Article

Piwi Nuclear Localization and Its Regulatory
Mechanism in Drosophila Ovarian Somatic Cells
Graphical Abstract

Authors
Ryu Yashiro, Yukiko Murota,
Kazumichi M. Nishida, ..., Lumi Negishi,
Haruhiko Siomi, Mikiko C. Siomi

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

In Brief
Piwi represses transposonstranscriptionally to maintain genome
integrity. Yashiro et al. show that
Importina plays a pivotal role in Piwi
nuclear localization and that Piwi has abipartite nuclear localization signal at the
N terminus. Piwi autoregulates its nuclear
localization by exposing the nuclear
localization signal to Impa upon piRNAloading.

HighlightsdImportina plays a pivotal role in Piwi nuclear localization in
OSCsd

Piwi has a classical bipartite nuclear localization signal at the
N terminusdpiRNA loading triggers conformational change in Piwi

d

Piwi autoregulates its nuclear localization by exposing NLS toenvironment

Yashiro et al., 2018, Cell Reports 23, 3647–3657
June 19, 2018 ª 2018 The Author(s).
https://doi.org/10.1016/j.celrep.2018.05.051

Cell Reports

ArticlePiwi Nuclear Localization and Its Regulatory
Mechanism in Drosophila Ovarian Somatic Cells
Ryu Yashiro,1,5 Yukiko Murota,1,5 Kazumichi M. Nishida,1 Haruna Yamashiro,1 Kaede Fujii,2 Asuka Ogai,2
Soichiro Yamanaka,3 Lumi Negishi,4 Haruhiko Siomi,3 and Mikiko C. Siomi1,6,*
1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan
2Yokohama City University School of Medicine, Yokohama, Kanagawa 236-0004, Japan
3Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan
4Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan
5These authors contributed equally
6Lead Contact*Correspondence: siomim@bs.s.u-tokyo.ac.jp
https://doi.org/10.1016/j.celrep.2018.05.051

SUMMARY

In Drosophila ovarian somatic cells (OSCs), Piwi represses transposons transcriptionally to maintaingenome integrity. Piwi nuclear localization requires
the N terminus and PIWI-interacting RNA (piRNA)loading of Piwi. However, the underlying mechanism
remains unknown. Here, we show that Importina(Impa) plays a pivotal role in Piwi nuclear localizationand that Piwi has a bipartite nuclear localization
signal (NLS). Impa2 and Impa3 are highly expressedin OSCs, whereas Impa1 is the least expressed. Lossof Impa2 or Impa3 forces Piwi to be cytoplasmic,
which is rectified by overexpression of any Impa
members. Extension of Piwi-NLS with an additional
Piwi-NLS leads Piwi to be imported to the nucleus
in a piRNA-independent manner, whereas replacement of Piwi-NLS with SV40-NLS fails. Limited proteolysis analysis suggests that piRNA loading onto
Piwi triggers conformational change, exposing the
N terminus to the environment. These results suggest that Piwi autoregulates its nuclear localization
by exposing the NLS to Impa upon piRNA loading.
INTRODUCTIONPIWI proteins are a subclade of the Argonaute family of proteinsenriched in the gonads, where PIWI binds PIWI-interacting RNAs
(piRNAs) to assemble piRNA-induced silencing complexes(piRISCs) to repress transposons to maintain genome integrity(Aravin et al., 2007; Ghildiyal and Zamore, 2009; Iwasaki et al.,2015; Juliano et al., 2011; Malone and Hannon, 2009; Yamashiro
and Siomi, 2018). Loss-of-function mutations of PIWI proteinsand piRNA factors essential in the pathway cause defects in
the genome surveillance system, leading to transposon derepression, failures in gametogenesis, and infertility (Khurana and
Theurkauf, 2010).
Drosophila expresses three genes encoding members of the
PIWI protein family, Piwi, Aubergine (Aub), and Ago3 in bothovaries and testes (Cox et al., 1998; Li et al., 2009; Nagao

et al., 2011; Schmidt et al., 1999). In the ovaries, Aub and Ago3
are expressed specifically in germ cells, where they silencetransposons post-transcriptionally by cleaving their transcripts
using their endonuclease activity known as Slicer (Brennecke
et al., 2007; Gunawardane et al., 2007; Li et al., 2009; Nishida
et al., 2007). Aub- and Ago3-bound piRNAs are mainly antisense
and sense to transposon-coding sequences, respectively, and
Aub and Ago3 reciprocally cleave transposon mRNAs and antisense transcripts, respectively. The resultant RNA fragments arefurther processed to mature piRNAs; thereby Aub/Ago3-Slicerdependent transposon silencing in germ cells is considered tobe coupled with piRNA biogenesis.
Piwi is expressed in both germ cells and ovarian somatic cells(OSCs), and in both cell types, Piwi is localized to the nucleus
and silences transposons transcriptionally by inducing heterochromatinization at the target loci with co-factors such as Panoramix/Silencio, Asterix/GTSF1, Maelstrom (Mael), and linker histone H1 (Czech et al., 2013; Dönertas et al., 2013; Handler
et al., 2013; Iwasaki et al., 2016; Kalmykova et al., 2005; Le
Thomas et al., 2013; Muerdter et al., 2013; Ohtani et al., 2013;
Saito et al., 2006, 2010; Sienski et al., 2012, 2015; Yu et al.,2015). Piwi nuclear localization requires both the N-terminal
region of Piwi and piRNA loading onto Piwi, which may be particularly obvious in OSCs (Klenov et al., 2011; Saito et al., 2009,2010). A proportion of Piwi in germ (nurse) cells may be translocated to the nucleus even under conditions where the piRNAbinding activity was impeded by mutations (Le Thomas et al.,2013). This layered, ‘‘gatekeeping’’ mechanism ensures PiwipiRISC-mediated nuclear transposon silencing, because
piRNA-free Piwi in the nucleus would be useless with no ability
to find its targets (Ishizu et al., 2011). However, how piRNAloading-dependent Piwi nuclear localization is regulated mechanistically is still unclear. The nuclear import machinery requiredfor Piwi nuclear import also remains unclear.
The N-terminal region of Piwi, which has been shown to benecessary for its nuclear import, is rich in basic residues, acharacteristic typical of classical nuclear localization signals(NLSs) whose nuclear import is mediated by an Importin a/b(Impa/b) heterodimer (Görlich, 1998; Stewart, 2007). The NLSof SV40 large T antigen (SV40-NLS) (PKKKRKV) (Kalderon
et al., 1984) and that of Xenopus laevis nucleoplasmin (NPNLS) (KRPAATKKAGQAKKKK) (Robbins et al., 1991) are the

