Developmental Cell

Review
Small RNA-Mediated Quiescenceof Transposable Elements in Animals
Kuniaki Saito1,2,* and Mikiko C. Siomi1,3,*
1Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan
2JST, PRESTO, Saitama 332-0012, Japan
3JST, CREST, Saitama 332-0012, Japan*Correspondence: saito@z6.keio.jp (K.S.), siomim@sc.itc.keio.ac.jp (M.C.S.)
DOI 10.1016/j.devcel.2010.10.011

Transposable elements (TEs) are major components of the intergenic regions of the genome. However, TE
transposition has the potential to threaten the reproductive fitness of the organism; therefore, organisms
have evolved specialized molecular systems to sense and repress the expression of TEs to stop themfrom jumping to other genomic loci. Emerging evidence suggests that Argonaute proteins play a criticalrole in this process, in collaboration with two types of cellular small RNAs: PIWI-interacting RNAs (piRNAs)of the germline and endogenous small interfering RNAs (endo-siRNAs) of the soma, both of which are transcribed from TEs themselves.
Introduction
Transposable elements (TEs) were discovered in maize by
Barbara McClintock in the 1940s. Since then, various TEs andTE-like sequences have been identified in many species. Worldwide genome projects have revealed that TEs are actuallymajor genomic components. For example, TEs comprise 45%of the human genome (Lander et al., 2001) and 15%–22% of
the genome of Drosophila melanogaster (Kapitonov and Jurka,
2003; Biémont and Vieira, 2006). TEs can be separated into
Class 1 and Class 2, based on their structures and modes ofintegration (Gogvadze and Buzdin, 2009). Class 1 comprises
retrotransposons and retrotransposon-like elements, suchas LINEs and SINEs, which replicate via transcribed mRNAintermediates that are converted back into DNA through multiple
rounds of reverse transcription. The retrotransposon DNAs
thus transpose into the genome by a mechanism analogous to‘‘copy and paste.’’ Class 2 TEs consist of DNA transposons
only. Unlike retrotransposons, DNA transposons move directlyfrom place to place through a ‘‘cut and paste’’ process.
The significance and biological implications of TEs and TElike sequences have been thoroughly reviewed (Goodier andKazazian 2008; Gogvadze and Buzdin, 2009). TEs can occasionally play beneficial roles in organisms. Transposition ofLINE-1 elements contributes to genetic diversity in neuronalprogenitor cells during the development of the nervous system(Coufal et al., 2009). LINE-1 expression may contribute to
the establishment of X inactivation in ES cells (Chow et al.,2010). The human endogenous retrovirus (HERV-W) encodes
an envelope protein named syncytin, which plays importantroles in trophoblast cell fusion and placental morphogenesis(Mi et al., 2000). The telomeric ends of linear chromosomes
are protected by TEs in Drosophila (Pardue et al., 2005). Andin many organisms, heterochromatic centromeres, which are
crucial for the segregation of chromosomes during cell division, consist of repeats and TEs (e.g., Volpe et al., 2002).
However, the mobilization of TEs is often considered deleterious because it leads to genomic structural rearrangements,
such as deletions, duplications, and inversions. Organisms

have thus developed effective strategies that constrain TE
transposition.
One such mechanism is mediated by small RNAs, in a processthat is highly analogous to RNA interference (RNAi) or RNA
silencing (Ghildiyal and Zamore, 2009; Kim et al., 2009; Malone
and Hannon, 2009; Siomi et al., 2008; Siomi and Siomi, 2009;
Thomson and Lin, 2009). RNA silencing is triggered by small
RNAs 20–30 nucleotides (nt) in length, which are often, if notuniversally, processed from double-stranded RNAs (dsRNAs)by the enzyme Dicer. The small RNAs are then loaded ontomembers of the Argonaute family of proteins. Argonaute proteinsbind target transcripts by means of base pairing between thesmall RNA and the target; this interaction confers target specificity on Argonaute regulation of transcription, transcript stability,
and/or translation. Accumulating evidence indicates that TEs are
silenced by similar (transcriptional or posttranscriptional) mechanisms, in which small RNAs and Argonaute proteins play key
roles.
The numbers and types of Argonaute family members differ
between species (Cerutti et al., 2000). In the paradigmatic
example of Drosophila, five Argonaute proteins are expressed.
Among these, the AGO proteins (AGO1 and AGO2) are ubiquitous, whereas the others (AGO3, Aubergine [Aub], and Piwi)
are gonad specific (Williams and Rubin, 2002) and are genericallyreferred to as PIWI proteins (Carmell et al., 2002). PIWI-interacting small RNAs (piRNAs) are mainly derived from intergenic
regions containing TEs, TE remnants, and other repetitiveelements (O’Donnell and Boeke, 2007; Klattenhoff and Theurkauf, 2008; Siomi and Kuramochi-Miyagawa, 2009), and mutations in PIWI genes cause derepression of TEs in gonadal cells(Aravin et al., 2001; Kalmykova et al., 2005; Savitsky et al.,2006; Sarot et al., 2004; Vagin et al., 2006; Li et al., 2009).
Thus, PIWI/piRNA complexes protect the integrity of the germline by suppressing TE expression and mobilization there.
Conversely, AGO2-associated small RNAs have a similar rolein nongonadal somatic cells (Golden et al., 2008; Okamura andLai, 2008; Ghildiyal and Zamore, 2009). The endogenous small
RNAs associated with AGO2 also arise from intergenic elements
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Developmental Cell

