COGEDE-814; NO. OF PAGES 7

Available online at www.sciencedirect.com

Gatekeepers for Piwi–piRNA complexes to enter the nucleus
Hirotsugu Ishizu, Akihiro Nagao and Haruhiko Siomi
RNA silencing pathways are now recognized to participate inessential cellular functions ranging from the regulation of mRNA
turnover to the suppression of the activity of potentially
deleterious transposable elements (TEs). Piwi-interacting
RNAs (piRNAs) are germline-specific, small silencing RNAs thatsuppress TE activity and maintain genome integrity during
germline development. In Drosophila ovarian somatic cells,
piRNAs are processed from long single-stranded RNAs by a
Dicer-independent pathway and are loaded onto Piwi in thecytoplasm. The Piwi–piRNA complexes are then transported
into the nucleus to exert TE silencing. This mechanism involves
gatekeepers for a functional Piwi–piRNA complex to beimported, which parallels with the Tetrahymena Twi1p–scan
RNA pathway used to carry out the programmed DNA
elimination.
Address
Department of Molecular Biology, Keio University School of Medicine,
Shinjuku-ku, Tokyo 160-8582, Japan
Corresponding author: Siomi, Haruhiko (awa403@z2.keio.jp)

Current Opinion in Genetics & Development 2011, 21:1–7
This review comes from a themed issue on
Differentiation and gene regulation
Edited by Jessica Treisman and Joel Richter0959-437X/$ – see front matter# 2011 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.gde.2011.05.001

Introduction
In RNA silencing pathways, small non-coding RNAs of
20–30 nt function as specificity determinants for the
repressive activities of Argonaute-containing effector
complexes, termed RNA-induced silencing complexes(RISCs) [1–4]. These recognize their target RNAs bybase pairing and promote the inactivation of homologous
sequences by a variety of mechanisms in the cytoplasm
and the nucleus. RNA interference (RNAi), one form of
RNA silencing, was originally described as a silencing
phenomenon initiated by exogenous double-stranded
RNA (dsRNA) [5], and many classes of small-silencing
RNAs including microRNAs (miRNAs) and endogenous
small-interfering RNAs (endo-siRNAs) have been shown
to be processed by Dicer from their dsRNA precursors.
These precursors can be derived from various sources,
such as simultaneous sense and antisense transcription of
specific genomic loci, fold-back structured transcripts
www.sciencedirect.com

from repetitive sequences and viral replication intermediates [1–4,6]. We now have a good understanding of thebasic biogenesis of small-silencing RNAs, which involves
dsRNA precursors and Dicers. Deviations from this paradigm however have been increasingly observed [7,8].
Production of some classes of small-silencing RNAs is
Dicer-independent; with current interests also turning to
how biogenesis of small-silencing RNAs is physiologicallyregulated [9,10]. In principle, expression of small-silencing RNAs can be controlled by many mechanisms including transcriptional regulations and epigenetic
alterations. However, substantial regulation of maturesmall-silencing RNA accumulation also occurs post-transcriptionally. Recent studies have shown that many steps
in the maturation of small-silencing RNAs are subject toregulation, and that formation and turnover of functional
RISCs are also regulated. Therefore, post-transcriptional
regulation of small-silencing RNA biogenesis provides anadditional level of complexity to RNA-silencing pathways
and contributes to the modulation of different biologicalprocesses.
In the Drosophila ovary, Piwi-interacting RNAs (piRNAs)
arise from repetitive intergenic elements including transposable elements (TEs), via Dicer-independent pathways and function through the PIWI subfamily (AGO3,
Aubergine, and Piwi) of Argonautes to ensure silencing of
TEs [11,12]. In ovarian somas, Piwi is localized in the
nucleus, where it silences TEs; however, piRNA loading
occurs in the cytoplasm, which in turn is required for the
nuclear entry of Piwi. This suggests a model in which thestate of Piwi in ovarian somas is monitored and only a Piwi
loaded with mature piRNA is selectively transported into
the nucleus to exert TE silencing. Here, we review the
recent progress in our understanding of piRNA biogenesis in the Drosophila ovary and discuss the nuclear
transport system of Piwi–piRNA complexes, which has
striking parallels to that of Twi1p–scanRNA complexes in
Tetrahymena.

