REVIEWSPIWI-interacting small RNAs:
the vanguard of genome defence
Mikiko C. Siomi*‡, Kaoru Sato*, Dubravka Pezic§ and Alexei A. Aravin§

Abstract | PIWI-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs
that form the piRNA-induced silencing complex (piRISC) in the germ line of many animal
species. The piRISC protects the integrity of the genome from invasion by ‘genomic
parasites’ — transposable elements — by silencing them. Owing to their limited expression
in gonads and their sequence diversity, piRNAs have been the most mysterious class of
small non-coding RNAs regulating RNA silencing. Now, much progress is being made into
our understanding of their biogenesis and molecular functions, including the specificsubcellular compartmentalization of the piRNA pathway in granular cytoplasmic bodies.

*Keio University School of
Medicine, Tokyo 160‑8582,
Japan.
‡
Japan Science andTechnology Agency, Core
Research for Evolutional
Science and Technology(CREST), Saitama 332‑0012,
Japan.
§
Division of Biology,
California Institute ofTechnology, 1200
E. California Boulevard,
Pasadena, California 91125,
USA.
Correspondence
to M.C.S. and A.A.A.
e-mails: siomim@sc.itc.keio.
ac.jp; aaa@caltech.edu
doi:10.1038/nrm3089

Gene expression in individual cells of multicellularorganisms is elaborately controlled by various mechanisms to initiate and maintain cellular differentiation,
resulting in distinct gene expression profiles in each cell.
One of the regulatory mechanisms present in almost all
eukaryotic organisms is RNA silencing, which provides
highly specific inhibition of gene expression throughcomplementary recognition of RNA targets by small(20–30 nucleotide (nt)) RNA molecules. The RNAinduced silencing complex (RISC) forms the core of the
RNA silencing machinery, and consists of a protein fromthe Argonaute (AGO) family and a small RNA that acts toguide RISC to its targets (a ‘guide’ RNA)1–5. Once loadedwith a small RNA, AGO proteins inhibit the expression of
their targets, either by cleavage using Slicer endo­nucleaseactivity, or by attracting additional proteins that can affecttrans­lation, RNA stability or chromatin structure. The
majority of eukaryotic organisms possess more than one
AGO protein, and the functions of individual membersof the family are often non-redundant.
Small silencing RNAs in metazoa can be divided into
three classes: small interfering RNAs (siRNAs), micro­
RNAs (miRNAs) and PIWI-interacting RNAs (piRNA­s)1–3.
Compared with siRNAs and miRNAs, piRNA­s are theleast investigated class of small RNA molecules. However,
early on in the study of RNA silencing, an analysis of
how Stellate protein-coding gene repeats are silenced in
the Drosophila melanogaster male germ line provided ahint that piRNAs existed6. A small RNA profiling study
in the D. melanogaster testes and early embryos later
revealed an abundance of endo­genous 23–29 nt small
RNAs that were derived from repetitive genomic elements and were originally termed “repeat‑associatedsmall interfering RNAs”7.

Different classes of small RNAs arise by varying
biogenesis mechanisms. The processing of bothmiRNAs and siRNAs from their precursors requires
cleavage by the Dicer endonuclease before loading
onto an AGO protein (FIG. 1). piRNAs, by contrast, areprocessed through Dicer-independent mechanisms8–13(FIG. 1) , and this finding led to the realization thatthey are distinct from siRNAs and miRNAs. piRNAs
specifically associate with PIWI proteins, which
are germline-specific members of the AGO protein
family, whereas siRNAs and miRNAs associate withubiquitously expressed AGO proteins. In this regard,piRNAs can also be distinguished from siRNA­sand miRNAs.
In this Review, we summarize our understanding of
the biogenesis and function of piRNAs, mainly focusing
on studies done in two model systems, D. melanogasterand mice. We also review recent progress in our understanding of how the piRNA pathway is spatially controlled in cytoplasmic bodies that appear during gonadal
development.

Protective functions of piRNAs
The main function of piRNAs is to silence transposable elements (TEs) in the germ line, and this roleis highly conserved across animal species. TEs are
genomic para­sites that threaten the genomic integrityof the host genome; they can move to new sites byinsertion or transposition and thereby disrupt genes
and alter the genome14 (BOX 1). In animals, endo­genous
siRNAs also silence TEs15, but the piRNA pathway isat the forefront of defence against transposons in germ
cells. To achieve this goal, piRNAs associate with PIWI
proteins to form an active piRNA-induced silencing

246 | APRIL 2011 | VOLUME 12

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REVIEWSCUK40#DKQIGPGUKUERK40#DKQIGPGUKUDOK40#DKQIGPGUKU
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Figure 1 | Biogenesis of small RNAs in mammals. a | Small interfering RNA (siRNA) biogenesis. Transcripts that are
0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[
able to form double-stranded RNA (dsRNA) or long stem–loop structures serve as endogenous
siRNA (endo-siRNA)
precursors. Pseudogenes transcribed in an antisense orientation produce RNA that pairs with cognate gene mRNAs
as well as with transcripts that are derived from intergenic repetitive sequences on the genome, including transposons.
The processing of endo-siRNA requires Dicer, whereas the role of Dicer-binding proteins — such as TAR RNA-binding
protein (TRBP; also known as TARBP2) and PACT (also known as PRKRA) — remains undetermined. After maturation,endo-siRNAs (which are ~21 nucleotides (nt) in length) are loaded onto Argonaute 2 (AGO2). Whether endo-siRNAs areloaded onto other AGO members, such as AGO1, AGO3 and AGO4, remains to be determined. Exogenous siRNAs(exo-siRNAs) are derived from exogenous dsRNAs by Dicer–TRBP (or Dicer–PACT). exo-siRNAs are loaded onto AGO1,
AGO2, AGO3 and AGO4; however, only the AGO2–siRNA complex functions in RNA interference, as other AGO
members lack Slicer activity. b | MicroRNA (miRNA) biogenesis. The primary transcripts of miRNAs (pri-miRNAs) aretranscribed by RNA polymerase II from miRNA genes on the genome. pri-miRNAs form hairpin structures and areprocessed to ~60–70 nt miRNA precursors (pre-miRNAs) by the microprocessor Drosha–DGCR8 (DiGeorge syndrome
critical region 8) complex in the nucleus. After being exported by exportin 5 and RanGTP, pre-miRNAs are further
processed to ~22 nt miRNA–miRNA* duplexes (in which miRNA* is the passenger strand that is degraded) by the
Dicer–TRBP (or Dicer–PACT) complex. Mature miRNAs are then loaded onto AGO1, AGO2, AGO3 and AGO4.
c | PIWI-interacting RNA (piRNA) biogenesis. piRNAs (which are 24–32 nt in length) are processed from single-stranded
RNA precursors that are transcribed largely from particular intergenic repetitive elements known as piRNA clusters.
Unlike miRNAs and siRNAs, piRNAs do not require Dicer for their processing. The requirement for Drosha has not beenformally tested. First, primary piRNAs are produced through the primary processing pathway and are amplified throughthe ping-pong pathway, which requires Slicer activity of PIWI proteins. Primary piRNA processing and loading onto
mouse PIWI proteins might occur in the cytoplasm. MIWI2 (also known as PIWI-like protein 4) specifically associates withsecondary piRNAs that are processed through the amplification loop, and is localized in the nucleus to exert its silencing
function. (A)n indicates the poly(A) tail formed during translation, and m7G indicates the 5′-terminal cap of the mRNA.

complex (piRISC) that can recognize and silence complementary RNA targets. Indeed, genetic evidence has
shown that, in addition to piRNAs, PIWI proteins arenecessary for TE silencing and have crucial roles in
gonadal development.

Silencing of transposable elements. In D. melanogaster,
mutations in each of the three members of the PIWI
family — PIWI, Aubergine (AUB) and AGO3 — leadto transposon derepression in the germ line, indicatingthat they act non-redundantly during TE silencing 16–19.

NATURE REVIEWS | MOLECULAR CELL BIOLOGYVOLUME 12 | APRIL 2011 | 247© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS
Box 1 | Life cycles of transposable elements
DNA transposons (see the figure, part a (green bar)) move around the genome mostly by a‘cut and paste’ mechanism. DNA transposons encode a transposase enzyme (green ovals)
that catalyses the movement of DNA transposons. Transposases enter the nucleus andbind the ends of DNA transposons at inverted/direct repeats (IR/DRs) to excise themfrom the genome. An excised DNA element is then integrated into a new site on the
genome through ‘target capture’. Long terminal repeat (LTR) retrotransposons (see thefigure, part b (pink bar)) move around the genome by a ‘copy and paste’ mechanism.
LTR retrotransposons contain LTRs at both ends and encode integrases (pink ovals) andglycosaminoglycan (GAG) proteins (grey circles). GAG proteins form virus-like particles,in which retrotransposon mRNAs are captured. Reverse transcription of the viral mRNAs
occurs in the virus-like particles to generate the DNA intermediates. This reaction takes
place in the cytoplasm. The DNA intermediates associated with integrases emerge
from the particles, and are imported into the nucleus and integrated into the genome.
Non-LTR retrotransposons (see the figure, part c (blue bar)) also move around the genome
by a ‘copy and paste’ mechanism. Non-LTR retrotransposons do not have LTRs and can bedivided into two subtypes, long interspersed nuclear elements (LINEs; dark blue) andshort interspersed nuclear elements (SINEs; light blue). LINEs encode two proteins, ORF1
(light blue ovals) and ORF2 (dark blue ovals), both of which have a reverse transcriptase
domain. ORF1 and ORF2 form ribonucleoprotein (RNP) complexes with their own
mRNAs in the cytoplasm. The RNP complexes are transported to the nucleus andthe mRNAs are integrated into new sites on the genome by target-primed reverse
transcription. SINEs do not encode a functional reverse transcriptase and therefore use
the enzyme of LINEs, or other transposable elements, for their transposition14. Orange
ovals represent ribosomes and (A)n indicates the poly(A) tail formed during translation.