Review
uni-strand cluster

dual-strand cluster

The uni-strand clusters are transcribed in only
one direction whereas the dual-strand clusters
produce piRNAs from both strands. The sense(blue) and antisense (red) transcripts are complementary to each other. However, piRNAs areproduced in a manner independent of Dicer
activity; thus, it is implied that they are produced
from single-stranded TE precursors (green, red,and blue). After processing, mature piRNAs areloaded onto PIWI proteins (Aub, Piwi, and AGO3)
and silence TEs by mechanisms whose details
remain elusive.

genometranscription

processing

loading

Piwi

AGO3

Figure 1. The piRNA Clusters Form Two
Groups: Uni-Strand and Dual-Strand
Clusters

Aub

TE silencingthat contain TE fragments and repeats, and are now referred
to as endogenous small interfering RNAs (endo-siRNAs or
esiRNAs). AGO/endo-siRNA components and PIWI/piRNA components are highly conserved in vertebrates and invertebrates,
and they all function in TE silencing.
In this review, we discuss how piRNAs and endo-siRNAs
are produced and how these small RNAs silence TEs, focusing
on studies in Drosophila melanogaster and then comparing
and contrasting with other organisms such as mice, frogs, andzebrafish.
PIWI Proteins in Drosophila
The piwi gene was originally discovered as an essential factor
for germline stem cell self-renewal in Drosophila (Lin and
Spradling, 1997; Cox et al., 1998). Further investigation
showed that mutations in piwi cause hyperactivation of retrotransposons (Vagin et al., 2006; Klenov et al., 2007). Likewise,
the paralogous aub gene is necessary for pole cell formation(Harris and Macdonald, 2001) and transposon silencing (Vagin
et al., 2006), and PIWI genes are commonly required forfemale fertility (Cox et al., 1998; Schmidt et al., 1999; Liet al., 2009).
The PIWI proteins show different subcellular localization andexpression patterns, suggesting distinct and discrete roles.
Among the PIWI proteins, only Piwi is localized in the nucleus(Cox et al., 2000), and its primary function in TE silencing appears
to be in the nucleus (Saito et al., 2010). In ovaries, Piwi can be detected in both somatic and germ cells (Cox et al., 2000; Saito
et al., 2006; Brennecke et al., 2007; Nishida et al., 2007). In thetestes, however, Piwi is barely detectable in germ cells but isstrongly expressed in somatic niche cells, which associate withand maintain the germline stem cells. Aub and AGO3 areexpressed in the germ cells of both males and females, but areabsent in gonadal somatic cells. Unlike Piwi, Aub and AGO3
are exclusively detected in the cytoplasm at steady-state levels
688 Developmental Cell 19, November 16, 2010 ª2010 Elsevier Inc.

and accumulate in the nuage (Brennecke
et al., 2007; Gunawardane et al., 2007;
Nishida et al., 2007; Li et al., 2009),
an electron-dense perinuclear structure
characteristic of germline cells in diverse
animals (al-Mukhtar and Webb, 1971;
Eddy, 1975). These observations indicatethat the PIWI proteins possess distinct
functions and utilize different systems for expression andsubcellular localization.
piRNAs in Drosophila
The first direct link between PIWI proteins and TEs was thediscovery of piRNAs, defined as small RNAs associated withPIWI proteins in any organism. Typically, piRNAs are 23–30 nt
long. Sequencing of piRNAs in Drosophila has revealed thatthey mainly originate from TEs and TE-related genomic elements(Saito et al., 2006; Brennecke et al., 2007; Gunawardane et al.,2007; Yin and Lin, 2007). Whereas miRNAs are processed by
Dicer from double-stranded primary precursors (Lee et al.,2004), piRNAs are produced in a Dicer-independent mannerfrom single-stranded precursors (Vagin et al., 2006). piRNAs
are considerably more heterogeneous than miRNAs. More than
70% of piRNAs have been cloned only once, indicating thatthe piRNA population is very complex, in clear contrast to themiRNA population, which consists of approximately 170 species
in Drosophila (Brennecke et al., 2007; Yin and Lin, 2007).
Mapping of piRNAs on the Drosophila genome revealed thatseveral hundred genomic regions could be referred to as piRNA
clusters. These clusters contribute to the generation of piRNAs,
most of which encode TEs and TE remnants and are mainly
localized in the pericentromeric and subtelomeric heterochromatin regions.
The piRNA clusters fall into two groups: dual-strand and unistrand clusters (Figure 1). The dual-strand clusters produce
piRNAs from both genomic strands, with the representative
being 42AB on chromosome 2. The uni-strand clusters aretranscribed in only one direction and are represented by theflamenco (flam) locus on chromosome X. The flam locus was
first discovered as a regulator of the activity of the retroviral
gypsy, idefix, and ZAM elements (Pélisson et al., 1994; Desset
et al., 2008). Antisense-oriented copies of these retro-elementsin the flam locus result in the production of mainly antisenseDevelopmental Cell

