyw

IP from ovary
Total
RNA

400 nt50 nt-

100 nt50 nt-

30 nt-DM297 piRNA

20 nt-

30 nt20 nt-- Minisatellite#1
piRNA

30 nt- I-element
20 nt-

Figure 6 Northern blot analyses for piRNAs associated with Aub and AGO3. (A) Northern blot analyses of RNAs illustrated in Figure 5B. DNA
oligo probes for a roo piRNA, roo#4 piRNA, and HeT-A piRNAs were used. Although the total amount of small RNAs associated with Aub in tud
mutant ovaries was two-fold greater compared with that in wt ovaries (Figure 5B), roo#4 and HeT-A-derived piRNAs in the Aub complex in tud
mutant ovaries were at much lower levels compared with those in wt ovaries. Interestingly, roo piRNA precursor-like signals were observed toaccumulate in tud mutant ovaries. (B) An alkaline-treated RNA probe (B200 nt) for I-element-derived piRNAs and a DNA oligo for DM297derived piRNA were used. (C) DNA oligos for R1DM- and DMRT1B-derived piRNAs were used. (D) Northern blot analyses of RNAs illustrated
in Figure 5B (right). A DNA oligo that recognized a piRNA, minisatellite#1, was used. Similar signals were detected in the AGO3 lanes of both
wt and tud mutant.

RNAs associated with Aub in dprmt5 and wt ovaries showedthat loss of sDMA modification led to lower levels of piRNAs
associated with Aub (Figure 7A), although the amounts of
Aub in both immunoprecipitates were equal to each other(Figure 2C). Northern blot analysis for roo#4 piRNA confirmed that piRNA loading onto Aub was severely lowered by
dprmt5 mutations (Figure 7B). These results imply that thesDMA modification itself has an impact on the association ofpiRNAs with Aub.
3826 The EMBO Journal VOL 28 | NO 24 | 2009

Aub localization in tud and dprmt5 mutants
How does the impairment in binding Tud and Aub in dpmrt5
and tud mutants affect the localization of Aub in egg chambers? To investigate this question, we immunostained ovaries
of dprmt5 and tud mutants using anti-Aub and anti-Tud
antibodies. Earlier studies have shown that both Aub andTud are highly enriched both in the nuage, a germline-specificelectron-dense material associated with nurse cell nuclei, andthe posterior pole (Bardsley et al, 1993; Snee and Macdonald,& 2009 European Molecular Biology Organization

Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et al

IP from ovary

dprmt5 – /–AntiAub

yw

yw

n.i.

dprmt5 – /–yw

dprmt5 – /–Anti-Aubyw

dprmt5 – /–

yw

n.i.

Total
RNA

dprmt5 – /–IP from ovary

50 nt 100 nt 40 nt 50 nt 30 nt 30 nt - roo#4 piRNA
20 nt -

Tud

Aub

Tud

dprmt5 – /–dprmt5 – /+tud – /–

tud – /+

Aub

20 nt -

Figure 7 Loss of dprmt5 function reduces the loading of piRNAs onto Aub in ovaries. (A) Small RNAs associated with Aub in wt and dprmt5
mutants were visualized by 32P-labelling. Immunoprecipitation of approximately equal amounts of Aub were checked by western blot analysis(as in Figure 2C). The total amount of small RNAs associated with Aub in dprmt5 mutant ovaries was approximately three-fold less comparedwith that in wt ovaries (the calculation was done with three individual sets of experimental data). (B) Northern blot analyses of RNAs
illustrated in (A). A DNA oligo that recognized roo#4 piRNA was used. The signal was hardly detected in the dprmt5 mutant lanes. Like tud,loss of dprmt5 function seems to cause aberrant piRNA loading onto Aub, but the extent was lower than that in tud mutants. (C)Immunohistochemical analyses of tud and dprmt5 mutant egg chambers using anti-Aub and anti-Tud antibodies. In tud mutants, accumulationof Aub and Tud in the nurse cell nuage (indicated with an arrow) was severely impaired and the two proteins were distributed more evenly inthe cytoplasm. In stage 10 egg chambers of dprmt5 and tud mutants, Aub accumulates to similar extents at the posterior pole; thus, Aubaccumulation at the posterior pole (indicated with an arrow head) is not entirely dependent on both sDMA modification and association with
Tud. Tud accumulation at the posterior pole in dprmt5 mutants was totally abolished as is seen in vls mutants (Anne and Mechler, 2005).