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Indeed, all PIWI proteins have crucial roles in gonad
development: both piwi and aub are required for maleand female fertility 20–22 and Ago3 is required for female
fertility, but only partially for male fertility 19. The nonredundant function of PIWI can be at least partly
explained by the fact that, unlike AUB and AGO3, it isexpressed in follicular cells that have somatic origins andsurround the developing germline cells20,23,24.
Deficiency in two members of the mouse PIWI protein family, MILI (also known as PIWI-like protein 2)
and MIWI2 (also known as PIWI-like protein 4), but not
the third member, MIWI (also known as PIWI-like protein 1), leads to the activation of long interspersed nuclear
element (LINE) and long terminal repeat (LTR) retrotransposons in the male germ line (BOX 1), as well as arrestof gametogenesis and complete sterility in males25–27.
However, contrary to the situation in D. melanogaster,
piwi mutations do not affect the female germ line
in mice, despite the fact that at least one membe­r,
Mili, is expressed in female germ cells together witht­ransposon‑derived piRNAs28,29.
Severe defects in gametogenesis observed in piwideficient flies and Mili- and Miwi2‑knockout miceare probabl­y directly caused by TE activation. Thede­repression of TEs that occurs as a result of loss of piwi
functions leads to the generation of double-stranded
DNA breaks during abortive or successful transposition,and this activates a DNA-damage checkpoint. In fact,
mutations in components of the DNA-damage checkpoint suppress some gametogenesis defects observed inflies deficient in piRNA-mediated TE silencing, although
they cannot prevent formation of double-stranded
breaks and the eventual sterility of piwi mutants30.
Consistent with their function in TE repression, allmembers of the PIWI family in D. melanogaster, and MILI
and MIWI2 in mice, are associated with transposonderived piRNAs. PIWI proteins loaded with piRNAs
that are antisense to TE mRNAs can specifically recognize and cleave transposon transcripts31,32. Similarly to
AGO proteins, recombinant fly PIWI proteins exhibit
Slicer endonuclease activity that cleaves target RNAs
that are complementary to the guide small RNAs32,33.
In mice, the role of PIWI proteins and piRNAs seems
to extend beyond post-transcriptional silencing. CpG
DNA methylation, which is required for efficient trans­
criptional silencing of LINE and LTR retrotransposons
in the genome, is decreased in the male germ line ofMili and Miwi2 mutants. Genetic evidence suggests thatboth mutants fail to establish de novo methylation of
TE sequences during spermatogenesis, leading to the
hypothesis that the piRISC can also guide the de novo
methylation machinery to TE loci26,27. MIWI2, loadedwith anti-transposon piRNAs, is present in the pro­spermatogonia nuclei (FIG. 1c) during the short period inembryogenesis when de novo DNA methylation takes
place in the male germ line26,27. piRNAs may present a
perfect guide for discriminating TE sequences from normal protein-coding genes and marking them for DNA
methylation; however, the biochemical details of how
these two mechanisms of piRNA action might be linked
have not yet been revealed.

0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[© 2011 Macmillan Publishers Limited. All rights reserved

www.nature.com/reviews/molcellbio

REVIEWS
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Figure 2 | Putative roles for piRNAs during silencing of protein-coding genes. a | Stellate (Ste) silencing by Suppressor
0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[of Stellate PIWI-interacting RNAs (Su(ste)-piRNAs) in Drosophila melanogaster testis.
Su(ste)-piRNAs are derived from theSu(ste) locus on the Y chromosome36. Su(ste) shows a strong homology to Ste on the X chromosome at the nucleotide
sequence level. Su(ste)-piRNAs bind both Aubergine (AUB) and Argonaute 3 (AGO3) and have a role in Ste silencing33,61.
Without Su(ste)-piRNAs, Ste is derepressed, which results in the aggregation of STE, or STE crystals, which consequently
causes male infertility. b | Fasciclin 3 (Fas3) silencing by traffic jam (tj)-piRNAs in D. melanogaster ovary. PIWI expression isdriven by TJ, a large MAF transcription factor encoded by the tj gene44. piRNAs derived from the 3′ untranslated region(UTR) of tj mRNAs are loaded onto PIWI, and the resultant complex functions in silencing Fas3 (REF. 44). The mRNA levelof Fas3 is upregulated in piwi mutants, as in tj mutant clones, resulting in an SGP (somatic gonadal precursor)-only
cell cluster and a failure to intermingle with PGCs (primordial germ cells)44. c | vasa silencing by AT‑chX-piRNAs in
D. melanogaster testis. AT‑chX-piRNAs are derived from the AT‑chX locus on the X chromosome (Chr X) and associatewith both AUB and AGO3 (REFS 33,61). AT‑chX-piRNAs direct AUB to vasa mRNAs to downregulate vasa. Increased
levels of Vasa protein result in oversized apexes in testes, where spermatogonial cells and spermatocytes are located33.
Mutations in Ago3 cause little or no change in the expression level of Vasa61. This may be because the expression level of
AGO3 in testes is much lower than that of AUB61. d | nanos (nos) mRNA decay driven by roo and 412‑derived piRNAs in
D. melanogaster embryos45. A failure in piRNA-induced nos mRNA deadenylation and translational repression results inhead development defects45. (A)n indicates the poly(A) tail formed during translation. CCR4, C-C chemokine receptor 4(also known as Twin); ORF, open reading frame; SMG, Smaug.

MorphogenA signalling moleculethat provides positional
information to cells by forming
a concentration gradientacross the developmental
field, and elicits multiple
different cellular responses tospecify cell fates in a concentration-dependent manner.

Maternal–zygotic transition
A developmental switch during
embryogenesis from control
by maternally provided geneproducts to control by
zygotically provided geneproducts. During this transition,embryonic transcription is
initiated and many maternal
products are degraded.

PIWI proteins and piRNAs in D. melanogaster may
also affect transcriptional silencing through effects
on chromatin5. In particular, PIWI is present in the
nucleus in the steady state9,20, and its nuclear localiz­ation is crucial for its function in TE silencing 34. A
Slicer-deficient PIWI mutant, however, rescued TE
silencing 34, indicating that PIWI-mediated silencing is
independent of Slicer. Some results suggested that PIWI
influences position-effect variegation in somatic cells,
wherein gene silencing depends on the genomic loci, bydirecting Heterochromatin protein 1A (HP1A) localiz­ation to heterochromatin35,36. It was also proposed thatPIWI can activate expression of telomeric sequences,which is dependent on the chromatin context 37. Other
evidence has shown, however, that HP1 recruitment toheterochromatin does not depend on PIWI38, leaving
this issue open to further investigation.
Endogenous siRNAs (endo-siRNAs) also partici­
pate in transposon silencing, as TEs are prone to producing double-stranded RNAs that are channelledtowards Dicer for processing 15. In fact, endo-siRNAs
repress TEs in D. melanogaster somatic cells that lackthe piRNA pathway 39,40 and, in mice, endo-siRNAs
also target TEs in the female germ line41,42. This might
explain why piwi deficiency in females has no phenotypicconsequences.

Control of non-transposable elements. Some piRNAs may
target non-transposable elements (non-TEs). Indeed, thefirst piRNAs that were described in D. melanogaster were
found to target repetitive, but protein-coding, Stellate
genes6 (FIG. 2a). Several studies later suggested that a specific population of D. melanogaster piRNAs might also be
involved in the silencing of non-repetitive, protein-coding
genes. For example, Fasciclin 3 (Fas3), a gene encoding
an immunoglobulin-like cell adhesion molecule43, might
be targeted by PIWI-associated piRNAs in follicle cells of
fly ovaries44 (FIG. 2b). The expression of Fas3 increases in
piwi mutant gonads. Overexpression of Fas3 prevents theintermingling of germline and somatic cells in the ovaries,
resulting in oogenesis defects43. Germline and somatic
cells also failed to intermingle in a piwi mutant 44. Thus,
it was suggested that the PIWI–piRNA complex represses
Fas3 transcripts44. Similarly, AUB-associated piRNAs
in the fly testes might target the essential maternaleffect gene vasa 33 (FIG. 2c). Some piRNAs may induce
degradation of maternally deposited mRNAs, such as
Nanos, the D. melanogaster posterior morphogen, during the maternal–zygotic trans­ition in the D. melanogasterembryo45 (FIG. 2d). These results suggest that piRNA function extends beyond transposon silencing. However, thecomplementarities between these piRNAs and theirpotential targets are marginal. Furthermore, the number

NATURE REVIEWS | MOLECULAR CELL BIOLOGYVOLUME 12 | APRIL 2011 | 249© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWSof piRNAs for which effects on protein-coding genes
have been shown in vivo is rather low. Therefore, additional studies are necessary to conclude that piRNAs canindeed target non-TEs. Protein-coding genes that areembedded in heterochromatin regions of the genome
often contain TE remnants in their introns and therefore have the potential to be targeted by nuclear piRNAs.
However, their expression is maintained in mutants thatshow a decrease in accumulation of piRNAs, suggesting that these genes may have a mechanism to prevent
piRNA‑mediated silencing 46.
The first piRNAs described in mammals — whichwere named pachytene piRNAs because they are firstexpressed during the pachytene stage of meiosis — do nottarget transposons47. Accordingly, deficiency in MIWI,
which associates with pachytene piRNAs, does not lead
to TE derepression. The targets of mammalian pachytene
piRNAs and MIWI are still unknown.

piRNA biogenesis
Extensive analyses of piRNAs associated with PIWI
proteins in flies and mice have identified the genomic
origins of piRNAs and led to the proposal of two bio­
genesis pathways: the primary processing pathway andthe ping-pong amplification loop (FIG. 3). Both mechanisms are important for mounting an effective defence
against transposons. First, the primary piRNA biogenesis
pathway provides an initial pool of piRNAs that target
multi­ple TEs. Next, the ping-pong cycle further shapes
the piRNA population by amplifying sequences that target active transposons. These two pathways are conservedin many animal species, including rats (Rattus norvegicus), zebrafish (Danio rerio), frogs (Xenopus laevis) andsilkworms (Bombyx mori)48–51.

Pachytene stage
A stage in meiotic prophasewhen homologous
chromosomes are completely
paired and chromosomal
crossover occurs.