Review
Figure 2. Models for piRNA Biogenesis in
Drosophila Ovaries(A) The piRNA 42AB locus on chromosome 2 istranscribed in both directions and the sense andantisense transcripts serve as piRNA precursors.
Rhino associates with DNA from this locus and isrequired for its transcription. The production of
42AB-derived piRNAs occurs in germline cells in
ovaries. These piRNAs are later amplified by the
ping-pong cycle.
(B) The flam piRNA locus on chromosome X gives
rise to transcripts in one direction. The flamderived piRNAs are loaded onto Piwi and are notamplified. These processes occur only in theovarian somatic cells.
(C) Amplification of piRNAs derived from theprimary processing pathway. The piRNAs deposited from the mother to the offspring are also
subject to the ping-pong cycle, specifically in the
germline cells of the ovary.
(D) Aub and Piwi associated with piRNAs accumulate at the posterior pole of the oocytes in
egg chambers. In developing embryos, the Auband Piwi-piRNA complexes are incorporated
into the primordial germ cells. AGO3 does notseem to be passed from the mother to theoffspring.

piRNAs (Figure 2; Brennecke et al., 2007). Although the mechanisms of transcription from the uni-strand piRNA clusters
remain unclear, recent chromatin immunoprecipitation analysis
revealed that the heterochromatin protein 1 (HP1) homolog
Rhino associates with DNA of the dual-strand 42AB cluster
and is required for its transcription. In rhino mutant ovaries,
the level of piRNAs was decreased by around 80% comparedwith wild-type ovaries, and in parallel, a significant reduction
in putative precursor RNA was observed, suggesting that
Rhino possibly promotes the transcription of all the dualstrand clusters, initiating piRNA biogenesis (Klattenhoff et al.,2009).
piRNA Biogenesis in Drosophila: Ping-Pong
Amplification
Examination of the nucleotide preferences of piRNAs associated with Piwi and Aub in Drosophila ovaries has shown thatthey have a strong bias for uracil (U) at their 50 end (1-U) andare mainly antisense to active TE transcripts (Saito et al.,2006; Brennecke et al., 2007; Gunawardane et al., 2007; Yinand Lin, 2007). In contrast, piRNAs bound to AGO3 are mostly
derived from the sense transcripts of TEs and show a strong

bias for adenosine (A) at the tenth nucleotide from their 50 end (10-A). In fact, thefirst 10 nucleotides of antisense piRNAs
frequently overlap with the sense
piRNAs. These findings (including the1-U/10-A relationship) are referred to as
the ping-pong signature; they imply that
AGO3-associated piRNAs may pair with
Aub- (and Piwi-) associated piRNAs
through their first 10 nucleotides (Brennecke et al., 2007; Gunawardane et al.,2007). Moreover, recombinant Piwi,
Aub, and AGO3 cleave complementary target RNAs between
nucleotides 10 and 11 of small guiding RNAs (Gunawardane
et al., 2007), suggesting an intriguing model in which PIWI
proteins generate 50 ends of piRNAs and amplify piRNAs fromlonger (e.g., conventional) transcripts in a Slicer-dependent
manner (Figure 2). This model is called the ping-pong cycle oramplification loop and explains why piRNAs are generated ina Dicer-independent manner, in contrast to siRNAs and miRNAs(Vagin et al., 2006).
In the ping-pong cycle, the Aub complex associated with anantisense piRNA specifically cleaves TE transcripts in the senseorientation. Therefore, the ping-pong cycle not only produces
piRNAs but also degrades TE mRNAs at the same time. Thismodel was confirmed by a recent study on ago3 mutant flies(Li et al., 2009). In ago3 mutants, the number of Aub-boundpiRNAs was markedly decreased and their antisense strand
bias was weakened from 71% to 41%. Some TEs were derepressed in ago3 mutants, as in aub mutants. These observations
support the notion that TE silencing and piRNA biogenesis arecoupled (Li et al., 2009). Thus, the seeds to initiate the pingpong cycle are supplied by the primary processed or maternallydeposited piRNAs (see below).
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Developmental Cell

