Article

Respective Functions of Two Distinct Siwi
Complexes Assembled during PIWI-Interacting RNA
Biogenesis in Bombyx Germ Cells
Graphical Abstract

Authors
Kazumichi M. Nishida,
Yuka W. Iwasaki, ..., Haruhiko Siomi,
Mikiko C. Siomi

Correspondence
siomim@bs.s.u-tokyo.ac.jp

In Brief
PIWI-interacting RNA (piRNA) biogenesis
consists of two sequential steps: primarypiRNA processing and the ping-pongcycle. The molecular functions of thefactors involved remain elusive. Nishida
et al. propose a mode for piRNAbiogenesis where the BmSpn-E/BmQin
dimer binds Siwi and functions in primarypiRNA processing, whereas BmVasa, by
associating with Siwi, ensures target RNA
release upon cleavage to facilitate theping-pong cycle.

HighlightsdSiwi forms distinct complexes with BmSpn-E/BmQin and
BmVasad

The requirements of Siwi/BmSpn-E/BmQin and Siwi/BmVasa
complexes differd

BmSpn-E, unlike BmVasa, is necessary for primary piRNAbiogenesisdTarget RNAs remaining on Siwi upon cleavage are releasedby BmVasa

Nishida et al., 2015, Cell Reports 10, 193–203
January 13, 2015 ª2015 The Authors
http://dx.doi.org/10.1016/j.celrep.2014.12.013

Accession Numbers
GSE58221

Cell Reports

ArticleRespective Functions of Two Distinct Siwi
Complexes Assembled during PIWI-Interacting
RNA Biogenesis in Bombyx Germ Cells
Kazumichi M. Nishida,1 Yuka W. Iwasaki,2 Yukiko Murota,1 Akihiro Nagao,2 Taro Mannen,2,3 Yumiko Kato,1Haruhiko Siomi,2 and Mikiko C. Siomi1,*
1Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan
2Keio University School of Medicine, Tokyo 160-8582, Japan
3Present address: Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido 060-0815, Japan*Correspondence: siomim@bs.s.u-tokyo.ac.jp
http://dx.doi.org/10.1016/j.celrep.2014.12.013
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

SUMMARYPIWI-interacting RNA (piRNA) biogenesis consists of
two sequential steps: primary piRNA processing andthe ping-pong cycle that depends on reciprocal
Slicer-mediated RNA cleavage by PIWI proteins.
However, the molecular functions of the factors
involved remain elusive. Here, we show that RNAs
cleaved by a Bombyx mori PIWI, Siwi, remain boundto the protein upon cleavage but are released by a
DEAD box protein BmVasa. BmVasa copurifies with
Siwi but not another PIWI BmAgo3. A lack of BmVasa
does not affect primary piRNA processing but abolishes the ping-pong cycle. Siwi also forms a complex
with BmSpn-E and BmQin. This complex is physically separable from the Siwi/BmVasa complex.
BmSpn-E, unlike BmVasa, is necessary for primarypiRNA production. We propose a model for piRNAbiogenesis, where the BmSpn-E/BmQin dimer binds
Siwi to function in primary piRNA processing,whereas BmVasa, by associating with Siwi, ensurestarget RNA release upon cleavage to facilitate theping-pong cycle.
INTRODUCTION
In germ cells, PIWI-interacting RNAs (piRNAs) arise primarily
from long noncoding RNAs transcribed from piRNA clusters
through primary piRNA processing and are amplified throughthe ping-pong cycle, enabling a continuous, substantial supplyof piRNA to cells (Pillai and Chuma, 2012; Senti and Brennecke,
2010; Siomi et al., 2011). The majority of piRNAs are highly
complementary to transposon transcripts and therefore act as
antisense oligos to silence them. piRNAs do not possess any
enzymatic activities for silencing but rather form piRNA-induced
silencing complexes (piRISCs) with PIWI proteins. Many PIWI
members have Slicer (endonuclease) activity with which theycleave target RNAs. Other PIWI members lack Slicer activity
and so collaborate with cofactors to accomplish the genesilencing. However, the underlying mechanisms remain largely
unknown.
The ping-pong cycle depends on reciprocal Slicer-mediatedRNA cleavage by two PIWI members (Brennecke et al., 2007;
Gunawardane et al., 2007). The current ping-pong model suggests that the process begins with target RNA cleavage by aparticular PIWI member loaded with primary piRNAs (the primarypiRISC). The Slicer-dependent cleavage on the targets occurs at
the position across from nucleotides 10 and 11 of the piRNAs.
Upon cleavage, the target RNAs are divided into two pieces:namely, 50 and 30 cleavage products. Of those, the 30 productis loaded onto the other PIWI member in the ping-pong cycle
and then trimmed down to the size of piRNAs (24–30 nt) fromits 30 end by an unidentified enzyme(s) (Kawaoka et al., 2011),
giving rise to the secondary piRISC. Because piRNAs within
the secondary piRISC originate from the targets of the primarypiRISC, the orientation opposes to that of piRNAs in the primarypiRISC. Moreover, piRNAs within the primary and secondary
piRISCs overlap through ten nucleotides from their 50 ends. Primary piRNAs show a strong bias for the first U (1U), whereas
secondary piRNAs show a 10A bias. These characteristics thatpiRNAs show in germ cells are referred to as the ping-pong
signature (Brennecke et al., 2007; Gunawardane et al., 2007).
The piRNA factors functioning in the ping-pong cycle include
Vasa, a germ-specific DEAD box RNA helicase that is highly
conserved in animal species and is necessary for germ cell
development (Schupbach and Wieschaus, 1986; Lasko andAshburner, 1988; Tanaka et al., 2000; Ishizu et al., 2012).
Drosophila melanogaster Vasa (DmVasa) is localized to the posterior pole in oocytes, where it associates with target mRNAs to
locally regulate their translation (Lasko and Ashburner, 1990).
Recent studies have shown that DmVasa and mouse Vasa homolog (MVH) are pivotal in piRNA biogenesis in the germline(Lim and Kai, 2007; Malone et al., 2009; Kuramochi-Miyagawa
et al., 2010; Nagao et al., 2010). In MVH mutant mouse testes,
as in wild-type (WT) testes, MILI, a mouse PIWI member (Aravin
et al., 2007; Pillai and Chuma 2012; Siomi et al., 2011), associates with primary piRNAs (Kuramochi-Miyagawa et al., 2010).
In contrast, MIWI2, one of three PIWI members and secondary
piRNA binder, is devoid of secondary piRNAs in MVH mutant
testes. When not loaded with piRNAs, MIWI2 fails to localize to
Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors 193