piRNA biogenesis in Drosophila
The Drosophila genome encodes three PIWI family
proteins: AGO3, Aubergine (Aub) and Piwi. Mutationsin the PIWI genes lead to defects in germline development and derepression of TEs [13–17]. PIWI subfamily proteins bind to a distinct population of smallRNAs, which mainly correspond to TEs, and are now
known as piRNAs [18–21]. They are produced via
Dicer-independent pathways from long single-stranded
RNAs, many of which are transcribed from TE-richclusters in the genome, and guide TE silencing in trans[17–21,22,23,24]. Two main pathways exist to generate
Current Opinion in Genetics & Development 2011, 21:1–7

Please cite this article in press as: Ishizu H, et al. Gatekeepers for Piwi–piRNA complexes to enter the nucleus, Curr Opin Genet Dev (2011), doi:10.1016/j.gde.2011.05.001

COGEDE-814; NO. OF PAGES 7

2 Differentiation and gene regulation

Figure 1(a)Follicle cell

egg chamber

OocyteNurse cell(b)Germ cell
Nucleus(c)

Somatic cell
piRNA cluster transcript

Cytoplasm
Nuage
Aub

transposon

Yb body

primary
processing

Piwi
Aub

ping-pong AGO3

AGO3

Cytoplasm

NucleusPiwi

transposon

Silencing
Current Opinion in Genetics & Development

The piRNA pathways in the Drosophila ovary. The three PIWI proteins — Piwi, Argonaute3 (Ago3), and Aubergine (Aub) — are expressed in Drosophila
ovaries. (a) Diagram of a mid-stage egg chamber of the Drosophila ovary. (b) Aub and Ago3 reside in the nuage, a cytoplasmic structure located in theperinuclear region of nurse cells. Multiple components of the piRNA pathway are enriched in nuage and therefore the ping-pong pathway may operate
there. Sense and antisense transcripts from TE loci within the genome of the Drosophila ovary are transcribed and do not always form dsRNAs. Slicer
activity of Aub and Ago3 is involved in the ping-pong amplification pathway, which amplifies a small initial pool of maternal and primary piRNAs.
Amplification only occurs between a pair of piRNAs with a ping-pong signature, thereby avoiding self-directed reactions. Aub associated with
antisense piRNA cleaves piRNA precursors in the sense strand. This reaction generates sense piRNAs that are loaded onto Ago3. Ago3 associatedwith sense piRNA cleaves target antisense transcripts, generating antisense piRNAs that are subsequently loaded into Aub. This reaction also depends
on the ongoing presence of a target and therefore steers piRNA production toward active elements. (c) In somatic follicle cells, piRNA clustertranscripts are processed into piRNAs without ping-pong amplification. Mature primary piRNAs are generated in Yb bodies, which are cytoplasmic
non-membranous structures found in follicle cells. Processed piRNAs are loaded into Piwi and guide the repression of somatically expressedtransposons. Somatic piRNAs corresponding to TEs are almost exclusively derived from the flamenco locus. The mechanism(s) by which primary
flamenco transcripts, approximately 150 kb, are exported to the cytoplasm and selected to be a piRNA precursor remains to be elucidated.

piRNAs in the Drosophila ovary [11,12], the ping-pong
amplification and the primary pathways (Figure 1). The
ping-pong pathway operates specifically in germline
cells and mainly engages AGO3 and Aub, both of which
are accumulated in nuage [20,21,22,23]. Nuage areperinuclear structures found at the cytoplasmic face ofthe nuclear envelope in animal germline cells, wheremultiple components of the piRNA pathway areenriched (Figure 1b) [21,22,23,25]. The pathway
depends on the endoribonuclease or Slicer activity of
AGO3 and Aub, which act catalytically, thereby leading
to repeated rounds of piRNA production [20,21]. However, there must be primary piRNAs that initiate thispathway and thus piRNAs produced by the ping-pong
pathway are often referred to as secondary piRNAs. It isbelieved that the ping-pong pathway is primed at least
partly by maternally contributed piRNAs [26].
Current Opinion in Genetics & Development 2011, 21:1–7

piRNA biogenesis and nuclear transport of
Piwi in ovarian somatic cells
Although AGO3 and Aub are cytoplasmic proteins
expressed only in germline cells in the ovaries, Piwi isa nuclear protein expressed in both germline, and somaticcell, such as the follicle cells of the ovaries [18,22,23].
The mechanism(s) by which Piwi represses TEs in the
nucleus remains to be elucidated, but previous studies
suggested that Piwi may play a role in heterochromatin
formation [27]. Alternatively, Piwi–piRNA complexes
may associate with the nuclear RNA turnover machinery,providing them with the target specificity for the degradation of TE transcripts.
Ovarian somatic piRNAs that correspond to TEs arealmost exclusively derived from one genomic cluster
named the flamenco locus, by a pathway independent of
www.sciencedirect.com

Please cite this article in press as: Ishizu H, et al. Gatekeepers for Piwi–piRNA complexes to enter the nucleus, Curr Opin Genet Dev (2011), doi:10.1016/j.gde.2011.05.001

COGEDE-814; NO. OF PAGES 7

Gatekeepers for Piwi–piRNA complexes to enter the nucleus Ishizu, Nagao and Siomi 3

Figure 2(a) Drosophila

piRNA precursors

primary piRNA pathway

Nucleus1?

