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Biology of PIWI-interacting RNAs: new insights into biogenesis andfunction inside and outside of germlines
Hirotsugu Ishizu, Haruhiko Siomi and Mikiko C. Siomi
Genes Dev. 2012 26: 2361-2373
Access the most recent version at doi:10.1101/gad.203786.112

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REVIEW

Biology of PIWI-interacting RNAs: new
insights into biogenesis and function
inside and outside of germlines
Hirotsugu Ishizu,1,2 Haruhiko Siomi,2 and Mikiko C. Siomi1,2,3,4
1
Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan; 2Keio University School of Medicine, Tokyo
160-8582, Japan; 3Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST),
Saitama 332-0012, Japan

PIWI-interacting RNAs (piRNAs) are endogenous small
noncoding RNAs that act as guardians of the genome,
protecting it from invasive transposable elements in the
germline. Animals lacking piRNA functions show defects
in gametogenesis and exhibit sterility. Their descendants
are also predisposed to inheriting mutations. Thus, thepiRNA pathway has evolved to repress transposons posttranscriptionally and/or transcriptionally. A growing number of studies on piRNAs have investigated piRNA-mediated gene silencing, including piRNA biogenesis. However,piRNAs remain the most enigmatic among all of thesilencing-inducing small RNAs because of their complexity and uniqueness. Although piRNAs have beenpreviously suggested to be germline-specific, recent studies have shown that piRNAs also play crucial roles innongonadal cells. Furthermore, piRNAs have also recently
been shown to have roles in multigenerational epigenetic
phenomena in worms. The purpose of this review is tohighlight new piRNA factors and novel insights in thepiRNA world.
RNA silencing controls gene expression in a spatiotemporal-specific manner to tightly regulate the development
and homeostasis of living bodies (Bartel 2009; Malone andHannon 2009). The key components of RNA silencing—small noncoding RNAs and Argonaute proteins—associate
stoichiometrically to form RNA-induced silencing complexes (RISCs), the effector complexes in RNA silencing(Siomi and Siomi 2009). RISCs are directed to the target
genes based on the complementarities between small
RNAs and target gene transcripts and inhibit their expression by cleaving the transcripts or inducing translational
inhibition, RNA instability, and/or heterochromatinization (Moazed 2009; Siomi et al. 2011).
Animal species express three types of endogenous
silencing-inducing small RNAs: microRNAs (miRNAs),
endogenous siRNAs (endo-siRNAs), and PIWI-interacting[Keywords: PIWI; germline; piRNA]
4
Corresponding author
E-mail siomim@biochem.s.u-tokyo.ac.jp
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.203786.112.

RNAs (piRNAs). These small RNAs can be classified on
the basis of their origins, processing factors, and Argonaute-binding partners (Kim et al. 2009). piRNAs are
generated from single-stranded precursors in a manner
independent of RNase III enzymes (Vagin et al. 2006;
Brennecke et al. 2007; Houwing et al. 2007), which are, incontrast, necessary for endo-siRNA and miRNA biogenesis. piRNAs associate with PIWI subfamily members of
the Argonaute family of proteins, while endo-siRNAs andmiRNAs associate with AGO subfamily members. piRNAs
are normally 24–32 nucleotides (nt) long, but endo-siRNAs
and miRNAs are 20–23 nt in most cases. Thus, theuniqueness of piRNAs is obvious.
piRNAs arise from intergenic repetitive elements in
the genome called piRNA clusters (Brennecke et al. 2007).
piRNA clusters span a wide region of the genome, occasionally consisting of >100,000 bases, and are mostly
comprised of various transposable DNA elements and theirremnants. Thus, piRNAs, especially those in Drosophila
gonads and mouse prepachytene piRNAs (piRNAs expressed before the pachytene stage of meiosis in spermatogenesis), are enriched in transposon sequences. Most ofthese piRNAs have an antisense orientation to transposon transcripts and hence can induce silencing by
hybridizing with them (Saito and Siomi 2010). Loss-offunction mutations in piRNAs and their cofactors, PIWI
proteins, derepress transposons, allowing them to insert
copies of themselves or relocate within the genome in arandom fashion (Kalmykova et al. 2005). This selfish event
activates the Chk2 DNA damage checkpoint, resulting in
defects of, for example, microtubule organization and axis
specification during gonadal development, which often
lead to infertility (Khurana and Theurkauf 2010).
The unique characteristics of piRNAs have attracted
many researchers, encouraging them to unveil themystery of piRNAs. Remarkable progress has been
made, especially in the area of biogenesis. A comprehensive computational analysis of piRNA populationsin the gonads of various animals—both wild type andmutants showing defects in oogenesis and/or spermatogenesis—led to two models for piRNA biogenesis:
the primary processing pathway and the amplification

