Article

Krimper Enforces an Antisense Bias on piRNA Pools
by Binding AGO3 in the Drosophila Germline
Graphical Abstract

Authors
Kaoru Sato, Yuka W. Iwasaki,
Aoi Shibuya, ..., Hirotsugu Ishizu,
Mikiko C. Siomi, Haruhiko Siomi

Correspondence
siomim@bs.s.u-tokyo.ac.jp (M.C.S.),
awa403@keio.jp (H.S.)

In Brief
Sato et al. show that Krimper, a Tudordomain protein, interacts withunmethylated AGO3 to promote AGO3
methylation, localization to nuage, and
Aub-AGO3 piRNA amplification. Krimperalso blocks primary piRNA loading onto
AGO3, enforcing an antisense bias on thepiRNA pool.

Highlightsd

Krimp binds unmethylated AGO3 to ensure sDMA
modification and heterotypic ping-pongdLoss of Aub allows primary piRNA loading onto AGO3d

Loss of Krimp in OSC cells allows AGO3 primary piRNAloadingd

Krimp blocks access of AGO3 to primary piRNAs enforcing anantisense bias on piRNAs

Sato et al., 2015, Molecular Cell 59, 553–563
August 20, 2015 ª2015 Elsevier Inc.
http://dx.doi.org/10.1016/j.molcel.2015.06.024

Accession Numbers
GSE69293

Molecular Cell

ArticleKrimper Enforces an Antisense Biason piRNA Pools by Binding AGO3
in the Drosophila Germline
Kaoru Sato,1,4 Yuka W. Iwasaki,2,4 Aoi Shibuya,2 Piero Carninci,3 Yuuta Tsuchizawa,1 Hirotsugu Ishizu,1 Mikiko C. Siomi,1,*
and Haruhiko Siomi2,*
1Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan
2Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan
3RIKEN Center for Life Science Technologies, Division of Genomic Technologies, RIKEN Yokohama Campus, Yokohama 230-0045, Japan
4Co-first author*Correspondence: siomim@bs.s.u-tokyo.ac.jp (M.C.S.), awa403@keio.jp (H.S.)
http://dx.doi.org/10.1016/j.molcel.2015.06.024

SUMMARY

Piwi-interacting RNAs (piRNAs) suppress transposon
activity in animal germ cells. In the Drosophila ovary,
primary Aubergine (Aub)-bound antisense piRNAs
initiate the ping-pong cycle to produce secondary
AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that
can act to destroy transposon mRNAs. Here weshow that Krimper (Krimp), a Tudor-domain protein,
directly interacts with piRNA-free AGO3 to promote
symmetrical dimethylarginine (sDMA) modification,
ensuring sense piRNA-loading onto sDMA-modified
AGO3. In aub mutant ovaries, AGO3 associates withping-pong signature piRNAs, suggesting AGO30 s
compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp
bodies in cultured ovarian somatic cells (OSCs), in
which only the primary piRNA pathway operates.
Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs,
further showing the capacity of AGO3 for primarypiRNA loading. We propose that Krimp enforces anantisense bias on piRNA pools by binding AGO3 andblocking its access to primary piRNAs.

INTRODUCTION
In most animals, the PIWI clade of Argonaute family proteins isrequired for fertility and silencing of germline transposable elements (TEs) (Ghildiyal and Zamore, 2009; Ishizu et al., 2012;
Juliano et al., 2011; Kim et al., 2009; Siomi et al., 2011). PIWI proteins are loaded with Piwi-interacting RNAs (piRNAs) of 25 to 35 nt
in length to form piRNA-induced silencing complexes (piRISCs). In
these, piRNAs guide the repressive activities of PIWI proteins toward TEs by means of base pairing to silence TEs and thereby
maintain genome integrity in the gonad (Siomi et al., 2011).
Two main pathways exist to generate piRNAs in the Drosophila
ovary: the primary processing pathway and the ping-pong ampli-

fication cycle (Brennecke et al., 2007; Gunawardane et al., 2007;
Ishizu et al., 2012; Malone et al., 2009; Siomi et al., 2011). In the
primary processing pathway, which operates in both germline
cells and surrounding somatic cells, the majority of piRNAs
are produced from long single-stranded RNAs, which are transcribed from TE-rich genomic regions called piRNA clusters.
The majority of primary piRNAs are antisense with respect to
TE coding strands. These piRNAs guide TE silencing in trans(Malone et al., 2009; Saito et al., 2009; Yamanaka et al., 2014).
Studies in cultured ovarian somatic cells (OSCs) revealed thatthe somatic primary pathway is a linear pathway in which piRNA
precursors are cleaved into intermediates by one or more nucleases, including Zucchini (Zuc). These intermediates are loaded
exclusively onto Piwi in the cytoplasmic Yb bodies, and subsequently, their 30 ends are defined, probably by trimming, andmethylated, yielding mature piRISCs, which are then transported
into the nucleus to exert TE silencing (Haase et al., 2010; Ipsaro
et al., 2012; Ishizu et al., 2012; Olivieri et al., 2010; Saito et al.,2010; Qi et al., 2011; Nishimasu et al., 2012). The primary processing pathway in germline cells produces piRNAs that areloaded onto both Piwi and Aub but not AGO3 (Brennecke
et al., 2007; Li et al., 2009; Malone et al., 2009; Siomi et al.,2011). Although primary piRNAs in both germline and surrounding somatic cells show a strong antisense bias (with respect to
TE coding strands) and exhibit a preference for a 50 uridine(1U) residue (Saito et al., 2006; Brennecke et al., 2007; Gunawardane et al., 2007; Malone et al., 2009; Siomi et al., 2011), theprimary piRNA pathways in the two cell types do not seem tobe identical, because the requirement of piRNA factors differs
between the two. How primary piRNAs in germline cells are produced and how these sequence and strand biases are enforced
remains to be elucidated.
The ping-pong cycle operates specifically in germline cells
and engages AGO3 and Aub, both of which accumulate in thenuage, perinuclear structures found at the cytoplasmic face ofthe nuclear envelope in animal germline cells (Brennecke et al.,2007; Gunawardane et al., 2007; Lim and Kai, 2007; Nishida
et al., 2007). Multiple components of the piRNA pathway areenriched in the nuage, causing this structure to be implicatedin piRNA production and TE silencing (Lim and Kai, 2007). Piwi
does not participate in this pathway, probably because of its
Molecular Cell 59, 553–563, August 20, 2015 ª2015 Elsevier Inc. 553