the nucleus, where the MIWI2–piRISC complex would normallyotherwise repress transposons by inducing DNA methylation oftarget genes (Kuramochi-Miyagawa et al., 2008). Thus, transposons are derepressed in MVH mutant testes, resulting in maleinfertility (Kuramochi-Miyagawa et al., 2010).
A more-recent study using BmN4 cells, a cultured germ cellline derived from B. mori (silkworm) ovaries, showed that
B. mori Vasa (BmVasa), when it lacks the ATP-hydrolyzing activity owing to a single mutation in the DEAD box (E339Q mutant),transiently associates with Siwi and BmAgo3, two PIWI members, and BmQin (also known as BmKumo), a Tudor domain
protein, to assemble a piRNA amplifier complex in the pingpong cycle (Xiol et al., 2014). The requirement of Qin/Kumo in
piRNA biogenesis was originally examined in Drosophila (Anandand Kai, 2012; Zhang et al., 2011). The amplifier complex contained three types of RNAs: antisense piRNAs, sense transposontranscripts, and 12 nt RNAs. The 12 nt RNAs showed a strong
bias for sense strand transposon transcripts and were considered to be Vasa footprints, as the recombinant BmVasa E339Q
mutant also bound 6–10 nt RNAs in E. coli (Xiol et al., 2014).
These results supported the idea that the amplifier is thebiochemical platform for generating BmAgo3-bound secondary
piRNAs from sense transposon precursors. However, the amplifier was detected only when the BmVasa DEAD box (E339Q)
mutant was ectopically expressed in BmN4 cells. The underlying
mechanism by which the amplifier facilitates secondary piRNA
generation also remains unclear.
In this study, by performing in vitro target RNA cleavage assays using immunopurified endogenous piRISCs, we show thattarget RNAs cleaved by Siwi remain bound to the protein uponcleavage but are released in the presence of BmVasa andATP. Target RNAs cleaved by Drosophila melanogaster Ago2
(DmAgo2) come off the RISC without the need for additional factors. Under normal conditions, BmVasa copurifies with Siwi, but
not with BmAgo3. However, the BmVasa E339Q mutant, whichlacks ATP-hydrolyzing and RNA-unwinding activities, forms acomplex with Siwi and BmAgo3. The complex contains transposon transcripts in the sense orientation, the sources of
BmAgo3-piRNAs, as has been observed (Xiol et al., 2014). However, no 12 nt RNAs were detected on endogenous BmVasa or
the E339Q mutant complex. Immunoprecipitation with an antiBmSpindle-E (BmSpn-E) antibody showed that BmSpn-E copurifies with Siwi and BmQin. Previous studies showed that
Spn-E, a DExH box/Tudor domain protein (Gillespie and Berg,
1995), is necessary for piRNA biogenesis in Drosophila (Aravin
et al., 2004; Vagin et al., 2006; Ishizu et al., 2012). The Siwi/
BmSpn-E/BmQin complex excludes the presence of BmVasa
and BmAgo3. Depletion of BmVasa does not affect the SiwipiRNA association, but BmAgo3 is devoid of piRNAs. In contrast,
depletion of BmSpn-E caused a severe reduction in primarypiRNA accumulation, resulting in Siwi and BmAgo3 not being
loaded with piRNAs. Based on these observations, we propose
a model for piRNA biogenesis in germ cells, where the BmSpn-E/
BmQin dimer binds Siwi to function in primary piRNA processing, whereas BmVasa, despite its association with Siwi, isdispensable for primary piRNA processing but is required forthe ping-pong cycle, where it ensures target RNA release from
Siwi upon cleavage.
194 Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors

RESULTS AND DISCUSSION
To thoroughly elucidate the mechanism underlying piRNAbiogenesis in a germ cell line BmN4, we raised monoclonal
antibodies for Siwi and BmAgo3, two silkworm PIWI proteins
involved in the pathway (Kawaoka et al., 2009). Western blottingon BmN4 cell lysates prepared before and after depletion of Siwi
and BmAgo3 revealed the specificity of the antibodies (Figure 1A). We then used these antibodies to immunoisolate endogenous Siwi-piRISC and BmAgo3-piRISC complexes from BmN4
cells. The piRNAs isolated from these complexes showed aslight size difference, apparently only a nucleotide apart, on an
acrylamide gel (Figure 1B), showing the high specificity of theimmunoprecipitation. Sequencing and bioinformatic analysis of
the small RNA pools revealed that both SIWI- and BmAgo3bound piRNAs are frequently mapped to transposons (Figure S1)and that they show typical ping-pong signatures; namely, piRNAs loaded onto endogenous Siwi (the largest population ofwhich has a length of 28 nt) are predominantly antisense andshow a strong 1U bias, whereas piRNAs with endogenous
BmAgo3 (the largest population of which has a length of 27 nt)
are predominantly sense and show a 10A bias (Figures 1C andS1), as has been previously observed (Xiol et al., 2012).
The hierarchy between Siwi and BmAgo3 in the piRNA amplification process, to determine the primary and secondary piRISCs, has not been examined. It is notable that the piRNA
strand bias that individual PIWI proteins show does not help us
to understand their hierarchy. For example, mouse MILI, which
is higher in the hierarchy for the piRNA amplification cycle thanMIWI2 (i.e., MILI loaded with primary piRNAs [MILI-piRISC] initiates the amplification cycle in which MIWI2 is loaded with secondary piRNAs produced by MILI-piRISC in the pathway), showsa strong bias for sense piRNAs (Aravin et al., 2008; De Fazio
et al., 2011), whereas a MILI homolog in Drosophila, Aubergine,shows a strong bias for antisense piRNAs (Brennecke et al.,2007; Gunawardane et al., 2007). Ago3, a second PIWI member
in Drosophila, is loaded solely with secondary piRNAs, the products of the ping-pong cycle, as is MIWI2 in mouse testes (Brennecke et al., 2007; Gunawardane et al., 2007). However, piRNAs
loaded onto Ago3 are predominantly sense, whereas thoseloaded onto MIWI2 are biased toward antisense. Likewise, wecannot exclude the possibility that primary piRNAs in silkworm
show a 10A bias. Thus, we set out to determine the hierarchy between Siwi and BmAgo3 in the ping-pong cycle by expressing
FLAG-Siwi and FLAG-BmAgo3 in BmAgo3- and Siwi-depleted
BmN4 cells, respectively (Figure S2A). RNAs isolated from thecomplexes were visualized by 32P-labeling at the 50 end (Figure 1D). FLAG-Siwi expressed in BmN4 cells upon effective
depletion of BmAgo3 was loaded with piRNAs, although thesignal was slightly lower (80%) than that in control cells (siLuc).
These piRNAs loaded onto FLAG-Siwi in BmAgo3-depleted andcontrol cells were sequenced and analyzed bioinformatically(Figure S2B). In sharp contrast, piRNA loading onto FLAGBmAgo3 was severely impaired by Siwi loss (10%; Figure 1D).
These results reveal that Siwi is higher in the hierarchy for the
ping-pong cycle than BmAgo3; i.e., primary piRNAs originatingfrom piRNA clusters, whose expression is independent of
BmAgo3-Slicer (target RNA cleavage) activity, are loaded onto