Piwi
piRNA intermediates
Armi
Yb body
Armi

Armi
Piwi

2

Piwi

3

Piwi

5

Zuc?

4

Piwi
piRNA
cytoplasm

Micronucleus(b) Tetrahymena

Parental Macronucleus

ncRNA

scnRNA pathway
1

Dcl1p

Giw1p

Twi1p

5

scnRNA

Giw1p

Twi1p

2

Twi1p

3

Twi1p

4

Twi1p

Giw1p
cytoplasm
Current Opinion in Genetics & Development

Models of gatekeepers for the nuclear entry of the functional Argonaute–small RNA complexes. (a) In Drosophila melanogaster primary piRNA
pathway, the nuclear localization of Piwi is required for silencing of target transposons. Step 1: piRNA cluster transcripts are processed into piRNAintermediates by unknown mechanisms. Step 2: Armi binds to Piwi and localizes it to Yb bodies. Step 3: piRNA intermediates are assembled into
Armi–Piwi complexes at the Yb bodies. Step 4: piRNA intermediates are processed into piRNA and mature piRNA are loaded into Piwi. Zuc, a putative
nuclease, is required for primary piRNA processing. Step 5: The functional Piwi–piRNA complex enters the nucleus. (b) In Tetrahymena, the nuclear
localization of Twi1p is required for RNAi-based DNA scanning and elimination. Step 1: In the sexual process of conjugation, the bi-directionallytranscribed ncRNAs are processed into scnRNAs by Dcl1p. Step 2: scnRNAs are exported to the cytoplasm, where Twi1p loads scnRNAs. Step 3: The
slicer activity of Twi1p removes scnRNA passenger strands. Step 4: Giw1p senses the state of Twi1p–scnRNA complexes and selectively binds withfunctional Twi1p–scnRNA complexes. Step 5: The Twi1p–scnRNA complex enters the parental macronucleus. Giw1p shows no obvious similarity withany known protein [44].

the ping-pong cycle and loaded onto Piwi [22,23,28].
This pathway is called the primary piRNA pathway(Figure 1c) [11,12]. The somatic piRNA pathway does
not require Slicer activity of Piwi but instead requires
the RNA helicase Armitage (Armi), the Tudor domain
containing RNA helicase Yb, and the mitochondrial
phospholipase D homolog Zucchini (Zuc), as essential
factors for primary piRNA biogenesis (Figure 2a)[23,28,29,30,31]. Lack of any of these factors leads
to a massive decrease in piRNA levels and TE derepression. The cytoplasmic localization of all three proteinssuggests that piRNA biogenesis and loading occurs in the
www.sciencedirect.com

cytoplasm. Yb is only expressed in the soma, but thenaccumulates in cytoplasmic foci as ‘Yb bodies’, which
localize in close proximity to mitochondria [32]. Armi
forms a complex with Piwi and Yb. Without Armi or Yb,
Piwi is not loaded with piRNAs and evenly localizes
throughout the cytoplasm. Without Zuc, Piwi is notloaded with piRNAs and accumulates at Yb bodies.
These findings suggest a model where piRNA precursors
are processed in the cytoplasm and formation of a functional Piwi–piRNA complex occurs in Yb bodies before
nuclear entry to exert TE silencing. Recent studies haveshown that the conformation of Argonaute proteins
Current Opinion in Genetics & Development 2011, 21:1–7

Please cite this article in press as: Ishizu H, et al. Gatekeepers for Piwi–piRNA complexes to enter the nucleus, Curr Opin Genet Dev (2011), doi:10.1016/j.gde.2011.05.001