Cell Reports 23, 3647–3657, June 19, 2018 ª 2018 The Author(s). 3647
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

prototypic monopartite and bipartite NLSs, respectively (Goldfarb et al., 2004; Stewart, 2007). Monopartite NLSs consist of asingle stretch of basic residues, while bipartite NLSs containtwo stretches of basic residues with a linker region between
them. In the Impa/b pathway, the adaptor molecule Impa protein
first binds cargos in the cytoplasm through their NLSs. Then,
Impb associates with the heterodimer through the Impb binding(IBB) domain of Impa to assemble a NLS-cargo/Impa/Impb
trimeric complex, which facilitates the nucleocytoplasmic translocation of the complex across the nuclear pores located on the
nuclear envelope. Upon transport, RanGTP in the nucleus binds
and disassembles the complex, releasing the cargos to the
nucleoplasm, where they exert their nuclear functions (Görlich,
1998; Stewart, 2007).
Members of the Impa family of proteins can be divided into
three phylogenetic clades, a1, a2, and a3 (Mason et al., 2009;
Pumroy and Cingolani, 2015). Drosophila contains a single repre€ssel andsentative of each clade, Impa1, Impa2, and Impa3 (KuFrasch, 1995; Goldfarb et al., 2004; Török et al., 1995; Mason
et al., 2002, 2003). All Impa proteins contain one IBB domain
at the N-terminal end, and the rest of the sequence contains
10 Armadillo (ARM) repeats, which were originally identified in
the protein encoded by the Drosophila segment polarity genearmadillo (Riggleman et al., 1989). While the IBB domain binds
Impb during nuclear import through the nuclear pore, the ARM
repeats assemble a scaffold structure that can accommodate
monopartite or bipartite NLSs (Conti et al., 1998; Fontes et al.,2003). When the ARM repeats are not bound with cargos, theIBB domain binds the ARM repeats intramolecularly, autoinhibiting nuclear import of cargo-free Impa (Kobe, 1999; Stewart,2007). Drosophila possibly contains a fourth Impa member,Impa4, which, however, lacks the IBB domain and is testis specific (Phadnis et al., 2012). A single Impb gene known as ketel
was found in the Drosophila genome (Lippai et al., 2000).
Drosophila Impa1 null flies developed to adulthood but showed
severe defects in gametogenesis and infertility (Ratan et al.,2008). Similarly, homozygous Impa2 mutants developed normally to adulthood, but mutant females, and not males, werecompletely sterile (Mason et al., 2002). Impa3 mutants showedembryonic lethality (Mason et al., 2003).
On the basis of all these earlier findings, we speculated that
Impa may be responsible for Piwi nuclear localization. To test
this, we first examined the levels of Impa proteins in cultured
OSCs. To this end, we produced monoclonal antibodies foreach Impa member and performed western blotting, which revealed that Impa2 and Impa3 were fairly highly expressed in
OSCs, whereas Impa1 was the least expressed. Loss of Impa2or Impa3 by RNAi treatment disturbed nuclear localization of
Piwi. However, nuclear localization of SV40-NLS-cargo was
not affected, suggesting that Piwi-NLS may have lower nuclear
localization efficiency than SV40-NLS. We defined the N-terminal 36 residues of Piwi as a bipartite NLS. Interestingly, bipartite
NP-NLS-cargo behaved similarly to Piwi in OSCs lacking Impa2or Impa3. Impa proteins may prioritize monopartite NLSs overbipartite NLSs, at least in OSCs. Replacement of Piwi-NLS
with SV40-NLS failed to obviate the requirement of piRNAloading in Piwi nuclear localization. However, Piwi was imported
to the nucleus piRNA independently upon duplication of the3648 Cell Reports 23, 3647–3657, June 19, 2018

Piwi-NLS. Substitution of the second NLS with a peptide thatlacks the NLS activity little changed the outcome. Limited proteolysis assays showed that about 90 residues in the N-terminalend of Piwi were inaccessible when Piwi was free from piRNAs,
but piRNA loading made the region accessible to the environment, most likely through a conformational change in Piwi.
piRNA loading increased the ability of Piwi to interact with
Impa2 and Impa3. Thus, Piwi autoregulates its nuclear localization by exposing the NLS to Impa upon piRNA loading. Additionof Piwi-NLS caused the otherwise cytoplasmic Aub to beimported to the nucleus in a piRNA-dependent manner, as was
Piwi. Piwi-NLS may have a unique property to be ‘‘hidden’’ intramolecularly prior to piRNA loading, possibly by domains
conserved in PIWI members.
RESULTS
Our previous RNA-sequencing data (Sumiyoshi et al., 2016)
suggested that Impa2 was relatively highly expressed in OSCs,
accounting for 70.7% of the total Impa expression, while theexpression level of Impa1 was much lower (4.0%) (Figure S1A).
The expression level of Impa3 was between those of Impa2
and Impa1 (25.3%) (Figure S1A). To estimate the levels of the
Impa proteins by western blotting, we first produced monoclonal
antibodies against each of the proteins (Figures S1B and S1C).
Comparison of the signals of the endogenous Impa proteins in
OSCs with those of recombinant Impa proteins suggested thatthe ratio of abundance levels of Impa1, Impa2, and Impa3 proteins in OSCs was about 5:77.5:17.5 (Figures S1A and S1D).
The corresponding monoclonal antibodies detected each Impa
protein in OSCs in immunofluorescence analyses (Figure S1E).
To examine whether Impa was responsible for Piwi nuclear
localization, we expressed myc-Piwi in OSCs in which each
Impa had been individually depleted by RNAi. The efficiency
and specificity of the RNAi treatment were determined by western blotting (Figure S2A). Immunofluorescence showed thatmyc-Piwi still accumulated in the nucleus in Impa1-depleted
OSCs, as it did in normal OSCs (Figure 1A). We considered theseresults to be reasonable because Impa1 was the least abundant
Impa in the OSCs (Figure S1A), and Impa2 and Impa3 remained
highly abundant in OSCs even after RNAi treatment (Figure S2A).
However, myc-Piwi was no longer restricted in the nucleus andwas detected in the cytoplasm in Impa2- and Impa3-depleted
OSCs (Figures 1B and 1C). piRNA loading of myc-Piwi wasbarely affected by lack of Impa2 and Impa3 (Figure S2B; data
not shown). These results support our original idea that Impa
plays a role in Piwi nuclear localization.
Impa2 accounted for about 78% of total Impa proteins in the
OSCs (Figure S1A). Therefore, after depletion of Impa2, theamounts of Impa1 and Impa3 proteins in the OSCs may not beenough to import all cargos to the nucleus. Indeed, depletionof Impa2 strongly affected the cellular localization of Piwi (Figure 1B). However, after depletion of Impa3, the amount of
Impa2 remained high in OSCs (Figure S2A). Therefore, thefinding that Piwi nuclear localization was affected by loss of
Impa3 (Figure 1C) was somewhat confusing. The simplest explanation for this result is that Piwi nuclear localization requires both
Impa2 and Impa3 simultaneously. To test this hypothesis, weImp 1