sequestration into the nucleus upon piRNA loading (Brennecke
et al., 2007; Gunawardane et al., 2007; Saito et al., 2010). The
pathway depends on the endoribonuclease or Slicer activities
of AGO3 and Aub, which cleave antisense and sense transcripts
of both piRNA clusters and active TEs to produce two classes ofpiRNAs: piRNAs associated with Aub, which are derived mainly
from the antisense strands of TEs and show a strong preference
for U at their 50 ends, and piRNAs associated with AGO3, whicharise mainly from the sense strands and show a preference foradenine (A) at nucleotide 10 (10A) but no 50 nucleotide preference(Brennecke et al., 2007; Gunawardane et al., 2007; Siomi et al.,2011). The two classes of piRNAs overlap by precisely 10 nt attheir 50 ends, which is evidence of piRNA amplification by reciprocal cycles of Slicer-mediated cleavage (Brennecke et al., 2007;
Gunawardane et al., 2007; Siomi et al., 2011). These features ofpiRNAs (1U/10A partners with a 10 nt, 50 overlap) are often
referred to as the ping-pong signature (Malone et al., 2009).
Both AGO3 and Aub act catalytically, thereby mediating not
only repeated rounds of piRNA production but also post-transcriptional silencing of TEs by consuming TE transcripts (Brennecke et al., 2007; Gunawardane et al., 2007). The ping-pong
pathway is believed to be primed by both maternally deposited
Aub-bound piRNAs and zygotic primary piRNAs that are loaded
onto Aub (Brennecke et al., 2007, 2008; Gunawardane et al.,2007; Siomi et al., 2011).
Amplification of the silencing signal in the ping-pong cycle requires a system that acts to enforce an antisense bias on piRNA
pools to efficiently silence TEs by cleaving TE sense transcripts
or mRNAs. Indeed, sense piRNAs are less abundant and disproportionately bound to AGO3, which enforces the characteristic
antisense bias of Aub-bound piRNAs. This increases the numberof piRNAs that can act to destroy TE mRNAs (Gunawardane
et al., 2007; Huang et al., 2014; Li et al., 2009). There are at least
two possible scenarios for how piRNA pools in AGO3 complexes
display a sense bias. First, AGO3 is actively prevented from being loaded with primary piRNAs. Second, the primary pathway
is inherently incompatible with AGO3 loading. A recent study
suggests that the latter may be the case (Olivieri et al., 2012);
however, several findings argue against this scenario. For
example, ectopic expression of AGO3 in OSCs increases theexpression of the gypsy transposon, which is normally silenced
by the primary piRNA processing pathway in OSCs (Li et al.,2009). This has been interpreted to mean that the primarypathway is compatible with AGO3 loading, meaning that
AGO3 can compete with Piwi for piRNAs, but unlike Piwi, itcannot act directly to silence TEs. Furthermore, a large numberof piRNAs with exactly the same sequences, derived from antisense strands of two loci, Su(Ste) and AT-chX, are associatedwith both AGO3 and Aub in the fly testis (Nagao et al., 2010).
This also implies that AGO3 can be loaded with the same primaryand secondary piRNAs that are loaded onto Aub. These results
suggest that AGO3 is actively prevented from being loadedwith primary piRNAs in the fly ovary. Thus, it is tempting to speculate that a factor(s) exists that interacts with AGO3 to blockloading of primary piRNAs onto AGO3.
Besides AGO3 and Aub, the ping-pong cycle in the fly ovary
requires the functions of many other factors, including the RNA
helicases Spindle-E (Spn-E) and Vasa and the Tudor domain
554 Molecular Cell 59, 553–563, August 20, 2015 ª2015 Elsevier Inc.