Review
piRNA Biogenesis in Drosophila: The Primary
Processing Pathway
Although Aub- and AGO3-associated piRNAs are mainly
produced by the ping-pong cycle, Piwi-associated piRNAs
show few ping-pong signatures, suggesting a ping-pong-independent piRNA biogenesis pathway (Brennecke et al., 2007).
Piwi is required for the silencing of a subset of TEs in gonadal
somatic cells. In fact, the majority of Piwi-associated piRNAs in
ovaries are derived from the flam uni-strand cluster, which isexpressed only in the soma (Brennecke et al., 2007). Further
studies suggested the idea that flam piRNAs are loaded onto
Piwi by a mechanism distinct from the amplification cycle(Malone et al., 2009; Li et al., 2009). In the ovarian somatic cell
(OSC) line, which contains only somatic cells of fly ovaries,
Piwi is expressed, but Aub and AGO3 are not, suggesting thatthe piRNAs in OSCs are produced by the primary processing
pathway but are not amplified further by the amplification cycle(Saito et al., 2009). In OSCs, Piwi is associated with abundantpiRNAs, including flam-derived piRNAs, confirming that flampiRNAs are produced only by the primary pathway (Figure 2).
Mechanisms of PIWI-piRNA-Mediated Silencing
Aub and AGO3 show Slicer activity (the RNaseH-like endonuclease activity of their PIWI domains) in vitro (Gunawardane
et al., 2007) and localize in the cytoplasm in vivo (Nishida et al.,2007; Li et al., 2009). Therefore, Aub-piRNA and AGO3-piRNAcomplexes most probably silence TEs posttranscriptionally bycleaving their transcripts. The Aub-piRNA complex may also
induce mRNA decay to silence target genes such as nanos, theembryonic posterior morphogen, by recruiting deadenylation
enzyme CCR4 and the RNA-binding protein Smaug to the target
mRNA (Rouget et al., 2010). How then do Piwi and the somatic
primary piRNAs act to silence TEs? One model is Slicer-mediatedRNA cleavage, because recombinant Piwi produced from E. coli
possesses Slicer activity (Saito et al., 2006). Another feasiblemodel is that Piwi is involved in heterochromatic gene silencing.
This model was suggested from the observations that Piwi influences position-effect variegation (PEV) (Pal-Bhadra et al., 2004).
PEV is a probabilistic variegation in gene silencing that depends
on the genomic locus; for example, if a euchromatic gene wererelocated in close proximity to heterochromatin through a genetic
rearrangement such as inversion or translocation, there is a highchance that this gene will be silenced by the heterochromatin.
It has also been shown that HP1a and Piwi associate specificallywith heterochromatin-containing TEs, such as 1360 and F
element. Piwi may direct HP1a on heterochromatin to silence
TEs in early-stage embryos (Pal-Bhadra et al., 2004). However,piwi mutations resulted in an increase of HP1 association withthe 3R-TAS subtelomeric region in ovaries and subsequently
increased transcriptional silencing, suggesting that Piwi mightact as an activator of TEs at 3R-TAS (Yin and Lin, 2007). In
addition, a recent study showed that HP1 recruitment on heterochromatin does not depend on Piwi (Moshkovich and Lei, 2010).
The mechanism by which the piRNA pathway contributes toheterochromatin regulation thus remains controversial.
piRNA-Mediated TE Repression in Vertebrates
PIWI proteins are conserved in vertebrates (Figure 3) and haveconserved functions in TE silencing and germ cell development.
690 Developmental Cell 19, November 16, 2010 ª2010 Elsevier Inc.

Xiwi1aXiwi1b
Miwi
Ziwi
Miwi2 (nucleus)
Xiwi2
Mili
Xili
Zili
Ago3
Piwi (nucleus)
Aub

Drosophila
zebrafish
Xenopusmouse

Figure 3. Phylogenetic Tree of Piwi Proteins in Fleis, Mice, Frogs,
and Zebrafish
Colors: flies, orange; mice, dark blue; frogs, pink; and zebrafish, sky blue. The
tree was based on ClustalW alignment of the predicted peptide sequences of
PIWI proteins. Piwi: D. melanogaster NP_476875; Aub: D. melanogaster
CAA64320; Ago3: D. melanogaster ABO27430; Ziwi: D. rerio NP_899181;
Zili: D. rerio ABM46842; Miwi: M. musculus NM_021311; Miwi2: M. musculus
NM_177905; Mili: M. musculus NM_021308. The peptide sequences of Xiwi1a,
Xiwi1b, Xiwi2, and Xili were retrieved from Wilczynska et al. (2009).