Phasing
An endonucleolytic processing
pattern with an equal
periodicity. Small interfering
RNAs are endonucleolyticallyprocessed (or ‘diced’) by Dicer
from individual precursors at
certain intervals to produce atypical size of ~21 nucleotides.
Dicer acts as a ‘ruler’ during
processing.

Tudor domain
A conserved protein motif
typically consisting of ~50
amino-acid residues. It is
commonly found in proteins,
such as RNA-binding proteins.
Some Tudor domains
specifically recognize and bindsymmetrically dimethylated
arginines.

The primary piRNA processing pathway. Initial anal­y­sis of piRNA sequences revealed their extremely highcomplexity: whereas individual miRNA species numberin their hundreds, there are hundreds of thousands,
if not millions, of individual piRNA sequences10,11,31.
Furthermore, there are no specific sequence motifs inpiRNAs, except for a strong bias for uridine at position 1(1U bias). Despite their diversity, most piRNAs can bemapped to a relatively small number of genomic regions
called piRNA clusters10,11,31. piRNA clusters extend fromseveral to more than 200 kilobases, and each cluster contains multiple sequences that generate piRNAs. Unlike
siRNAs, piRNAs do not show any phasing within a cluster
sequence and can overlap with each other. Furthermore,no significant secondary structures, reminiscent of thestem–loop structures in miRNA precursors, have been
detected in regions surrounding mature piRNAs. In someclusters in both D. melanogaster and mice, piRNAs map
to both genomic strands, suggesting bidirectional trans­
cription29,31. However, other clusters, such as flamenco(flam) in D. melanogaster and pachytene piRNA clusters
in mice, produce piRNA from a single genomic strand(although some pachytene clusters are bidirectionallytranscribed in opposite directions from a central promoter)11,31. Accordingly, it is likely that piRNA precursors are single-stranded and therefore do not require

Dicer for their processing. Some evidence from reverse
transcription PCR and expressed sequence tag databases
supports the notion that long, single-stranded RNAs areindeed transcribed from piRNA clusters10,11,26,31. Genetic
data from D. melanogaster suggest that long RNA molecules, spanning the whole piRNA cluster, are processed
into mature piRNAs: P‑element insertion at the 5′ endof flam that disrupts transcription of the long RNA also
leads to elimination of all piRNAs from this cluster, easily
more than 100 kilobases from the site of insertion24,31. The
cause of 1U bias in piRNA is currently unknown. One
possibility is that the 1U bias arises during processing ofcluster transcripts to mature piRNAs. Another possibility,
at least in follicle cells, is that initial piRNA processing
occurs in a completely random fashion, but that PIWI,
the only recipient of mature flam-piRNAs in follicle cells(where AUB and AGO3 are not present), preferentiallyassociates with 1U piRNAs. This would be in accordance with findings from the plant Arabidopsis thaliana,
in which each AGO protein shows a unique 5′ nucleotide
bias in the small RNAs with which it associates52.
It is currently unclear how primary piRNAs are produced from piRNA clusters (FIG. 3a). Deficiency in PIWI
proteins, for example PIWI in D. melanogaster and
MILI and MIWI in mice, eliminates primary piRNA­s.
However, it is unlikely that PIWI proteins are directly
involved in primary processing. Interestingly, piRNAs
that associate with each member of the PIWI protein
family have a distinct size (~26 nt, ~28 nt and ~ 30 nt
for MILI, MIWI2 and MIWI, respectively, in mice and~25 nt, ~24 nt and ~23 nt for PIWI, AUB and AGO3,
respectively, in D. melanogaster), suggesting that PIWI
proteins can act as ‘rulers’ that define the size of maturepiRNAs. Recent studies have identified several additional
proteins that are involved in primary piRNA biogenesis
in D. melanogaster, including the putative RNA helicase
Armitage (ARMI), the putative nuclease Zucchini (ZUC),
and the Tudor domain and helicase domain protein Female
sterile (1) Yb (FS(1)YB; also known as Yb). Mutations in
and/or depletion of any of these three proteins eliminates
primary piRNAs associated with PIWI from somatic follicle cells of the ovaries, and from the ovarian somatic
sheet cells or the ovarian somatic cell (OSC) cultured cellline derived from ovarian somatic sheet cells34,53–55. ARMI
and ZUC are also required for piRNA accumulation in
germline cells in ovaries51, whereas FS(1)YB is exclusively
expressed in follicle cells56.
piRNA clusters work as repositories of information,
enabling production of many mature piRNAs that target
diverse TEs. piRNA clusters in D. melanogaster, as well
as clusters expressed during mouse embryogenesis — the‘pre-pachytene piRNA clusters’ — are extremely rich infull-length and truncated copies of TEs29,31.47. As a result,up to 90% of piRNAs originating from such clusters
match transposon sequences. Furthermore, the orientation of piRNAs exclusively derived from the uni-stranded
clusters (the piRNA clusters trans­cribed in only one direction) is usually antisense to that of TE mRNAs24,29,31. ThesepiRNAs can recognize and destroy TE transcripts derived
from multiple sites spread throughout the genome. The
best-characterized example of this is the flam locus in

250 | APRIL 2011 | VOLUME 12

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REVIEWSC2TKOCT[RTQEGUUKPID#ORNKȮECVKQPNQQR RKPIRQPIE[ENG

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Figure 3 | Two pathways for piRNA biogenesis. a | In the Drosophila melanogaster primary PIWI-interacting RNA(piRNA) processing pathway, the primary antisense transcripts transcribed from transposons
and/or the piRNA
0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[
clusters are processed to piRNAs by unknown mechanisms and are loaded onto Aubergine (AUB) or PIWI19,24,45. piRNAs
derived from the flamingo (flam) locus are exclusively loaded onto PIWI because flam is active only in ovarian somas
where only PIWI is expressed. piRNA-induced silencing complexes (piRISCs) produced through this mechanism act asa ‘trigger’ of the amplification loop. b | This amplification mechanism (also known as the ping-pong cycle) is most likely
to involve Slicer activity of AUB and Argonaute 3 (AGO3)31,32, but not PIWI itself24. AUB associated with antisense
piRNA cleaves piRNA precursors in the sense strand. This reaction determines and forms the 5′ end of piRNAs that areloaded onto AGO3. AGO3 associated with sense piRNA cleaves antisense piRNA precursors, generating the 5′ end ofantisense piRNAs that subsequently are loaded onto AUB. The 3′ end of piRNA is formed by an unknown nuclease (ornucleases), which is followed by 2′‑O-methylation mediated by HEN1. piRNAs that induce the amplification loop may
also be deposited from the mother. c | During the ping-pong cycle in mouse prospermatogonia, primary piRNAs areprocessed from transposable elements (TEs) and cluster transcripts, and associate primarily with MILI (also known as
PIWI-like protein 2), which resides in discrete cytoplasmic granules termed pi-bodies. MILI loaded with sense piRNA
cleaves antisense transcripts, generating 5′ ends of secondary piRNAs that are loaded into MIWI2 (also known as
PIWI-like protein 4). MIWI2 in turn cleaves sense transcripts, generating new sense piRNAs that are loaded into MILI.
MIWI2 resides in cellular bodies that also contain components of processing bodies (P-bodies), and are therefore
called piP-bodies. MIWI2 and its partners in turn translocate into the nucleus, where they are involved in de novo
methylation of TE sequences. 3′OMe, 2′-O-methylation of the piRNA 3′ terminus.

D. melanogaster: flam is expressed in follicle cells of theovaries and contains copies of gypsy, Idefix and ZAM TEs.
Accordingly, specific disruption of this cluster activatesthese three transposons, as well as other elements that canbe targeted by flam, in follicle cells57–60. The sequences ofpiRNA clusters are likely to be in constant flux, allowing
adjustments that are tailored to new active transposons
invading the species. Indeed, analysis of piRNA clusters
in different Drosophila species has shown that, although
the clusters’ locations are conserved, their sequence content has evolved very quickly 24. An un­explored questionis whether the sequence content of piRNA clusters is
updated by random integration of new TEs or by active
mechanisms that target these new insertions specificallyto the piRNA loci.
piRNAs are also generated from mRNAs of active
TEs as well as some protein-coding genes29,31,51. This
processing probably occurs using the same machinerythat processes piRNA cluster transcripts. For proteincoding genes, such as the traffic jam (tj) locus in
D. melanogaster, most piRNAs are derived from the 3′
untranslated region31,51, suggesting that the translational

NATURE REVIEWS | MOLECULAR CELL BIOLOGYVOLUME 12 | APRIL 2011 | 251© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS
machinery affects processing (FIG. 2b). As only a small
fraction of protein-coding mRNAs generates piRNAs,
there must be a specific process that discriminates thesetranscripts. However, the rules that govern such selection
are not clear. The function, if any, of piRNAs originating from gene loci is also mysterious: it is unlikely thatthey regulate expression of host genes, and neither dothey have perfectly complementary targets. Some genederived piRNAs, such as those derived from tj, seem tomake use of their imperfect complementarity and repress
other protein-coding genes, including Fas3 as describedabove44 (FIG. 2b).
In some cell types, primary piRNA biogenesis is the
only mechanism that generates piRNAs. In particular, allpiRNAs in somatic follicle cells of the D. melanogaster
ovary and pachytene piRNAs expressed during meiosis inmouse spermatocytes are generated by primary processing 10,11,24,44,48. However, in other cell types, such as germline cells of the D. melanogaster ovary and pre-meiotic
spermatogonia in mice, there is another mechanism called
the ping-pong cycle that amplifies specific sequences
generated by the primary biogenesis pathway.

Hypervariable antibody
repertoire
The antibody specificities thatcan potentially be produced byan individual. A huge repertoireof different antibodies isgenerated in a single individual.

