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Seminars in Cell & Developmental Biology 21 (2010) 754–759

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology
journal homepage: www.elsevier.com/locate/semcdb

Review

piRNA-mediated silencing in Drosophila germlines
Mikiko C. Siomi a,b,∗ , Tomohiro Miyoshi a , Haruhiko Siomi aabKeio University School of Medicine, Tokyo 160-8582, Japan
JST, CREST, Saitama 332-0012, Japana r t i c l e

i n f oArticle history:
Available online 18 January 2010
Keywords:
piRNA
PIWI
RNA silencing
Germline
Drosophilaa b s t r a c t
PIWI interacting RNAs (piRNAs) are a subset of small RNAs predominantly expressed in the germlinesof various species. In Drosophila, the main sources of piRNAs are transcripts of mobile DNA elements,
including active transposons and their wreckage, found on the genome. After maturing from the primary transcripts, piRNAs are speciﬁcally loaded onto germline-speciﬁc Argonaute proteins – Argonaute3,
Aubergine and Piwi – collectively referred to as PIWI proteins. Loss of function of PIWI proteins and/or thepiRNA loci on the genome lead to derepression of transposons and causes severe defects in gametogenesis and fertility. The necessity for both PIWI proteins and piRNAs in protecting the genome of the gametes
from mischievous mobile genomic elements is thus obvious. There have been extensive biochemical andgenetic studies on PIWI proteins and piRNAs. These have shed light not only on the molecular mechanisms of gene silencing mediated by piRNAs and PIWI proteins, but also on their intriguing relationshipwith cellular genes that have been shown to be important for gametogenesis and fertility.
© 2010 Elsevier Ltd. All rights reserved.

Contents
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8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
piRNA biogenesis; the ampliﬁcation loop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
piRNA biogenesis; the primary processing pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
piRNA modiﬁcation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Regulation of PIWI expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Post-translational modiﬁcation of PIWI proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mode of function of the PIWI–piRNA complex in gene silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
To date, the accumulated evidence clearly indicates that in
Drosophila three sorts of small RNAs are expressed endogenously;
endogenous small interfering RNAs (endo-siRNAs or esiRNAs),
microRNAs (miRNAs) and piRNAs [1–3]. Although all these small
RNAs are considered fundamentally separable, certain commonalities can be found in all. First, they are short ribonucleic acidmolecules consisting of only 20–30 nucleotides (nt). Second, they

∗ Corresponding author at: Department of Molecular Biology, Keio University
School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan.
Tel.: +81 3 5363 3498.
E-mail address: siomim@sc.itc.keio.ac.jp (M.C. Siomi).
1084-9521/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcdb.2010.01.011

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all are not expressed ‘as is’; rather, they are matured from much
longer RNA precursors through processing pathways consisting ofmultiple, sequential RNA cleavage steps, which are accomplishedby particular kinds of RNase enzymes [4]. Third, all these small
RNAs function as important players in gene silencing machineries by associating directly with Argonaute proteins [1,5], which areanother set of important players in the silencing mechanisms. The
gene silencing pathways mediated particularly with small RNAsand Argonaute proteins are referred to collectively as RNA silencing[1]. The association between small RNAs and Argonaute proteinsis direct and occurs stoichiometrically [6,7]. In addition, they do
not cross-interact basically; namely, miRNAs and esiRNAs associatewith the ubiquitously expressed Argonaute proteins, AGO1 andAGO2, respectively (although some minor miRNAs were recently
shown to be associated with AGO2) [8–13], whereas piRNAs asso-