In mice, three PIWI proteins, mouse Piwi (Miwi), Miwi-like (Mili),and Miwi2 (Kuramochi-Miyagawa et al., 2001, 2004; Deng andLin, 2002; Carmell et al., 2007), have been characterized, andloss of each leads to derepression of TEs and spermatogenic
arrest (Kuramochi-Miyagawa et al., 2001, 2004; Aravin et al.,2007a; Carmell et al., 2007). Mili expression begins in primordial
germ cells at embryonic day (E) 12.5 and continues throughout
adult spermatogenesis, up to the round spermatid stage (Kuramochi-Miyagawa et al., 2004). Miwi2 is transiently expressed inembryonic gonocytes from E15.5, with its expression decreasing
after birth, prior to meiosis (Aravin et al., 2008, Kuramochi-Miyagawa et al., 2008). Miwi is specific to meiotic and postmeiotic
spermatogenesis of the adult; it is expressed from pachytene
to round spermatid stages (Kuramochi-Miyagawa et al., 2001;
Deng and Lin, 2002). piRNAs isolated from PIWI proteins at
each developmental stage can be divided into three groups:
prenatal piRNAs, prepachytene piRNAs, and pachytene piRNAs.
Mili and Miwi2 associate with prenatal piRNAs, largely corresponding to TEs in fetal gonads (Aravin et al., 2007a, 2008;
Kuramochi-Miyagawa et al., 2008). At the prepachytene stage,
Mili, the only Piwi protein expressed at this stage, associateswith prepachytene piRNAs containing both TE and non-TE
sequences (Aravin et al., 2007a, 2008; Kuramochi-Miyagawa
et al., 2008). Miwi associates with pachytene piRNAs derived
from non-TE elements, with only 12%–17% of pachytene
piRNAs corresponding to TEs (Aravin et al., 2006; Girard et al.,2006; Grivna et al., 2006; Watanabe et al., 2006). Consistent
with the distinct temporal profiles of each paralog and its associated piRNAs, the loss of zebrafish Mili (Zili) leads to the expression of TEs in the developing gonad and early failure of germline
development, while the loss of zebrafish Miwi (Ziwi) causes
apoptosis later in the germline, at the premeiotic stage. In zebrafish and Xenopus, the majority of piRNAs correspond to TEs(Houwing et al., 2008; Kirino et al., 2009; Lau et al., 2009), similar
to piRNAs in Drosophila. Unlike Drosophila and mice, theDevelopmental Cell

Review
Xenopus genome contains numerous DNA transposons (Jurka
et al., 2005), and thus the proportion of piRNAs targeting DNA
transposons is high (Lau et al., 2009). These studies indicatethat piRNA content differs with development and the genomic
context.
Accumulating evidence has demonstrated that piRNA biogenesis occurs through the primary piRNA processing and theamplification loop in mice, frogs, and zebrafish (Aravin et al.,2007b; Ghildiyal and Zamore, 2009; Houwing et al., 2007; Kimet al., 2009; Malone and Hannon, 2009). In gonocytes of fetal
mice, where both Mili and Miwi2 are expressed, the ping-pong
signature is obvious between Mili-associating sense 10-A
piRNAs and Miwi2-associating antisense 1-U piRNAs. Moreover, the ping-pong signature is found in zebrafish and Xenopus(Kirino et al., 2009; Lau et al., 2009; Houwing et al., 2008), whichstrongly supports the existence of conserved mechanisms ofbiogenesis in vertebrates. Interestingly, piRNA amplification is
not limited to cycles between distinct PIWI proteins. At the prepachytene stage, Mili shows a tendency to associate with both
1-U and 10-A piRNAs during a certain period (Aravin et al.,2007a). Similar to Mili, Ziwi may be able to amplify piRNAs
without Zili expression (Houwing et al., 2008), suggesting thatPIWI proteins self-amplify piRNAs via the amplification loop.
Taken together, these studies indicate that, as in Drosophila,
TE mRNAs in vertebrates are degraded by PIWI proteins via
a ping-pong cycle.
Intriguingly, Mili and Miwi2 mutant mice exhibit loss of DNA
methylation in TEs. Further studies have clarified that the Miliand Miwi2-piRNA complexes are involved in de novo DNA methylation of TEs (Aravin et al., 2008; Kuramochi-Miyagawa et al.,2008). In general, CpG DNA methylation causes stable repression of transcription (Bourc’his and Bestor, 2004). However, itremains unclear how piRNAs are involved in DNA methylation.
Scaffolds and Modifiers of piRNA Pathways
Tudor (Tud) was identified as a nuage component required forgametogenesis in classical genetic analyses of the Drosophila
germline (Boswell and Mahowald, 1985; Thomson and Lasko,
2004, 2005; Arkov et al., 2006). More recently, biochemical andstructural studies have revealed that the Tudor domains of Tudbind directly to symmetrical dimethyl arginines (sDMAs) in PIWI
proteins (Kirino et al., 2009; Nishida et al., 2009; Liu et al.,2010). This arginine methylation is mediated by the methyltransferase PRMT5 (also known as Dart5/Capsuleen) (Kirino et al.,2009), which is also required for germline development.
Complexes consisting of Tud, Aub, and AGO3 are heteromericand contain RNAs resembling the precursors for both sense
and antisense piRNAs (Nishida et al., 2009). This suggests theinvolvement of Tud in piRNA biogenesis, e.g., as a platform forpiRNA amplification. Loss of Tud function in ovaries causes
Aub to be associated with a greater abundance of piRNAs
compared with Aub in wild-type ovaries (Nishida et al., 2009).
The population of TE-derived piRNAs was significantly alteredby loss of Tud function, although an obvious change in the strand
bias was not seen in the mutants (Nishida et al., 2009). Loss of
dprmt5 diminishes piRNA association with Aub (Nishida et al.,2009), suggesting that sDMA modification itself influences theRNA-binding capacity of PIWI proteins, independently of Tudor
domain proteins.