The ping-pong amplification loop. In D. melanogaster,
primary piRNAs are associated with PIWI and AUB,
and are mostly antisense to transposon mRNAs9,31–33,37.
Surprisingly, cloning of piRNAs associated with AGO3
in D. melanogaster revealed that these sequences do nothave a 1U bias. Instead, they have a strong bias for adenosine at the tenth nucleotide from their 5′ end (10A) andare in the sense orientation. Furthermore, significant
fractions of AGO3- and AUB-associated piRNAs are able
to pair through their first ten nucleotides31,32. In addition,
recombinant PIWI proteins exhibit Slicer activity, whichcleaves target RNAs between their tenth and eleventh
nucleotides relative to the ‘guide’ small RNAs32. Theseobservations have led to a ‘ping-pong’ model of piRNAbiogenesis, in which PIWI proteins amplify piRNAs in a
Slicer-dependent amplification cycle (FIG. 3b). This model
suggests that cleavage of complementary transcripts targeted by AUB-bound 1U primary piRNA leads to thegeneration of the 5′ end of new secondary piRNAs thathave a 10A bias, and accordingly can be loaded onto
AGO3 (REFS 31,32). The enzymatic activity that generates the 3′ end of these secondary piRNAs is unknown.
AGO3, loaded with secondary piRNAs, is able to thenrecognize and cleave complementary transcripts. This
results in the generation of further piRNAs that exactly
correspond to the original primary piRNA sequences
and are loaded onto AUB. The ping-pong pathway also
amplifies piRNA­s in D. melanogaster testes, especiallythose originating from TEs. Non-TE-derived piRNAs —such as Suppressor of Stellate (Su(ste))-derived piRNAs,
which silence Stellate — seem to be barely amplified by
the amplification loop61.
The distinct functions of AUB and AGO3 in the pingpong cycle of D. melanogaster explain their non-redundantroles in the silencing of the same set of transposons19,31.
MILI and MIWI2 in mice also have non-redundant rolesin the ping-pong cycle and trans­poson repression, withMILI preferentially associating with primary piRNAs and

MIWI2 with secondary piRNAs29 (FIG. 3c). However, theorientation of piRNAs in mice is reversed compared withfruitflies, with the majority of mouse primary piRNAs
being sense and secondary piRNA­s being antisense relative to transposons. The operation of an amplification
loop that requires two PIWI members is supported bygenetic data: in Mili-deficient mice that fail to generate
primary piRNAs, MIWI2 also remains unloaded19,29; andin fly Ago3 mutants, the number of AUB-bound piRNAs
was markedly decreased and their antisense strand bias
was weakened19. However, it is also possible that a single
PIWI protein might have dual roles in the amplification
loop by accepting both primary and secondary piRNA­s,
as has been shown for MILI expressed in postnatal
spermato­gonia without its MIWI2 partner 29.
It has been proposed that the biological role of theping-pong cycle is to fine-tune the piRNA population
so that active TEs can be targeted29,31. Indeed, the cycle
amplifies piRNAs only if corresponding sense and antisense transcripts are present within the cell. Whereas
antisense transcripts are usually derived from piRNA
clusters, sense transcripts often arise from mRNAs ofactive transposons29. The two steps of piRNA biogenesis— the primary processing pathway and the amplification
loop — and their function in mounting defence against
TEs can be compared with the function of the adaptive
immune system in protecting against pathogens. The
primary piRNA biogenesis pathway resembles the initial
generation of the hypervariable antibody repertoire, whereas
the amplification loop is analogous to antigen-directedclonal expansion of antibody-producing lympho­cytes
during the acute immune response.
The importance of the amplification loop in defence
against transposons is underlined by studies of hybrid
dysgenesis in D. melanogaster 62 (BOX 2). This pheno­menon is observed in crosses between two different fly
stocks, which result in sterile progeny with severe gonadal
dystrophy and activation of specific transposons, such as
P- and I-elements. The peculiarity of hybrid dysgenesis
is that it is observed only when males of one stock arecrossed to females of the other, and no abnormalities areobserved for the reciprocal cross. As the genotypes of theprogeny of both crosses are identical, epigenetic factors
must be responsible for the hybrid dysgenesis phenotype.
It was revealed that the transposon activation that results
is caused by a failure to maternally deposit piRNAs thattarget specific, paternally deposited transposons62,63.
Maternal deposition of piRISCs into the egg provides
the embryo with an initial piRNA pool to initiate theamplification cycle and build up resistance to TEs.

Regulation by PIWI-interacting proteinspiRNAs are tightly bound to PIWI proteins that canrepress complementary target loci and generate secon­
dary piRNAs using their Slicer endonuclease activity. However, genetic studies in both D. melanogasterand mice have revealed several other proteins thatare required for efficient piRNA-mediated silencing.
Furthermore, some of these proteins interact directlywith PIWI proteins. Analysis of the post-translational
modification of PIWI proteins has revealed that they

252 | APRIL 2011 | VOLUME 12

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REVIEWS
Box 2 | Hybrid dysgenesis and piRNA silencing
When two DrosophilaC*[DTKFF[UIGPGUKUETQUU D4GEKRTQECNETQUU
melanogaster stocks are(GOCNG/CNG(GOCNG/CNG
crossed together, the genome
0Q2GNGOGPV 2GNGOGPV2GNGOGPV 0Q2GNGOGPV
of one stock (shown with redeyes in the figure) contains
P‑element transposons, theactivity of which is suppressedby corresponding
PIWI-interacting RNAs
(piRNAs). Another stock(shown with white eyes in the2GNGOGPV
RK40#
figure) is devoid of P‑elements(and cognate piRNAs. In a‘hybrid dysgenesis’ cross (parta of the figure), piRNAs
cannot be deposited into theembryo through the paternal
germ line, which results intransposon activation andsterility. By contrast, in a‘reciprocal’ cross (part b of2GNGOGPVCEVKXG
2GNGOGPVUKNGPEGF(NKGUUVGTKNG(NKGUHGTVKNG
the figure), maternal
deposition of piRNAs into the
0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[
embryo through the female germ line initiates the
ping-pong cycle of piRNA
amplification, which provides resistance against P‑elements in the progeny and resultsin flies that are fertile62.

contain conserved symmetrical dimethyl arginine(sDMA) residues that provide a binding platform for
Tudor-domain-containing proteins64,65 (FIG. 4).
Tudor-domain proteins. Biochemical analysis of proteinsthat co-purify with PIWI proteins in D. melanogasterand mice has revealed that PIWIs associate with protein
arginine N-methyltransferase 5 (PRMT5; also known as
DART5 or Capsuleen in D. melanogaster)66–68. In conjunction with methylosome protein 50 (MEP50; also
known as Valois in D. melanogaster), PRMT5 symmetrically methylates conserved arginine residues present at
the amino termini of PIWI proteins in multiple animal
species67–70. Interestingly, these residues are absent in
AGO proteins, suggesting that arginine dimethylation isa molecular signature of the piRNA pathway. Proteomic
studies have revealed that mouse PIWI complexes contain several proteins that have single or multiple Tudor
domains that are known to interact with sDMAs68–70.
The three mouse PIWI members associate with several
Tudor-domain proteins that have varying expression patterns during gametogenesis69–72 (FIG. 4a). Furthermore,
Tudor and PIWI proteins interact when expressed ina heterologous cell culture system, and this interaction
requires the presence of sDMAs68. Biochemical studies
have suggested the existence of a complex recognitioncode between members of the PIWI and Tudor families68,73–75 (FIG. 4b). In D. melanogaster, Tudor (TUD), a
factor that is necessary for gametogenesis and contains
11 Tudor domains76–79 (FIG. 4c), associates with AUB andAGO3 (REFS 66,67). D. melanogaster PIWI also contains
sDMAs; however, it is unknown which Tudor-domain
protein binds to these66. The structural basis for thesDMA-dependent interaction between PIWI proteins
and Tudor domains was recently determined, which
revealed that the five carboxy‑terminal tandem Tudor
domains of TUD (TUD7 to TUD11) are necessary andsufficient for AUB binding and germ cell formation, andthat the binding characteristics of eTUD11 (a fragment
encompassing the eleventh TUD domain of Tudor)
resembles those of TUD7 to TUD11. Four aromatic residues that are located in eTUD11 — Phe2403, Tyr2410,
Phe2427 and Phe2430 — were determined to form thesDMA binding cage80,81. Several other proteins that are
required for piRNA-mediated silencing in flies, such as
Spindle-E (SPN-E), FS(1)YB, Krimper (KRIMP) andTejas, have a single Tudor domain, although their directbinding partners are not yet known (FIG. 4c). The interaction between Tejas and AUB is sDMA-independent75,
suggesting another mechanism by which PIWI proteins
can interact with Tudor-domain-containing proteins.
Proteins that contain multiple Tudor domains havethe potential to interact simultaneously with several
proteins through sDMAs and function as a scaffolding
platform for the assembly of multi-protein complexes.
This might be particularly important for the ping-pongmechanism, which requires close association between
two PIWI proteins. Indeed, an estimation based on structural studies suggested that TUD might bind several AUB
molecules simultaneously 80. Accordingly, analysis of thepiRNA pathway in Tudor domain containing 1 (Tdrd1)deficient mice demonstrated that although piRNAs arestill present, the ping-pong cycle for generating secondary piRNAs is impaired68. Another study produced different results indicating that, in Tdrd1‑deficient mice,
MILI starts to accept small RNAs originating from ribo­
somal RNAs and protein-coding RNAs, both exonic andintronic, although the underlying mechanism remains
unclear 70. Similarly, loss of Tud function in Drosophila
ovaries did not eliminate piRNAs; however, the population of transposon-derived piRNAs was substantiallychanged67. Similarly to deficiencies in MILI and MIWI2,
deficiencies in the Tudor proteins TDRD1 and TDRD9
lead to a failure in de novo methylation of TEs in mouse
germline cells, and consequent TE overexpression70,71.
Overall, it seems that deficiencies in individual Tudordomain proteins significantly impair the piRNA pathway,
especially its ping-pong components, but do not abolish
it. It is plausible that several Tudor-domain-containing
proteins have partially redundant roles in the piRNA
pathway, explaining the relatively mild phenotypes of
the individual mutants. Some Tudor-domain-containing
proteins, such as SPN‑E and FS(1)YB, are crucial for thepiRNA pathway 24,34,54,56,82. These proteins also contain a
DEAH-type RNA helicase domain, but their molecularfunctions in the PIWI complex are unclear.
Additional regulators of piRNA-mediated silencing.
There are several proteins that do not contain Tudor
domains but are required for piRNA-mediated silencing. In particular, the putative RNA helicase ARMI
is crucial for primary piRNA biogenesis in D. melanogaster, and its mammalian homologue MOV10L1
seems to have a similar role in mice54,82–84. It is unknown,