B

（kDa）

Figure 1. Siwi Is Required for BmAgo3-piRISC Formation in the Ping-Pong Cycle

IP

（kDa）
(nt)

n.i.
anti-Siwi
anti-BmAgo3

siLuc
siSiwisiLuc
siBmAgo3

A(A) Western blotting shows the specificity of antiSiwi and anti-BmAgo3 monoclonal antibodies
raised in this study. Tubulin: a loading control.
(B) piRNAs isolated from endogenous Siwi and
BmAgo3 immunopurified from BmN4 cells using
specific antibodies were labeled with 32P for
visualization. IP, immunoprecipitation; n.i., nonimmune IgG.
(C) Characterization of piRNAs loaded onto
endogenous Siwi and BmAgo3 in BmN4 cells.
(D) A lack of BmAgo3 slightly affected the SiwipiRNA association, whereas piRNA loading onto
FLAG-BmAgo3 was severely impaired by Siwi
loss, suggesting that Siwi is higher in the hierarchy
than BmAgo3 in the ping-pong cycle. Upper andlower panels show western blotting results and32
P-labeled piRNAs, respectively.
(E) Placement of Siwi and BmAgo3 in the pingpong cycle.

100 175 -175 -- BmAgo3- Siwi80 -70 60 50 -80 40 -

58 -

58 -

46 -

46 -

30 -

piRNA

20 -

- Tubulin- Tubulin

D
IP-Flag

Ratio of small RNA reads (%)

30
20

IP-Flag-FlagSiwi

siSiwisiLucSiwi-piRNAs
40

siLuc

50

siBm
Ago3

C

-FlagBmAgo3

to Siwi. RNase A treatment of lysate prior
to immunoprecipitation did not affect the-piRNA-piRNA
20
25
30
35
interaction between BmVasa and Siwi
20 50
(Figure 2C). Thus, the Siwi-BmVasa interBmAgo3-piRNAs
20 40
action is RNA independent. It is noted that30
this finding agrees with the previous
20
observation that MVH interacts with MILI
E
10through the N-terminal region, which prePrimary piRNA processing0
sumably has no RNA-binding property
20
25
30
35
(Kuramochi-Miyagawa et al., 2004).
small RNA length (nt)Siwi
To examine whether BmVasa would
2
Siwi-piRNAs
preferably bind piRNA-free Siwi or Siwi1
piRISC, Siwi was immunoprecipitated0
BmAgo3individually using anti-Siwi and anti5101520BmVasa antibodies. Preferable binding
2
BmAgo3-piRNAs
piRNA amplificationof BmVasa to piRNA-free Siwi would sug1
gest an involvement of BmVasa in primary0piRNA processing. An alternative idea
5
10
15
20
Nucleotide
may be that BmVasa plays a role in Siwi
loading with secondary piRNAs producedby BmAgo3-piRISC in the ping-pong
Siwi, whereas secondary piRNAs, the products of Siwi-piRISC pathway. After equalizing the amount of Siwi in both immunoprecleavage in the ping-pong cycle, are loaded onto BmAgo3 cipitates by adequate dilution, RNAs were isolated from the fractions and 32P labeled at the 50 end. Siwi in the BmVasa complex(Figure 1E).
To understand the molecular function of BmVasa in the ping- was loaded with piRNAs to a similar extent to that in the anti-Siwi
pong cycle, we generated an anti-BmVasa monoclonal antibody immunoprecipitates (Figure 2D). This suggests that BmVasa(Figure 2A) and used it for immunoprecipitation. The protein binds Siwi-piRISC. FLAG-BmAgo3 expressed in BmVasacomponents were visualized by silver staining (Figure 2B). depleted BmN4 cells was scarcely associated with piRNAs,
The existence of BmVasa (75 kDa) in the complex was whereas piRNA loading onto FLAG-Siwi was hardly affectedconfirmed by western blotting (Figure 2B). Besides BmVasa, (Figures 2E and S3B). Moreover, deep sequencing of piRNAs
an 100 kDa protein band (the expected size of Siwi) appeared loaded onto FLAG-Siwi upon deletions of BmVasa and BmAgo3
on the silver-stained gel. Western blotting confirmed it to be revealed a similar sequence variety of Siwi-bound piRNAs in
Siwi (Figure 2B). Notably, BmAgo3 was not detected in the com- BmVasa- and BmAgo3-depleted BmN4 cells (Figures S2C–
plex (Figure 2B). Under more-stringent conditions for immuno- S2E). A unilateral collaboration of BmVasa with Siwi-piRISC in
precipitation (i.e., high salt), which presumably disrupt most pro- the ping-pong cycle is suggested (Figure 2F).
The cellular localization of BmVasa along with Siwi andtein-protein interactions, Siwi disappeared from the complex(Figure S3A), excluding cross-reactivity of anti-BmVasa antibody BmAgo3 in BmN4 cells was examined. Although Siwi, but not
10

(nt)
30 -

0

U
A

CA

U

U
A

(nt)
30 -

U
A

bits

G

U
A
C

A

U
C

U

G

G

Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors 195

-Siwi160 --BmAgo3

120 -

58 -

-BmVasa-RNase A-Siwi

100 90 46 --Siwi

-BmVasa

80 --BmVasa

IP
input
n.i.
anti-Siwi
anti-BmVasa

IP

n.i.
anti-BmVasa

input

(kDa)

175 -

C

IP

anti-BmVasa

(kDa)

n.i.