COGEDE-814; NO. OF PAGES 7

4 Differentiation and gene regulation

Figure 3

Giw1p

IES

Ema1p

Parental Mac
Early/Mid Stage

Parental Mic

ncRNA
Dcl1p
scnRNA

Twi1p

Parental Mac

New Mac

IES

Late Stage

New Mic

Current Opinion in Genetics & Development

A model of small RNA-directed DNA scanning and elimination in Tetrahymena. In the early conjugation stages, the genome of the micronucleus (Mic),
including the internal eliminated sequences (IESs), is bi-directionally transcribed, followed by the formation of double-stranded non-coding RNAs
(ncRNAs). The ncRNAs are processed by Dicer-like protein Dcl1p to generate scnRNA duplexes. The scnRNAs are exported from the Mic to thecytoplasm, where they are loaded into Twi1p and processed into single-stranded scnRNAs by the slicer activity of Twi1p. Giw1p binds to the Twi1p–
scnRNA complex and transfers the complex to the parental macronucleus (Mac). Ema1p is required for the interaction between scnRNA and ncRNA in
the parental Mac, and induces the degradation of scnRNAs homologous to the DNA sequence. In the later conjugation stages, Twi1p binding with
scnRNAs that are not degraded in the parental Mac is transferred to the new developing Mac, followed by the elimination of IESs. In this system, therecognition of germline (with TEs) versus macronuclear specific sequences (without TEs) is achieved by a developmental comparison of parental anddeveloping genomes, guided by small RNAs, in trans.

undergoes a dynamic change during loading of small
RNAs to accommodate them [33,34]. This conformational change is likely assisted by several of the chaperoninsthat interact with Argonaute proteins [35–37]. Hsp90
interacts with Piwi and is required for piRNA accumulation [38,39], thereby suggesting that it is a piRNAloading factor. However, a protein(s) that senses thefunctional Piwi–piRNA complex has not been identified.

Scan RNA biogenesis and transport in
Tetrahymena
The nuclear transport of Piwi–piRNA complexes in ovarian somatic cells is likened to that of Twi1p–scnRNAcomplexes in the single-cell ciliate Tetrahymena. Like
most ciliated protozoa, Tetrahymena thermophila possesses
two distinctly different types of nuclei in each cell: thelarger somatic macronucleus contributes to gene expression; and the smaller germline micronucleus performs
reproductive functions [40,41]. Both are descendants ofthe same zygotic nucleus, but only the micronucleus
produces both new micronuclei and macronuclei during
sexual reproduction. In the developing new macronucleus, programmed DNA elimination occurs, which
restructures the somatic genome to become drastically
Current Opinion in Genetics & Development 2011, 21:1–7

different from its germline origin. The approximately
6000 internal eliminated sequences (IESs) mainly consistof repeated sequences that contain TEs. The DNA
elimination is mediated by the interaction of germlineproduced small RNAs with approximately 28–29 nt,
termed scan RNAs (scnRNAs), and transcripts from themacronucleus (Figure 3) [42].
The scnRNA duplexes are processed in the micronucleus
by the Dicer-like protein, Dcl1p, from long dsRNAs
produced by bidirectional transcription of the micronuclear genome early during conjugation [43]. The duplexes
are transported into the cytoplasm, where they are converted into a single-stranded form as they are loaded onto
Twi1p, a Piwi protein. Unwinding of the scnRNA duplex
is facilitated by slicing the unincorporated strand by
Twi1p [44], similar to the function of AGO2 on siRNA
duplexes for the formation of RISCs [45,46]. This resultsin the production of scnRNAs for all types of sequences
including cellular genes and TEs. Because Twi1p is a
Piwi family protein, scnRNAs can be considered as theciliate equivalent of piRNAs. The Twi1p–scnRNA complexes are initially channeled to the parental macronucleus, which requires Giw1p (Figure 2b) [44]. Loadingwww.sciencedirect.com

Please cite this article in press as: Ishizu H, et al. Gatekeepers for Piwi–piRNA complexes to enter the nucleus, Curr Opin Genet Dev (2011), doi:10.1016/j.gde.2011.05.001

COGEDE-814; NO. OF PAGES 7

Gatekeepers for Piwi–piRNA complexes to enter the nucleus Ishizu, Nagao and Siomi 5of scnRNA promotes association of Twi1p with Giw1p
and thereby nuclear localization of Twi1p–scnRNA complexes. In the parental macronucleus, it has been proposed that the existing rearranged genome be scanned forhomology [42]. The scnRNAs that pair with maternalsequences are removed from the pool of active complexes, most likely, by recognizing nascent long transcripts with the help of the RNA helicase Ema1p [47],while the micronucleus-specific scnRNAs such as IESspecific and TE-specific scnRNAs are enriched. Given
the recent findings that extensive pairing between atarget RNA and an Argonaute-bound small guide RNA
triggers the destabilization of the small RNA [48,49], theremoval of scnRNAs by long transcripts is probably
achieved by similar pairing-induced degradation of
scnRNAs. Finally, Twi1p–scnRNA complexes areexported from the parental macronucleus and reimported into the developing zygotic macronucleus,
where they pair with homologous sequences and induce
the elimination of the marked DNA sequences throughtargeted histone H3K9 methylation [50,51].