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ciate only with the PIWI proteins Piwi and Aubergine (Aub; also
known as Sting) and Argonaute 3 (AGO3) [14–18]. The partnership
between small RNAs and Argonaute proteins is well regulated.
Regardless of the commonalities described above, the threesmall RNAs are considered distinct because each shows speciﬁcfeatures. Thus, piRNAs are germline-speciﬁc whereas miRNAs andesiRNAs are ubiquitous. Whereas miRNAs originate mainly fromtheir own miRNA coding genes [4], esiRNAs do not have theirown genes but are derived mainly from various transposons, theirwreckage and other intergenic repetitive elements [2]. In thisregard, esiRNAs are considered relatively closer to piRNAs than are
miRNAs.
Although their origins are barely distinguishable, esiRNAs andpiRNAs are considered distinct groups. One of the main reasons isbecause esiRNAs and piRNAs are loaded onto different Argonaute
proteins [1–3]. Factors required for the biogenesis of esiRNAs andpiRNAs also differ from one another. Recent studies have shownthat esiRNAs are processed from long RNA precursors by an RNase
III enzyme, Dicer2 [8–10,19]. In the esiRNA processing pathway,
Dicer2 is associated with one particular isoform of Loquacious(Loqs), Loqs-PD (the loqs gene gives rise to four isoforms, Loqs-PA
to Loqs-PD) [20–22]. Dicer2 was originally discovered as a factor
excising siRNA duplexes from exogenous long double-stranded (ds)
RNAs [23]. In this exogenous siRNA (exo-siRNA) excision, Dicer2 is
known to associate with R2D2 [24]. The most recent understanding regarding siRNA processing is that R2D2 and Loqs-PD associatewith Dicer2 simultaneously, in part [22] and act sequentially in both
esiRNA and exo-siRNA processing pathways [25]. The two isoformsof Loqs, Loqs-PA and Loqs-PB, function in the miRNA pathway by
associating speciﬁcally with Dicer1 [22,26,27].
piRNA biogenesis does not require dicer genes [28]. It is therefore
obvious that esiRNAs and piRNAs are produced through individual
pathways, although their precursors cannot be classiﬁed scrupulously. This individuality of their processing is also supported by
the notion that the size distribution of esiRNAs and piRNAs clearly
differs; esiRNAs are about 21 nt while piRNAs range from 24 to 30 nt[1–3]. The esiRNA pathway is ubiquitous while the piRNA pathway
is germline-speciﬁc. It is peculiar that only germlines possess boththe pathways. The two mechanisms do not seem to be redundant ingermlines; this is because loss of the piRNA pathway causes severe
defects in gametogenesis [29,30] while loss of the esiRNA pathway
showed some mild defects in embryogenesis [31].
In this review, we summarize ﬁndings obtained so far about
Drosophila piRNAs and PIWI proteins. This article includes biogenesis and the 3 -terminal modiﬁcation of piRNAs, expressional
regulation and post-translational modiﬁcation of PIWI proteins
and the mode of function of the PIWI–piRNA complexes in silencing transposable genomic elements and non-transposable genes inovaries and testes.

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Fig. 1. Ampliﬁcation loop and primary processing pathways for piRNA production.
Transcripts of transposons in antisense orientation are processed to piRNAs throughprimary processing and loaded onto Piwi and Aub. piRNAs loaded onto Aub are
ampliﬁed through the ampliﬁcation loop. However, piRNAs loaded onto AGO3 arethought to be produced only through the ampliﬁcation loop. Pimet/dmHEN1 associates with PIWI proteins while 2 -O-methylates piRNAs that are already loaded
onto proteins.

age activity of Aub and AGO3 in the processing pathway [15,16].
This new idea correlated well with the earlier observation that dicer
genes are not required for piRNA production [28]. The basic concept of this model is that reciprocal target RNA cleavage reactions
by Aub and AGO3 circulate and constantly bear abundant piRNAs
in germline cells. The potentiality that Aub and AGO3 possess atarget RNA cleavage activity, as in the case of other Argonaute proteins – another fundamental concept required for instituting thismodel – was demonstrated experimentally using in vitro assay systems [16,18]. Based on these observations, the ampliﬁcation loop
model was constructed (Fig. 1). In parallel with piRNA production,transposons should be silenced efﬁciently because the majority ofpiRNA precursors are transposon transcripts. This concept further
strengthened the plausibility of the ampliﬁcation loop model. A
recent study shows that the rhino gene, encoding a nuclear heterochromatin protein 1 (HP1) homolog, might also be involved in
the ampliﬁcation loop [32], but the detailed mechanism remains
unknown.