proteins Tudor (Tud), Tejas (Tej), Qin/Kumo, and Krimper (Krimp),
as well as Maelstrom (Mael), a component of the microtubuleorganizing center (Lim and Kai, 2007; Malone et al., 2009; Nishida et al., 2009; Patil and Kai, 2010; Anand and Kai, 2012; Satoet al., 2011; Zhang et al., 2011, 2014). All of these factors accumulate in the nuage (Findley et al., 2003; Lim and Kai, 2007; Malone et al., 2009; Nishida et al., 2009; Patil and Kai, 2010; Anandand Kai, 2012; Zhang et al., 2011; Sato et al., 2011). Amongthem, proteins that contain multiple Tudor domains are known
to possess the potential to interact simultaneously with several
proteins through symmetrical dimethylarginines (sDMAs) andto function as a scaffolding platform for the assembly of multiprotein complexes (Chen et al., 2011; Siomi et al., 2011). Thismight be particularly important for the ping-pong mechanism,
which requires close association between two PIWI proteins(Nishida et al., 2009). Here, we show that Krimp, a Tudor-domain
protein, specifically interacts with AGO3. AGO3 in Krimp-associated complexes is mainly in a non-sDMA form and free frompiRNAs. In krimp mutant ovaries, AGO3 is not sDMA modified.
Loss of Krimp has pronounced impacts on piRNA levels andping-pong signatures; a striking depletion of AGO3-associatedpiRNAs manifests, Aub-Aub homotypic ping-pong prevails,
and there is a relative increase in Aub-bound sense piRNAs. In
aub mutant ovaries, piRNAs with ping-pong signatures are still
loaded onto AGO3. This suggests that primary piRNAs can beloaded onto AGO3, although their AGO3 loading is normally
blocked in the presence of Aub. In OSCs, where only the primarypathway operates and neither AGO3 nor Aub are expressed(Saito et al., 2009), Krimp accumulates at cytoplasmic granules
termed Krimp bodies, and transfected AGO3 is sequesteredinto these bodies (Olivieri et al., 2012). However, interestingly,
upon krimp RNAi in OSCs, AGO3 becomes dispersed in thecytoplasm and loaded with primary piRNAs. Together, thesefindings suggest a model in which Krimp prevents AGO3 frombecoming loaded with primary piRNAs, thereby enforcing anantisense bias on piRNA pools in the ping-pong cycle.
RESULTS
Krimp Binds to Unmethylated AGO3
In krimp mutant ovaries, AGO3 loses its ability to localize to the nuage (Nagao et al., 2011), while Aub remains accumulated in thenuage as in the control (Lim and Kai 2007; Nagao et al., 2011).
These findings prompted us to examine if Krimp has a specificphysical interaction with AGO3 in the ovary. We immunopurified
Krimp-associated complexes from ovary lysates and determined
if AGO3 was present in the complexes. As shown in Figure 1A,
Krimp co-purified with AGO3, but not with other PIWI proteins,suggesting a specific interaction of Krimp with AGO3.
Krimp is a Tudor-domain protein. PIWI proteins containsDMAs, which are incorporated by the dPRMT5/Capsuleen/
Dart5 enzyme and which can be recognized by Tudor-domain
proteins (Kirino et al., 2009; Nishida et al., 2009). Thus, we wanted
to determine if the association of Krimp with AGO3 is sDMA modification dependent. To this end, we produced a monoclonal
antibody that specifically recognizes sDMA-containing AGO3(sDMA-AGO3) (Figures S1A and S1B). Using the sDMA-specific antibody, we compared the amounts of sDMA-AGO3 inFigure 1. Krimp Specifically Associateswith AGO3 in a sDMA-Independent Manner(A) Krimp-IP complexes from Oregon-R ovary
lysate were immunoreacted using specificantibodies against Piwi subfamily proteins. ‘‘n.i.’’
indicates non-immune IgG used as a negative
control.
(B) Krimp- and AGO3-IP complexes from OregonR ovary lysate were immunoreacted using antibodies against AGO3 and sDMA-modified AGO3(sDMA-AGO3). The ratio of sDMA-AGO3/AGO3
signal intensity was calculated.
(C) Krimp-IP complexes from ovary lysates ofOregon-R and dart5 mutant were immunoreactedusing antibodies against Krimp and AGO3 (leftpanel). Ovary lysates of Oregon-R and dart5
mutant were immunoreacted using antibodies
against AGO3 and sDMA-AGO3 (right panel). The
ratio of sDMA-AGO3/AGO3 signal intensity wascalculated.
(D) GST pull-down assay using recombinant GST,
GST-AGO3, and Krimp.
(E) Summary of the functional domains required
for the Krimp-AGO3 interaction (for more detail,see Figures S1E–S1I). See also Figure S1 and S2.

immunoprecipitates obtained with an anti-Krimp antibody and ananti-AGO3 antibody that recognizes both sDMA and unmethylated AGO3. The results showed that AGO3 in Krimp complexes
was mostly devoid of sDMA modification (Figure 1B). We further
found that Krimp interacted with AGO3 in dart5 mutant ovaries(Figure 1C), where AGO3 was not sDMA-modified (Figure 1C).
To confirm these results, we performed glutathione S-transferase(GST) pull-down assays using GST-AGO3 and Krimp (nontagged) proteins expressed in and purified from E. coli. GSTAGO3 purified from E. coli was not sDMA-modified (Figure S1A).
Krimp expressed in E. coli specifically associated with GSTAGO3, but not with GST itself (Figure 1D). Together, these results
show that Krimp is capable of interacting with unmethylated
AGO3 and also show that this interaction is direct, not requiringother factors.
To corroborate these findings, we performed GST pull-down
experiments with synthetic oligo-peptides with/without sDMA
modification (AGO3-2 and AGO3-2-sDMA) (Nishida et al., 2009).
Only an unmodified (AGO3-2) but not a sDMA-modified (AGO32-sDMA) oligo-peptide bound to GST-Krimp (Figure S1C),

showing that the Krimp interaction is sensitive to the methylation status of AGO3.
We also performed peptide pull-down experiments using ovary lysates and foundthat Tud interacts only with AGO3-2sDMA (Figure S1D), confirming our previous findings that Tud interacts specificallywith sDMA-modified AGO3 (Nishida et al.,2009). Krimp in ovary lysates interacts
with both AGO3-2 and AGO3-2-sDMA(Figure S1D). Together these results suggest that Krimp initially forms a complex
with unmethylated AGO3, in whichsDMA modification of AGO3 occurs. sDMA modified AGO3 thendissociates from Krimp to interact with Tud.
We undertook domain mapping of both AGO3 and Krimp
using GST fusion proteins and a series of in-vitro-translateddeletion products to identify the regions responsible for theirinteraction. Krimp is predicted to contain N-terminal coiled-coildomains, a zinc finger, and a canonical Tudor domain (Figures
1E and S1E). We found that the C-terminal region containing
the canonical Tudor domain of Krimp is required for the interaction with non-sDMA-modified AGO3 (Figure S1F). A series of
AGO3 deletion products revealed that an N-terminal portion containing 82 aa is required for the interaction with Krimp (Figures
S1G–S1I). These results demonstrate that AGO3 directly interacts with the C-terminal Tudor domain-containing region of
Krimp through its N-terminal region, which contains sDMAmodifiable arginine residues (Figure 1E) (Nishida et al., 2009).
Krimp-Associated AGO3 Is Devoid of piRNAs
Recent findings indicate that Krimp has no significant impact
on primary piRNA biogenesis but is required for the nuage
Molecular Cell 59, 553–563, August 20, 2015 ª2015 Elsevier Inc. 555

Figure 2. AGO3 Coimmunoprecipitatedwith Krimp Is Not Loaded with piRNAs(A) Krimp- and AGO3-IP complexes from
Oregon-R ovary lysate were immunoreactedusing an antibody against AGO3. The amount of
Krimp-associated AGO3 was approximately
equal to that of AGO3 immunoprecipitated with
anti-AGO3 after a 1:6 dilution. ‘‘n.i.’’ indicates nonimmune IgG used as a negative control.
(B and C) Quantification of piRNAs in the antiKrimp and anti-AGO3 IP complexes by northernblot analysis (B) and real-time PCR (C). Minisatellite#1, AGO3-pilger, and MaxEllement are
AGO3-bound piRNAs (Gunawardane et al., 2007).
idefix piRNA is a Piwi-bound somatic piRNA (Saito
et al., 2009). miR-310, let-7, and bantam are
miRNAs. N = 3; error bars indicate SEM.