myc-Piwi

merge & DAPI

Imp 2

myc-Piwi

merge & DAPI

Imp 3

myc-Piwi

merge & DAPI

Imp 1 RNAi

controlA

Imp 2 RNAi

controlB

Imp 3 RNAi

controlC

Figure 1. Requirement of Impa in Piwi Nuclear Localization in OSCs
(A) Nuclear localization of myc-Piwi (red) was not affected by loss of Impa1(green) in OSCs. EGFP siRNA was used as a control. DAPI signals (blue) show
the nuclei. Scale bars, 5 mm.
(B) Nuclear localization of myc-Piwi (red) was affected by loss of Impa2 (green)in OSCs. EGFP siRNA was used as a control. DAPI signals (blue) show thenuclei. Scale bars, 5 mm.
(C) Loss of Impa3 (green) also affected nuclear localization of myc-Piwi (red) in
OSCs. EGFP siRNA was used as a control. DAPI signals (blue) show the nuclei.
Scale bars, 5 mm.
See also Figures S1 and S2 and Table S1.

performed rescue experiments. First, Impa2- and Impa3-lacking
OSCs were prepared; then each Impa gene was expressed bycotransfection with FLAG-Piwi in the cells. If both Impa2 andImpa3 were simultaneously required for Piwi nuclear localization,ectopic expression of Impa3 in Impa2-lacking OSCs would notrestore Piwi nuclear localization, while ectopic expression of
Impa2 should rescue the defective phenotype. Examination revealed that not only Impa2 expression, but also Impa1 andImpa3 expression (all Impa genes were RNAi-resistant) rectified
Piwi nuclear localization (Figure 2A). Similarly, ectopic expression of all individual Impa genes rescued the defective phenotype caused by depletion of Impa3 (Figure 2B). Therefore, theidea that Impa2 and Impa3 were simultaneously required for
Piwi nuclear localization was excluded.
An alternative explanation may be that the binding activity of
Piwi-NLS to Impa2 was relatively low, and therefore Impa2 prioritized cargos other than Piwi in nuclear import when the total
amount of Impa proteins was reduced, albeit only by about
18%, as a consequence of, for instance, Impa3 depletion. To
check this, we examined whether the subcellular localization of
SV40-NLS-cargo was affected by lack of Impa3 as well as
Impa2. To this end, Mael fused with a myc-tag, and a monopartite SV40-NLS at the N- and C-terminal ends of Mael, respectively, was employed. Mael is one of the piRNA factors requiredin germ cells, but is dispensable for the somatic piRNA pathway
in OSCs (Matsumoto et al., 2015; Sienski et al., 2012). We foundthat depletion of neither Impa2 nor Impa3 affected cellular localization of myc-Mael-SV40-NLS (cargo-SV40-NLS in Figures 3A
and 3B), suggesting that efficiency of nuclear import of PiwiNLS was somewhat lower than that of monopartite SV40-NLS.
Previously, we showed that a Piwi mutant Piwi-DN that lacked
72 amino acid residues from the N-terminal end was mislocalized to the cytoplasm, although Piwi-DN formed piRISCs with
piRNAs as well as wild-type (WT) Piwi (Saito et al., 2009). The
72 residues were very basic, and arginine and lysine residues
together accounted for 23.6% (17 out of 72 residues), which isa typical feature of classical NLSs bound with Impa proteins (Figure 4A). We fused the peptide consisting of the 72 residues to
EGFP and found that this fusion protein was localized exclusively
to the nucleus (Figure 4B). However, the peptide was too long to
be defined as a monopartite or bipartite NLS. Thus, we deleted
36 N-terminal residues from Piwi and examined the cellular localization. This mutant Piwi-DN36 failed to accumulate in the
nucleus (Figure 4C). We then fused the 36 residues to EGFPand found that this fusion protein, Piwi-N36-EGFP, strongly
accumulated in the nucleus similar to Piwi-N72-EGFP (Figure 4C). In contrast, Piwi-N20-EGFP, which contained only 20
residues from the N-terminal end of Piwi, was found to bethroughout the cells, the same as EGFP alone (Figures 4B and
4C), although the 20 residues included a basic stretch
7-RGRRR-11. Based on these results, we defined the 1–36 residues at the N-terminal end of Piwi as the Piwi-NLS.
Next, we produced a series of Piwi-NLS mutants in the context
of full-length Piwi. We first mutated four residues 9-RRRP-12 ofPiwi to four alanine residues. This mutant Piwi-NLS-M1 appeared mostly in the cytoplasm, but was slightly detected also
in the nucleus (Saito et al., 2009) (Figure 4D). Piwi-NLS-M2,
where Arg22 was mutated to alanine, was nearly exclusively