2. piRNA biogenesis; the ampliﬁcation loop

3. piRNA biogenesis; the primary processing pathway

Analyses of the piRNAs that associate with PIWI proteins in
Drosophila germlines have led to two models for piRNA biogenesis; the ampliﬁcation loop pathway (also known as the Ping–Pong
pathway) and the primary processing pathway [1,4].
In Drosophila ovaries, Aub is associated mainly with antisense
piRNAs that predominantly contain uracil at their 5 end (1st-U)[15,16]. By contrast, AGO3 is associated mainly with sense piRNAs
that show a strong bias for adenosine at the 10th nucleotide fromthe 5 end (10th-A) [15,16]. In fact, piRNA sequences found in the
Aub- and AGO3-associated small RNA libraries often form ‘pairs’
through 10 nucleotides from their 5 ends. Unique characteristics
shown by these piRNA sequences prompted an intriguing idea for
piRNA production; namely, an involvement of the target RNA cleav-piRNAs derived from the ﬂamenco (ﬂam) locus on the X chromosome are exclusively loaded onto Piwi in Drosophila ovaries [33].
Expression of ﬂam has been shown to occur only in ovarian follicle cells where the expressions of Aub and AGO3 are undetectable[15,16,34]. From all these results, it was inferred that there should
be another system for the piRNA production, besides the ampliﬁcation loop pathway [15,35]. This piRNA biogenesis system is referred
to as the ‘primary processing pathway’ (Fig. 1). Recently, thorough piRNA sequence analyses have been carried out using various
mutants of the piRNA pathway genes, including ago3 and others.
These revealed that the primary piRNA processing does not require
AGO3 and that requirements for the piRNA pathway genes in the
primary and secondary piRNA processing pathways differ from one

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another [36,37]. For instance, it was found that zucchini (zuc), a
putative cytoplasmic nuclease, is likely involved predominantly inthe primary piRNA processing while spindle-E (spn-E; also known as
homeless), a protein containing Tudor and RNA helicase domains,might be involved predominantly in the ampliﬁcation loop [37].
Exclusion of Aub and AGO3 from the primary processing pathway
was further supported by the notion that, even in established celllines, piRNAs are abundantly expressed in ovarian somatic sheets(OSSs) [35] and ovarian somatic cells (OSCs) [38], where the expressions of Aub and AGO3 are undetectable. The requirement of zucfor primary piRNA processing was conﬁrmed by RNA interference(RNAi)-mediated knockdown of zuc and following deep sequencing analysis of piRNAs in OSCs [38]. To date, other factors necessary
for the primary processing remain undetermined. A Piwi mutant
that lacks its putative nuclear localization signal and thus is now
localized exclusively in the cytoplasm, still associates with piRNAs
to the same extent as the full-length Piwi protein [38]. This clearly
indicates that factors required for primary piRNA processing might
exist in the cytoplasm and that Piwi is imported into the nucleus
after being loaded with mature piRNAs in the cytoplasm. In Schneider 2 (S2) cells, Piwi expressed by transfection is localized in the
nucleus, although Piwi in the environment is not loaded with piRNAs (personal communication with K. Saito). It seems that Piwi
does not have to be bound with piRNAs to be imported into the
nucleus, at least in S2 cells. The vasa gene (see below) might beunnecessary for primary processing because OSSs and OSCs do not
express Vasa at a level detectable by immunoblotting [38,39].
4. piRNA modiﬁcation
Drosophila piRNAs but not miRNAs show resistance to particular chemical reactions; periodate oxidation and ␤ elimination[28,40]. This infers that either the 2 or 3 terminal hydroxyl
group on the ribose at the 3 end of piRNAs is chemically modiﬁed. Moreover, as in the case of piRNAs in mice, Drosophila
piRNAs contain 2 -O-methyl groups at their 3 ends [40,41] (Fig. 1).
The enzyme responsible for this modiﬁcation was determined
to be dmHEN1/Pimet, the Drosophila homolog of Arabidopsis
HEN1 that catalyzes the 2 -O-methyl modiﬁcation for miRNAs[40,41] (Fig. 1). HEN1 in Arabidopsis and in other plants suchas rice, contains two functional domains: dsRNA-binding andmethyltransferase domains [42,43]. In contrast, dmHEN1/Pimet
has methyltransferase domain but lacks dsRNA-binding domain.
Thus, dmHEN1/Pimet is just about half the size of Arabidopsis HEN1.
In plants, miRNAs become 2 -O-methyl modiﬁed when they arestill in a duplex form with miRNA* molecules in the nucleus [42].
Drosophila piRNAs are 2 -O-methyl modiﬁed after being loaded
onto PIWI proteins via the direct interaction of dmHEN1/Pimet
with PIWI proteins [40,41]. It could then be speculated thatdmHEN1/Pimet discarded the dsRNA-binding domain through evolution because it became unnecessary to be retained for its function.
In plants, miRNAs are stabilized by the 2 -O-methyl modiﬁcation[42,44]. However, the physiological signiﬁcance of 2 -O-methyl
modiﬁcation of piRNAs in Drosophila germlines remains obscure[40].
5. Regulation of PIWI expression
The extent of piRNA accumulation in germlines is largely inﬂuenced by the expression levels of parental DNA elements (i.e.,transposons, other intergenic repetitive elements and the piRNAloci such as ﬂam). The availability of these piRNA processing factors might also inﬂuence the piRNA level in germlines. How, then,is the expression of PIWI proteins regulated? Studies in the OSC
line revealed that the expression of Piwi protein – one of the PIWI