localization of AGO3, suggesting a specific role in the ping-pongcycle (Malone et al., 2009; Zhang et al., 2011). Our findings that
Krimp preferentially interacts with unmethylated AGO3 suggestthat Krimp may well regulate sDMA modification of AGO3 and,
subsequently, the ping-pong cycle.
We first addressed whether AGO3 in Krimp complexes isloaded with piRNAs. We compared the amounts of piRNAs in
anti-Krimp and anti-AGO3 immunoprecipitates. To equalize
AGO3 quantities, the anti-AGO3 immunoprecipitates weresequentially diluted before western blot analysis. The results
showed that a 1:6 dilution equalized the amount of AGO3 inthe anti-AGO3 and anti-Krimp immunoprecipitates (Figure 2A).
We then performed northern blot analysis to examine the amountof minisatellite#1 piRNA in the complexes. Minisatellite#1 piRNA,a sense piRNA known to be specifically loaded onto AGO3 (Nishida et al., 2009), was detected in the AGO3 complexes (which
had been diluted 1:6 before analysis) but not in the Krimp complexes (Figure 2B). Additionally, piRNA detection by qRT-PCR
revealed that other piRNAs that are loaded onto AGO3 are notpresent in anti-Krimp immunoprecipitates (Figure 2C). These results show that most AGO3, in a form associated with Krimp, is
not loaded with piRNAs. Thus, Krimp interacts with AGO3 before
AGO3 enters the ping-pong cycle, which likely occurs in thenuage.
Krimp Is Required for sDMA Modification of AGO3 andSecondary piRNA Loading
AGO3 in Krimp complexes is mostly not sDMA modified and freefrom piRNAs. We thus asked if AGO3 is sDMA modified in krimp
mutants. We found that the majority of AGO3 is not sDMA modified (Figures 3A and S2A), but that both Aub and Piwi remain
sDMA modified in krimp ovaries (Figures S2B and S2C), which
556 Molecular Cell 59, 553–563, August 20, 2015 ª2015 Elsevier Inc.

suggests that Dart5 is functional in
krimp mutants. As a consequence,sDMA-dependent interaction between
AGO3 and Tud is impaired in krimp
mutant ovaries, where Aub still interacts
with Tud (Figures S2D and S2E). These
results suggest that Krimp is required
for sDMA modification of AGO3. We
found no significant association of Krimper with Dart5 and Valois, which are components of arginine-methylation complexes(methylosomes), (Anne and Mechler, 2005) (Figures S2F and
S2G), suggesting that Krimp-AGO3 complexes only transiently
interact with methylosomes. Because AGO3 lost its ability tolocalize to the nuage in krimp mutant ovaries (Malone et al.,2009; Nagao et al., 2011; Zhang et al., 2011), Krimp is likely topromote sDMA modification of AGO3 by localizing AGO3 in nuage where methylosome components are also accumulated(Anne and Mechler, 2005).
The requirement of Krimp in the sDMA modification of AGO3
prompted us to ask how piRNA loading onto individual PIWI proteins is affected in krimp mutant ovaries. We immunopurified
AGO3, Aub, and Piwi from control (krimp heterozygous, krimp/
CyO) and krimp mutant (trans-heterozygous, krimp/Df) ovaries(Figure S3) and examined the piRNAs that are associated withequivalent amounts of each protein (Figure 3B). Although theamounts of piRNAs associated with Piwi were only slightly
reduced in krimp mutant ovaries, Aub- and AGO3-bound piRNAs
were markedly reduced. In particular, almost no AGO3-associated piRNAs were detected in krimp mutant ovaries. Thus, ourdata show that Krimp has a huge impact on the abundance of
germline piRNAs bound to Aub/AGO3, especially their binding
to AGO3. Our results also explain the previous findings showingthat loss of krimp causes a substantial reduction in overall piRNA
populations and also results in a slight shift in size toward longer
piRNAs, characteristic of Piwi-bound piRNAs (Malone et al.,2009).
Loss of Krimp Permits Aub-Aub Homotypic Ping-Pong
We then performed deep sequencing of AGO3/Aub/Piwi-boundpiRNAs in control and krimp mutant ovaries and compared them

Figure 3. krimp Is Required for sDMA Modification and the Aub-AGO3 HeterotypicPing-Pong Amplification Loop
(A) The level of AGO3 sDMA modification in the
krimp mutant. AGO3-IP complexes from ovary
lysates of control (krimp/CyO) and krimp mutant(krimp/Df) were immunoreacted using antibodies
against AGO3 and sDMA-AGO3. The ratio ofsDMA-AGO3/AGO3 signal intensity was calculated. (B) IPs were performed from ovary lysates ofkrimp/CyO and krimp/Df using antibodies against
each Piwi subfamily protein. Equal amounts ofRNA molecules were isolated from complexes
shown in (Figure S3) and then 32P labeled.
(C) Strand-bias was compared with Piwi-bias foreach piRNA associated with Aub in krimp/CyO
and krimp/Df. The plots are colored according totheir Piwi-bias; piRNAs bound to Piwi indicating
primary piRNAs (higher Piwi-bias; blue dots),piRNAs bound to Aub/Ago3 indicating ping-pongpiRNAs (lower Piwi-bias; red dots), and piRNAs
with intermediate Piwi-bias (white dots).
(D) Heatmaps of the ping-pong signature (leftpanel) and ping-pong z score (right panel)indicating the degree to which complementary 50
10-mers are found in pairwise library comparisons, with a key to the intensity of the signal
shown below. Ping-pong signals were frequently
observed between Aub and AGO3 in krimp/CyO,
whereas Aub-Aub homotypic ping-pong wasobserved to prevail in krimp/Df.
(E) Sequence logos of Piwi-, Aub-, and AGO3bound total piRNAs in krimp/CyO and krimp/Df.
See also Figures S3 and S4.