Cell Reports 23, 3647–3657, June 19, 2018 3649

EGFP

Imp 1

Imp 2

Piwi

Imp 2 RNAimerge & DAPI
EGFP

Imp 1

Imp 2

Piwi
merge & DAPIImp 3 RNAi

myc

B

Imp 3

Figure 2. All Three Impa Members Restore
Piwi Nuclear Localization Affected by Lossof Impa2 and Impa3 in OSCs
(A) Expression of individual Impa members(green), but not EGFP, in Impa2-depleted OSCs
restored nuclear localization of Piwi (red). DAPI
signals (blue) show the nuclei. Scale bars, 5 mm.
(B) Expression of individual Impa proteins (green)in Impa3-depleted OSCs restored nuclear localization of Piwi (red). DAPI signals (blue) show thenuclei. Scale bars, 5 mm.
See also Table S1.

myc

A

localized to the nucleus (Figure 4D), and Piwi-NLS-M3, where
Arg34 was mutated to alanine, was found in both the nucleus
and the cytoplasm nearly evenly. However, Piwi-NLS-M4 andPiwi-NLS-M5, where Lys31 and Lys31/Arg34 were mutated toalanine residues, respectively, were not localized to the nucleus(Figure 4D). Piwi-NLS-M1 and Piwi-NLS-M5 only weakly boundto Impa3 (Figure S2C). In summary, two small basic islands
around 9-RRRP-12 and 31-KVFR-34 within the 36-residue
Piwi-NLS sequence were necessary for Piwi nuclear localization.
Thus, we defined Piwi-NLS as a bipartite NLS.
Mass spectrometric analysis of Piwi in OSCs showed that Arg82
might be deeply modified to be a symmetric dimethyl arginine

3650 Cell Reports 23, 3647–3657, June 19, 2018

(sDMA) (Figure S3A). sDMA is one of the
post-translational modifications that are
observed in the majority of PIWI proteins
across species (Yamashiro and Siomi,2018). Arginines within Piwi-NLS may
also be sDMA-modified, but only weakly(Figure S3A). Piwi was undetected with
Y12 and SYM11, the antibodies specifically reacting to sDMAs (Figure S3B andImp 3
data not shown). These results suggestthat sDMA modification of Piwi hardly impacts on Piwi nuclear localization in OSCs.
We then took NP-NLS, a representativeof bipartite NLSs, and fused it to EGFP to
produce NP-NLS-EGFP. In contrast to
SV40-NLS-containing cargo (Figures 3A
and 3B), NP-NLS-EGFP was localized
exclusively to the nucleus of normal
OSCs, as expected, but was detected in
the cytoplasm under conditions where
Impa2 or Impa3 had been depleted (NPNLS-cargo in Figures 4E and 4F), similar
to Piwi (Figures 1B and 1C). These results
suggest that the sensitivity that Piwi
showed to a subtle reduction in the total
amount of Impa proteins in OSCs was
not unique to Piwi, and that monopartite
NLSs likely have higher efficiency in nuclear transport than bipartite NLSs, atleast in OSCs.
Piwi nuclear localization requires not
only its NLS but also piRNA loading, suggesting that Piwi-NLS is not accessible for Impa in the cytosol
until Piwi is loaded with piRNA. One hypothesis was that an
unknown protein (temporally termed as protein X) specificallyoccupied the NLS of piRNA-free Piwi, blocking Impa binding to
the NLS. Then, piRNA loading onto Piwi triggered displacementof the protein X from Piwi-NLS, allowing Piwi-piRISC to be successfully localized to the nucleus. To test this model, we
exchanged Piwi-NLS with SV40-NLS to determine whether the
Piwi mutant SV40-NLS-Piwi-DN required piRNA loading or not
to be localized to the nucleus. Because SV40-NLS-containing
cargo was localized to the nucleus with no specific treatments(Figures 3A and 3B), we assumed that SV40-NLS-Piwi-DN wouldImpα2cargo
SV40-NLS

merge & DAPI

Impα3

cargo
SV40-NLS

merge & DAPI

Impα2 RNAi

controlA

Impα3 RNAi

control

B

Figure 3. Effects of Impa Depletion on SV40-NLS Localization in
OSCs(A and B) Depletion of Impa2 (green in A) and Impa3 (green in B) only marginallyaffected cellular localization of cargo-SV40-NLS (myc-Mael-SV40-NLS) (red)in OSCs. DAPI signals (blue) show the nuclei. Scale bars, 5 mm.
See also Table S1.

also be translocated to the nucleus without piRNA loading. However, against our expectation, the mutant failed to be localized to
the nucleus when piRNA biogenesis had been compromised by
Yb depletion (Figure 5A). Thus, the ‘‘protein X’’ hypothesis was
unlikely.
An alternative model is that Piwi can hide its own NLS intramolecularly from Impa until piRNA is loaded onto Piwi. To examine
this possibility, we fused another Piwi-NLS composed of 36 residues to the N-terminal end of WT Piwi (Figure 5B). This processextended the NLS by duplication of the sequence in the context
of full-length Piwi by fusing Piwi-NLS to the N terminus of WT
Piwi. If this additional Piwi-NLS projected into the cellular environment beyond the surface of piRNA-free Piwi, we considered
Impa would bind to it and translocate the mutant Piwi-NLS-Piwi
to the nucleus, regardless of piRNA loading. Indeed, Piwi-NLSPiwi was localized to the nucleus even after Yb depletion. Two
independent Piwi-NLS-Piwi mutants in which the second NLS
was mutated to impede the activity as NLS behaved similarly