proteins – in ovarian somatic cells must be tightly regulated by the
trafﬁc jam (tj) gene that encodes a large Maf transcriptional factor,
Trafﬁc jam [38] (Fig. 2). This upregulation of piwi expression by tj
should also take place in ovaries because in tj mutants only somatic
cells but not germ cells (ovarian stem cells and their developing
cells) lose piwi expression [38]. This observation regarding the cell
type-dependent expression of piwi correlates well with the fact thatthe tj gene is exclusively expressed in somatic cells in ovaries [45].
The factors required for driving the expression of Piwi – as wellas Aub and AGO3 – in ovarian germ cells remains undetermined.
It should be noted that Piwi expression in somatic niche cells is
controlled Yb, encoding a protein containing Tudor and RNA helicase domains [46]. The mechanisms through which the expressionsof Aub and AGO3 are repressed in the ovarian somatic cells also
remain unknown. Depletion of HP1, a nuclear protein involved inheterochromatinization, upregulates Aub expression but not Piwi
expression in S2 cells (M.C.S., unpublished data). Therefore, a direct
involvement of HP1 in aub repression is likely.
6. Post-translational modiﬁcation of PIWI proteins
PIWI proteins in Drosophila have been determined to containsymmetric dimethyl arginines (sDMAs) [47,48]. The factor mediating this post-translational modiﬁcation was also determined as amethyltransferase, PRMT5 [47] (Fig. 2). Earlier studies showed that
Sm proteins in U snRNP complexes, such as SmB/B’, contain sDMAs
and that sDMAs of Sm proteins are recognized by and bound with a
Tudor domain-containing protein, Survival Motor Neuron (SMN)[49,50]. This association of Sm with SMN promotes the recruitment of U snRNA and leads to the efﬁcient assembly of U snRNP.
Through this sequential system, the quality of U snRNP is maintained [49]. It has thus been inferred that functions of PIWI proteins,
as well as piRNA loading onto PIWI proteins, would be controlled
by an sDMA-dependent association of Tudor domain-containing
protein(s) with PIWI proteins. In fact, it was revealed that Tudor,
one of 19 Drosophila Tudor family members, associates with Aub
and AGO3 speciﬁcally thorough their sDMAs and that the sDMAspeciﬁc association maintains the population of piRNAs associatedwith Aub and AGO3 in ovaries [48]. A speciﬁc fraction of Aub that
was associated with Tudor was loaded with far less mature piRNAs
but instead held piRNA precursor like RNA molecules within thecomplex, compared with the Tudor-free state of Aub. Thus, Tudor in
the piRNA pathway might act like SMN in U snRNP assembly. SMN
contains one Tudor domain and binds with the Sm protein. Thismeans that one Tudor domain might be sufﬁcient to associate withsingle or multiple sDMAs on a target protein. Tudor contains eleven
Tudor domains and thus it is plausible that one Tudor molecule canassociate with both Aub and AGO3 at the same time. Indeed, theTudor complex immunoisolated from ovary lysates contains both
Aub and AGO3. Aub and AGO3 could dimerize and this association does not require Tudor. Dimerization of Aub is not detectable.
Together these results suggest that Tudor might function as a platform for the ampliﬁcation loop of piRNA production by recruiting
the piRNA precursors to the Aub–AGO3 complex efﬁciently (Fig. 2).
Whether Tudor can be associated with Piwi in an sDMA-speciﬁcmanner and/or if Tudor is also involved in the primary piRNA processing remains unclear and further investigations will be needed
to answer these questions.
7. Mode of function of the PIWI–piRNA complex in genesilencing
The PIWI–piRNA complexes target transposon transcripts tosilence them and to protect the genomes of gametes from theirinvasion. However, targets of the PIWI–piRNA complexes are notAuthor's personal copy
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Fig. 2. Schematic of RNA silencing in somatic and germline cells in ovaries. In somatic cells, Piwi protein expression is driven by the tj gene. piRNAs derived from the 3 UTR
of protein-coding genes including tj are loaded onto Piwi protein. This complex of Piwi and piRNA functions in silencing target genes such as fasIII. Transposon silencing
mediated by Piwi also occurs in somatic cells. In ovarian germline cells, Piwi is expressed but may be unnecessary for silencing [58]; thus Piwi is omitted here. Tudor can
associate with Aub and AGO3 at the same time in germline cells, putting the two proteins in a very proximal region. By doing this, Tudor might promote piRNA production
through the ampliﬁcation loop pathway. There is evidence that Tudor may also functions to recruit piRNAs precursors to the complex.