NATURE REVIEWS | MOLECULAR CELL BIOLOGYVOLUME 12 | APRIL 2011 | 253© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS
however, whether the RNA helicase domain exhibits
genuine enzymatic activity and how this affects piRNAbiogenesis. Armi may also be necessary for secondary
piRNA amplification, because mutations in Armi caused
a reduction in PIWI- and AUB-associating piRNAs;C)GTOEGNNOKITCVKQP')GPQOGFGOGVJ[NCVKQP'%GNNE[ENGCTTGUVFGPQXQOGVJ[NCVKQP

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Figure 4 | PIWI and Tudor proteins regulate the piRNA pathway. a | Expression of
0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[PIWI, Tudor proteins and PIWI-interacting RNAs (piRNAs)
during mouse spermatogenesis.
MIWI2 (also known as PIWI-like protein 4) is the only mouse PIWI protein that, together
with Tudor domain containing 9 (TDRD9), localizes in the nucleus when de novo
methylation is established (between embryonic day 15.5 (E15.5) and postnatal day 2 (P2)).
Pre-pachytene piRNAs are enriched in sequences that match transposable elements(TEs) and associate with MILI (also known as PIWI-like protein 2) and MIWI2. Pachytene
piRNAs that are expressed starting at the pachytene stage of meiosis (P14) are notenriched in TE sequences, are expressed from a different set of piRNA clusters and areassociated with MIWI (also known as PIWI-like protein 1) and MILI. Expression of different
Tudor-domain proteins also starts at different stages of spermatogenesis. b | Biochemicalinteractions between mouse PIWI and Tudor proteins. The domain structure of Tudor
proteins is shown on the left64 and the PIWI proteins that these proteins interact with are
shown on the right. c | Biochemical interactions between D. melanogaster PIWI and Tudor
proteins. The domain structures of Tudor proteins are shown on the left and PIWI proteinsthat interact with these proteins are shown on the right. AGO, Argonaute; AUB,
Aubergine; DEXDc, DEXDc helicase domain; FS(1)YB, Female sterile (1) Yb (also known asYb); HA2, helicase-associated domain; HELICc, helicase superfamily carboxy‑terminal
domain; KH, K homology RNA-binding domain; KRIMP, Krimper; MYND, MYND (myeloid,
Nervy, DEAF‑1)-type zinc finger domain; ND, not determined; RING, RING-type zincfinger domain; SPN-E, Spindle-E; TUD, Tudor.

this was also observed for mutations in rhino, which
encodes an HP1 homologue that associates with DNA
of the 42AB piRNA cluster (a dual strand cluster), andRhino is required for its transcription and secondary
piRNA accumulation46.
Vasa and its murine homologue, MVH (also known
as DDX4), are conserved components of germline
granules and have been implicated in the piRNA pathway 61,72,82,85–87. Vasa contains an RNA helicase domain,which has RNA helicase activity 88, but the importanceof this domain in the piRNA pathway is still unknown.
Similarly to PIWI proteins, Vasa has sDMAs at its
N terminus that can be recognized by Tudor domains74.
However, the proteins that interact with Vasa specificallythrough its sDMAs remain undetermined.
Maelstrom (MAEL), a high mobility group box (HMGbo­x) protein, is involved in the piRNA pathway in bothD. melanogaster and mice82,89–92, although its molecularfunctions during piRNA biogenesis are not yet fully
understood. In mice, the MILI-interacting protein
GASZ (also known as ASZ1), a structural protein thatis crucial for MILI stability and mediates formation of
the cellular bodies occupied by MILI, has also been
implicated in the biogenesis of pachytene piRNAs93.
Squash (SQU), a putative nuclease in flies, might also
be involved in the piRNA pathway, particularly at the‘effector’ (silencing) step; this is in contrast to ARMI
and ZUC, both of which function in the ‘initiation’(piRNA biogenesis) phase of the piRNA pathway 53. Thisis because mutations in squ show little impact on piRNA
populations24 but display an effect on TE silencing 53 inmutant ovaries. The precise molecular functions ofmany PIWI-interacting proteins in the piRNA pathwa­yawait further investigation.

Spatial control of piRNA biogenesis
Different PIWI proteins are localized to either thecytoplasm or the nucleoplasm. In the cytoplasm, PIWI
proteins and many of their interactors colocalize in distinct granular cytoplasmic structures that are present in
the germline cells of many metazoa species. It shouldbe emphasized that, even for PIWI members that arepresent in the nucleus, such as PIWI in D. melanogasterand MIWI2 in mice, there is accumulating evidence
that production and loading of piRNAs occurs in thecytoplasmic granules15,29,44,54,92. Germline granules that
contain components of the piRNA pathway tend to belocated in close proximity to the nucleus15,33,61,67,71,75,86,90–97,
suggesting that they serve as structures that recognize
and process piRNA precursors exported from the nucleus
to the cytoplasm.
Germline granules for piRNA production. Developing
germ cells of the D. melanogaster ovary contain nuage,
an electron-dense, non-membranous perinuclear structure98,99 (FIG. 5a). Nuage can be detected in nurse cells but
is absent from the transcriptionally quiescent oocyte;
thus, the presence of nuage seems to correlate withactive transcription. Many nuage components, including PIWIs loaded with piRNAs, are supplied by nurse
cells to the oocyte and localize at its posterior end33,66,94.

254 | APRIL 2011 | VOLUME 12

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REVIEWSC

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Germline granule
A specialized electron-dense
structure that comprises
particular RNAs and proteinsthat are indispensable forgermline development.
Vasa, an RNA helicase, is awell-characterized componentof germline granules.

High mobility group box(HMG-box). A protein domain
involved in DNA binding thatcomprises ~80 amino-acidresidues that form three
α‑helices.

(5  ;$;DDQF[;DDQF[(5  ;$

Figure 5 | piRNA biogenesis takes place in cellular bodies. a | A schematic representationof Drosophila melanogaster
0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[
egg chambers. At mid-stage oogenesis, the D. melanogaster egg chamber consists of one oocyte and 15 nurse cells andsomatic follicle cells. Nuage is a non-membranous structure that is located in the perinuclear region of nurse cells.
Yb-bodies are cytoplasmic non-membranous structures that are found only in somatic follicle cells, and these can also be
observed in ovarian somatic cells (OSCs) derived from follicle cells. b | Cellular localization of Aubergine (AUB; shown ingreen) in a stage‑6 egg chamber33, which clearly shows that AUB accumulates at nuage. c | Nuage is probably where theamplification loop takes place. In nurse cells, PIWI-interacting RNA (piRNA) factors such as AUB and Argonaute 3 (AGO3)
localize to the nuage. Tudor (TUD) associates with AUB and AGO3 simultaneously in germ cells. Through this association,
Tudor might activate the amplification loop. Tudor may also have a role in recruiting piRNA precursors to piRNA-induced
silencing complexes (piRISCs). Vasa, Spindle-E (SPN‑E), Krimper (KRIMP), Tejas and Maelstrom (MAEL) localize to nuage,
which suggests that they are involved in this pathway. However, their functions in piRNA biogenesis still remain unclear.
Unlike AUB and AGO3, PIWI shows steady-state localization to the nucleus in nurse cells. d | In mouse prospermatogonia,
MILI (also known as PIWI-like protein 2) localizes in multiple small granules termed pi-bodies, whereas MIWI2 (also known
as PIWI-like protein 4) is present in fewer and bigger granules, known as piP-bodies, that are often in contact with MILI
granules. e | The composition of pi- and piP-bodies. Strong and weak genetic interactions between different componentsthat guarantee correct localization of corresponding proteins are shown as solid and dashed arrows, respectively.
f | Immunofluorescence of zucchini (zuc)-depleted OSCs stained with Female sterile (1) Yb (FS(1)YB; also known asYb)-specific antibodies shows that PIWI is located in Yb-bodies in OSCs34. g | Yb-bodies are the sites where primarypiRNAs are produced in follicle cells of the ovary, where PIWI, but not AUB or AGO3, is expressed. Armitage (ARMI)
associates with PIWI and localizes it to Yb-bodies34,54. FS(1)YB is the main component of Yb-bodies. The piRNAintermediates, which are partially processed from the primary precursors, are loaded onto the complex comprising
ARMI, PIWI and FS(1)YB at Yb-bodies and processed into mature piRNAs. ZUC, a putative nuclease that localizes tomitochondria, is also required for the primary piRNA processing. The majority of ZUC-positive mitochondria are in fact
located close to Yb-bodies34. Without piRNA loading, PIWI is not localized to the nucleus. These findings support the ideathat Yb-bodies not only generate primary piRNAs for PIWI but also inspect the functional piRNA–PIWI complex before it
enters the nucleus. DCP1A, Decapping protein 1A; DDX6, DEAD-box protein 6; GASZ, also known as ASZ1; GW182,
also known as TNRC6; MVH, also known as DDX4; Oo, oocyte; TDRD, Tudor domain containing. Part b is reproduced, withpermission, from REF. 33 © (2007) Cold Spring Harbor Laboratory Press. Part d is reproduced, with permission, from REF. 29© (2008) Elsevier. Part f is reproduced, with permission, from REF. 34 © (2010) Cold Spring Harbor Laboratory Press.