2004; Thomson and Lasko, 2004). In the germarium, wildtype staining patterns of Aub were seen in dpmrt5 and tud
mutants (data not shown). However, in stage 8 nurse cells,
the accumulation of Aub in the nuage was greatly impaired inboth mutants and Aub was evenly dispersed in the cytoplasmof mutant nurse cells (Figure 7C). These data suggest thatboth the sDMA modification and the association with Tud are
required for Aub to accumulate in the nuage. Furthermore,
Tud accumulated at very low levels in the dprmt5 nuage,as is also seen in valois (vls) mutants (Anne and Mechler,2005). Valois belongs to the superfamily of WD-repeat
proteins and forms a complex with dPRMT5. Valois also
accumulates in the nuage and the posterior pole in egg
chambers and associates with Tud in vitro (Anne andMechler, 2005). Although it is not known if Aub is unmethy& 2009 European Molecular Biology Organization

lated in vls mutants, as it is in dprmt5 mutants (this study
and Kirino et al, 2009), it is speculated that Tud accumulationin the nuage might be reciprocally dependent on the sDMA
modification of Aub and/or AGO3. However, in stage 10 egg
chambers of dprmt5 and tud mutants, we observed that
Aub accumulates at the posterior pole to a similar extent tothat in wt; thus, Aub accumulation at the posterior pole
only partially depends on both sDMA modification andassociation with Tud (Figure 7C). Tud accumulation toposterior pole in dprmt5 mutants was totally abolished(Figure 7C), as is seen in vls mutants (Anne and Mechler,2005), meaning that Tud accumulation at the posteriorpole depends on the sDMA modification of Aub, or of other,still unknown but dPRMT5-dependently sDMA-modified
proteins.
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Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et al

Discussion
Argonaute proteins are non-sequence-specific RNA-binding
proteins. In an in vitro environment, recombinant Argonaute
produced from Escherichia coli can be artificially loaded withsmall RNAs of a variety of sequences by simple incubation insolution (Miyoshi et al, 2005; Rivas et al, 2005). This incubation
results in the formation of an ‘active enzyme’ that can cleave
any given RNA molecule harboring a sequence completely
complementary to the small RNA. If this disorderly process
occurred in vivo, it would instantly threaten the life of a cell bypossibly targeting important cellular RNAs. Therefore, to avoid
such a dangerous situation, the RNA silencing system mediatedby Argonaute proteins and small RNAs requires ‘gatekeepers’
to ensure that Argonaute proteins are loaded with functional
small RNAs. An important step towards a detailed understanding of piRNA biogenesis in vivo is to elucidate how Argonaute
proteins specifically distinguish their guide small RNAs among
the myriad of RNA sequences in cells. In this study, we
biochemically characterized germline-specific Argonaute proteins, Aub and AGO3, and demonstrated that, at least in
ovaries, a regulatory system to control piRNA association, orloading, is available and that the system is elaborate. It consistsof two consecutive steps: the post-translational modification of
Argonaute proteins, namely dPRMT5-mediated sDMA modification of Aub and AGO3, and the selective association of Aub
and AGO3 with a specific protein, namely sDMA-dependentassociation of Aub and AGO3 with Tud.
Without dPRMT5, Aub is not modified to contain sDMAs.
Loss of sDMA modification does not change the expression
levels of Aub in ovaries, suggesting that sDMA itself does not
function as an index for Aub stabilization in the cellularenvironment. However, in dprmt5 mutants, although Tud isstill present, Aub is not recognized by Tud as a binding
substrate and instead of accumulating in the nuage of nurse
cells, Aub disperses in the cytosol, where it would be free tobind, in a rather non-specific manner, with single-stranded
RNA molecules. Interestingly, deep-sequencing results showthat the majority of the single-stranded RNA molecules
selected by Aub are fragments of transposon transcripts.
These RNAs captured by Aub might then be trimmed byunknown ribonucleases to the size of piRNAs in the same
way that piRNA precursors are processed by ribonucleases in
the processing pathway. Even in dprmt5 mutants, Aub shows
substantial association with small RNAs the size of piRNAs.
This small RNA population, however, seems to be different
from that seen in the wt as northern blot analysis revealed that
Aub in dprmt5 mutants is loaded with fewer transposonoriginating piRNAs. Once Aub is loaded with small RNAs,
regardless of whether they are ‘genuine’ or ‘aberrant’, it
accumulates at the posterior pole of oocytes. This suggeststhat the transport machinery for accumulating Aub at theposterior pole cannot distinguish between Aub associatedwith genuine piRNA and Aub associated with other nongenuine piRNA molecules. Like Aub, Tud also lost the ability
to accumulate in the nuage of dprmt5 mutants, indicating thatthe ability of Tud to accumulate in the nuage depends on its
association with Aub (and AGO3) through sDMA modification.
How does Aub behave in tud mutants? Our data showedthat in tud mutant ovaries, Aub is as stable as it is in wt
ovaries. Also, Aub in tud mutants was sDMA modified to thesame extent as Aub in wt; thus, in tud mutants, Aub, and most3828 The EMBO Journal VOL 28 | NO 24 | 2009