limited to transposons. Studies in testes showed that piRNAs canalso target protein-coding genes, stellate (ste) and vasa (Fig. 3).
The ste gene encodes Stellate protein that is homologous to Casein
kinase ␤ subunit [50,51]. Once expressed in testes under conditions without any silencing regulation, the Stellate protein forms
needle-shaped aggregates and these aggregates interfere with
spermatogenesis by unknown mechanisms [51,52]. To overcome
this defect, Drosophila has, through evolution, acquired a silencing system for the ste gene that relies on piRNAs derived from therepetitive suppressor of stellate [su(ste)] elements that are highly
homologous to the ste gene [28,53]. The su(ste)-derived piRNAs areloaded onto Aub and block ste expression, most likely by cleav-Fig. 3. RNA silencing mediated by Aub in ﬂy testes. There is clear evidence that Aubin testes, by associating with piRNAs derived from non-transposable DNA elements,
functions for silencing of not only transposons but also protein-coding genes suchas vasa and ste.

ing its transcripts. This was inferred from the observation that the
Aub–piRNA complex immunopuriﬁed from testes could cleave theste transcript in vitro [18]. Notably, su(ste)-piRNAs are the mostabundant forms associated with Aub in testes. It is very unlikelythat ovaries express su(ste)-piRNAs because they have not beenfound in the Aub small RNA library and other libraries made fromtotal ovarian small RNAs.
The vasa gene encodes a putative RNA helicase, Vasa [54].
Genetic studies revealed that loss of vasa function caused severe
defects in oogenesis [55]. However, any importance for Vasa functions in testes was not reported. However, it was later reported
that the protein level of Vasa in aub mutant testes was increased
by about twofold compared with those in wild type testes and thataub mutants resulted in oversized apexes in the testes, where thespermatogonial cells and spermatocytes are located [18]. The same
phenomenon was also observed by vasa overexpression [18]. Aubassociated piRNAs in testes included AT-chX-piRNAs derived fromtwo tiny intergenic elements on chromosome X, both of whichshowed strong complementarities to vasa transcripts. The biological consequences of vasa gene silencing by the Aub–piRNA complex
in the testis remain unknown, but it seems likely that Aub regulates
vasa gene expression in concert with AT-chX-piRNAs in testes, most
likely at the post-transcriptional level because the vasa mRNA level
was not changed in aub mutant testes.
In ovaries, in addition to transposon transcripts, piRNAs arealso derived from the 3 UTR (untranslated region) of tj transcripts
and loaded onto Piwi in OSC and OSS lines [38,56]. It seems that
the Piwi-tj-derived piRNA complex functions in downregulating
particular protein-coding genes in ovarian somatic cells, such as
fascicline III (fasIII) [38]. FasIII is a cell adhesion molecule necessary for gametogenesis [57]. Thus, the tj gene in ovarian somatic
cells has at least dual roles regulating piwi functions; ﬁrst as a transcriptional factor that drives the piwi expression; and second as a