IP

input
n.i.
anti-Siwi
anti-BmVasa

B

siBmVasa

siLuc

A

80 -

-BmVasa+RNase AG

30 -BmAgo3/Siwi

70 60 -

-Tubulin

-h.c.
50 -

D

F
Primary piRNA processing

-Siwi

10 µm

5 µm

10 µm

5 µm

10 µm

5 µm

Siwi/BmVasa

40 -

Siwi
BmVasa-BmVasa(nt)
30 --piRNA

BmAgo320 -piRNA amplification

BmAgo3/BmVasa

anti-Siwi

anti-BmVasa

n.i.

input

IP

E

30 20 -siLuc

siBmVasa

siLuc
(nt)

-FlagSiwi-piRNA(nt)
30 -siBmVasa

Flag-IP

Flag-IP-FlagBmAgo3-piRNA

20 -Figure 2. BmVasa Binds Siwi, but Not BmAgo3, in the Ping-Pong Cycle(A) Western blotting shows the specificity of anti-BmVasa monoclonal antibody raised in this study.
(B) BmVasa copurifies with Siwi, but not BmAgo3, from BmN4 cells. Left: silver staining of the protein components in the anti-BmVasa immunoprecipitatedcomplex. Right: western blotting shows that BmVasa binds Siwi, but not BmAgo3. h.c., heavy chains of the antibodies.
(C) RNase A treatment of the lysate prior to immunoprecipitation barely affected the interaction between BmVasa and Siwi.
(D) BmVasa associates with Siwi, regardless of its association with piRNAs. Top and middle panels show western blotting results. Bottom panel shows piRNAs
labeled with 32P.
(E) A lack of BmVasa causes BmAgo3 to be devoid of piRNAs. piRNA loading onto Siwi was not affected by BmVasa loss. Upper and lower panels show western
blotting results and piRNAs labeled with 32P, respectively.
(F) A model for BmVasa function with Siwi in the ping-pong cycle.
(G) BmVasa strongly colocalizes with Siwi at the nuage in BmN4 cells.

196 Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors

160 -

(nt)

120 -

- Siwi100 -Dmutantwt

mutantmutant(nt)
80 -- piRNA-4
target

70 -

50 -

60 -

40 -

50 -

- cleavage
product

40 -

30 -

Figure 3. RNAs Cleaved by Siwi-piRISC
Remain Associated with the Complex upon
Cleavage

Siwiinput control piRISC

wt

IP

wt

B

total RNAn.i.
anti-Siwi
anti-BmAgo3

anti-Siwi

n.i.

(kDa)

IP

anti-Siwi onlyA

- piRNA-4

20 -

40 -

60 50 40 -

F

Beads
Sup

Siwi-piRISC(nt)- piRNA-4
targetcontrol

(nt)
70 -

cf.

input

- h.c.
50 -E

BmAgo3-piRISC

input

60 -

control

70 -Siwi-piRISC

C

80 -

- piRNA-4
target70 60 -- cleavage
product

50 -(A) A silver-stained gel showing Siwi immunoisolated from BmN4 cells under stringent conditions.
(B) Northern blotting confirms the specific loadingof piRNA-4 onto Siwi.
(C) Siwi-piRISC, but not BmAgo3-piRISC, cleaves
the piRNA-4 target in in vitro target RNA cleavage
assays. Control: nonimmune IgG.
(D) Siwi-piRISC cleaves the piRNA-4 wild-typetarget (wt), but not the mutant target (mutant).
Control: nonimmune IgG.
(E) The cleaved piRNA-4 target remains bound with
Siwi-piRISC upon cleavage. Control: nonimmune
IgG. cf., centrifuge.
(F) Cleaved piRNA-4 target dissociates from
DmAgo2-siRISC upon cleavage.
(G) Cleaved target remains bound with BmAgo3piRISC upon cleavage.

- cleavage
product

40 -G

the center were mutated (Figure S3G),
could not be cleaved by Siwi-piRISC (Figure 3D). Thus, piRNA-4 triggers piRNA-4
DmAgo2Siwi
target cleavage when it is loaded onto
(nt)
Siwi.
cleavagecleavage- piRNA
150 (nt)
targetDmAgo2
Siwi
Fractionation of the supernatant from70 - piRNA-4
thebeads with Siwi-piRISC attached,target60 100 Sup
Beads
Sup Beads
upon cleavage, revealed that the vast ma- cleavage
50 90 DmAgo2
Siwi
jority of both 50 and 30 products remained
product- cleavage
80 40 product70 bound to Siwi-piRISC (Figure 3E; data
not shown). We examined if this was also
the case with DmAgo2, a ubiquitously
BmAgo3, localizes diffusely in the cytosol, localization of both expressed Argonaute protein in Drosophila (Miyoshi et al.,PIWI proteins at the nuage (Eddy, 1975; Mahowald, 1971), elec- 2005). A 21 nt small interfering RNA (siRNA) duplex, whose guidetron-dense, nonmembranous perinuclear structures considered strand is identical to that of piRNA-4 in sequence over 21 nt fromto be the center for piRNA biogenesis, was clearly observed the 50 end, was incubated with Schneider 2 cell lysate to form the(Figure 2G). The Siwi-positive and BmAgo3-positive nuage sig- DmAgo2-siRISC. The resultant siRISC was immunopurified using
nals only partially overlapped (Figures 2G and S3C). BmVasa anti-DmAgo2 antibody (Miyoshi et al., 2005). The 50 productwas strongly colocalized with Siwi at the nuage, whereas its co- cleaved by DmAgo2 was detected mostly in the supernatant (Figlocalization with BmAgo3 was partial (Figures 2G and S3C). A ure 3F). It should be noted that, after cleavage, the state of theunilateral collaboration of BmVasa with Siwi-piRISC is further ten-base overlap between the small RNA (either piRNA or siRNA)and the 30 cleaved product ought to be identical in both cases
supported.
(FigureS3H). The 30 cleaved product of BmAgo3-piRISC (FigThe current model of the ping-pong cycle involves Siwi- and
BmAgo3-Slicer activity, although the endonuclease activity of ure S3I) was also retained on the effector complex (Figure 3G).
neither protein has been examined. Thus, in vitro target RNA These results indicate a uniqueness of piRISC; i.e., piRISC, unlike
cleavage assays were performed using Siwi-piRISC immuno- siRISC, requires cofactor(s) to release cleaved RNAs after slicing
purified from BmN4 cells using the anti-Siwi antibody under event. Without this step, the piRISC fails to be free from target
high salt conditions. The purity of the complex was examined RNAs, and therefore, the piRNA amplification cycle cannot proby silver staining and western blotting (Figures 3A and S3D). ceed properly, leaving BmAgo3 devoid of secondary piRNAs.
BmVasa is a DEAD box protein that tightly interacts with SiwiThe RNA target (a piRNA-4 target) used in this assay contains
a sequence fully complementary to piRNA-4 (Figure S3E), one piRISC in BmN4 cells (Figure 2B). We hypothesized that BmVasaof the piRNAs associated with Siwi in BmN4 cells. Northern blot- might act as the factor releasing cleaved RNAs from Siwi-piRting confirmed the existence of piRNA-4 in Siwi-piRISC, but not ISC. To test this hypothesis, FLAG-BmVasa was purified fromin BmAgo3-piRISC (Figure 3B). As expected, the piRNA-4 target BmN4 cells after its ectopic expression by transfection (Figwas cleaved by Siwi-piRISC, but BmAgo3-piRISC failed to do so ure S4A) and incubated with Siwi-piRISC bound to cleavage(Figure 3C). The cleavage required Mg2+ ions (Figure S3F), as has products. FLAG-BmVasa, but not FLAG-EGFP employed as abeen shown with DmAgo2-Slicer activity (Schwarz et al., 2004; negative control, released significant amounts of the 30 cleavage
Miyoshi et al., 2005). A mutant piRNA-4 target, in which 3 nt at product from the effector complex (Figure 4A). In the absence of
Sup