likely AGO3 as well, maintain the ability to be recognized asbinding substrates by Tud. However, Tud itself is not available.
This is why the tud mutant is largely a phenocopy of the
dprmt5 mutant, although the severity of the phenotype washigher in the dprmt5 mutant (MCS and KMN, unpublisheddata). The only clear phenotypic difference found between tud
and dprmt5 mutants in our study was that the total amount ofsmall RNAs loaded onto Aub in tud mutants was much greatercompared with that in dprmt5 mutants. One clear difference at
the molecular level between tud and dprmt5 mutants was thesDMA modification; in tud mutants, Aub is sDMA modified, as
in wt, whereas Aub is not sDMA modified in dprmt5 mutants.
These data suggest that the RNA-binding ability of Aub issignificantly affected by the presence or absence of sDMA
modification. More precisely, Aub sDMA modification elevatesthe affinity of Aub for piRNAs.
As described earlier, in the piRNA biogenesis system, andespecially in the amplification loop system, Aub and AGO3
should be located in intimate proximity to each other toenable the reciprocal passing of piRNA intermediates thathave been cleaved from their primary transposon transcripts
by the Slicer (endonuclease) activity of Aub and AGO3. This
Aub and AGO3-mediated RNA cleavage is thought to benecessary for determining and forming the 50 ends ofpiRNAs. However, the physical interaction between Aub
and AGO3 in Drosophila has not been demonstrated. In thisstudy, we show for the first time that in ovaries, Aub and
AGO3 are able to be simultaneously associated with Tud.
Even in tud mutants, Aub and AGO3 interacted with eachother, indicating that Tud is not constantly required for the
association between Aub and AGO3. However, in tud mutants, Aub does not accumulate in the nuage of nurse cells
and Aub-associated piRNAs showed a clear difference inpopulation from those of the wt control. Thus, Tud is clearlynecessary for maintaining the proper association of piRNAs
with PIWI proteins. It should be emphasized that Tud function does not equate to that of Tdrd1 in mice, because Mili
piRNAs devoid of Tdrd1 accept the entry of abundant cellulartranscripts, such as rRNA and tRNA, into the piRNA pathway(Reuter et al, 2009).
The heteromeric complexes consisting of Tud, Aub, and/or
AGO3 held piRNA precursor-like molecules a few hundred nt
long. This size range suggests that they do not correspond to
the primary precursors of piRNAs or to the primary transcripts of transposons. However, the specificity shown by thenorthern blot analysis indicates that they might correspond topiRNA precursors that are ready to be processed by Aub andAGO3. This was further confirmed by RT–PCR experiments
using primers for certain transposon transcripts. These findings suggest that Tud stringently scrutinizes RNAs for their
specific features and passes them to PIWI proteins that arephysically associated with Tud. Thus, the cooperation ofdPRMT5 with Tud can be envisaged as a two-step regulatory
unit for PIWI function in germline cells. The requirement of
Tud in the primary piRNA processing pathway remains
unclear, and is under investigation in our laboratory.