Some of the nuage components give rise to the pole
plasm, the structure that is both necessary and sufficient to induce formation of primordial germ cells inthe developing embryo100.
Nuage contains many other proteins that are genetically required for piRNA-mediated silencing, suchas SPN‑E, MAEL, Vasa, KRIMP, Tejas and TUD75,82(FIG. 5b,c). Deficiencies in these proteins eliminate piR-

NAs or distort the piRNA profile. Furthermore, they
can cause other components from nuage to delocalize
or even cause the structure to disintegrate. Many nuage
components, including TUD, SPN‑E, KRIMP and Tejas,
have Tudor domains, whereas others, such as PIWI
proteins and Vasa, have sDMAs that are recognized by

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REVIEWS
Tudor domains. Accordingly, protein–protein interactions within nuage are often mediated by Tudor–sDMA
associations. It has been proposed that nuage might bea site for piRNA biogenesis and/or for the recognitionand destruction of piRNA targets82.
In mice, PIWI proteins are expressed at different
stages of testis development, but all are localized in thecytoplasm of germ cells in electron-dense granular structures that resemble nuage in D. melanogaster 28,29,72,92,93,95.
These granules have been termed ‘intermitochondrial
cement’ or ‘chromatoid bodies’ (CBs), depending on theparticular stage of spermatogenesis. Some components ofthese granules, such as MVH, are present at all stages96,98.
MILI and MIWI2 are co-expressed during embryonic
testis development; however, unlike AUB and AGO3 in
D. melanogaster, they are localized in two distinct types of
granule. MILI granules, which were originally describedas intermitochondrial cement, contain the Tudor-domaincontaining protein TDRD1 in addition to MVH70,87,98 and
GASZ93 (FIG. 5d,e). MIWI2 granules, which are often foundin a close proximity to MILI granules, include another
Tudor protein, TDRD9, and MAEL. MIWI2 granules
contain all of the known components of processing bodies (P-bodies), which are cytoplasmic granules that are
responsible for mRNA storage and decay in somatic cells(FIG. 5d,e). Accordingly, they have been dubbed ‘piP-bodies’.
As MILI granules or intermito­chondrial cement harbour
components that are exclusively involved in the piRNA
pathway, they are also called ‘pi-bodies’98. MILI, but not
MIWI or MIWI2, has been reported to be expressed in
the female germ line, where it localizes in cytoplasmic
granules in both arrested and growing oocytes29.
The localization of many components of pi- and piPbodies is co-dependent. For example, pi-bodies do notseem to form in Mvh and Gasz mutants87,93. The presence of MILI in pi-bodies depends on TDRD1 (REF. 68).
Furthermore, TDRD1 can attract MILI into the granules when both proteins are co-expressed in a hetero­logous cell culture system, underlining the pivotal roleof the Tudor–sDMA interaction in granule formation68.
Currently, it is unknown whether there is functionalcrosstalk between components of the piRNA and P‑bodypathways within individual piP-bodies, although pi- andpiP-bodies do physically and functionally interact witheach other. Disruption of MILI-containing pi-bodies
also disintegrates MIWI2‑containing piP-bodies, but
not vice versa29. This agrees with the notion that MIWI2
is downstream of MILI in the ping-pong mechanism(FIG. 3c). Disruption of pi-bodies also prevents loading ofpiRNAs onto MIWI2 and prevents it from entering thenucleus. This suggests that the key steps of piRNA biogenesis occur in these cytoplasmic granules. Disruptionof the pi-bodies and piP-bodies also results in overexpression of TEs and loss of de novo DNA methylation of theircorresponding genomic loci. Thus, the correct structureof these compartments heavily influences the effici­encyof TE silencing. After birth, MIWI2 expression ceases,
but pi-bodies are still present in spermatogonia andearly spermatocytes. Expression of MIWI commences
during meiosis, followed by the formation of CBs. A CB
is a single large granule present in haploid spermatids

and contains MILI, MIWI, MVH, MAEL and multiple
Tudor-domain-containing proteins, such as TDRD1 andTDRD6 (REFS 72,95,97). Similarly to piP-bodies, CBs harbour some P‑body components, such as AGO proteins101.
The CB is probably the site where pachytene piRNAs,in complex with MIWI and MILI, exert their function.
However, as their targets are unknown, this assumption
still awaits experimental verification.
Yb-bodies in the D. melanogaster ovary. Primary piRNAbiogenesis and silencing of TEs take place in somatic follicle cells of the D. melanogaster ovary and in the cultured
OSC line derived from D. melanogaster follicle cells24,34,44,54(FIG. 5a). However, these follicle cells are devoid of typicalnuage as they do not express a nuage marker, Vasa, as wellas the two PIWI proteins AUB and AGO3. Many proteins that are required for the piRNA pathway in germline
cells, such as SPN‑E, Vasa, MAEL, TUD and KRIMP, aredispensable for primary piRNA production in somatic
cells, suggesting that the piRNA pathway uses different
machineries in germline and somatic cells. Interestingly,
recent studies have demonstrated that, similarly to germline cells, the piRNA pathway in somatic follicle cells
is tightly associated with cytoplasmic granules called
Yb-bodies34,54,55 (FIG. 5a,f,g). Yb-bodies do not overlap
with known cytoplasmic granules such as P‑bodies or
U‑bodies (the latter of which are implicated in snRNP(RNA-protein complex) metabolism) and have beenpreviously implicated in RNA metabolism56.
Genetic and RNA interference-based screens inflies and OSCs, respectively, have identified PIWI andthree other proteins, ARMI, ZUC and FS(1)YB, as being
required for piRNA-mediated TE silencing and primarypiRNA accumulation34,54,55 (FIG. 5f,g). ARMI and FS(1)YB
colocalize at Yb-bodies. However, FS(1)YB seems to be
the core factor in Yb-body formation: ARMI localization in Yb-bodies requires FS(1)YB, but FS(1)YB does
not require ARMI for localizing to Yb-bodies. BothPIWI and FS(1)YB interact with ARMI. Without FS(1)YB or ARMI, PIWI localizes to neither Yb-bodies nor the
nucleus. Thus, PIWI’s subcellular localization depends
on ARMI. In the absence of ZUC, PIWI accumulates
together with ARMI in the Yb-bodies and does not enter
the nucleus (FIG. 5f,g). Upon depletion of ARMI or FS(1)YB, PIWI is no longer loaded with piRNAs and does not
localize to the nucleus, suggesting that piRNA loading isrequired for PIWI nuclear entry. It is plausible that cytoplasmic Yb-bodies are the sites at which piRISC function is checked before nuclear entry is permitted (FIG. 5g).
Taken together, these data indicate that cytoplasmic
granular structures are indispensable for piRNA-related
mechanisms in both germline and somatic follicular cells,
although the composition of the granules is different inthese two cell types.

Conclusions
piRNAs, in complex with PIWI proteins, form a conserved silencing pathway that is related to other small
RNA silencing pathways but is clearly distinct in its
mechanism and functions. piRNAs provide an elaborate system that protects the germline and gonadal

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REVIEWS
somatic cells against the harmful expression of TEs.
Several reports indicate that piRNAs might also target and regulate expression of normal host proteincoding genes. However, these data should be further
verified. piRNA­s also mediate de novo methylationof TE sequences in the mammalian male germ line.
This provides a tantalizing link between two modesof epigenetic regulation of gene expression: posttranscriptional silencing by small RNAs and transcriptional repression by local chromatin structure.
Further studies should reveal the molecular mechanism by which piRNAs affect DNA methylation andpotentially expand our use of small RNAs as tools in
research and therapy.
Compared with other small RNA silencing mechanisms, the piRNA pathway remains poorly investigated, mainly because it primarily operates in germline
cells. Biochemical and genetic studies in flies and mice
have identified the protein partners of PIWI proteins;

1.
2.
3.
4.
5.
6.

7.
8.

9.

10.
11.

12.

13.
14.
15.

Ghildiyal, M. & Zamore, P. D. Small silencing RNAs:
an expanding universe. Nature Rev. Genet. 10,
94–108 (2009).
Kim, N. V., Han, J. & Siomi M. C. Biogenesis of small
RNAs in animals. Nature Rev. Mol. Cell Biol. 10,
126–139 (2009).
Malone, C. D. & Hannon, G. J. Small RNAs as
guardians of the genome. Cell 136, 656–668(2009).
Siomi, H. & Siomi, M. C. On the road to reading
the RNA-interference code. Nature 457, 396–404(2009).
Thomson, T. & Lin, H. The biogenesis and function of
PIWI proteins and piRNAs: progress and prospect.
Annu. Rev. Cell Dev. Biol. 25, 355–376 (2009).
Aravin, A. A. et al. Double-stranded RNA-mediatedsilencing of genomic tandem repeats and transposable
elements in the D. melanogaster germline. Curr. Biol.
11, 1017–1027 (2001).
Aravin, A. A. et al. The small RNA profile during
Drosophila melanogaster development. Dev. Cell
5, 337–350 (2003).
Vagin, V. V., Sigova, A., Li, C., Seitz, H., Gvozdev, V. &Zamore, P. D. A distinct small RNA pathway silences
selfish genetic elements in the germline. Science
313, 320–324 (2006).
This paper hinted for the first time that Dicer
endonuclease activity — which is essential for
miRNA and siRNA biogenesis — might bedispensable for piRNA biogenesis in
D. melanogaster.
Saito, K. et al. Specific association of Piwi with
rasiRNAs derived from retrotransposon andheterochromatic regions in the Drosophila genome.
Genes Dev. 20, 2214–2222 (2006).
Aravin, A. et al. A novel class of small RNAs bind to
MILI protein in mouse testes. Nature 442, 203–207(2006).
Girard, A., Sachidanandam, R., Hannon, G. J. &Carmell, M. A. A germline-specific class of small
RNAs binds mammalian Piwi proteins. Nature 442,
199–202 (2006).
Grivna, S. T., Beyret, E., Wang, Z. & Lin, H. A novel
class of small RNAs in the mouse spermatogenic cells.
Genes Dev. 20, 1709–1714 (2006).
References 10–12 describe the discovery ofpiRNAs in the mammalian male germ line. ThesepiRNAs were not enriched in transposon-derived
sequences and were later named ‘pachytene
piRNAs’ because they are initially expressedat the pachytene stage of meiosis.
Houwing, S. et al. A role for Piwi and piRNAs in
germ cell maintenance and transposon silencing in
Zebrafish. Cell 129, 69–82 (2007).
Kazazian, H. H. Jr. Mobile elements: drivers of genome
evolution. Science 303, 1626–1632 (2004).
Saito, K. & Siomi, M. C. Small RNA-mediatedquiescence of transposable elements in animals.
Dev. Cell 19, 687–697 (2010).

however, crucial questions regarding the enzymatic
machineries that process piRNAs and the rules thatdefine the recognition of precursor transcripts remainunanswered. Future research should explain how
the transcription of piRNA clusters, which occupy
a substantial portion of our genomes, is regulated.
Furthermore, understanding the molecular control ofthe piRNA pathway will hopefully allow researchers to
direct piRNAs to new artificial targets. Studies in flies
and mice have revealed a tight association between
the piRNA machinery and germline granules (specific cytoplasmic organelles that are present in animal
gonads). These reports have raised the idea, whichshould be explored further, that nuage is a processing
site for piRNAs and/or the place where silencing takes
place. Overall, the hope is that future studies will fill inthese major gaps in our understanding of how piRNAbiogenesis and function is governed and, undoubtedly,
yield many exciting discoveries.