to Piwi-NLS-Piwi (Figures S4A and S4B), suggesting the internal
NLS in Piwi-NLS-Piwi is dispensable for rendering the piRNA-independent, nuclear localization activity to the protein.
Human Ago2 (hAgo2) that was not bound with guide RNA wasfragmented to domains in limited proteolysis assays, but guide
RNA loading made hAgo2 resistant to the proteolysis treatment(Elkayam et al., 2012). We tested whether Piwi behaved similarlyin the assays. To this end, we expressed Piwi in cultured
Drosophila Schneider 2 (S2) cells, which are non-gonadal somatic cells, so they do not operate the piRNA pathway. Indeed,
Piwi expressed in S2 cells was devoid of piRNAs or any otherRNAs, and was cytoplasmic (Figures S5A and S5B). We then
immunopurified piRNA-free Piwi from S2 cells using anti-Piwi
monoclonal antibody 3G11. In parallel, Piwi-piRISC was immunopurified from OSCs using 3G11. The epitope of 3G11 waswithin the 20 residues at the N-terminal end that corresponded
to the first half of the Piwi-NLS (Figure S5C). In accordancewith this fact, we found that immunopurification of piRNA-free
Piwi from S2 cells was much more difficult than immunopurification of Piwi-piRISC from OSCs, so to obtain comparable
amounts of Piwi, we prolonged the duration of immunoprecipitation from S2 cells. Also, the cell number of S2 cells used wasabout 10 times higher compared with that of OSCs. Both Piwi
fractions were subjected to limited proteolysis assays. First,
we used thermolysin because it was employed in the originalhAgo2 study (Elkayam et al., 2012). However, even a low final
thermolysin concentration of 0.08 ng/mL nearly completelydegraded even Piwi-piRISC (data not shown). We then usedchymotrypsin, which we found successfully released a large
portion of Piwi-piRISC to the supernatant (Figure 5C), as was
the case for Siwi-piRISC in our previous study (Siwi is one of
two PIWI members in silkworm; Matsumoto et al., 2016). The
large portion of Piwi in the supernatant was detected by anotheranti-Piwi monoclonal antibody 4D2 (Saito et al., 2009) by western
blotting. The epitope of 4D2 was determined to be located between Trp72 and Lys89 (Figure S5D), suggesting that the proteolysis occurred upstream of the 4D2 epitope. In contrast, forpiRNA-free Piwi, multiple 4D2-positive bands appeared exclusively in the bead fraction, implying that proteolysis occurredat multiple sites downstream of the 4D2 epitope. These results
strongly suggest that the N-terminal region of approximately
90 residues of Piwi (including Piwi-NLS and 3G11/4D2 epitopes)
was hardly accessible when Piwi was devoid of piRNAs, but
piRNA loading induced a major conformational change in Piwi,
as in hAgo2, exposing the N-terminal region to the environment.
Similar results were obtained when Piwi (with no tag) exogenously expressed in OSCs before and after depletion of Gasz,
an essential piRNA biogenesis factor (Handler et al., 2013),
was employed (Figure 5D). In this particular experiment, weknocked down Gasz, but not Yb, because piRNA-free Piwi
was somewhat more stable than that in Yb-depleted cells(data not shown). Thus, our earlier observation that Piwi-piRISC(from OSCs) and piRNA-free Piwi (from S2 cells) showed distinct
proteolytic patterns (Figure 5C) was not because of use of two
different cell lines in the assays.
We then examined whether Piwi binding with Impa is affectedby piRNA loading status of Piwi. To this end, we expressed
FLAG-Piwi in Gasz-depleted OSCs, immunoprecipitated the

Cell Reports 23, 3647–3657, June 19, 2018 3651

A

Figure 4. Piwi Contains a Bipartite NLS at
the N-Terminal End

D(A) Domain structure and amino acid sequence of
the N-terminal 72 residues of Piwi (N72) are
shown. L0 domain: 38 residues (Y76–R113);
N domain: 73 residues (I114–I186); L1 domain:
S187–T263 (77 residues); PAZ domain: I264–T371(108 residues); L2 domain: G372–T470 (99 residues); MID domain: P471–I601 (131 residues);
PIWI domain: E602–L843 (242 residues). Three
residues, 73-GDQ-75, do not belong to any of thedomains indicated. The domain structure wasdefined by sequence alignment with the peptide
sequence of Siwi (Matsumoto et al., 2016). Lysine
and arginine residues are shown in red.
(B) myc-Piwi-N72-EGFP (green), but not mycEGFP, was exclusively localized to the nucleus.
EGFP has a small molecular weight (about
25 kDa), which allows EGFP to diffuse passively inand out of the nucleus in a receptor-independent
fashion. DAPI signals (blue) show the nuclei. Scalebars, 5 mm.
(C) Deletion of 36 residues from the N-terminal endof Piwi caused mislocalization of Piwi (green: mycPiwi-DN36) in the cytoplasm. Piwi-N36-EGFP(green), but not Piwi-N20-EGFP, was nearlyexclusively localized to the nucleus. DAPI signals(blue) show the nuclei. Scale bars, 5 mm.
(D) myc-Piwi and myc-Piwi-NLS-M2 were nearlyexclusively localized to the nucleus. Piwi-NLS-M3
can be detected in both the nucleus and thecytoplasm evenly in OSCs. Piwi-NLS-M1, -M4,
and -M5 strongly accumulated in the cytoplasm.
DAPI signals (blue) show the nuclei. Scale bars,
5 mm.
(E and F) Depletion of Impa2 (in E) and Impa3 (in F)
affects cellular localization of NP-NLS-cargo(green: myc-NP-NLS-EGFP) in OSCs. DAPI signals (blue) show the nuclei. Scale bars, 5 mm.
See also Figures S2 and S3 and Table S1.

B

C

E

F

Impa3 complex, and probed it with anti-FLAG antibody. As
expected, depletion of Gasz severely reduced the levels of
Piwi-Impa3 interaction (Figure 5E). The level of Piwi-Impa2 interaction was similarly reduced (Figure S5E). These results strongly
support our original idea that Piwi-piRNA loading is necessary for
Piwi being tightly associated with Impa in vivo. It was experimentally confirmed that Piwi exogenously expressed in Gaszdepleted OSCs was loaded with piRNAs to a much lesser extent
than Piwi expressed in normal OSCs (Figures S5F and S5G).
Aub exogenously expressed in OSCs by transfection was
loaded with piRNAs that are normally loaded onto endogenous
Piwi in the cells (Sato et al., 2015). However, Aub-piRISC remained to be in the cytoplasm, as was the case in germ cells,
because it did not possess an NLS. We then fused Piwi-NLS to
Aub and found that the Piwi-NLS-Aub protein was imported to
the nucleus in OSCs (Figure 6A). However, Aub was forced
to be in the cytoplasm when Yb was depleted to block piRNA