Beads

Sup

Beads

DmAgo2Siwi

BmAgo3control piRISC
Beads
Sup
Beads
Sup

cf.

cf.

Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors 197

Acleavage
Beads
FlagBmVasa

Siwi

Beads

Sup

Flag- FlagEGFP BmVasa

Flag- FlagEGFP BmVasa- + - +

- + - +

(nt)

-piRNA-4
target70 FlagEGFP

Sup

60 50 -

-cleavage
product70 -myc-IP-BmAgo3-BmAgo3-mycBmVasa
-h.c.

IP

mycBmVasa-Siwi-piRNA-4
target60 -Danti-BmAgo3

mycBmVasa

E339Q

wt

D338A

Flag-EGFP

FlagBmVasa

E339Q

Sup

FlagBmVasa
D338A

myc-IP

mycBmVasa

myc-EGFP
wt
D338A
E339Q
myc-EGFP
wt
D338A
E339Q

AEAD
DQAD

Beads

wt

Flag-EGFP

(nt)

input

DEAD

n.i.

C

BmVasa wt
D338A
E339Q

wt

B

E339Q

40 -

myc-EGFP

Beads

ATP

-myc-BmVasa(nt)
40 30 20 40 30 20 --PiggyBacpiRNA-yamato
piRNA

50 -

-cleavage
product

40 -

-l.c.

E

-mycEGFP

myc-IP

myc-IP

(nt)
50 -

(nt)

(nt)

100 -

100 -

40 -

70 -

70 -

30 -

(nt)
40 -

-piRNA20 -Bmmar6

(nt)

100 -

70 -

70 -

10 -

L1Bm

20 -

10 -

yamato

(nt)

(nt)

100 -

100 -

70 -

30 -

Pao

(nt)

100 -

myc-BmVasa
E339Q

R2Bm

n.i.

BmRT3

IP

myc-EGFPF

myc-EGFP
wt
E339Q

mycBmVasa

myc-EGFP
wt
E339Q

mycBmVasa

anti-BmVasa

myc-IP70 -antisense

sense

Figure 4. The RNA-Unwinding Activity of BmVasa Plays a Role in Releasing the Cleaved Targets from Siwi-piRISC upon Cleavage(A) FLAG-BmVasa releases the cleaved targets from Siwi-piRISC in the presence of ATP.
(B) D338A and E339Q mutations in BmVasa impair the RNA-releasing activity of BmVasa.
(C) The BmVasa E339Q mutant forms a complex with not only Siwi but also BmAgo3. l.c., light chains of the antibodies.

(legend continued on next page)