Regulated nuclear transport pathways of Piwi
proteins
Nuclear transport of Piwi in Drosophila ovarian somatic
cells and Twi1p in Tetrahymena requires small RNA
loading (Figure 2). N-terminally truncated Piwi that is
loaded with piRNAs cannot translocate into the nucleus
and thus cannot silence TEs [28,29]. There are at least
two possible scenarios concerning the nuclear import of
Piwi. First, because the N-terminal portion of the protein
contains a nuclear localization signal (NLS) that functionsin the nongonadal somatic S2 cell line where piRNAs are
not produced ([28]; K Saito, unpublished observation),
the NLS may remain masked by intermolecular folding orinteraction with another protein when Piwi is not loadedwith piRNAs in ovarian somatic cells. As the conformation of Argonaute proteins changes at a small RNA
loading step [33,34], piRNA loading may also induce
conformational change of Piwi to dissociate a protein thatmasks the NLS at the N-terminal part of Piwi. This isakin to the Schizosaccharomyces pombe protein Mei2.
Accumulation of Mei2 in the nucleus requires a noncoding RNA termed meiRNA [52]. Second, conformational changes following piRNA loading may allow
binding of a protein(s) that brings Piwi into the nucleus byproviding its specific import signal. This model is related
to the Tetrahymena Giw1p [44]. Genetically distinct
nuclei in each Tetrahymena cell share the same cytoplasm
and therefore share the products of most genes that may
be equally distributed within the cytoplasm. Binding of
Giw1p brings a Twi1p–scnRNA complex into parental
macronuclei at the early stages of conjugation. Thus
Giw1p not only senses the state of Twi1p but may also
supply an initial import signal(s) that channels Twi1p to
specific nuclei during sequential transport events. Reimport of Twi1p into developing macronuclei likely
www.sciencedirect.com

involves a different mechanism. Nuclear transport of Piwi
in the ovarian soma depends on the cytoplasmic Yb body,which is not observed in germline cells. Instead, nuclear
accumulation of Piwi in germline cells appears to dependon the presence of Aub and AGO3 ([22]; A Nagao,
unpublished observation). Lack of Aub or AGO3 results
in interruption of the ping-pong cycle, leading to the lossof germline piRNAs [22,23]. Therefore, nuclear transport of Piwi in germline cells may also depend on piRNAloading. How the state of Piwi (with/without piRNA) in
germline cells is sensed remains to be elucidated; clearly
the sensing system differs from that of the soma, at least
concerning the presence and absence of Yb. The mechanisms involved in Piwi’s nuclear entry are distinct between the germline and somatic cells in the Drosophila
ovary.

Conclusions
The state of the protein to be transported can be modified
by interaction with a regulatory protein, which either
blocks or promotes association of the machinery thatallows for importing of proteins [53]. Given that thenuclear transport of some Argonaute proteins in Caenorhabditis elegans [54] and mice [55] is also dependent uponsmall RNA loading, an important recurring theme is thatcells have systems that sense the state of Argonaute/Piwi
proteins and transport only functional RISCs into the
nucleus. In the future, it will be important to identify
factors that recognize only a functional RISC among allother intermediates during RISC maturation. It would
also be advantageous to identify the factors that supply
specific signals to the RISC for recognition by nuclear
transport machineries, and those that act to keep RISC
intermediates in the cytoplasm so that nuclear import ofpremature RISCs is avoided.

Acknowledgements
We thank Mikiko Siomi, Kuniaki Saito and other members of the Siomi
laboratory for comments on the manuscript. This work was supported by
MEXT (Ministry of Education, Culture, Sports, Science, and Technologyof Japan) grants to HS. HI is supported by the JSPS (Japan Society for the
Promotion of Science).

References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1.

Siomi H, Siomi MC: On the road to reading the RNAinterference code. Nature 2009, 457:396-404.

2.

Kim NV, Han J, Siomi MC: Biogenesis of small RNAs in animals.
Nat Rev Mol Cell Biol 2009, 10:126-139.