with piRNAs in total small RNAs (Figures 3C–3E and S4; Table
S1). Total small RNA libraries were normalized to endo-siRNAreads, and AGO3/Aub/Piwi-IP libraries were normalized to identical numbers of reads. We first confirmed that loss of krimp results in both a substantial reduction in overall piRNA populations
and a shift in size toward Piwi-bound piRNAs (Malone et al.,2009) (Figure S4A). For the piRNAs mapped to TEs, loss of krimp

affected the populations bound to Aub/
AGO3 rather than those bound to Piwi
(Figures S4B–S4D).
We then quantified the ping-pong
signature (10 nt overlap at their 50 ends)
of krimp heterozygous and mutant piRNA
reads mapped to TEs by calculating
the likelihood of piRNAs to have a complementary ping-pong partner with theindicated overlapping base pairs (Figure S4E). A strong ping-pong signature
among piRNAs, which correlates with
germline silencing, was observed inpiRNAs from krimp heterozygous flies.
Interestingly, piRNAs isolated from krimp
mutant ovaries also showed a weak but
evident ping-pong signature. From thedata showing that AGO3 piRNAs arealmost completely ablated in krimp
mutant ovaries (Figure 3B), we hypothesized that this may becaused by the ability of Aub piRNAs to engage in piRNA amplification by homotypic Aub-Aub cycling. The disruption of Aub/
AGO3 ping-pong amplification was also observed by focusing
on strand bias of piRNAs bound to Aub. The antisense strand
bias of Aub, which was clearly seen in krimp heterozygous files,
could no longer be detected in mutant flies (Figure S4F), and Aub
Molecular Cell 59, 553–563, August 20, 2015 ª2015 Elsevier Inc. 557

started to load sense piRNAs rather than antisense piRNAs. Thiswas particularly observed for piRNAs originating from ping-pong
amplification rather than primary biogenesis, based on the resultthat piRNAs from transposons with a low Piwi-bias were significantly biased toward the sense strand only within krimp mutants
compared with those in heterozygous flies (p < 0.001) (Figure 3C).
In contrast to Aub-bound, ping-pong participating piRNAs
having a bias for the sense strand, the strand bias of Piwi-boundprimary piRNAs remained unchanged (Figure S4F). To test our
hypothesis that the Aub-mediated ping-pong amplification operates in krimp mutant ovaries, we analyzed the complementary
relationships between the first ten0 nucleotides of total small
RNAs and AGO3/Aub/Piwi-bound piRNAs (Figure 3D). In krimp
heterozygous libraries, we observed that >52% (z score >106.5) of small RNAs in the Aub library have complementary
partners in the AGO3 library. In contrast, Aub-AGO3 heterotypicping-pong was not present in krimp mutant ovaries, but significantly, the self-complementarity among Aub-bound piRNAs
was observed in krimp mutant ovaries (>57%, z score = 30.9;
Figure 3D). The identification of a nucleotide bias for piRNAs in
krimp mutants also supports the possibility of Aub-Aub selfping-pong amplification. Primary piRNAs, which are known tobind to both Aub and Piwi (Brennecke et al., 2008), show a strong
antisense bias and typically begin with U (1U). Sense piRNAs
typically bear an A at position 10 (10A) and bind AGO3 (Figure 3E). Although this 1U-bias is seen in the krimp mutant Aub library, the degree was much lower than that in heterozygous flies.
Moreover, a 10A-bias was also observed, indicating that an AubAub homotypic ping-pong amplification loop was active in krimp
mutants (Figure 3E). This shows that Aub-Aub homotypic pingpong dominates in krimp mutants, resulting in an increased
piRNA sense fraction (Figure 3C and Figure S4F), thereby leading
to derepression of TEs (Lim and Kai, 2007). Together, our data
indicate that Krimp is required for the biogenesis of AGO3-boundpiRNAs and piRNAs generated by the Aub-AGO3 ping-pong
amplification loop, while its effects on primary biogenesis and
Aub-Aub homotypic ping-pong are limited.
Primary piRNAs Are Loaded onto AGO3 in the Absenceof AubLoss of aub impairs the production of germline piRNAs, and theremaining piRNAs display no ping-pong cycle (Malone et al.,2009). This suggests that only primary piRNAs are produced,
which are then loaded onto Piwi and/or AGO3 in aub mutant
ovaries. To test if remaining primary piRNAs in aub mutant
ovaries are loaded onto AGO3, we immunopurified AGO3 complexes from aub mutant ovaries. This revealed that small RNAs
were still loaded onto AGO3 in aub mutant ovaries (Figure 4A),
although the amounts of associated small RNAs were reduced.
Sequencing of these AGO3-associated small RNAs further revealed that characteristic features of AGO3-associated piRNAs
are largely lost; they showed a decrease of 10A with a concomitant increase of 1U (Figure 4B). Additionally, piRNAs derived
from both germline-specific dual-stranded piRNA cluster,
42AB, and uni-stranded primary piRNA cluster, Cluster 2, were
loaded onto AGO3 in aub mutant ovaries (Figure 4C). The degreeof ping-pong amplification of AGO3-bound piRNAs mapped to
the 42AB cluster was reduced, compared with control (Fig558 Molecular Cell 59, 553–563, August 20, 2015 ª2015 Elsevier Inc.