198 Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors

ATP, the activity was negligible (Figure 4A). The 50 cleavage
product was also released from the piRISC upon incubation
with BmVasa (data not shown). It seems that uncleaved, fulllength target RNAs were also released from piRISC after incubation with FLAG-BmVasa in the presence of ATP (Figure 4A). The
mutant piRNA-4 target that was not cleaved by Siwi-Slicer (Figure 3D), and therefore remained bound with the complex, wasalso released upon incubation with BmVasa (Figure S4B), suggesting that BmVasa liberates Siwi-piRISC from the complex
containing uncleavable target RNAs.
The crystal structure of DmVasa bound to an ATP analog and asingle-stranded RNA revealed that Glu400 (the second aminoacid in the DEAD motif), His575, and Gly552 interact with a watermolecule necessary for ATP hydrolysis (Sengoku et al., 2006).
Eukaryotic translation initiation factor 4A (eIF4A), another
DEAD box protein, facilitates translation by relaxing the 50 end
secondary structure of mRNA substrates (Gingras et al., 1999).
A Glu183 mutation to glutamine in eIF4A, which is equivalent toGlu400 in DmVasa, significantly reduced the ATP hydrolysis
and RNA-unwinding activities of eIF4A (Pause and Sonenberg,
1992). Therefore, we altered Glu339 in BmVasa (corresponding
to Glu400 in DmVasa) to a glutamine and examined how this mutation affects the BmVasa activity. Incubation of FLAG-BmVasa
E339Q (Figure S4A) with Siwi-piRISC failed to release the 30
product from the effector complex (Figure 4B). We also mutated
Asp338 in BmVasa to alanine (the first amino acid in the DEAD
motif). Asp338 in BmVasa corresponds to Asp399 in DmVasa,
which is necessary for ATP binding. This mutant behaved similarly to the E339Q mutant (Figures 4B and S4A). These results
indicate that the ATP-binding/hydrolysis and RNA-unwinding
activities of BmVasa are required to displace the cleavage products from Siwi-piRISC. It is notable that BmVasa WT, but not its
mutants, also displaced uncleaved target RNAs from Siwi-piRISC (Figure 4B).
To further characterize the BmVasa mutants, coimmunoprecipitation experiments were performed. The BmVasa E339Q
complex contained much more Siwi than did the BmVasa WT
complex (Figures 4C and S4C). An 105 kDa protein was present in the E339Q complex (Figure S4C) and was confirmed tobe BmAgo3 by western blotting (Figure 4C), consistent withearlier observations (Xiol et al., 2014). Northern blotting showedthat BmAgo3 within the E339Q complex was loaded with muchless piRNAs compared with BmAgo3 immunoprecipitated using
anti-BmAgo3 antibody from the crude lysate (Figure 4D), suggesting that BmAgo3 bound to the BmVasa mutant complex is
not yet in the piRISC form. Sense, but not antisense, piRNA intermediates were detected in the E339Q mutant complex (Figures
4E and S4D). The sense piRNA intermediates were observed
also in the BmVasa WT complex, although the signal strengths
were significantly lower and the lengths of the RNAs may belonger, suggesting aberrant accumulation of the molecules in
the mutant complex. The amplifier complex composed of
BmVasa E339Q mutant, Siwi, and BmAgo3 contained RNAs of12 nt (Vasa footprints; Xiol et al., 2014). However, in our study,neither the BmVasa E339Q mutant complex nor the endogenous
BmVasa complex contained 12 nt RNAs (Figure 4F). This
discrepancy may have been caused by differences in the tags
and antibodies used for immunoprecipitation.
Spn-E is a DExH box/Tudor domain protein necessary foraccumulating piRNAs in Drosophila ovaries (Aravin et al., 2004;
Vagin et al., 2006; Malone et al., 2009; Ishizu et al., 2012). Depletion of BmSpn-E in BmN4 cells caused a severe reduction in theaccumulation of sense (secondary) piRNAs associated with
BmAgo3 (Figure 5A). Depletion of Siwi and BmAgo3 produced
similar results (Figure 5A). Thus, the requirement of BmSpn-E
in the ping-pong cycle is conserved in silkworm.
To understand the function of BmSpn-E in the ping-pong cycle, we raised an anti-BmSpn-E antibody (Figure S5A) and performed immunoprecipitation experiments. Mass spectrometricanalysis revealed that BmSpn-E forms a complex with Siwi
and BmQin (Figure 5B). Western blotting on the complex components confirmed the existence of Siwi and BmQin in the complex(Figures 5B and S5B). Because Siwi was detected in the complex, we assumed the presence of BmVasa in the complex.
However, this was not the case. Western blotting detected no
BmVasa (Figure 5B). It seems that Siwi assembles two distinct
complexes: one with BmVasa and one with the BmSpn-E/BmQin
dimer. This idea was further supported by the observationthat the Siwi/BmVasa complex (Figure 2B) contains neither
BmSpn-E nor BmQin (Figure 5C). Immunofluorescence using
monoclonal antibodies specific to BmVasa, BmSpn-E, and
BmQin showed that the BmVasa-positive nuage hardly overlapped with the BmSpn-E/BmQin-positive nuage, although the signals for BmSpn-E and BmQin overlapped considerably (Figures
5D and S5C). These results also suggest that the Siwi/BmSpn-E/
BmQin complex is separable from the Siwi/BmVasa complex.
Our finding that Siwi forms two distinct complexes in BmN4
cells prompted us to examine if BmSpn-E also plays a differentrole in primary piRNA processing. Northern blotting experiments
revealed that a lack of BmSpn-E, but not a loss of BmVasa,
causes a severe decrease in Siwi-bound piRNA accumulation(Figure 5E). We then examined the amounts of piRNAs loaded
onto Siwi and BmAgo3 under conditions where BmSpn-E wasdepleted (Figure S5D). Not only BmAgo3-piRNAs but also
Siwi-bound piRNAs were hardly detected by 32P labeling underthese conditions (Figure 5F). These results suggest that the requirements for BmVasa and BmSpn-E in piRNA biogenesis ingerm cells differ: whereas BmVasa is required specifically for
production of BmAgo3-secondary piRNAs, BmSpn-E is necessary for primary piRNA biogenesis. Thus, the ping-pong cycle,
which largely depends on the existence of primary piRNAs, isalso abolished by loss of BmSpn-E. Siwi residing in the
BmSpn-E complex was devoid of piRNAs (Figure 5G), supporting the idea that BmSpn-E binds Siwi prior to its loading with primary piRNAs. This is consistent with the original idea that
BmSpn-E functions in primary piRNA biogenesis in germ cells.

(D) BmAgo3 residing in the BmVasa E339Q complex is nearly devoid of mature piRNAs. The top two panels show western blotting results. The bottom two panels
show northern blotting results.
(E) Northern blotting showed that the BmVasa E339Q complex preferably binds sense transposon transcripts.
(F) piRNAs present in the BmVasa E339Q mutant complex and with endogenous BmVasa.

Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors 199

BmN4+ - + lysate-yamatopiRNA-U6 snRNA

siBmVasa

siBmSpn-E

siBmAgo3

siSiwisiLucanti-BmSpn-E

E

input
n.i.

n.i.
-L1Bm
piRNA

anti-BmSpn-E

B

siBmSpn-E

siLuc

siSiwisiBmAgo3

A-piRNA-4+ - + BmN4 lysate

(kDa)
220 -

-BmQin-BmSpn-E-piggybac
piRNA

160 -

-BmSpn-E-BmQin-BmRT3piRNA-Bmmar6piRNA-U6 snRNA

-Siwi

C

120 -

-BmVasa-Siwi

100 90 -

F

input
n.i.
anti-BmSpn-E
anti-Siwi-BmQinsiBmSpn-E

siLuc

70 -

60 -

-Siwi
-BmVasa

-h.c.

50 -

-FlagSiwi

(nt)

IP

Flag-IPsiLuc-BmSpn-E

G
Flag-IP

80 -siBmSpn-E

anti-BmVasa

n.i.

input

IP

30 -

-BmSpn-E-FlagBmAgo3

(nt)
30 -

-Siwi
-piRNA-piRNA(nt)
30 --piRNA
20 -

20 -

20 -

H

D
BmVasa

BmQin

BmVasa/BmQin/DAPI

BmSpn-E
wt

DEVH

TUDOR

D250A

AEVH

TUDOR

E251Q

DQVH

TUDOR

siBmSpn-E

E251Q

D250A

mycBmSpn-E

wt

BmSpn-E/BmVasa/DAPI
siLuc

BmVasa

myc-EGFP

1µm

BmSpn-E-mycBmSpn-E
1µm

BmSpn-E

BmQin-Tubulin

BmSpn-E/BmQin/DAPI-piRNA-4-BmRT3piRNA-Bmmar6piRNA
1µm-bantammiRNA

Figure 5. Siwi Forms Two Distinct Complexes in piRNA Biogenesis in Germ Cells(A) Depletion of BmSpn-E (siBmSpn-E) causes a severe reduction in sense (secondary) piRNA accumulation in BmN4 cells. Depletion of Siwi and BmAgo3
produced a similar result. U6 small nuclear RNA (snRNA): a loading control.
(B) Left: silver staining of the protein components in the anti-BmSpn-E immunoprecipitated complex. Right: western blotting shows that Siwi forms a complex
with BmSpn-E and BmQin. BmVasa was not detected in the complex.
(C) Western blotting shows that the Siwi/BmVasa complex (Figure 2B) involves neither BmSpn-E nor BmQin.
(D) Immunofluorescence shows that BmSpn-E and BmQin only partially colocalize with BmVasa. The signals for BmSpn-E and BmQin largely overlap, as expected.