3.

Carthew RW, Sontheimer EJ: Origins and mechanisms ofmiRNAs and siRNAs. Cell 2009, 136:642-655.

4.

Moazed D: Small RNAs in transcriptional gene silencing andgenome defence. Nature 2009, 457:413-420.

5.

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC:
Potent and specific genetic interference by double-stranded
RNA in Caenorhabditis elegans. Nature 1998, 391:806-811.
Current Opinion in Genetics & Development 2011, 21:1–7

Please cite this article in press as: Ishizu H, et al. Gatekeepers for Piwi–piRNA complexes to enter the nucleus, Curr Opin Genet Dev (2011), doi:10.1016/j.gde.2011.05.001

COGEDE-814; NO. OF PAGES 7

6 Differentiation and gene regulation

6.

Okamura K, Lai EC: Endogenous small interfering RNAs in
animals. Nat Rev Mol Cell Biol 2008, 9:673-678.

7.

Miyoshi K, Miyoshi T, Siomi H: Many ways to generate
microRNA-like small RNAs: non-canonical pathways for
microRNA production. Mol Genet Genomics 2010, 284:95-103.

8.

Ketting RF: The many faces of RNAi. Dev Cell 2011, 20:148-161.

9.

Siomi H, Siomi MC: Posttranscriptional regulation of miRNAbiogenesis in animals. Mol Cell 2010, 38:323-332.

10. Davis BN, Hata A: Regulation of MicroRNA biogenesis: amiRiad of mechanisms. Cell Commun Signal 2009, 7:18.
11. Siomi MC, Sato K, Pezic D, Aravin AA: PIWI-interacting small
RNAs: the vanguard of genome defence. Nat Rev Mol Cell Biol
2011, 12:246-258.
12. Malone CD, Hannon GJ: Small RNAs as guardians of the
genome. Cell 2009, 136:656-668.
13. Cox DN, Chao A, Lin H: piwi encodes a nucleoplasmic factor
whose activity modulates the number and division rate ofgermline stem cells. Development 2000, 127:503-514.
14. Kalmykova AI, Klenov MS, Gvozdev VA: Argonaute protein PIWI
controls mobilization of retrotransposons in the Drosophilamale germline. Nucleic Acids Res 2005, 33:2052-2059.
15. Aravin AA, Naumova NM, Tulin AV, Vagin VV, Rozovsky YM,
Gvozdev VA: Double-stranded RNA-mediated silencing ofgenomic tandem repeats and transposable elements in the D.
melanogaster germline. Curr Biol 2001, 11:1017-1027.
16. Harris AN, Macdonald PM: Aubergine encodes a Drosophilapolar granule component required for pole cell formation andrelated to eIF2C. Development 2001, 128:2823-2832.
17. Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD: A
distinct small RNA pathway silences selfish genetic elementsin the germline. Science 2006, 313:320-324.
18. Saito K, Nishida KM, Mori T, Kawamura Y, Miyoshi K,
Nagami T, Siomi H, Siomi MC: Specific association of Piwi with
rasiRNAs derived from retrotransposon and heterochromatic
regions in the Drosophila genome. Genes Dev 2006,
20:2214-2222.
19. Nishida KM, Saito K, Mori T, Kawamura Y, Nagami-Okada T,
Inagaki S, Siomi H, Siomi MC: Gene silencing mechanisms
mediated by Aubergine piRNA complexes in Drosophila malegonad. RNA 2007, 13:1911-1922.
20. Gunawardane LS, Saito K, Nishida KM, Miyoshi K, Kawamura Y,
Nagami T, Siomi H, Siomi MC: A slicer-mediated mechanism for
repeat-associated siRNA 50 end formation in Drosophila.
Science 2007, 315:1587-1590.
21. Brennecke J, Aravin AA, Stark A, Dus M, Kellis M,
Sachidanandam R, Hannon GJ: Discrete small RNA-generatingloci as master regulators of transposon activity in Drosophila.
Cell 2007, 128:1089-1103.
22. Li C, Vagin VV, Lee S, Xu J, Ma S, Xi H, Seitz H, Horwich MD,Syrzycka M, Honda BM et al.: Collapse of germline piRNAs in
the absence of Argonaute3 reveals somatic piRNAs in flies.
Cell 2009, 137:509-521.
By analyzing Drosophila piRNA pathway mutants for their impacts on
piRNA populations, this work, together with Ref. [23], demonstrates thatat least two distinct pathways for piRNA biogenesis with different components function in ovarian germ and somatic cells.
23. Malone CD, Brennecke J, Dus M, Stark A, McCombie WR,Sachidanandam R, Hannon GJ: Specialized piRNA pathways
act in germline and somatic tissues of the Drosophila ovary.
Cell 2009, 137:522-535.
See annotation to Ref. [22].
24. Yin H, Lin H: An epigenetic activation role of Piwi and a Piwiassociated piRNA in Drosophila melanogaster. Nature 2007,
450:304-308.
25. Lim AK, Kai T: Unique germ-line organelle, nuage, functions torepress selfish genetic elements in Drosophila melanogaster.
Proc Natl Acad Sci U S A 2007, 104:6714-6719.
Current Opinion in Genetics & Development 2011, 21:1–7