In mice too, Tudor-domain-containing proteins (or Tdrds) associate with PIWI proteins specifically through sDMA modification(Chen et al., 2009; Kojima et al., 2009; Reuter et al., 2009; Shojiet al., 2009; Vagin et al., 2009; Wang et al., 2009). Tdrd1 is required
for efficient operation of the ping-pong cycle (Vagin et al., 2009). In
Tdrd1 and Tdrd9 mutants, the sequence profile of piRNAs isaltered (Reuter et al., 2009; Shoji et al., 2009). Thus, Tudor domain
proteins have conserved roles in the quality control of transposon-derived piRNAs (Table 1; Siomi et al., 2010).
Tejas, another Tud-domain protein expressed in Drosophila
ovaries, also associates with Aub (Patil and Kai, 2010). However,unlike Tudor, Tejas does not seem to require sDMA modification
to associate with Aub. In Tejas mutants, steady-state levels of
piRNAs were low and TEs reactivated; thus, Tejas modulates
piRNA biogenesis. krimper (Lim and Kai, 2007), zucchini (zuc)(Pane et al., 2007), spn-E (Vagin et al., 2006), and maelstrom(mael) (Findley et al., 2003; Lim and Kai, 2007) also affect piRNA
accumulation, and TEs are derepressed in the ovaries of mutants
for all these genes (Table 1; Vagin et al., 2006; Lim and Kai, 2007;
Pane et al., 2007). Deep-sequencing of piRNAs in these mutants
has shown distinct requirements: for example, spn-E mutations
reduce not only piRNA amplification but also the production ofprimary piRNAs originating from cluster 2, one of ping-pongindependent uni-strand clusters (Malone et al., 2009). However,
spn-E is apparently unnecessary for producing primary piRNAs
from flam in ovary (Malone et al., 2009). On the other hand,zuc, which encodes a putative nuclease, is necessary forsomatic primary piRNA processing but not for the ping-pongcycle (Haase et al., 2010; Malone et al., 2009; Olivieri et al.,2010; Saito et al., 2010). These observations suggest that piRNA
pathway components perform different and specific roles inpiRNA biogenesis in Drosophila. Krimper and Spn-E containTud domains; however, whether they interact with PIWI proteinsthrough sDMAs remain unknown.
Krimper, Zuc, and Mael localize to the nuage, suggesting thatnuage might be an important site of piRNA processing in germ
cells. Recent studies indicate that Yb bodies consisting of Yb
and Armitage (Armi) are the sites for somatic primary piRNA processing (Olivieri et al., 2010; Saito et al., 2010). Both Armi and Yb
contain an RNA helicase domain and therefore are implicated in
RNA metabolism. In fact, mutations in Armi cause a severe
decrease in piRNA accumulation in ovaries (Vagin et al., 2006).
In Armi- or Yb-depleted cells of the ovarian soma, Piwi is not
associated with piRNAs and mislocalizes to the cytoplasm,
resulting in a general loss of piRNAs, and suggesting that Armiand Yb are piRNA biogenesis modulators. Yb contains a Tud
domain; however, its function remains undetermined. In mice,piRNA pathway components exhibit differential localization.
Miwi2/Tdrd9/Mael colocalizes with the components of Processing bodies referred to as piP-bodies, whereas Mili/Tdrd1/
MVH/GASZ associates with the intermitochondrial cement or
pi-bodies (Aravin et al., 2009; Ma et al., 2009; Shoji et al.,2009; Kuramochi-Miyagawa et al., 2010). piP-body formation
depends on pi-bodies, and Miwi2 fails to associate with piRNAs
when pi-bodies are not formed. On the other hand, pi-bodyformation is not affected by piP-body disruption, suggesting
a possible progression of piRNA processing from pi-body to
piP-body. Thus, both piP-bodies and pi-bodies are implicatedas the sites for piRNA biogenesis in mice. It will be interesting
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Table 1. Features of TE-Derived Small RNAs in Animals
Small RNA

Mouse

Frog

Zebrafish

Fly

piRNA

piRNA

piRNA

piRNA

Function/Motif

References
reviewed in Klattenhoff
and Theurkauf, 2008;
Siomi and KuramochiMiyagawa, 2009;
Ghildiyal and Zamore,
2009; Kim et al., 2009;
Malone and Hannon,
2009; Thomson and Lin,
2009; Lau, 2010; Senti
and Brennecke, 2010

Size (nt)

25–30

26–31

27–30

24–30

see entry above

Nucleotide
preference

5’U; 10th A

5’U; 10th A

5’U; 10th A

5’U; 10th A

see entry above

AssociatedArgonaute
protein

Miwi; Mili;
Miwi2

Xiwi1a,1b;
Xili; Xiwi2

Ziwi; Zili

Piwi; Aub;AGO3

see entry above

Amplificationof small RNA(5’U-10thA)

Mili-Miwi2;
Mili-Mili

not determined

Ziwi-Zili

Piwi-AGO3;
Aub-AGO3

see entry above

Other factors
required for small
RNA-mediatedTE silencing

Tdrd-1

Tdrd-1*

Tdrd-1*

Tdrd-9Tdrd-9*Tdrd-9*Maelstrom

Maelstrom*

Maelstrom*Tdrd-6*

Tdrd-6*

Tdrd-6*

Mov10l1

Mov10l1*

Mov10l1*

Tudor, MYND

Vagin et al., 2009;
Reuter et al., 2009

Spindle E

Tudor, DEXDc

Shoji et al., 2009;
Vagin et al., 2006

Maelstrom

HMG

Soper et al., 2008;
Lim and Kai, 2007

Krimper

Tudor

Lim and Kai, 2007

Armitage

Helicase?