Materials and methods
Trypsin digestion and LC-MS/MS analysis
The method for trypsin digestion of protein has been describedearlier (Fujinoki et al, 2003). LC-MS/MS analysis was performed& 2009 European Molecular Biology Organization

Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et alusing a LTQ orbitrap XL electron transfer dissociation (ETD) mass
spectrometer (Thermo Fisher Scientific). The methods used for
LC-MS/MS were slightly modified from those described earlier(Fujii et al, 2004). The mass spectrometer was operated in a datadependent acquisition mode in which the MS acquisition with amass range of m/z 420–1600 was automatically switched to MS/MS
acquisition under the automated control of Xcalibur software. The
top four precursor ions in an MS scan were selected by Orbitrap,with resolution R ¼ 60 000 and in subsequent MS/MS scans by ion
trap in the automated gain control (AGC) mode where the AGC
values were 5.00 105 and 1.00 104 for full MS and MS/MS,
respectively. To analyse dimethylation sites, ETD was used.

anti-Spn-E monoclonal antibodies, the N-terminal region of Spn-E(150 amino acids), fused with a GST peptide, was produced in E.
coli and used to immunize mice. To produce anti-Tud monoclonal
antibodies (Figures 3C and 5A), the C-terminal region of Tud(amino acids 2189-stop codon), fused with a His peptide, wasproduced in E. coli and used to immunize mice. The plasmid for
His-tagged recombinant Tud was a kind gift from K HanyuNakamura (Kobe-RIKEN CDB). Hybridomas producing anti-Spn-E
and anti-Tud monoclonal antibodies were prepared essentially as
described earlier (Ishizuka et al, 2002). Anti-sDMA antibody(SYM11) was purchased from Upstate and used according to the
manufacturer’s instructions.

Database searching and protein identification
Database searches were performed using the MASCOT searchengine (Matrix Science) against the NCBInr_20090513 database(selected for Drosophila), assuming trypsin as the digestion enzyme
and allowing for trypsin specificity of up to four missed cleavages.
The database was searched with a fragment ion mass tolerance of0.60 Da and a parent ion tolerance of 3.0 p.p.m. The iodoacetamide
derivative of cysteine was specified as a fixed modification andmethylation of arginine, oxidation of methionine, dimethylation ofarginine and acetylation of N-termini were specified as variable
modifications. Scaffold (version Scaffold_2_02_03; Proteome Software) was used to validate MS/MS-based peptide and protein
identifications. We accepted the peptide identifications when thePeptide Prophet algorithm (Keller et al, 2002) specified probabilities
at 495.0%. Sequence coverage was defined as percentage of theprotein in the identified peptide sequence.

Immunoprecipitation
Immunoprecipitation was performed using anti-Aub, anti-AGO3(polyclonal), and anti-Tud antibodies immobilized on Dynabeads
ProteinG (Invitrogen). The buffer used was Binding buffer. In
Figures 2D and 4A, 1 ml ovary lysate (4 mg/ml) was used per
immunoprecipitation. In Figures 2C and 5A and B, 1 ml ovary lysate(2 mg/ml) was used per immunoprecipitation. After incubation
with ovary lysate, the bead fractions were extensively washed with
Binding buffer and the protein pools bound to the beads were eluted
with SDS–PAGE sample buffer. After heating to 701C for 10 min,protein samples were run on SDS–PAGE gels and processed forwestern blot analysis. In all, 2% of the ovary lysate used for each
immunoprecipitation was run as an input sample. Anti-AGO3
polyclonal antibodies were generated by immunizing mice with a
His-tagged recombinant AGO3 peptide (amino acids 1–289) that
had been produced in E. coli.

Drosophila strains
The yellow white (y w) strain was used as a wild-type strain. The
dprmt5 allele used was csule00797 (a kind gift from AG Matera). The
PBac{RB}csule00797 insertion is located within exon 2, which
generates a protein without the methyltransferase domain (Gonsalvez
et al, 2006). The tud allele used was tud1 bw sp/CyO l(2)DTS5131(Drosophila Genetic Resource Center stock number; 106505). tud1 hasa stop codon (Lys1036UAG) mutation, which effectively abolishes
protein expression, presumably because of the instability of theTud fragment or because of degradation of tud mRNA throughnonsense-mediated RNA decay (Arkov et al, 2006). All stocks weremaintained at 251C.