26. Brennecke J, Malone CD, Aravin AA, Sachidanandam R, Stark A,
Hannon GJ: An epigenetic role for maternally inherited piRNAs
in transposon silencing. Science 2008, 322:1387-1392.
27. Pal-Bhadra M, Leibovitch BA, Gandhi SG, Rao M, Bhadra U,
Birchler JA, Elgin SC: Heterochromatic silencing and HP1
localization in Drosophila are dependent on the RNAi
machinery. Science 2004, 303:669-672.
28. Saito K, Inagaki S, Mituyama T, Kawamura Y, Ono Y, Sakota E,Kotani H, Asai K, Siomi H, Siomi MC: A regulatory circuit for piwi
by the large Maf gene traffic jam in Drosophila. Nature 2009,
461:1296-1299.
Using a Drosophila ovarian somatic cell line where Piwi, but not Aub or
AGO3, is expressed, this work identifies the primary piRNAs that are
produced by a ping-pong independent pathway.
29. Saito K, Ishizu H, Komai M, Kotani H, Kawamura Y, Nishida KM,
 Siomi H, Siomi MC: Roles for the Yb body components
Armitage and Yb in primary piRNA biogenesis in Drosophila.
Genes Dev 2010, 24:2493-2498.
Together with Ref. [30], this work identifies several components of the
primary piRNA pathway and provides evidence that supports a model inwhich a functional Piwi–piRNA complex is formed in cytoplasmic Ybbodies before its nuclear entry to exert transposon silencing.
30. Olivieri D, Sykora MM, Sachidanandam R, Mechtler K,
 Brennecke J: An in vivo RNAi assay identifies major genetic
and cellular requirements for primary piRNA biogenesis in
Drosophila. EMBO J 2010, 29:3301-3317.
See annotation to Ref. [29].
31. Qi H, Watanabe T, Ku HY, Liu N, Zhong M, Lin H: The Yb body, amajor site for Piwi-associated RNA biogenesis and a gateway
for Piwi expression and transport to the nucleus in somatic
cells. J Biol Chem 2011, 286:3789-3797.
32. Szakmary A, Reedy M, Qi H, Lin H: The Yb protein defines anovel organelle and regulates male germline stem cell
self-renewal in Drosophila melanogaster. J Cell Biol 2009,
185:613-627.
33. Wang Y, Juranek S, Li H, Sheng G, Tuschl T, Patel DJ: Structure ofan argonaute silencing complex with a seed-containing guideDNA and target RNA duplex. Nature 2008, 456:921-926.
34. Parker JS: How to slice: snapshots of Argonaute in action.
Silence 2010, 1:3.
35. Iki T et al.: In vitro assembly of plant RNA-induced silencing
complexes facilitated by molecular chaperone HSP90. Mol Cell
2010, 39:282-291.
36. Iwasaki S et al.: Hsc70/Hsp90 chaperone machinery mediates
ATP-dependent RISC loading of small RNA duplexes. Mol Cell
2010, 39:292-299.
37. Miyoshi T, Takeuchi A, Siomi H, Siomi MC: A direct role for Hsp90
in pre-RISC formation in Drosophila. Nat Struct Mol Biol 2010,
17:1024-1026.
38. Specchia V et al.: Hsp90 prevents phenotypic variation by
suppressing the mutagenic activity of transposons. Nature
2010, 463:662-665.
39. Gangaraju VK, Yin H, Weiner MM, Wang J, Huang XA, Lin H:
Drosophila Piwi functions in Hsp90-mediated suppression ofphenotypic variation. Nat Genet 2011, 43:153-158.
40. Yao MC, Chao JL: RNA-guided DNA deletion in Tetrahymena:an RNAi-based mechanism for programmed genome
rearrangements. Annu Rev Genet 2005, 39:537-559.
41. Kurth HM, Mochizuki K: Non-coding RNA: a bridge between
small RNA and DNA. RNA Biol 2009, 6:138-140.
42. Mochizuki K, Fine NA, Fujisawa T, Gorovsky MA: Analysis of apiwi-related gene implicates small RNAs in genome
rearrangement in tetrahymena. Cell 2002, 110:689-699.
43. Mochizuki K, Gorovsky MA: A Dicer-like protein in Tetrahymena
has distinct functions in genome rearrangement,
chromosome segregation, and meiotic prophase. Genes Dev
2005, 19:77-89.
44. Noto T, Kurth HM, Kataoka K, Aronica L, DeSouza LV, Siu KW,
 Pearlman RE, Gorovsky MA, Mochizuki K: The Tetrahymena
www.sciencedirect.com