3652 Cell Reports 23, 3647–3657, June 19, 2018

biogenesis, as was found for Piwi (Figure 6A). Similar results were obtained
when Aub was fused to SV40-NLS(Figure 6A). Both Piwi-NLS-EGFP and SV40-NLS-EGFP wereinsensitive to Yb depletion and localized to the nucleus in Ybdepleted OSCs (Figure 6B). Addition of another Piwi-NLS to
Piwi-NLS-Aub caused the protein to be nuclear localized in amanner independent of piRNAs (Figure S5H). These results suggest that piRNA loading-driven conformational change, which
controls the accessibility of the PIWI N terminus, is a common
feature of PIWI proteins.
DISCUSSION
In this study, we revealed that Impa played a vital role in Piwi nuclear localization in OSCs, and that Piwi autoregulated its cellular
localization by controlling the accessibility of its own N-terminal
bipartite NLS to Impa, by changing the structure in a piRNAloading-dependent manner. With this ‘‘layered’’ regulation, Piwi
was strictly localized to the cytoplasm before piRNA loading.

Figure 5. Addition of Piwi-NLS to FullLength Piwi Achieves piRNA-Independent
Piwi Nuclear Localization(A) SV40-NLS-Piwi-DN (green) requires piRNAloading for Piwi to be localized to the nucleus in
OSCs. Yb was detected in the control using antiYb antibody (red dots). DAPI signals (blue) show
the nuclei. Scale bars, 5 mm.
(B) Piwi-NLS-Piwi (red) is imported to the nucleus
piRNA independently in OSCs. Yb was detected in
the control using anti-Yb antibody (green dots).
Scale bars, 5 mm. The proposed models are shown(bottom).
(C) Limited proteolysis of Piwi immunoisolated
from S2 cells (i.e., piRNA-free Piwi) and OSCs (i.e.,
piRNA-bound Piwi). The anti-Piwi antibody 3G11
that reacts with the N-terminal end (1–20 residues)of Piwi (Figure S5C) was used for immunoprecipitation. Western blotting using 4D2 whose epitopewas between Try72–Lys89 of Piwi (Figure S5D)
shows that piRNA-bound Piwi was digested between the 3G11 and 4D2 epitopes, so the majorityof the core region of Piwi positive for 4D2 wasdetected in the supernatant after chymotrypsin
treatment. piRNA-free Piwi was digested downstream of the 4D2 epitope, so the N-terminal
regions containing both 3G11 and 4D2 epitopes
mostly remained in the bead fraction (Piwi peptides), suggesting that the N-terminal region containing approximately 90 residues of Piwi wasdifficult to access when Piwi was free from piRNA.
The bands indicated with asterisks correspond to
full-length Piwi.
(D) Limited proteolysis of Piwi exogenously expressed (with no tag) in and immunoisolated from
OSCs using anti-Piwi antibody before (PiwipiRISC) and after (piRNA-free Piwi) Gasz depletion.
It should be noted that the latter material also
contains endogenous Piwi-piRISC (50%) thatwas formed before Gasz depletion.
(E) Depletion of Gasz severely reduced the activityof Piwi to interact with Impa3 in OSCs.
See also Figures S4 and S5 and Table S1.

However, once piRNA was loaded onto Piwi, Piwi-NLS was
exposed to the cellular environment, where the adaptor protein
Impa bound to it and triggered the nuclear import of Piwi-piRISC
together with Impb. Unfortunately, depletion of Impb in OSCs by
RNAi caused severe damage to the cells; thus, the necessity of
Impb in the processing remains unclear.
The limited proteolysis analysis provided compelling evidence
that Piwi drastically changed the conformation in a piRNAdependent manner, similar to what was reported for hAgo2.
However, direct evidence is still missing. For this, crystal structural analysis of Piwi in the presence and absence of piRNAs isdesirable. However, this is currently difficult because it isextremely challenging to purify a large amount of recombinant
PIWI proteins without piRNAs owing to their high instability incellular environments. Nonetheless, we recently solved the crystal structure of Siwi-piRISC (Matsumoto et al., 2016). To makethe crystals, we collected endogenous Siwi-piRISC by immunoi-

solating the complex from cultured BmN4 cells using anti-Siwi
antibody. Because the vast majority of Siwi in BmN4 cells areloaded with piRNAs and the interaction between them is so tight,
removing piRNAs from the piRISCs was the issue to be solved.
Using a powerful electron-cryo-microscopy may be anotherway to go; however, the molecular mass of PIWI proteins isaround 90–100 kDa, which may be too small for this type of analysis. We are currently exploring gentle but efficient ways ofremoving piRNAs from PIWI-piRISCs.
Our rescue experiments revealed that all three Impa proteins
could individually rescue mislocalization of Piwi to the cytoplasm
caused by loss of Impa2 and Impa3 in OSCs. This clearlyexcludes a paralog-specific role of Impa in Piwi nuclear localization. However, previous genetic studies showed that sterility
caused by Impa2 mutations was not rescued by ectopic expression of Impa1 or Impa3, although the levels of Impa mRNAs (andImpa proteins) ectopically expressed in the mutant ovaries were

Cell Reports 23, 3647–3657, June 19, 2018 3653

A

B

Figure 6. SV40-NLS-Aub and Piwi-NLS-Aub
Were Imported to the Nucleus in a piRNADependent Manner, Similar to Piwi, in OSCs
(A) Both Piwi-NLS-Aub and SV40-NLS-Aub(green) were imported to the nucleus in OSCs.
Their import was blocked when piRNA biogenesis
was impaired by Yb RNAi, so Piwi-NLS-Aub andSV40-NLS-Aub were localized in the cytoplasm.
Yb was detected in the control using anti-Yb
antibody (red dots). DAPI signals (blue) show thenuclei. Scale bars, 5 mm.
(B) Nuclear localization of Piwi-NLS-EGFP and
SV40-NLS-EGFP (green) was unaffected by Yb
depletion in OSCs. Scale bars, 5 mm.
See also Figure S5 and Table S1.