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precursor of piRNAs that are speciﬁcally loaded onto Piwi protein
to silence speciﬁc target genes including fasIII. It should be notedthat the 3 UTRs of many other genes also act as sources for piRNAs
in OSC and OSS lines [38,56].
8. Conclusions
Extensive studies on Drosophila piRNAs and PIWI proteins haveso far revealed many unique and interesting aspects of how themolecules function in RNA silencing mechanisms occurring specifically in germlines. On the other hands we also realize that manyother aspects of PIWI proteins and piRNAs molecules remain
unclear and therefore investigation on this particular topic needs
to be continued. The remaining important questions include; what
are the factors necessary for piRNA production? Candidate genes
include armitage, spn-E, maelstrom, krimper, rhino, vasa and squash.
Deﬁcient ﬂy lines of each of these genes and/or OSC and OSS lines
from which each gene function is depleted by RNAi will be very useful tools to address this particular question and related questions.
sDMA modiﬁcation of PIWI proteins is another important issue inthis germline-speciﬁc RNA silencing ﬁeld. What are the dynamics
of sDMA modiﬁcation through the piRNA pathway? Is there any
enzyme that removes the dimethyl groups from the sDMAs of PIWI
proteins? When does conversion of sDMA to regular arginines – if it
occurs – take place during the development? What is the physiological meaning of this reaction? Expectations for the accomplishmentof future research are endless.
Acknowledgments
We thank Kuniaki Saito and Kazumichi Nishida for useful discussions. H.S. is supported by MEXT (Ministry of education, Culture,
Sports, Science and Technology). M.C.S. is supported by NEDO (New
Energy and Industrial Technology Development Organization) andCREST (Core Research for Evolutional Science and Technology) fromJST (the Japanese Science and Technology Agency).
References[1] Siomi H, Siomi MC. On the road to reading the RNA-interference code. Nature
2009;457:396–404.
[2] Okamura K, Lai EC. Endogenous small interfering RNAs in animals. Nat Rev Mol
Cell Biol 2008;9:673–8.
[3] Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev
Genet 2009;10:94–108.
[4] Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell
Biol 2009;10:126–39.
[5] Hutvagner G, Simard MJ. Argonaute proteins: key players in RNA silencing. Nat
Rev Mol Cell Biol 2008;9:22–32.
[6] Wang Y, Sheng G, Juranek S, Tuschl T, Patel DJ. Structure of the guide-strandcontaining argonaute silencing complex. Nature 2008;456:209–13.
[7] Wang Y, Juranek S, Li H, Sheng G, Tuschl T, Patel DJ. Structure of an argonaute
silencing complex with a seed-containing guide DNA and target RNA duplex.
Nature 2008;456:921–6.
[8] Czech B, Malone CD, Zhou R, Stark A, Schlingeheyde C, Dus M, et al. An endogenous small interfering RNA pathway in Drosophila. Nature 2008;453:798–802.
[9] Kawamura Y, Saito K, Kin T, Ono Y, Asai K, Sunohara T, et al. Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 2008;453:793–7.
[10] Ghildiyal M, Seitz H, Horwich MD, Li C, Du T, Lee S, et al. Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science
2008;320:1077–81.
[11] Förstemann K, Horwich MD, Wee L, Tomari Y, Zamore PD. Drosophila microRNAs are sorted into functionally distinct argonaute complexes after production
by dicer-1. Cell 2007;130:287–97.
[12] Okamura K, Liu N, Lai EC. Distinct mechanisms for microRNA strand selection
by Drosophila Argonautes. Mol Cell 2009;36:431–44.
[13] Czech B, Zhou R, Erlich Y, Brennecke J, Binari R, Villalta C, et al. Hierarchicalrules for Argonaute loading in Drosophila. Mol Cell 2009;36:445–56.
[14] Saito K, Nishida KM, Mori T, Kawamura Y, Miyoshi K, Nagami T, et al. Speciﬁc
association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev 2006;20:2214–22.
[15] Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R, et al. Discrete small RNA-generating loci as master regulators of transposon activity in
Drosophila. Cell 2007;128:1089–103.