Please cite this article in press as: Ishizu H, et al. Gatekeepers for Piwi–piRNA complexes to enter the nucleus, Curr Opin Genet Dev (2011), doi:10.1016/j.gde.2011.05.001

COGEDE-814; NO. OF PAGES 7

Gatekeepers for Piwi–piRNA complexes to enter the nucleus Ishizu, Nagao and Siomi 7

argonaute-binding protein Giw1p directs a mature argonautesiRNA complex to the nucleus. Cell 2010, 140:692-703.
This work identifies Giw1p, which binds to Twi1p and is required for its
nuclear localization, and provides evidence that suggests a model inwhich Giw1p senses the state of Twi1p-associated scnRNAs and selectively transports the mature Twi1p–scnRNA complex into the nucleus.
45. Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD: Passengerstrand cleavage facilitates assembly of siRNA into Ago2containing RNAi enzyme complexes. Cell 2005, 123:607-620.
46. Miyoshi K, Tsukumo H, Nagami T, Siomi H, Siomi MC: Slicer
function of Drosophila Argonautes and its involvement in RISC
formation. Genes Dev 2005, 19:2837-2848.
47. Aronica L, Bednenko J, Noto T, DeSouza LV, Siu KW, Loidl J,
Pearlman RE, Gorovsky MA, Mochizuki K: Study of an RNA
helicase implicates small RNA-noncoding RNA interactions inprogrammed DNA elimination in Tetrahymena. Genes Dev
2008, 22:2228-2241.
48. Ameres SL, Horwich MD, Hung JH, Xu J, Ghildiyal M, Weng Z,
Zamore PD: Target RNA-directed trimming and tailing of small
silencing RNAs. Science 2010, 328:1534-1539.
49. Cazalla D, Yario T, Steitz JA: Down-regulation of a hostmicroRNA by a Herpesvirus saimiri noncoding RNA. Science
2010, 328:1563-1566.

www.sciencedirect.com

50. Madireddi MT, Coyne RS, Smothers JF, Mickey KM, Yao MC,
Allis CD: Pdd1p, a novel chromodomain-containing protein,links heterochromatin assembly and DNA elimination in
Tetrahymena. Cell 1996, 87:75-84.
51. Liu Y, Taverna SD, Muratore TL, Shabanowitz J, Hunt DF, Allis CD:
RNAi-dependent H3K27 methylation is required forheterochromatin formation and DNA elimination in
Tetrahymena. Genes Dev 2007, 21:530-1545.
52. Yamashita A, Watanabe Y, Nukina N, Yamamoto M: RNAassisted nuclear transport of the meiotic regulator Mei2p infission yeast. Cell 1998, 95:115-123.
53. Mason DA, Goldfarb DS: The nuclear transport machinery as aregulator of Drosophila development. Semin Cell Dev Biol 2009,
20:582-589.
54. Guang S, Bochner AF, Pavelec DM, Burkhart KB, Harding S,
Lachowiec J, Kennedy S: An Argonaute transports siRNAs
from the cytoplasm to the nucleus. Science 2008,
321:537-541.
55. Aravin AA, Sachidanandam R, Bourc’his D, Schaefer C, Pezic D,
Toth KF, Bastor T, Hannon GJ: A piRNA pathway primed byindividual transposons is linked to de novo DNA methylation in
mice. Mol Cell 2008, 31:785-799.

Current Opinion in Genetics & Development 2011, 21:1–7

Please cite this article in press as: Ishizu H, et al. Gatekeepers for Piwi–piRNA complexes to enter the nucleus, Curr Opin Genet Dev (2011), doi:10.1016/j.gde.2011.05.001