comparable with endogenous ones (Mason et al., 2002). Embryonic lethality of Impa3 mutants was also not rescued by overexpression of Impa1 or Impa2 (Mason et al., 2003). These results
suggest that some cargo proteins, crucial for gonadal development and fertility in Drosophila, were forced to remain cytoplasmic under the circumstance where other Impa isoforms
were expressed in the mutant ovaries. Identification of cargos
for which nuclear import is tightly controlled by a particular
Impa protein is awaited.
Piwi bears a bona fide bipartite NLS. Why a bipartite, notmonopartite, NLS evolved in Piwi is still unknown. Our results revealed that bipartite NLSs, from both Piwi and NP, showed lessefficiency in nuclear import than monopartite NLSs, at least in
OSCs. Therefore, it might be expected that having a monopartite
NLS would simply be more effective and secure for exerting Piwi
function in the nucleus. However, interestingly, our results also
showed that Piwi containing its own NLS was clearly more sensitive to loss of piRNA than Piwi bearing SV40-NLS instead of its
own NLS. This result suggests that Piwi autoregulation may be
more controllable with bipartite NLSs than with monopartite
NLSs, and this is most likely the reason why Piwi evolutionally acquired a bipartite NLS rather than a monopartite NLS. Notably,
the N-terminal sequence of MIWI2, one of three PIWI proteins
expressed in mouse testis whose nuclear localization and subsequent piRNA-mediated transposon silencing in the nucleus
also requires piRNA loading (Carmell et al., 2007; Siomi et al.,2011), is similar to the N-terminal sequence of Piwi, including
the NLS. It is likely that MIWI2 also evolutionally acquired a bipartite NLS rather than a monopartite NLS (Figure S5I).
Piwi-NLS-Aub was able to be localized in the OSCs in apiRNA-dependent manner, similar to endogenous Piwi protein.
To accomplish transposon transcriptional silencing, Piwi-piRISC
has to target and remain bound with nascent transcripts that are
attached to the target loci upon transcription to efficiently induce

3654 Cell Reports 23, 3647–3657, June 19, 2018

heterochromatinization of the loci. Aub
exhibits a strong Slicer activity, so nuclear Aub-piRISC may immediately
cleave the target RNAs upon association.
If this were the case, Aub-piRISC, evenafter being successfully localized to
the nucleus in a similar way as PiwipiRISC, would not induce transcriptionalsilencing, as opposed to Piwi-piRISC, which can. However,
Aub-piRISC would efficiently cleave transposon mRNAs Slicer
dependently even in the nucleus, which should lead to post-transcriptional silencing of transposons. We are currently examining
the effect of nuclear Aub-piRISC in transposon silencing.
A recent Arabidopsis study showed that heterochromatic
small interfering RNAs (siRNAs) (hc-siRNAs) drive AGO4, a ubiquitously expressed Argonaute member, into the nucleus to facilitate target gene silencing via DNA methylation (Ye et al., 2012).
In the study, however, not only AGO4 WT but also AGO4 mutant
incapable of unwinding hc-siRNA duplexes to single-strand
remained to be cytoplasmic, suggesting that the siRNA duplex
loading onto AGO4 was insufficient for driving nuclear localization of AGO4. Rather, it further required AGO4 endonucleasedependent, passenger strand cleavage and displacement, i.e.,
activation of AGO4-RISC. The Ye et al. (2012) report also
revealed that AGO4 contains a monopartite NLS at Asp176Pro183 residues (DRKRLRRP), and the relocation of NLS to thevery N-terminal end of AGO4 drove AGO4 nuclear localizationin an hc-siRNA-independent manner. Furthermore, dissociationof a protein chaperone Hsc70/HSP90 from AGO4 was also
necessary for AGO4-RISC nuclear localization. Therefore,
Drosophila Piwi and Arabidopsis AGO4 show rather distinct
features regarding their nuclear localization, although both
Argonaute proteins are key components of nuclear transcriptional silencing in the respective species.

EXPERIMENTAL PROCEDURES
Cell Culture, RNAi, and Transfection with Plasmids
OSCs and S2 cells were cultured as described previously (Miyoshi et al., 2005;
Saito et al., 2009). To perform RNAi, we transfected OSCs with 200 pmol
siRNAs using Nucleofector 2b (Lonza). The siRNA sequences used in thisstudy are listed in Table S1. OSC transfection with plasmids was carried out

using Xfect Transfection Reagent (Clontech) as described previously (Murota
et al., 2014). S2 cells were transfected using Lipofectamine Transfection
Reagents (Invitrogen).
Production of Anti-Impa Monoclonal Antibodies
Recombinant proteins of full-length Impa1, Impa2, and Impa3 fused to glutathione S-transferase (GST) (GST-Impa1, GST-Impa2, and GST-Impa3) wereexpressed in and purified from E. coli and then injected into mice for immunization. Production and selection of hybridomas that produced anti-Impa
monoclonal antibodies were performed as described previously (Nishida
et al., 2015). To examine the specificity of anti-Impa monoclonal antibodies
raised in this study, we performed western blotting as described previously(Miyoshi et al., 2005) using OSC lysates before and after RNAi treatment foreach Impa member. b-Tubulin (bTub) was detected as a loading control using
anti-bTub mouse monoclonal antibody (1:2,500 dilution; obtained from theDevelopmental Studies Hybridoma Bank).
Plasmid Construction
We used plasmids pAcM-Piwi and pAcM-EGFP that were constructed andused previously (Saito et al., 2009) to express myc-Piwi and myc-EGFP in
OSCs. To yield pAcF-Piwi for expressing FLAG-Piwi in OSCs and S2 cells,
we exchanged the region encoding a myc peptide in pAcM-Piwi with a DNA
fragment encoding a FLAG peptide using NEBuilder HiFi DNA Assembly
Master Mix (NEB). The PCR primers used were F-Piwi-I-F/F-Piwi-I-R and
F-Piwi-V-F/F-Piwi-V-R (Table S1). Construction of all other plasmids is
described in the Supplemental Information.