[16] Gunawardane LS, Saito K, Nishida KM, Miyoshi K, Kawamura Y, Nagami T, et al.
A slicer-mediated mechanism for repeat-associated siRNA 5 end formation in
Drosophila. Science 2007;315:1587–90.
[17] Yin H, Lin H. An epigenetic activation role of Piwi and a Piwi-associated piRNAin Drosophila melanogaster. Nature 2007;450:304–8.
[18] Nishida KM, Saito K, Mori T, Kawamura Y, Nagami-Okada T, Inagaki S, et al. Genesilencing mechanisms mediated by Aubergine piRNA complexes in Drosophilamale gonad. RNA 2007;13:1911–22.
[19] Okamura K, Chung WJ, Ruby JG, Guo H, Bartel DP, Lai EC. The Drosophila
hairpin RNA pathway generates endogenous short interfering RNAs. Nature
2008;453:803–6.
[20] Hartig JV, Esslinger S, Böttcher R, Saito K, Förstemann K. Endo-siRNAs depend
on a new isoform of loquacious and target artiﬁcially introduced, high-copy
sequences. EMBO J 2009;28:2932–44.
[21] Zhou R, Czech B, Brennecke J, Sachidanandam R, Wohlschlegel JA, Perrimon N,
et al. Processing of Drosophila endo-siRNAs depends on a speciﬁc Loquacious
isoform. RNA 2009;15:1886–95.
[22] Miyoshi K, Miyoshi T, Hartig JV, Siomi H, Siomi MC. Molecular mechanisms thatfunnel RNA precursors into endogenous small-interfering RNA and micro RNA
biogenesis pathways in Drosophila. RNA, 2010, doi:10.1261/rna.1952110.
[23] Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001;409:363–6.
[24] Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, et al. R2D2, a bridge
between the initiation and effector steps of the Drosophila RNAi pathway. Science 2003;301:1921–5.
[25] Marques JT, Kim K, Wu PH, Alleyne TM, Jafari N, Carthew RW. Loqs and R2D2
act sequentially in the siRNA pathway in Drosophila. Nat Struct Mol Biol 2009:1.
[26] Förstemann K, Tomari Y, Du T, Vagin VV, Denli AM, Bratu DP, et al. Normal
microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol 2005;3:e236.
[27] Saito K, Ishizuka A, Siomi H, Siomi MC. Processing of pre-microRNAs by the
Dicer-1-Loquacious complex in Drosophila cells. PLoS Biol 2005;3:e235.
[28] Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD. A distinct small
RNA pathway silences selﬁsh genetic elements in the germline. Science
2006;313:320–4.
[29] Lin H, Spradling AC. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development
1997;124:2463–76.
[30] Schüpbach T, Wieschaus E. Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering
egg morphology. Genetics 1991;129:1119–36.
[31] Deshpande G, Calhoun G, Schedl P. Drosophila argonaute-2 is required earlyin embryogenesis for the assembly of centric/centromeric heterochromatin,
nuclear division, nuclear migration, and germ-cell formation. Genes Dev
2005;19:1680–5.
[32] Klattenhoff C, Xi H, Li C, Lee S, Xu J, Khurana JS, et al. The Drosophila HP1 homolog
Rhino is required for transposon silencing and piRNA production by dual-strand
clusters. Cell 2009;138:1137–49.
[33] Sarot E, Payen-Groschêne G, Bucheton A, Pélisson A. Evidence for a piwidependent RNA silencing of the gypsy endogenous retrovirus by the Drosophilamelanogaster ﬂamenco gene. Genetics 2004;166:1313–21.
[34] Pélisson A, Song SU, Prud’homme N, Smith PA, Bucheton A, Corces VG. Gypsy
transposition correlates with the production of a retroviral envelope-like protein under the tissue-speciﬁc control of the Drosophila ﬂamenco gene. EMBO J
1994;13:4401–11.
[35] Lau NC, Robine N, Martin R, Chung WJ, Niki Y, Berezikov E, et al. Abundantprimary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line.
Genome Res 2009;19:1776–85.
[36] Li C, Vagin VV, Lee S, Xu J, Ma S, Xi H, et al. Collapse of germline piRNAs
in the absence of Argonaute3 reveals somatic piRNAs in ﬂies. Cell 2009;137:509–21.
[37] Malone CD, Brennecke J, Dus M, Stark A, McCombie WR, Sachidanandam R,
et al. Specialized piRNA pathways act in germline and somatic tissues of the
Drosophila ovary. Cell 2009;137:522–35.
[38] Saito K, Inagaki S, Mituyama T, Kawamura Y, Ono Y, Sakota E, et al. A regulatory circuit for piwi by the large Maf gene trafﬁc jam in Drosophila. Nature
2009;461:1296–9.
[39] Niki Y, Yamaguchi T, Mahowald AP. Establishment of stable cell lines ofDrosophila germ-line stem cells. Proc Natl Acad Sci USA 2006;103:16325–30.
[40] Saito K, Sakaguchi Y, Suzuki T, Suzuki T, Siomi H, Siomi MC. Pimet, the Drosophila
homolog of HEN1, mediates 2’-O-methylation of Piwi-interacting RNAs at their3 ends. Genes Dev 2007;21:1603–8.
[41] Horwich MD, Li C, Matranga C, Vagin V, Farley G, Wang P, et al. The
Drosophila RNA methyltransferase, DmHen1, modiﬁes germline piRNAs andsingle-stranded siRNAs in RISC. Curr Biol 2007;17:1265–72.
[42] Yu B, Yang Z, Li J, Minakhina S, Yang M, Padgett RW, et al. Methylation as a
crucial step in plant microRNA biogenesis. Science 2005;307:932–5.
[43] Chen X. MicroRNA biogenesis and function in plants. FEBS Lett
2005;579:5923–31.
[44] Li J, Yang Z, Yu B, Liu J, Chen X. Methylation protects miRNAs and siRNAs froma 3 -end uridylation activity in Arabidopsis. Curr Biol 2005;15:1501–7.
[45] Li MA, Alls JD, Avancini RM, Koo K, Godt D. The large Maf factor Trafﬁc Jam
controls gonad morphogenesis in Drosophila. Nat Cell Biol 2003;5:994–1000.
[46] Szakmary A, Reedy M, Qi H, Lin H. The Yb protein deﬁnes a novel organelle andregulates male germline stem cell self-renewal in Drosophila melanogaster. J
Cell Biol 2009;185:613–27.