Northern blot analysis
Immunoprecipitation was performed using anti-Aub and anti-AGO3(polyclonal) antibodies immobilized on Dynabeads ProteinG(Invitrogen). The buffer used was Binding buffer. In Figure 4B,
1 ml ovary lysate (4 mg/ml) was used per immunoprecipitation. In
Figures 5B and C and 6A and B, 1 ml ovary lysate (2 mg/ml) wasused per immunoprecipitation. After immunoprecipitation, total
RNAs were isolated from the immunopurified complexes by phenol:
chloroform extraction and precipitated with ethanol. RNAs were
dephosphorylated with CIP (NEB) and labelled for visualization
with [g-32P]ATP by T4 polynucleotide kinase (TaKaRa). Total RNAs
from either y w, tud, or dprmt5 ovaries were isolated using ISOGEN(Nippon Gene). Total RNAs (10 mg) were used in Figures 6 and 7B.
Northern blot analysis was performed as reported earlier (Saito
et al, 2006). The probe used to recognize roo#4 piRNA was as
described earlier (Nishida et al, 2007). Probes used in Figure 4B todetect antisense roo piRNAs were as follows:

Pull-down assay
Aub, AGO3-1, and AGO3-2 peptides, with and without sDMA
modification, were synthesized by Operon. All peptides were
biotinylated at their N-terminal ends. The synthesized peptides(2 mg) were bound with 30 ml of streptavidin Dynabeads (Invitrogen) and incubated for 2 h with ovary lysates at 41C. Ovaries lysates(from about 200 ovaries) were prepared by crushing in 200 ml ofBinding buffer [30 mM HEPES-KOH (pH 7.3), 150 mM KOAc, 5 mM
MgOAc, 5 mM DTT, 0.1% NP-40, 2 mg/ml Pepstatin, 2 mg/ml
Leupeptin, and 0.5% Aprotinin]. After centrifugation at 14 000 g
for 1 min at 41C, the supernatant was transferred to a new
microcentrifuge tube and kept on ice. The pellet was then crushed
again in 200 ml of Binding buffer. After centrifugation as above, thesupernatant was combined with the first supernatant and kept on
ice. These steps were repeated several times. The protein
concentration of the lysates was adjusted each time to 4 mg/ml
with Binding buffer. After incubation with 1 ml of ovary lysate, thebead fractions were extensively washed with Binding buffer and theprotein pools bound to the beads were eluted with SDS–PAGE
sample buffer. After heating to 701C for 10 min, protein samples
were run on SDS–PAGE gels and either silver-stained or processed
for western blot analysis. In all, 2% of the ovary lysate used foreach pull-down assay was run as an input sample.
Western blot analysis
Western blot analysis was performed as described earlier(Miyoshi
et al, 2005). Anti-Aub monoclonal antibody (Nishida et al, 2007)
was used at 1 mg/ml. Culture supernatant of hybridomas producing
anti-AGO3 monoclonal antibody (Gunawardane et al, 2007) wasused without dilution as a primary antibody. Anti-tubulin antibody
was obtained from the Developmental Studies Hybridoma Bank andused at 1:1000 dilution. Anti-Tud polyclonal antibodies were kindgifts from K Hanyu-Nakamura (Kobe-RIKEN CDB) and P Lasko(McGill University) and were used at 1:2000 dilution. To produce& 2009 European Molecular Biology Organizationroo#1 piRNA-as:roo#3 piRNA-as:roo#7 piRNA-as:
roo-primer-1701:
roo-primer-1731:
roo-primer-1761:
roo-primer-1791:

50 -TGGGCTCCGTTCATATCTTATG-30
50 -TGAGAGTTCGCTATTCGAAGAA-30
50 -TCTGAGGCATCCGTTTGGTAAA-30
50 -TATCTAGAAGATACGTCTAAACTAATAGAC-30
50 -AGTAGTCCAGATTTCCTTAAAATAAGGAAA-30
50 -AATAAAATTGAATTTTTATGGCATAAAATA-30
50 -GATAACCTGATTGAACAGGTGAATAGTCGT-30 .