16. Vagin, V. V. et al. The RNA interference proteinsand vasa locus are involved in the silencing ofretrotransposons in the female germline of Drosophilamelanogaster. RNA Biol. 1, 54–58 (2004).
17. Kalmykova, A. I., Klenov, M. S. & Gvozdev, V. A.
Argonaute protein PIWI controls mobilization ofretrotransposons in the Drosophila male germline.
Nucleic Acids Res. 33, 2052–2059 (2005).
18. Savitsky, M., Kwon, D., Georgiev, P., Kalmykova, A. &Gvozdev, V. Telomere elongation is under the controlof the RNAi-based mechanism in the Drosophila
germline. Genes Dev. 20, 345–354 (2006).
19. Li, C. et al. Collapse of germline piRNAs in theabsence of Argonaute3 reveals somatic piRNAs inflies. Cell 137, 509–521 (2009).
20. Cox, D. N. et al. A novel class of evolutionarily
conserved genes defined by piwi are essential for stem
cell self-renewal. Genes Dev. 12, 3715–3727 (1998).
21. Lin, H. & Spradling, A. C. A novel group of pumilio
mutations affects the asymmetric division of germline
stem cells in the Drosophila ovary. Development 124,
2463–2476 (1997).
22. Schmidt, A. et al. Genetic and molecularcharacterization of sting, a gene involved in crystal
formation and meiotic drive in the male germ line of
Drosophila melanogaster. Genetics 151, 749–760(1999).
23. Cox, D. N., Chao, A. & Lin, H. piwi encodes anucleoplasmic factor whose activity modulates thenumber and division rate of germline stem cells.
Development 127, 503–514 (2000).
24. Malone, C. D. et al. Specialized piRNA pathways act ingermline and somatic tissues of the Drosophila ovary.
Cell 137, 522–535 (2009).
25. Carmell, M. A. et al. MIWI2 is essential for
spermatogenesis and repression of transposons in themouse male germline. Dev. Cell 12, 503–514 (2007).
26. Aravin, A. A., Sachidanandam, R., Girard, A.,
Fejes-Toth, K. & Hannon, G. J. Developmentally
regulated piRNA clusters implicate MILI in transposon
control. Science 316, 744–747 (2007).
27. Kuramochi-Miyagawa, S. et al. DNA methylation ofretrotransposon genes is regulated by Piwi family
members MILI and MIWI2 in murine fetal testes.
Genes Dev. 22, 908–917 (2008).
28. Kuramochi-Miyagawa, S. et al. Two mouse piwi-related
genes: miwi and mili. Mech. Dev. 108, 121–133(2001).
29. Aravin, A. A. et al. A piRNA pathway primed byindividual transposons is linked to de novo DNA
methylation in mice. Mol. Cell 31, 785–799 (2008).
References 26, 27 and 29 identify the link between
piRNA silencing of transposable elements andCpG methylation of their genomic sequences in
the male germ line, which is probably mediatedby the nuclear MIWI2 complex. As the piRNAs
that potentially guide this DNA methylation are
expressed early during mouse gametogenesis,
they were named ‘pre-pachytene piRNAs’.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY

30. Klattenhoff, C. et al. Drosophila rasiRNA pathway
mutations disrupt embryonic axis specification
through activation of an ATR/Chk2 DNA damage
response. Dev. Cell 12, 45–55 (2007).
31. Brennecke, J. et al. Discrete small RNA-generatingloci as master regulators of transposon activity in
Drosophila. Cell 128, 1089–1103 (2007).
32. Gunawardane, L. S. et al. A slicer-mediatedmechanism for repeat-associated siRNA 5′ endformation in Drosophila. Science 315, 1587–1590(2007).
References 31 and 32 proposed the amplification
loop — also known as the ‘ping-pong model’ — forpiRNA biogenesis by revealing the specific featuresor ‘signature’ of piRNAs that are associated withPIWI proteins in D. melanogaster ovaries.
33. Nishida, K. M. et al. Gene silencing mechanisms
mediated by Aubergine piRNA complexes in
Drosophila male gonad. RNA 13, 1911–1922 (2007).
34. Saito, K. et al. Roles for the Yb body components
Armitage and Yb in primary piRNA biogenesis in
Drosophila. Genes Dev. 24, 2493–2498 (2010).
35. Pal-Bhadra. M. et al. Heterochromatic silencing and
HP1 localization in Drosophila are dependent on theRNAi machinery. Science 303, 669–672 (2004).
36. Brower-Toland. B. et al. Drosophila PIWI associateswith chromatin and interacts directly with HP1a.
Genes Dev. 21, 2300–2311 (2007).
37. Yin, H. & Lin, H. An epigenetic activation role of Piwi
and a Piwi-associated piRNA in Drosophilamelanogaster. Nature 450, 304–308 (2007).
38. Moshkovich. N. & Lei. E. P. HP1 recruitment in theabsence of argonaute proteins in Drosophila.
PLoS Genet. 6, e1000880 (2010).
39. Ghildiyal, M. et al. Endogenous siRNAs derived fromtransposons and mRNAs in Drosophila somatic cells.
Science 320, 1077–1081 (2008).
40. Kawamura, Y. et al. Drosophila endogenous small
RNAs bind to Argonaute 2 in somatic cells. Nature
453, 793–797 (2008).
41. Tam, O. H. et al. Pseudogene-derived small interfering
RNAs regulate gene expression in mouse oocytes.
Nature 453, 534–538 (2008).
42. Watanabe, T. et al. Endogenous siRNAs from naturally
formed dsRNAs regulate transcripts in mouse oocytes.
Nature 453, 539–543 (2008).
43. Li, M. A., Alls, J. D., Avancini, R. M., Koo, K. & Godt, D.
The large Maf factor Traffic Jam controls gonadmorphogenesis in Drosophila. Nature Cell Biol.
5, 994–1000 (2003).
44. Saito, K. et al. A regulatory circuit for piwi by thelarge Maf gene traffic jam in Drosophila. Nature
461, 1296–1299 (2009).
References 19, 24 and 44 provided new insights
into piRNAs that are specifically expressed in
ovarian somatic cells, mainly by providing evidence
that the origins of, and processing factors
regulating, these somatic piRNAs are uniquefrom those of germline piRNAs.

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REVIEWS
45. Rouget, C. et al. Maternal mRNA deadenylation anddecay by the piRNA pathway in the early Drosophila
embryo. Nature 467, 1128–1132 (2010).
46. Klattenhoff. C. et al. The Drosophila HP1 homolog
Rhino is required for transposon silencing and piRNA
production by dual-strand clusters. Cell 138,
1137–1149 (2009).
47. Aravin, A. A., Hannon, G. J. & Brennecke, J. The Piwi–
piRNA pathway provides an adaptive defense in thetransposon arms race. Science 318, 761–764 (2007).
48. Lau, N. C. et al. Characterization of the piRNAcomplex from rat testes. Science 313, 363–367(2006).
49. Houwing, S., Berezikov, E. & Ketting, R. F. Zili isrequired for germ cell differentiation and meiosis inzebrafish. EMBO J. 27, 2702–2711 (2008).
50. Kawaoka S. et al. The Bombyx ovary-derived cell line
endogenously expresses PIWI/PIWI-interacting RNA
complexes. RNA 15, 1258–1264 (2009).
51. Robine, N. et al. A broadly conserved pathway
generates 3’UTR-directed primary piRNAs. Curr. Biol.
19, 2066–2076 (2009).
52. Mi, S. et al. Sorting of small RNAs into Arabidopsis
argonaute complexes is directed by the 5′ terminal
nucleotide. Cell 133, 116–127 (2008).
53. Haase, A. D. et al. Probing the initiation and effector
phases of the somatic piRNA pathway in Drosophila.
Genes Dev. 24, 2499–2504 (2010).
54. Olivieri, D., Sykora, M. M., Sachidanandam, R.,
Mechtler, K. & Brennecke, J. An in vivo RNAi assay
identifies major genetic and cellular requirements forprimary piRNA biogenesis in Drosophila. EMBO J. 29,
3301–3317 (2010).
55. Qi. H. et al. The Yb body, a major site for Piwiassociated RNA biogenesis and a gateway for Piwi
expression and transport to the nucleus in somatic
cells. J. Biol. Chem. 286, 3789–3797 (2011).
References 34, 54 and 55 provided evidence thatcytoplasmic Yb-bodies are the sites for primarypiRNA biogenesis in D. melanogaster. Bothbiochemical and genetic approaches suggested arequirement for ARMI, FS(1)YB and other factors,
such as ZUC, in the primary piRNA pathway.
56. Szakmary, A., Reedy, M., Qi, H. & Lin, H. The Yb
protein defines a novel organelle and regulates malegermline stem cell self-renewal in Drosophilamelanogaster. J. Cell Biol. 185, 613–627 (2009).
57. Prud’homme, N., Gans, M., Masson, M., Terzian, C. &Bucheton, A. Flamenco, a gene controlling the gypsy
retrovirus of Drosophila melanogaster. Genetics 139,
697–711 (1995).
58. Desset, S., Meignin, C., Dastugue, B. & Vaury, C.
COM, a heterochromatic locus governing the control
of independent endogenous retroviruses from
Drosophila melanogaster. Genetics 164, 501–509(2003).
59. Mével-Ninio, M., Pelisson, A., Kinder, J., Campos, A. R.
& Bucheton, A. The flamenco locus controls the gypsy
and ZAM retroviruses and is required for Drosophila
oogenesis. Genetics 175, 1615–1624 (2007).
60. Desset, S., Buchon, N., Meignin, C., Coiffet, M. &Vaury, C. In Drosophila melanogaster the COM locus
directs the somatic silencing of two retrotransposons
through both Piwi-dependent and -independent
pathways. PLoS ONE 3, e1526 (2008).
61. Nagao, A., Mituyama, T., Huang, H., Chen, D.,
Siomi, M. C. & Siomi, H. Biogenesis pathways ofpiRNAs loaded onto AGO3 in the Drosophila testis.
RNA 16, 2503–2515 (2010).
62. Brennecke, J. et al. An epigenetic role for maternallyinherited piRNAs in transposon silencing. Science
322, 1387–1392 (2008).
63. Bourc’his, D. & Voinnet, O. A small-RNA perspective
on gametogenesis, fertilization, and early zygotic
development. Science 330, 617–622 (2010).
64. Vagin, V. V. Hannon, G. J. & Aravin, A. A. Arginine
methylation as a molecular signature of the Piwi
small RNA pathway. Cell Cycle 8, 4003–4004(2009).
65. Siomi, M. C., Mannen, T. & Siomi, H. How does theroyal family of Tudor rule the PIWI-interacting RNA
pathway? Genes Dev. 24, 636–646 (2010).
66. Kirino, Y. et al. Arginine methylation of Piwi proteinscatalysed by dPRMT5 is required for Ago3 and Aub
stability. Nature Cell Biol. 11, 652–658 (2009).