(legend continued on next page)200 Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authorsprimary piRNAbiogenesis

BmQin

Bm
Spn-E
when
the R BmSpn-E
NA-unwind lacks !
ing activitySiwi

Ping-Pong cycle(secondary piRNA biogenesis)

Siwi

Siwi
Bm
Vasa

Vasa
mut

target binding

target binding

Siwi
Bm
Vasa

Siwi
Vasa
mut

target cleavage

Siwi

Siwi

target cleavage

Siwi
Bm
Vasa
!
and BmAgo3 loading

Vasa
mut
BmAgo3

BmAgo3piRNA maturation

BmAgo3X

Figure 6. A New Model for piRNA Biogenesis in Germ Cells Consisting of Two Sequential Steps: Primary piRNA Processing and thePing-Pong Cycle
The BmSpn-E/BmQin dimer binds Siwi to function in primary piRNA processing, whereas BmVasa, via associating with Siwi, ensures target RNA
release from the protein upon cleavage to facilitate the ping-pong cycle. A
BmVasa-like protein (shown as X) may dissociate from the BmAgo3-piRISC
complex in the process. BmAgo3 remains associated with the Siwi-BmVasa
E339Q mutant complex.

A recent study showed that the DExH box helicase domain of
Spn-E, which most likely is required for the ATP-binding/hydrolysis and RNA-unwinding activities of Spn-E (Lüking et al., 1998),
is necessary for transposon silencing in Drosophila germline (Ottet al., 2014). Thus, we asked if BmSpn-E’s DExH box is required
for Siwi-bound piRNA production in BmN4 cells. Loss of BmSpnE drastically decreased the amount of Siwi-bound piRNAs (Figures 5E and 5F). Ectopic expression of myc-tagged BmSpn-E
WT in BmSpn-E-depleted BmN4 cells rescued this defect inpiRNA accumulation. However, two BmSpn-E DExH box
mutants, D250A and E251Q, failed to rescue the defective
phenotype, resulting in similar levels of piRNA accumulation to
myc-EGFP, which was used as a negative control (Figure 5H).
These results correlate well with the recent finding that the
Spn-E DExH box mutants including E239K (E239 in Drosophila
Spn-E is equivalent to E251 in BmSpn-E) showed considerably
higher levels of germ-specific transposons compared to theother alleles (Ott et al., 2014). Under conditions where BmSpnE WT binds BmQin, both BmSpn-E mutants D250A and E251Q

failed to bind BmQin (Figure S5E). CLIP experiments showedthat BmSpn-E WT bound directly and tightly with RNAs in
BmN4 cells. By contrast, the RNA-binding activity of BmSpn-E
D250A and E251Q was very low (Figure S5F). Their results suggest that the property of BmSpn-E to bind with RNA substrates
and BmQin is necessary for promoting primary piRNA biogenesis in the cells. We are currently engaged to identify RNA molecules bound with BmSpn-E in BmN4 cells.
These results are sufficient to propose a model for piRNAbiogenesis in germ cells in which the BmSpn-E/BmQin heterodimer and BmVasa mutually exclusively associate with Siwi toform two physically and functionally distinct Siwi complexes.
The Siwi/BmSpn-E/BmQin complex plays a role in Siwi-piRNAcomplex (primary piRISC) formation, whereas BmVasa, whileassociating with Siwi, actively releases target RNAs from Siwi
upon cleavage to facilitate BmAgo3-piRNA complex (secondary
piRISC) formation in the ping-pong cycle (Figure 6).
In this study, we showed for that BmVasa, a germ-specific
DEAD box RNA helicase, uses RNA-unwinding activity to releasetarget RNAs from Siwi-piRISC, upon cleavage, in the ping-pongcycle in germ cells. The BmVasa complex immunopurified from
BmN4 cells contained Siwi, but not BmAgo3 (Figure 2B). The
cleavage products of BmAgo3-piRISC are also retained on thecomplex upon cleavage (Figure 3G), suggesting that a protein
factor like BmVasa, but not BmVasa itself, is required to dissociate these products from the complex. Indeed, we showed by
performing in vitro assays that BmVasa failed to displace thecleavage products from BmAgo3-piRISC (Figure S5G). We are
currently aiming to identify BmAgo3-associating proteins. This
will hopefully help us understand the counterpart of BmVasa
functioning specifically for BmAgo3-piRISC in the ping-pong
pathway.
EXPERIMENTAL PROCEDURES
Production of Monoclonal Antibodies
Monoclonal antibodies for BmVasa, Siwi, BmAgo3, BmSpn-E, and BmQin
were produced essentially as previously described (Saito et al.,2006). Mice were immunized with glutathione S-transferase (GST)-tagged
BmVasa (full-length), the N-terminal region of Siwi (amino acids
2–100) tagged with GST, the N-terminal region of BmAgo3 (amino acids 2–100) tagged with GST, the N-terminal region of BmSpn-E (amino acids
1–124) tagged with GST, or the N-terminal region of BmQin (amino acids
11–173) tagged with GST.
Small RNA Isolation from the Immunoprecipitated Materials
Immunoprecipitation was performed essentially as described previously (Saito
et al., 2006). BmN4 lysates prepared in binding buffer (30 mM HEPES [pH 7.4],
150 mM potassium acetate, 5 mM magnesium acetate, 5 mM dithiothreitol,
0.1% Tergitol-type NP-40, 2 mg/ml pepstatin, 2 mg/ml leupeptin, and 0.5%
aprotinin) were incubated with antibodies bound to Dynabeads Protein G(Invitrogen). The beads were then washed three times with binding buffer containing 500 mM sodium chloride and twice with binding buffer with no supplement. Total RNAs were eluted from the beads by phenol-chloroform treatment
and precipitated with ethanol. RNAs were dephosphorylated with CIP (New(E) Northern blotting shows that BmSpn-E is necessary for Siwi-bound piRNA accumulation in BmN4 cells. U6 snRNA: a loading control.
(F) A lack of BmSpn-E drastically affected both Siwi-piRNA and BmAgo3-piRNA associations.
(G) Siwi in the BmSpn-E complex is devoid of piRNAs. The top two panels show western blotting results. The bottom panel shows piRNAs labeled with 32P.
(H) Ectopic expression of BmSpn-E WT, but not its DExH-box mutants, D250A and E251Q, increased the levels of Siwi-bound piRNAs in BmN4 cells lacking
endogenous BmSpn-E. The top two panels show western blotting results. The bottom four panels show northern blotting results.

Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors 201

England Biolabs) and radiolabeled with 32P-g-ATP using T4 PNK (NEB). RNA
cloning was carried out as previously described (Hirano et al., 2014).
Small RNA Sequencing and Data Analysis
Sequencing of the small RNA libraries generated from the Siwi and BmAgo3
immunoprecipitates was performed using MiSeq (Illumina), resulting in
2,048,705 Siwi small RNA reads and 2,916,872 BmAgo3 small RNA reads intotal. Adaptor sequences were removed from obtained reads and mapped
to the B. mori genomic sequence (Mita et al., 2004) with Bowtie2 (Langmeadand Salzberg, 2012), allowing zero mismatches. Using genome-mapped
reads, length distribution and nucleotide bias were calculated. Weblogo3
was used to produce sequence logos (Crooks et al., 2004). Sequences werealigned to the 50 end to search for nucleotide bias.
Plasmid Construction
Expression vectors for FLAG-Siwi, FLAG-BmAgo3, FLAG-BmVasa, and mycBmVasa were generated by Gateway Technology (Invitrogen) using the pIB-3
3 FLAG and pIB-myc vectors. The expression vector for myc-BmSpn-E wasgenerated with an In-Fusion HD Cloning kit (TaKaRa). The expression vectors
for FLAG-BmVasa mutants, myc-BmVasa mutants, and myc-BmSpn-E
siRNA-resistant and myc-BmSpn-E mutants were generated by inserting thecorresponding coding regions. The sequences of DNA oligos used are shownin Table S1.
RNAi and Transgene Expression in BmN4 Cells
BmN4 cells (4 3 105 cells) were transfected with 500 pmol of siRNA duplex in
10 ml of FuGENE HD transfection reagent (Promega). After transfection, cells
were incubated at 27 C for 72 hr. siRNA transfection was performed again
to ensure the RNAi effect. The sequences of the siRNAs used are summarizedin Table S1. Luc siRNA (Saito et al., 2009) was used as a control. To exogenously express proteins in BmN4 cells, the cells (6 3 105 cells) were transfected with 2 mg of plasmid in 5 ml of FuGENE HD transfection reagent. Aftertransfection, cells were incubated at 27 C for 48 hr.
Immunoprecipitation and Western Blotting
Siwi and BmAgo3 were immunopurified from BmN4 cells using anti-Siwi andanti-BmAgo3 antibodies, respectively. FLAG-Siwi and FLAG-BmAgo3 were
immunopurified from BmN4 cells using anti-FLAG M2 antibody (Sigma). After
immunoprecipitation, the beads were washed extensively with binding buffer
containing 500 mM sodium chloride and then with binding buffer. mycBmVasa (both WT and mutant) complexes were immunopurified from BmN4
using anti-myc antibody (Sigma-Aldrich). BmVasa and BmSpn-E were immunopurified from BmN4 cells using anti-BmVasa and anti-BmSpn-E antibodies.
After immunoprecipitation, the beads were washed extensively with binding
buffer. Western blotting was performed as described previously (Miyoshi
et al., 2005). To examine the RNA dependency of Siwi-BmVasa interaction,
BmN4 lysates were treated with 10 mg/ml RNaseA at 4 C for 30 min prior to
immunoprecipitation.
Immunofluorescence
Immunofluorescence was carried out essentially as described previously(Nishida et al., 2013). Cells were immunostained with anti-Siwi, anti-BmAgo3,
anti-BmVasa, anti-BmSpn-E, and anti-BmQin antibodies. Alexa 488/Alexa
Fluor 555 goat anti-mouse immunoglobulin G 1 (IgG1)/IgG2a antibodies (Invitrogen) were used as secondary antibodies. Images were taken at the same
exposure.
Northern Blot Analysis
Northern blot analysis was carried out essentially as described previously(Saito et al., 2010). The sequences of the DNA oligos used are shown in Table
S1. Templates for producing probes to detect BmRT3, Bmmar6, L1Bm,
R2Bm, Pao, and yamato transcripts were amplified by PCR using primers containing T7 and T3 promoter sequences. BmRT3, Bmmar6, and L1Bm RNAprobes were transcribed using a Megascript T3 kit (Life Technologies).
R2Bm, Pao, and yamato RNA probes were transcribed with a Megascript T7
kit (Life Technologies).

202 Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors

In Vitro Target RNA Cleavage Assay
Cleavage assays were performed as described previously (Miyoshi et al.,2005). To make the piRNA target template, piRNA target-F and piRNAtarget-R were annealed and inserted into pBlueScript vector treated with
KpnI and EcoRI. The piRNA target template was amplified by PCR using T7
and T3 promoter sequences. The piRNA target was transcribed using a Megascript T3 kit and then radiolabeled with [32P]-pCp using T4 RNA ligase 1 (NEB).
To make the piRNA-4 target, piRNA-4 target-30 was radiolabeled with 32P-gATP using T4 PNK and was ligated to piRNA-4 target-WT-50 or piRNA-4
target-mut-50 . RISC formation assays were performed as previously described(Nishida et al., 2007).
Cleaved RNA-Releasing Assays
The beads and supernatant were fractionated after target RNA cleavage
assays. The beads were mixed with FLAG-BmVasa WT, FLAG-BmVasa
mutants, or FLAG-EGFP (Figure S4A) in the presence of 4 mM ATP, 10 mM
creatine phosphate, and 30 mg/ml creatine kinase and then incubated for2 hr at 27 C.
ACCESSION NUMBERS
Deep sequencing data have been deposited in the NCBI Gene Expression
Omnibus and are available under accession number GSE58221.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,five figures, and one table and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2014.12.013.
ACKNOWLEDGMENTS
We are grateful to I. Sagawa and T. Watanabe for performing mass spectrometry and confocal microscopy, respectively. We also thank M. Kamatani, A.
Shibuya, M. Motomiya, and K. Miyakubi for their technical assistance andother members of the M.C.S. and H.S. laboratories for discussions and comments on the manuscript. This work was supported by grants from the Ministryof Education, Culture, Sports, Science and Technology of Japan to Y.W.I.,
H.S., and M.C.S. K.M.N. is supported by the Japan Society for the Promotionof Science.
Received: June 13, 2014
Revised: November 24, 2014
Accepted: December 5, 2014
Published: December 31, 2014
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Cell Reports 10, 193–203, January 13, 2015 ª2015 The Authors 203