The probe used to detect minisatellite#1 piRNA (Figure 6D) was
50 -TCGTGTATTGTCTTTTTGGGTTTGCG-30 .
The DNA fragments used to detect piRNAs originating from theI-element retrotransposon (accession number: M14954.2) weregenerated by PCR of Drosophila melanogaster genomic DNA withthe primers:
I-element-for: 50 -CAAGCAGAATACGATCGCTA-30
I-element-rev: 50 -TGGTCCAATTTGGGTGGGAT-30 . Amplified fragments were then cloned into pBS SK þ .
PCR was again performed using T7 and T3 promoter sequence
primers, and the PCR products were used as templates for in vitrotranscription using a MAXIscript T7 kit (Ambion) in the presence of
32
P-UTP. Transcribed RNAs were extracted with phenol:chloroform,
precipitated with ethanol, and partially hydrolyzed as describedearlier (Saito et al, 2006).
Northern blots for HeT-A, DM297, DMRT1B, and R1DM piRNAs(in Figure 6) were performed according to Pall and Hamilton(2008). Probes used were as follows:
HeT-A-2701:
HeT-A-2801:

50 -CTGCAGCCAAGCGGGATTTA-30
50 -TGCGGCACCCTGTGTCCCGG-30
The EMBO Journal

VOL 28 | NO 24 | 2009 3829

Functional involvement of Tud and dPRMT5 in Drosophila germlines
KM Nishida et al

HeT-A-2902:
DM297:DMRT1B:
R1DM:

50 -GCGCGACTTCCAACTTTGTAACTC-30
50 -TAGTCTTAAGCTGAGATCCAAAGAA-30
50 -GGGCAGAGTGCCAACACAAATGCT-30
50 -CACGGGTTGAGCAGCTATCCAAGA-30 .

The DNA fragments used to detect pre-piRNAs originating fromthe roo retrotransposon (Figure 4B, right) were generated by PCR of
D. melanogaster genomic DNA with the primers:
T7-roo-for:T3-roo-rev:

50 -TAATACGACTCACTATAGGGATATCTAGAAGATACGTCTA-30
50 -AATTAACCCTCACTAAAGGGCCGCCTTAACAACCGTCGAC-30 .

The PCR products were used as template for in vitro transcription
using a MAXIscript T3 kit (Ambion) in the presence of 32P-UTP.
Hybridizations were performed at 651C in 0.2 M sodium phosphate(pH 7.2), 7% SDS and 1 mM EDTA and washed at 651C in 2  saline
sodium citrate and 0.1% SDS.
Immunohistochemistry
Ovaries were dissected manually from adult flies in 1  PBS.
Immunostaining was performed following standard procedures. For
tud1, anti-Aub (1:1000 dilution) and anti-Tud (1:2000 dilution)
antibodies were used as primary antibodies. Alexa 488-conjugatedanti-rabbit IgG (Molecular Probes) (1:1000 dilution) and Cy3conjugated anti-mouse IgG (Sigma) (1:100 dilution) were used as
the secondary antibodies. For dprmt5 staining, anti-Aub antibody
was directly labelled using a Fluorescein Labeling kit-NH2 (Dojindo
Molecular Technologies). Anti-Tud antibody was used at a dilutionof 1:500. Cy3-conjugated anti-rabbit IgG (Sigma) (1:100 dilution)
was used as the secondary antibody. DNA was stained with DAPI.

All images were collected using a confocal microscope (Zeiss LSM5EXCITER).
Supplementary data
Supplementary data are available at The EMBO Journal Online(http://www.embojournal.org).

Acknowledgements
We thank AG Matera, K Hanyu-Nakamura, A Nakamura, andP Lasko for providing reagents. We also thank the Drosophila
Genetic Resource Center for kindly supplying Drosophila strains.
For assistance with MS analysis, we acknowledge I Sagawa at theInstitute of Health Biosciences University of Tokushima. We thank K
Saito and K Miyoshi for technical advice and assistance, and othermembers of the Siomi laboratory for discussions and comments on
the paper. This work was supported by MEXT (Ministry of education, Culture, Sports, Science, and Technology) grants to HS, andNEDO (New Energy and Industrial Technology Development
Organization) grants to MCS. MCS is supported by CREST (Core
Research for Evolutional Science and Technology) from JST (Japan
Science and Technology Agency). MKN is supported by JSPS (Japan
Society of the Promotion of Science). MCS is an Associate Professorof the Global Center of Excellence for Human Metabolomics
Systems Biology, funded by MEXT.

Conflict of interest
The authors declare that they have no conflict of interest.

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