ure 4D). These findings show that primary piRNAs derived frompiRNA clusters are loaded onto AGO3 in aub mutant ovaries
and are not significantly further amplified by AGO3-AGO3 homotypic ping-pong. Furthermore, these findings suggest that primary piRNAs can be loaded onto AGO3, but that this loadingis actively blocked in the presence of Aub. In aub mutant ovaries,
the nuage localization of both AGO3 and Krimp is lost; instead,
Krimp accumulates in large cytoplasmic foci termed Krimp
bodies, where AGO3 is also accumulated (Lim and Kai, 2007;
Nagao et al., 2011; Olivieri et al., 2012). Thus, the reduced
loading of piRNAs onto AGO3 is probably caused by the sequestration of AGO3 to Krimp bodies.
Recapitulation of AGO3-Primary piRNA Loading in OSCs
Krimp accumulates at Krimp body-like cytoplasmic foci in OSCs
(Figures 5A and 5B) (Olivieri et al., 2012) where neither Aub nor
AGO3 are expressed and, therefore, only the primary piRNAbiogenesis pathway operates (Saito et al., 2009). These foci in
OSCs are probably equivalent to the Krimp bodies in aub mutant
ovaries. Interestingly, although ectopically expressed Aub isdistributed almost evenly in the cytoplasm and does not change
Krimp body formation in OSCs (Olivieri et al., 2012), ectopically
expressed AGO3 colocalizes with Krimp bodies in OSCs (Figures 5A and 5B) (Olivieri et al., 2012), suggesting a physicalinteraction of Krimp with AGO3. Ectopically expressed Aub in
the heterologous system was efficiently loaded with primarypiRNAs, while ectopically expressed AGO3 was sequesteredinto Krimp bodies and did not appear to associate with piRNAs(Figures 5A–5C) (Olivieri et al., 2012). We surmised that AGO3
might also be loaded with primary piRNAs if Krimp was depleted,
thereby releasing AGO3 from Krimp bodies in OSCs. Ectopically
expressed Aub was found to co-purify with small RNAs in bothnormal and krimp-depleted OSCs (Figures 5C–5G). We thus
wanted to test whether AGO3 exists in a complex with primarypiRNAs in Krimp-depleted OSCs, where production of primarypiRNAs is not affected (Figure S5A). We first examined thecellular localization of myc-tagged AGO3 after depletion of
Krimp by RNAi. As shown in Figure 5A, AGO3 was distributed
throughout the cytoplasm in krimp RNAi knock-down OSCs.
We then immunopurified myc-AGO3 complexes from Krimpdepleted OSCs using an anti-AGO3 antibody and examined
whether small RNAs were present in purified complexes.
AGO3 in Krimp-depleted OSCs was found to co-purify with small
RNAs (Figure 5C).
Deep sequencing of the AGO3-associated small RNAs in OSC
cells revealed that they show characteristics of somatic primarypiRNAs (Figures 5D–5G and S5B): they possess a strong
1U-bias (with no 10A bias) and correspond to somatic piRNA
clusters, including flamenco and traffic jam, but not to germline
clusters such as 42AB. The profile of AGO3-bound piRNAs in
OSCs is almost identical to that of Piwi-bound piRNAs. This
clearly indicates that AGO3 was efficiently loaded with primarypiRNAs in OSCs when Krimp was depleted. Thus, AGO3 iscompatible with primary piRNA loading in OSCs, but Krimp
blocks the loading.
It was previously shown that ectopic expression of UASAGO3 driven by actin5c-Gal4 in the ovarian soma increases
the expression of the gypsy transposon, which is otherwise

Figure 4. piRNAs Are Loaded onto AGO3 in aub Mutant Ovaries(A) AGO3-interacting piRNAs are barely detectable in aub mutant ovaries. IPs were performed from ovary lysates of control and aub mutant (aub HN2/QC42) using
antibodies against AGO3.
(B) Sequence logos of AGO3-bound piRNAs in the control and aub mutant.
(C) Uniquely mapping piRNAs are plotted over the germline-specific dual-stranded piRNA cluster 42AB and the uni-stranded piRNA cluster Cluster 2 (sense andantisense piRNAs are indicated with peaks pointing upward and downward, respectively). Libraries were normalized to allow for a direct comparison of piRNAdensities between all libraries (see Experimental Procedures).
(D) Depiction of the ping-pong signature, defined as the value at nucleotide position 10. Graphs indicate the relative frequency with which a complementarypiRNA exists with a 50 end (y axis) at the indicated distance (x axis) for the uniquely mapped piRNAs plotted over the cluster 42AB.

silenced by the primary piRNA processing pathway in OSCs
(Li et al., 2009). This finding suggests that AGO3 can competewith Piwi for primary piRNAs but that, unlike Piwi, it cannot
act to efficiently silence TEs, probably caused by failure of
nuclear localization or the inefficiency of its Slicer activity. In
agreement with the observation by Li et al. (2009), we foundthat ectopic expression of Aub in OSCs resulted in derepression of the mdg1 transposon regardless of Krimp expression,
while AGO3 in the heterologous system caused derepressionof the mdg1 transposon only when Krimp was depleted (Figure 5H). These results show that both Aub and AGO3, when
they are loaded with piRNAs, increase the expression of TEs
in OSCs. This is probably because they can compete with
Piwi for primary piRNAs, but they cannot silence TEs. We previously demonstrated that the nuclear localization but not the
Slicer activity of Piwi is required for TE silencing in OSCs (Saito
et al., 2010), indicating that TE silencing via the somatic primarypiRNA pathway occurs only in the nucleus via a Slicer-independent mechanism. Because both Aub and AGO3 remain in thecytoplasm in transfected OSCs, they cannot silence TEs in
OSCs. Taken together, these results further support the ideathat AGO3 is competent to be loaded with primary piRNAs in
the absence of Krimp.

DISCUSSION
Our data suggest that Krimp regulates sDMA modification of
AGO3 through direct binding to the N-terminal portion of theprotein, thereby controlling interactions of other Tudor-domain
proteins, such as Tud (Figure 6A).
The Role of Krimp in the Ping-Pong Cycle
A recent study showed that a lack of Qin, another Tudor domain
protein, disrupts the interaction between AGO3 and Aub andtriggers Aub-Aub homotypic ping-pong in fly ovaries, resulting
in an increase in the abundance of sense piRNAs while the overall abundance of piRNAs is preserved (Zhang et al., 2011, 2014).
Therefore, Qin seems to act to suppress Aub-Aub homotypicping-pong by promoting the Aub-AGO3 interaction, which leads
to an increase in heterotypic Aub-AGO3 ping-pong (Figure 6B). A
considerable amount of Aub-Aub homotypic ping-pong occurs
even in controls, so Aub-Aub homotypic ping-pong is probably
a default pathway for piRNA biogenesis; however, this pathway
increases the proportion of sense piRNAs in the piRNA pool andso does not efficiently repress TEs. Therefore, there should be asystem(s) that promotes heterotypic Aub-AGO3 ping-pong andthat also increases the proportion of antisense piRNAs. Although
Molecular Cell 59, 553–563, August 20, 2015 ª2015 Elsevier Inc. 559