Zheng et al., 2010;
Frost et al., 2010;
Vagin et al., 2006

Tdrd-7*/Tdrd-5*

Tdrd-7*/Tdrd-5*

Tdrd-7*/Tdrd-5*

Tejas

Tudor

Patil and Kai, 2010

MVH

DDX4*

Vasa*

Vasa

DEAD

Kuramochi-Miyagawa
et al., 2010; Lim andKai, 2007

PLD6*

PLD6*

PLD6*

Zucchini

Nuclease?

Pane et al., 2007

Squash

Nuclease?

Pane et al., 2007

dPRMT5

Methyltransferase

Kirino et al., 2009

Fs(1)Yb

Tudor

Olivieri et al., 2010;
Saito et al., 2010

CG2183*

ANK repeat

Ma et al., 2009

PRMT5*GASZ

PRMT5*GASZ*

PRMT5*GASZ*

Genes marked with an asterisk show that the involvement on piRNA-mediated TE silencing has not been confirmed experimentally.

to determine what the specificity of these structures might be, forexample, whether primary and ping-pong processes are differentially localized between them.
Germline Transmission and Hybrid Dysgenesis
The complex relationship between nuage architecture, germline
development, and piRNA pathways suggests that the broader
implications of piRNA-mediated germline integrity control are
worth considering. During ovary development, Aub and Piwi
accumulate in specialized cytoplasm at the posterior pole ofthe oocyte, where germ cell determinants are localized (Rongoand Lehmann, 1996). Maternally deposited Aub and Piwi arethus incorporated into the primordial germ cells that inherit this692 Developmental Cell 19, November 16, 2010 ª2010 Elsevier Inc.

‘‘posterior pole plasm’’ and are transmitted to the next generation directly. In contrast, AGO3 does not seem to be passed
on from mother to offspring, suggesting that antisense piRNAs
may be selectively inherited, whereas sense piRNAs are not.
Brennecke et al. revealed that piRNAs are truly transmitted
from mother to offspring (Brennecke et al., 2008). This maternal
inheritance of piRNAs was shown to explain a long-standing
but poorly understood phenomenon of ‘‘hybrid dysgenesis,’’ inwhich the introduction of new genetic material via the malegermline induces genetic instability in female progeny (Kidwell
and Kidwell, 1976). The relevant genetic material was known to
include TEs for some time (e.g., Rubin et al., 1982), and a maternally transmitted epigenetic suppressor, called a cytotype, had

Developmental Cell

Review
been proposed (Engels, 1979), but it took several decades
before piRNA characterization progressed to a point where therealization could be made that the cytotype corresponds tomaternally inherited PIWI/piRNA complexes (Figure 2; Brennecke et al., 2008). These results extended the paradigm ofpiRNA-dependent germline integrity, to provide molecularinsight into the epigenetics of incompatibility between diverging
animal subpopulations.
Interestingly, fertility is enhanced by two environmental
factors, age and high temperature (Bucheton, 1978). Heat shockprotein 90 (Hsp90), a chaperone involved in several cellular anddevelopmental pathways, has been implicated in TE control
mediated by piRNAs (Specchia et al., 2010). Mutations affecting
Hsp90, or treatment with the specific Hsp90 inhibitor geldanamycin, decreased piRNA biogenesis and increased the expression levels and the mobilization rates for all types of TEs. Theseobservations suggest that Hsp90 may act as a piRNA factor andcouple the control of TEs to environmental inputs.