the RNAi effect. Plasmids to overexpress FLAG-Piwi, together with mycImpa1, myc-Impa2, or myc-Impa3, were simultaneously introduced into thecells. Myc-EGFP was expressed as a negative control. One day later, immunofluorescence was performed as described above (see Immunofluorescence).
Between 15 and 18 cells were analyzed per Impa member, and representative
images were shown in corresponding figures (Figures 2A and 2B).
Limited Proteolysis Assays
Piwi with no tags was expressed in S2 cells by transfection and then immunoprecipitated using anti-Piwi antibody 3G11, to obtain piRNA-free Piwi. Piwi-piRISC
was immunoprecipitated from OSCs using 3G11. After extensive washing,chymotrypsin was added to both immunoprecipitated samples to give a final
concentration of 2.0 ng/mL. After incubation at 37 C for 30 min, the bead fractions
were separated from the supernatants, and both fractions were subjected towestern blotting using 4D2. Western blotting was performed as described previously (Miyoshi et al., 2005). To perform RNAi, we transfected OSCs with 200 pmol
EGFP-siRNA or Gasz-siRNA using Nucleofector 96-well shuttle (Lonza). To
obtain piRNA-free Piwi and Piwi-piRISC, Piwi with no tag was expressed in
OSCs treated with EGFP-siRNA or Gasz-siRNA using Nucleofector 2b in Nucleofection Solution (5 mM KCl, 15 mM MgCl2, 50 mM D-mannitol) added to
180 mM Church phosphoric acid buffer (pH 7.2) instead of 60 mM Na2PO4 and60 mM NaH2PO4 (Nye et al., 2014) and then immunoprecipitated using antiPiwi antibody 3G11. After extensive washing, chymotrypsin was added to bothimmunoprecipitated samples to give a final concentration of 2.0 ng/mL. Afterincubation at 37 C for 30 min, the beads fractions were separated from the supernatants, and both fractions were subjected to western blotting using 4D2.

Estimation of Impa Protein Levels in OSCs
Recombinant GST-Impa proteins isolated from E. coli and BSA (NEB) were run
on protein gels. Protein concentrations of full-length GST-Impa in each solution were estimated by staining them with Coomassie Brilliant Blue (CBB)and comparing them with BSA stained with CBB. Western blotting was thenperformed using anti-Impa monoclonal antibodies (this study) on GST-Impa
and OSC whole lysates. Signal intensity was calculated using ImageJ (NIH).

SUPPLEMENTAL INFORMATION

Immunofluorescence
Immunofluorescence was performed as described previously (Saito et al.,2010). The primary antibodies used in this study were anti-Impa monoclonal
antibodies (this study), anti-myc rabbit polyclonal antibodies (1:500 dilution;
Sigma), anti-FLAG mouse monoclonal antibody (1:1,000 dilution; Sigma),anti-myc mouse monoclonal antibody (1:1,000 dilution; Sigma), and anti-Yb
monoclonal antibody (1:500 dilution) (Murota et al., 2014). The secondary
antibodies used were Alexa 488-conjugated anti-mouse immunoglobulin M(IgM; Abcam), Alexa 488-conjugated anti-mouse IgG (Molecular Probes),
Alexa 488-conjugated anti-rabbit IgG (Molecular Probes), Alexa 546-conjugated anti-mouse IgG (Molecular Probes), and Alexa 546-conjugated anti-rabbit IgG (Molecular Probes). Transfection of OSCs prior to immunofluorescencewith plasmids was performed as described previously (Saito et al., 2009). Impa
and Yb were depleted by RNAi by introducing Impa-siRNAs and Yb-siRNA into
OSCs prior to overexpression of myc-Piwi (Figure 1), myc-Mael-SV40-NLS(Figure 3), myc-NP-NLS-EGFP (Figures 4E and 4F), myc-SV40-NLS-Piwi-DN(Figure 5A), myc-Piwi, (Figure 5B), myc-Piwi-NLS-Piwi (Figure 5B), mycPiwi-NLS-Aub (Figure 6A), myc-SV40-NLS-Aub (Figure 6A), myc-Piwi-NLSEGFP (Figure 6B), and myc-SV40-NLS-EGFP (Figure 6B). EGFP-siRNA or
luciferase (luc)-siRNA was used as a negative control. Between 12 and 15 cells
were analyzed per Impa member, where individual Impa proteins were efficiently depleted, and representative images were shown in corresponding figures (Figures 1A–1C, 3A, 3B, 4E, and 4F). Between 10 and 16 cells wereanalyzed before and after Yb depletion, and representative images wereshown in corresponding figures (Figures 5A, 5B, 6A, 6B, S4A, S4B, andS5H). Approximately 10 cells were analyzed, and representative images
were shown in corresponding figures (Figures 4B–4D).

We thank H. Ohtani for anti-Gasz antibody, Y. Tsuchizawa for plasmid construction, T. Shima for technical advice, and M. Oka and Y. Yoneda for discussions about properties of Impa proteins. We also thank members of the Siomi
laboratories at the University of Tokyo and Keio University School of Medicine
for technical advice and intensive discussion. This work was supported by aGrant-in-Aid for Scientific Research (S) (17H06111) from MEXT to M.C.S.
Y.M., K.M.N., S.Y., and H.S. were supported by a Grant-in-Aid for Young Scientist (B) (16K18488), a Grant-in-Aid for Scientific Research (C) (15K06948), a
Grant-in-Aid for scientific research on innovative areas (26112513), and a
Grant-in-Aid for Scientific Research (S) (25221003), respectively, from MEXT.

Supplemental Information includes Supplemental Experimental Procedures,five figures, and one table and can be found with this article online at https://doi.org/10.1016/j.celrep.2018.05.051.
ACKNOWLEDGMENTS

AUTHOR CONTRIBUTIONS
R.Y., H.S., and M.C.S. conceived the project and designed the experiments.
R.Y., Y.M., K.M.N., H.Y., and S.Y. performed the experiments with K.F.’sand A.O.’s help. L.N. performed mass spectrometric analysis. R.Y., Y.M.,
K.M.N., H.S., and M.C.S. analyzed the data and wrote the paper.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: February 13, 2018
Revised: April 25, 2018
Accepted: May 16, 2018
Published June 19, 2018
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