Figure 5. AGO3 Is Compatible with the Primary piRNAs in Krimp-Depleted Somatic Cells(A) Cellular localization of AGO3 in Krimp-depleted OSCs. Ectopically expressed AGO3 was detected in Krimp bodies (indicated by an arrow). The distribution of
AGO3 became diffuse following the depletion of Krimp by RNAi.
(B) Cellular localization of Aub in OSCs. Krimp body was indicated by an arrow.
(C) Aub- and AGO3-IP complexes from OSC lysates overexpressing Myc-Aub or Myc-AGO3 with or without depletion of krimp were then immunoreacted using
Aub or AGO3 antibody. RNA molecules were isolated from complexes and then 32P labeled.
(D) Chart summarizing the annotation of Piwi-, Aub-, and AGO3-bound piRNA populations.
(E) siRNA-normalized small RNA size profiles are shown for Piwi-, Aub-, and AGO3-bound piRNAs.
(F) Sequence logos of Piwi-, Aub-, and AGO3-bound piRNAs.
(G) Uniquely mapping piRNAs are plotted over the somatic primary piRNA cluster flamenco, the genic piRNA locus traffic jam, and the germline-specificdual-stranded piRNA cluster 42AB (sense and antisense piRNAs are indicated with peaks pointing upward and downward, respectively).
(H) Expression levels of several transposons in OSCs overexpressing Aub or AGO3. mdg1 and Dm297, but not HeT-A, transposons are silenced by a
Piwi-dependent piRNA pathway in OSCs. rp49 was used as an internal control. See also Figure S5. N = 3; error bars indicate SEM.

560 Molecular Cell 59, 553–563, August 20, 2015 ª2015 Elsevier Inc.

Figure 6. Krimp Enforces an Antisense Biason piRNA Pools by Assembling AGO3 in the
Ping-Pong Cycle(A) During oogenesis, in nurse cells, Krimp associates with nascent, piRNA-, and sDMA-free
AGO3 and translocates it to the nuage, the location of dPRMT5-mediated sDMA modification.
Upon sDMA modification, Krimp might thendissociate from sDMA-modified AGO3 prior to its
entry into the ping-pong cycle. Additionally, fromobservations in the OSC system, Krimp keeps
AGO3 away from the primary piRNA pathway ingermline cells. TDRDs, Tudor domain-containing
proteins; SAM, S-adenosylmethionine.
(B) Piwi subfamily proteins potentially associatewith both sense and antisense piRNAs. Antisense
piRNAs but not sense piRNAs can act to repress
the activation of transposons and can be loaded
onto mainly Piwi and Aub in germ cells, because
Piwi and Aub but not AGO3 are transmitted to thenext generation to maintain germline development in Drosophila. The piRNA production shownby dotted lines is hardly observed in the normal
state.

Aub-Aub homotypic ping-pong also prevails in krimp mutant
ovaries, the overall abundance of piRNAs in germline cells is
dramatically decreased. This profile of piRNAs in krimp mutant
ovaries is very similar to that in ago3 mutant ovaries in which
the ping-pong cycle collapses (Huang et al., 2014; Li et al.,2009). The increase in Aub-Aub homotypic ping-pong observedin krimp mutant ovaries may largely reflect the loss of AGO3-Aub
heterotypic ping-pong, rather than a direct effect on promoting
Aub-Aub homotypic ping-pong. Therefore, Krimp plays a very
different role to Qin in the ping-pong cycle.
The ping-pong cycle is mediated by the Slicer-dependent
mutual cleavage of sense and antisense transcripts of a TE,
which should result in equal amounts of sense and antisense
piRNAs. Thus, an important question is this: what makes sense
piRNAs less abundant and disproportionately bound to AGO3
in the ping-pong cycle, enforcing the characteristic antisense
bias of Aub-bound piRNAs? In other words, why is Aub-Aub homotypic ping-pong not sufficient to produce the characteristic
antisense bias of Aub-bound piRNAs? In krimp mutant ovaries,
AGO3 is no longer sDMA modified and is free from piRNAs.
Therefore, Krimp not only masks the N-terminal portion of
AGO3 where sDMA-modifiable arginine residues reside, butalso mediates the sDMA modification that leads to interactionsof AGO3 with other Tudor domain proteins, including Tud, andto its nuage localization (Figure 6A). In
this way, Krimp promotes the pingpong cycle. Because sDMA modificationof Aub is not affected in krimp mutant
ovaries, complex formation between
Aub loaded with primary piRNAs andTudor domain proteins may occur before
sDMA-modified AGO3 joins the complexes to ensure Aub-AGO3 heterotypicping-pong. Qin may act in this step tosuppress Aub-Aub homotypic ping-pong to further promote
Aub-AGO3 heterotypic ping-pong.
AGO3 is clearly compatible with the primary piRNA pathway in
OSCs when Krimp is depleted. Therefore, Krimp may not only
promote the Aub-AGO3 heterotypic ping-pong cycle but also
actively prevent AGO3 from becoming loaded with primarypiRNAs in germline cells (Figure 6B). In contrast, in aub mutant
ovaries where AGO3 no longer accumulates at nuage but issequestered into Krimp bodies (Olivieri et al., 2012), AGO3 is still
loaded with reduced levels of piRNAs that have characteristics
of primary piRNAs (Figure 4). These results suggest that complete blockage of primary piRNA loading onto AGO3 in ovarian
germline cells requires an additional factor(s). An alternative
possibility may be that AGO3 does not selectively accept primary piRNAs when aub is mutated but is perhaps loaded in arather non-specific manner. Thus, together, these findings suggest that the characteristic antisense bias of Aub-bound piRNAs
is created by the sum of piRNAs produced in the ping-pong cycle
and the continual flow of primary piRNAs onto Aub. Aub ismuch more abundant than AGO3 in fly ovaries (Brennecke
et al., 2007; Gunawardane et al., 2007), further suggesting thatthe continuous flow of primary piRNAs to Aub should contributeto the antisense bias of Aub-bound piRNAs. Alternatively, it isalso conceivable that AGO3-sense piRNA complexes may be
Molecular Cell 59, 553–563, August 20, 2015 ª2015 Elsevier Inc. 561