67. Nishida, K. M. et al. Functional involvement of Tudorand dPRMT5 in the piRNA processing pathway in
Drosophila germlines. EMBO J. 28, 3820–3831(2009).
68. Vagin, V. V. et al. Proteomic analysis of murine Piwi
proteins reveals a role for arginine methylation in
specifying interaction with Tudor family members.
Genes Dev. 23, 1749–1762 (2009).
69. Chen, C. et al. Mouse Piwi interactome identifies
binding mechanism of Tdrkh Tudor domain to arginine
methylated Miwi. Proc. Natl Acad. Sci. USA 106,20336–20341 (2009).
70. Reuter, M. et al. Loss of the Mili-interacting Tudor
domain-containing protein‑1 activates transposons
and alters the Mili-associated small RNA profile.
Nature Struct. Mol. Biol. 16, 639–646 (2009).
71. Shoji, M. et al. The TDRD9‑MIWI2 complex is essential
for piRNA-mediated retrotransposon silencing in themouse male germline. Dev. Cell 17, 775–787 (2009).
72. Vasileva, A., Tiedau, D., Firooznia, A., Müller-Reichert, T.
& Jessberger, R. Tdrd6 is required for spermiogenesis,
chromatoid body architecture, and regulation of
miRNA expression. Curr. Biol. 19, 630–639 (2009).
73. Wang, J., Saxe, J. P., Tanaka, T., Chuma, S. & Lin, H.
Mili interacts with tudor domain-containing protein 1
in regulating spermatogenesis. Curr. Biol. 19,
640–644 (2009).
74. Kirino, Y. et al. Arginine methylation of vasa protein
is conserved across phyla. J. Biol. Chem. 285,
8148–8154 (2010).
References 66–74 identify direct interactions
between the N termini of PIWI proteins — whichcarry dimethylated arginine residues — andproteins with Tudor domains that recognize
this modification. The authors show that the
PIWI–Tudor protein interactions are crucial forthe proper intracellular localization of the piRNA
machinery and its silencing mechanism.
75. Patil, V. S. & Kai, T. Repression of retroelements in
Drosophila germline via piRNA pathway by the tudor
domain protein Tejas. Curr. Biol. 20, 724–730 (2010).
76. Boswell, R. E. & Mahowald, A. P. tudor, a gene
required for assembly of the germ plasm in Drosophilamelanogaster. Cell 43, 97–104 (1985).
77. Thomson, T. & Lasko, P. Drosophila tudor is essential
for polar granule assembly and pole cell specification,
but not for posterior patterning. Genesis 40,
164–170 (2004).
78. Thomson, T. & Lasko, P. Tudor and its domains: germ
cell formation from a Tudor perspective. Cell Res. 15,
281–291 (2005).
79. Arkov, A. L., Wang, J. Y. S., Ramos, A. & Lehmann, R.
The role of Tudor domains in germline development
and polar granule architecture. Development 133,
4053–4062 (2006).
80. Liu, H. et al. Structural basis for methylargininedependent recognition of Aubergine by Tudor.
Genes Dev. 24, 1876–1881 (2010).
81. Liu, K. et al. Structural basis for recognition ofarginine methylated Piwi proteins by the extended
Tudor domain. Proc. Natl Acad. Sci. USA 107,
18398–18403 (2010).
82. Lim, A. K & Kai, T. Unique germ-line organelle,nuage, functions to repress selfish genetic elements
in Drosophila melanogaster. Proc. Natl Acad. Sci. USA
104, 6714–6719 (2007).
83. Frost, R. J. et al. MOV10L1 is necessary for protectionof spermatocytes against retrotransposons by Piwiinteracting RNAs. Proc. Natl Acad. Sci. USA 107,
11847–11852 (2010).
84. Zheng, K. et al. Mouse MOV10L1 associates with Piwi
proteins and is an essential component of the Piwiinteracting RNA (piRNA) pathway. Proc. Natl Acad.
Sci. USA 107, 11841–11846 (2010).
85. Liang, L., Diehl-Jones, W. & Lasko, P. Localizationof vasa protein to the Drosophila pole plasm isindependent of its RNA-binding and helicase activities.
Development 120, 1201–1211 (1994).
86. Toyooka, Y. et al. Expression and intracellular
localization of mouse Vasa-homologue protein
during germ cell development. Mech. Dev. 93,
139–149 (2000).
87. Kuramochi-Miyagawa, S. et al. MVH in piRNA
processing and gene silencing of retrotransposons.
Genes Dev. 24, 887–892 (2010).

258 | APRIL 2011 | VOLUME 12

88. Sengoku. T., Nureki. O., Nakamura. A., Kobayashi. S.
& Yokoyama. S. Structural basis for RNA unwindingby the DEAD-box protein Drosophila Vasa. Cell
125, 287–300 (2006).
89. Soper, S. F. et al. Mouse maelstrom, a componentof nuage, is essential for spermatogenesis andtransposon repression in meiosis. Dev. Cell 15,
285–297 (2008).
90. Costa, Y. et al. Mouse MAELSTROM: the link between
meiotic silencing of unsynapsed chromatin andmicroRNA pathway? Hum. Mol. Genet. 15,
2324–2334 (2006).
91. Findley, S. D., Tamanaha, M., Clegg, N. J. &Ruohola-Baker, H. Maelstrom, a Drosophila spindleclass gene, encodes a protein that colocalizes with
Vasa and RDE1/AGO1 homolog, Aubergine, in nuage.
Development 130, 859–871 (2003).
92. Aravin, A. A. et al. Cytoplasmic compartmentalizationof the fetal piRNA pathway in mice. PLoS Genet. 5,e1000764 (2009).
93. Ma, L. et al. GASZ is essential for male meiosis
and suppression of retrotransposon expression in
the male germline. PLoS Genet. 5, e1000635 (2009).
94. Anne, J. & Mechler, B. M. Valois, a component of
the nuage and pole plasm, is involved in assemblyof these structures, and binds to Tudor and the
methyltransferase Capsuléen. Development 132,
2167–2177 (2005).
95. Kotaja, N., Lin, H., Parvinen, M. & Sassone-Corsi, P.
Interplay of PIWI/Argonaute protein MIWI and kinesin
KIF17b in chromatoid bodies of male germ cells.
J. Cell Sci. 119, 2819–2825 (2006).
96. Onohara, Y., Fujiwara, T., Yasukochi, T., Himeno, M. &Yokota, S. Localization of mouse vasa homolog protein
in chromatoid body and related nuage structures ofmammalian spermatogenic cells during
spermatogenesis. Histochem. Cell Biol. 133,
627–639 (2010).
References 92, 95 and 96 revealed that,in mouse germ cells, components of piRNA
machinery are localized in specific granularstructures that were originally known as CBsor intermitochondrial cement.
97. Kojima, K. et al. Associations between PIWI proteins
and TDRD1/MTR‑1 are critical for integrated
subcellular localization in murine male germ cells.
Genes Cells 14, 1155–1165 (2009).
98. al-Mukhtar, K. A. & Webb, A. C. An ultrastructural
study of primordial germ cells, oogonia and early
oocytes in Xenopus laevis. J. Embryol. Exp. Morphol.
26, 195–217 (1971).
99. Eddy, E. M. Germ plasm and the differentiationof the germ cell line. Int. Rev. Cytol. 43, 229–280(1975).
100. Illmensee, K. & Mahowald, A. P. Transplantation ofposterior polar plasm in Drosophila. Induction of germ
cells at the anterior pole of the egg. Proc. Natl Acad.
Sci. USA 71, 1016–1020 (1974).
101. Kotaja, N. et al. The chromatoid body of male germ
cells: similarity with processing bodies and presenceof Dicer and microRNA pathway components. Proc.
Natl Acad. Sci. USA 103, 2647–2652 (2006).

Acknowledgements

We thank H. Siomi, A. Nagao, A. Webster, N. Perkins and
I. Olovnikov for comments on the manuscript. The work in thelaboratory of M.C.S. is supported by Ministry of Education,
Culture, Sports, Science and Technology (MEXT) grants to K.S.
and M.C.S. M.C.S. is supported by the Core Research for
Evolutional Science and Technology (CREST) programme of theJapan Science and Technology Agency (JST). The work in thelaboratory of A.A.A. is supported by the US National Institutesof Health (grants DP2 OD007371A and R00 HD057233‑02
to A.A.A.) and the Ellison Medical Foundation.

Competing interests statement

The authors declare no competing financial interests.

FURTHER INFORMATION
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Alexei A. Aravin’s homepage: http://biology.caltech.edu/
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