Author's personal copy
M.C. Siomi et al. / Seminars in Cell & Developmental Biology 21 (2010) 754–759[47] Kirino Y, Kim N, de Planell-Saguer M, Khandros E, Chiorean S, Klein PS, et al.
Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3and Aub stability. Nat Cell Biol 2009;11:652–8.
[48] Nishida KM, Okada TN, Kawamura T, Mituyama T, Kawamura Y, Inagaki S, et al.
Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway
in Drosophila germlines. EMBO J 2009;28:3820–31.
[49] Meister G, Eggert C, Fischer U. SMN-mediated assembly of RNPs: a complex
story. Trends Cell Biol 2002;12:472–8.
[50] Battle DJ, Kasim M, Yong J, Lotti F, Lau CK, Mouaikel J, et al. The SMN complex: an assembly machine for RNPs. Cold Spring Harb Symp Quant Biol
2006;71:313–20.
[51] Bozzetti MP, Massari S, Finelli P, Meggio F, Pinna LA, Boldyreff B, et al. The Ste
locus, a component of the parasitic cry-Ste system of Drosophila melanogaster,
encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2. Proc Natl Acad Sci USA
1995;92:6067–71.
[52] Palumbo G, Bonaccorsi S, Robbins LG, Pimpinelli S. Genetic analysis of Stellateelements of Drosophila melanogaster. Genetics 1994;138:1181–97.

759[53] Aravin AA, Naumova NM, Tulin AV, Vagin VV, Rozovsky YM, Gvozdev
VA. Double-stranded RNA-mediated silencing of genomic tandem repeats
and transposable elements in the D. melanogaster germline. Curr Biol
2001;11:1017–27.
[54] Lasko PF, Ashburner M. The product of the Drosophila gene vasa is very similar
to eukaryotic initiation factor-4. Nature 1988;335:611–7.
[55] Styhler S, Nakamura A, Swan A, Suter B, Lasko P. vasa is required for GURKEN
accumulation in the oocyte, and is involved in oocyte differentiation andgermline cyst development. Development 1998;125:1569–78.
[56] Robine N, Lau NC, Balla S, Jin Z, Okamura K, Kuramochi-Miyagawa S, et al. A
broadly conserved pathway generates 3 UTR-directed primary piRNAs. Curr
Biol 2009.
[57] Snow PM, Bieber AJ, Goodman CS. Fasciclin III: a novel homophilic adhesion
molecule in Drosophila. Cell 1989;59:313–23.
[58] Cox DN, Chao A, Baker J, Chang L, Qiao D, Lin H. A novel class of evolutionarily
conserved genes deﬁned by piwi are essential for stem cell self-renewal. Genes
Dev 1998;12:3715–27.