TE
genometranscription
dsRNA
processing by Dicer2/Loqs

endo-siRNAloading
AGO2

slicing
Endogenous Small Interfering RNAs: Biogenesis
Recent studies have indicated that TE silencing by small RNAs is
not a gonad-specific event. Rather, TE transcripts are converted
into a subset of small RNAs in nongonadal somatic cells,
which display different characteristics from those of piRNAs.
These small RNAs are designated endo-siRNAs or esiRNAs.
In Drosophila, endo-siRNAs specifically bind to AGO2, theeffector of RNAi (Figure 4). AGO2 was originally demonstrated
to associate with exogenous siRNAs (exo-siRNAs) derived
from external dsRNAs in RNAi. The exo-siRNAs produced fromviral dsRNAs have been shown to serve as a defense mechanism
against viral infection (Galiana-Arnoux et al., 2006; van Rij
et al., 2006; Wang et al., 2006). However, the recent discovery
of endo-siRNAs clarified that AGO2 does not always wait for‘‘external’’ siRNAs, but rather plays particular roles in cellular
RNAi with endo-siRNAs.
Like piRNAs, endo-siRNAs are mainly derived from TEs,
heterochromatic regions, and intergenic elements of the
genome. The endo-siRNAs occasionally arise from proteincoding genes transcribed from overlapping 30 untranslated
regions (UTRs) or long hairpin RNA genes (Czech et al., 2008;
Ghildiyal et al., 2008; Kawamura et al., 2008; Okamura et al.,2008). Sequencing of small RNAs in Drosophila embryos,
ovaries, and the S2 cell line has revealed that endo-siRNAs are
21 nt long and broadly map across the locations of most TEs
in both the sense and antisense orientations (Figure 4). However,they do not show an obvious nucleotide preference at their 50
end. These characteristics, together with the observation thatendo-siRNAs show a typical phasing pattern when mapped on
the genome, suggest Dicer2 involvement in the processing
pathway. In fact, loss of dicer2 function abolished the productionof endo-siRNAs in vivo (Czech et al., 2008; Kawamura et al.,2008; Okamura et al., 2008). It had previously been shown that
Dicer1 partners with the dsRNA-binding partner Loquacious(Loqs) to generate miRNAs (Förstemann et al., 2005; Jiang
et al., 2005; Saito et al., 2005), whereas Dicer2 uses the related
protein R2D2 during exo-siRNA production (Liu et al., 2003). It
therefore came as a surprise to find that endo-siRNA biogenesis
occurs by an unusual combination of Dicer2 and Loqs (Czech
et al., 2008; Okamura et al., 2008). This confusion was resolved

TE silencing
Figure 4. Endo-siRNA Biogenesis
Like piRNAs, endo-siRNAs are mainly derived from TE transcripts. In thispathway, Dicer2 associates with Loqs-PD, both of which are required forendo-siRNA processing from their dsRNA precursors. Mature endo-siRNAs
are loaded onto AGO2.

when it was discovered that the loqs gene gives rise to four
isoforms, loqs-PA-loqs-PD (Förstemann et al., 2005; Hartig
et al., 2009; Zhou et al., 2009; Miyoshi et al., 2010), such that
Loqs-PB and Loqs-PD are involved in the miRNA and endosiRNA processing pathways, respectively (Hartig et al., 2009;
Miyoshi et al., 2010; Zhou et al., 2009). Indeed, Loqs-PB associates with Dicer1, whereas Loqs-PD associates with Dicer2.
These specific associations of Loqs isoforms with Dicer proteins
most probably confer the ability of Dicer proteins to discriminate
small RNA precursor substrates. The involvement of Loqs-PD in
TE silencing might be modest, because endo-siRNA production
depends less on Loqs than Dicer2 and AGO2 (Chung et al., 2008;
Czech et al., 2008).
Endo-siRNA-Mediated TE Silencing
In mouse oocytes, the endo-siRNA pathway plays importantroles in TE silencing (Tam et al., 2008; Watanabe et al., 2008;
Murchison et al., 2007). However, the effects of endo-siRNAs
are subtler in Drosophila, where TEs are derepressed (1.5- to
9-fold) when dicer2 or ago2 gene functions are lost, but onlya small subset of specific TEs are affected, and the mutant flies
are viable and fertile (Chung et al., 2008; Czech et al., 2008; Ghildiyal et al., 2008; Kawamura et al., 2008). It is plausible that theabsence of strong phenotypes may be due in part to redundancy
with piRNAs: Endo-siRNA factors are expressed in germline cells
where piRNA-mediated silencing occurs, and sequence analysis
has shown that AGO2-associated endo-siRNAs often map to
Developmental Cell 19, November 16, 2010 ª2010 Elsevier Inc. 693

Developmental Cell

Review
piRNA clusters. Thus, these regions of the genome are able toproduce both endo-siRNAs and piRNAs. This raises importantquestions about how sense and antisense strands from thesepiRNA clusters form dsRNAs to produce endo-siRNAs andhow they stay single stranded as piRNA precursors.
Conclusions
Despite their sequence diversity, piRNAs and endo-siRNAs do
not disturb the expression of protein-coding genes. Thus, there
must be strict cellular systems that discriminate TEs fromprotein-coding genes. How does this operate in vivo? One possibility is that TEs are identified at the transcription stage. Rhino
contributes specifically to piRNA production from dual-strand
piRNA clusters but not from uni-strand clusters, suggestingthat Rhino can discriminate between the two clusters. Because
Rhino is an HP1 homolog, it is most likely that Rhino functions
at the transcriptional level, although there is no direct evidence
to support this. The second possibility is that TEs are identified
at the posttranscriptional level. TEs are transcribed by RNA polymerase II and are poly(A)-tailed, suggesting that piRNA precursors and mRNAs may contain similar structural characteristics.
This would make it difficult to discriminate between TE transcripts and protein-coding transcripts. However, there shouldbe cellular mechanisms that can separate the two species. It
would be of interest to identify these mechanisms in Drosophila
and in other species, such as mice.
ACKNOWLEDGMENTS
We thank H. Siomi and other members of the Siomi laboratory for discussions
and comments on the manuscript. This work was supported by Ministry of
Education, Culture, Sports, Science, and Technology of Japan grants to
K.S. and M.C.S. K.S. is supported by Precursory Research for Embryonic
Science and Technology from Japan Science and Technology Agency.
M.C.S. is supported by Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency.

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