catalytically more active than Aub-antisense piRNA complexes,
potentially resulting in the antisense bias of Aub-bound piRNAs.
However, in OSCs, AGO3 is loaded with primary piRNAs when
Krimp is depleted while AGO3 in germline cells is not loadedwith piRNAs. This suggests that in addition to Krimp, a second/backup system may also operate to further prevent AGO3 fromassociating with primary piRNAs in germline cells.
In aub mutant ovaries, Krimp is no longer accumulated at thenuage but forms Krimp bodies (Lim and Kai, 2007; Nagao et al.,2011), suggesting that the nuage localization of Krimp is Aub
dependent. However, Krimp does not directly interact with
Aub, and Krimp in OSCs forms Krimp bodies when Aub is ectopically expressed. Therefore, there must be a germline-specificfactor(s) that links Aub and Krimp (Figure 6B). The N-terminal
coiled-coil domains of Krimp are required for Krimp body formation (data not shown). Thus, a factor(s) may exist to mask thecoiled-coil domains to prevent Krimp from forming the aggregates in germline cells. The function and/or stability of this putative factor could be Aub dependent in germline cells. The next
key challenge will be to identify such a factor that regulates
Krimp, thereby contributing to the operation of the ping-pongcycle.
Both AGO3 and Aub have the potential to load sense andantisense piRNAs, and the loading of these two proteins likely
relies on the functions of interacting proteins, such as Krimp.
We therefore hypothesized that AGO3 and Aub are functionallyvery similar and that their interacting partners play essential roles
in determining the behaviors of the two PIWI proteins, such thatthey effectively participate in ping-pong amplification. If both
Aub and AGO3 are compatible with the primary piRNA pathway,
why then are primary piRNAs loaded only onto Aub and Piwi?
Perhaps this is a system to ensure a heterotypic ping-pong
between Aub and AGO3 and some other as yet unknown mechanism to amplify antisense piRNAs. During Drosophila germline
development, Piwi and Aub, but not AGO3, are directly deposited from mother to offspring through germline transmission(Brennecke et al., 2007). Antisense piRNAs that can act torepress the activation of TEs and initiate the ping-pong cycle
are loaded onto Piwi and Aub (Figure 6B) (Le Thomas et al.,2014). The maternal loading of antisense piRNAs, together withthe functions of Tudor domain proteins such as Krimp and Qin,
could establish an antisense bias in the ping-pong amplification
loop.
There is no apparent homolog of Krimp in mammals. This may
be because the ping-pong amplification mechanism in mammals
is different from that in Drosophila. For example, the mouse
PIWI protein, MILI, loads sense rather than antisense primarypiRNAs. However, heterotypic ping-pong with ping-pong partner MIWI2 may not operate because, once loaded with secondary piRNAs, MIWI2 is imported into the nucleus to direct specific
DNA methylation of transposon loci (Aravin et al., 2007, 2008; De
Fazio et al., 2011). It is likely that a Krimp-like protein exists in
mammals to enforce a piRNA strand bias.

Production of an Anti-sDMA-AGO3 Monoclonal Antibody
An anti-sDMA-AGO3 monoclonal antibody was raised specifically against a
synthetic peptide corresponding to the N terminus of the protein. Details are
given in the Supplemental Information.
Immunoprecipitation, Pull-Down Assay, and Western Blot Analysis
Immunoprecipitation (IP) and western blot analysis were performed as
described previously (Nishida et al., 2009). Details of these and pull-down assays are given in the Supplemental Information.
Northern Blot Analysis and Total RNA LabelingNorthern blot analysis and total RNA labeling were performed as by Nishida
et al., (2009) and Saito et al., (2010). Details are given in the Supplemental
Information.
RNAi, Plasmid Transfection, Immunohistochemistry, and qRT-PCR
Analysis
OSC culture, RNAi, plasmid transfection, immunohistochemistry in OSCs, and
qRT-PCR analysis were performed as in Saito et al. (2006, 2009, 2010) andSato et al. (2011). Details are given in the Supplemental Information.
Cloning and Analysis of Small RNA Libraries
Cloning and analysis of small RNA libraries were performed as previously
described (Saito et al., 2009; Malone et al., 2009) with modifications. Details
are given in the Supplemental Information.
ACCESSION NUMBERS
The Gene Expression Omnibus (GEO) accession number for small RNA
sequencing data reported in this paper is GEO: GSE69293.
SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures, two tables, and Supplemental
Experimental Procedures and can be found with this article online at http://dx.
doi.org/10.1016/j.molcel.2015.06.024.
AUTHOR CONTRIBUTIONSK.S., Y.W.I., M.C.S., and H.S. conceived of the project and designed theexperiments. K.S. and Y.W.I. performed all the experiments with the help ofA.S., Y.T., P.C., and H.I. Y.W.I. performed the majority of computational analyses. K.S., Y.W.I., M.C.S., and H.S. analyzed the data and wrote the paper.
ACKNOWLEDGMENTS
We thank members of the Siomi laboratories for discussions and comments on
the manuscript, A. Shimizu and T. Mituyama for technical suggestions regarding
small RNA sequence analysis, and A. Nakamura and D. Chen for antibodies. This
work was supported by MEXT (Ministry of Education, Culture, Sports, Science,
and Technology) grants to Y.W.I., H.S., and M.C.S. P.C. is supported by theFunding Program for Next Generation World-Leading Researchers and by aresearch grant from MEXT to the RIKEN Center for Life Science Technologies,
and M.C.S. is supported by CREST (Core Research for Evolutional Scienceand Technology) from JST (Japanese Science and Technology Agency).
Received: February 9, 2015
Revised: May 4, 2015
Accepted: June 17, 2015
Published: July 23, 2015
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