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loop (see below, ‘‘Outline of the piRNA Biogenesis
Pathway’’).
However, the mechanisms underlying piRNA biogenesis and functions remain largely unknown, mainly
because the piRNA pathway has little in common withthe endo-siRNA and miRNA pathways as well as the
restriction of piRNA territories to the reproductive
tissues. Nonetheless, recent studies have suggested a possible function of piRNAs in nongonadal cells such asneuronal cells in Aplysia and mammals (Lee et al. 2011;
Rajasethupathy et al. 2012). Other studies have characterized new piRNA factors, Vreteno (Vret) and Shutdown(Shu) (Handler et al. 2011; Zamparini et al. 2011; Olivieri
et al. 2012; Preall et al. 2012; Xiol et al. 2012), and revealed
Zucchini (Zuc) as an endonuclease necessary for primarypiRNA biogenesis (Ipsaro et al. 2012; Nishimasu et al.
2012). Multigenerational epigenetic phenomena in nematodes are now known to involve piRNAs (Ashe et al.
2012; Shirayama et al. 2012). Thus, the visibility of thepiRNA field has rapidly broadened.
Outline of the piRNA biogenesis pathway
The primary processing pathway
Nascent transcripts arising from piRNA clusters areprocessed into piRNA-like molecules, which are thenloaded onto PIWI proteins (Fig. 1). The factors involved in
the transcription of piRNA clusters and its regulation
remain elusive. The current model proposes that the 59
ends of piRNAs are determined prior to loading onto PIWI
proteins. However, the 39 ends of piRNAs harbor extra
bases, which need to be trimmed upon association withPIWI proteins (Kawaoka et al. 2011; Vourekas et al. 2012).
The lengths of mature piRNAs are determined during
this step, largely depending on the sizes of the PIWI
proteins. Thus, piRNAs associated with individual PIWIs
show a similar but distinct size distribution (Siomi et al.
2011). The factors responsible for the 39 trimming
remain elusive. Upon maturation, the 39 ends of piRNAs
are 29-O-methylated by Hen1/Pimet, which is associatedwith PIWI proteins (Horwich et al. 2007; Saito et al. 2007).
This modification maintains the stability of piRNAs invivo (Horwich et al. 2007; Kamminga et al. 2010, 2012;
Billi et al. 2012). Hen1 was first discovered in Arabidopsis
as the enzyme responsible for 29-O-methylation of miRNAs(Chen 2005). However, in animals, Hen1 does not 29-Omethylate miRNAs. Although there is no direct evidence
for this, the high turnover rate for animals’ miRNAs may
serve to spatiotemporally regulate their function. Only
selected PIWI proteins are loaded with primary piRNAs.
For instance, Drosophila expresses three PIWI proteins:
Of these, Aubergine (Aub) and Piwi, but not Ago3, associate with primary piRNAs (Li et al. 2009; Malone et al.
2009; Saito et al. 2009). In mice, among three PIWI proteins, MIWI and MILI associate with primary piRNAs,
whereas MIWI2 mostly associates with secondary piRNAs,
as does Ago3 in flies (see below, ‘‘The Amplification Loop’’;Siomi et al. 2011). The mechanism underlying this PIWI
selection remains undetermined.

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The amplification loop
Primary piRNAs are subjected to an amplification system
to enforce the high expression of piRNAs in the germline:
This system is called the amplification loop or the PingPong cycle (Fig. 1; Brennecke et al. 2007; Gunawardane
et al. 2007; Houwing et al. 2008). In this system, Aub inflies and MILI in mice associated with primary piRNAs
cleave the target RNAs via their Slicer (endonuclease)activity (Saito et al. 2006; Gunawardane et al. 2007;
Nishida et al. 2007). This process forms the 59 ends of
secondary piRNAs. In Drosophila, maternally depositedpiRNAs can also trigger the Ping-Pong cycle, as do
primary piRNAs (Brennecke et al. 2008). The cleavage
products are then transferred onto other PIWI members,
Ago3 in flies and MIWI2 in mice, and are trimmed fromthe 39 end to give rise to mature piRNAs. This step ismechanistically equivalent to that observed in the primary processing pathway (Fig. 1), although the factors
required might differ. In turn, in flies, Ago3 associatedwith secondary piRNAs cleaves its targets, giving rise to
secondary piRNAs, which associate with Aub (Brennecke
et al. 2007; Gunawardane et al. 2007; Li et al. 2009;
Malone et al. 2009). Through these reciprocal Slicermediated cleavage reactions between Aub and Ago3, theheterotypic Ping-Pong cycle persists in Drosophila(Fig. 1). Piwi is translocated to the nucleus upon primarypiRNA loading (Haase et al. 2010; Olivieri et al. 2010;
Saito et al. 2010; Ishizu et al. 2011); therefore, Piwi barely
contributes to secondary piRNA production via the amplification loop (Malone et al. 2009; Olivieri et al. 2012).
In the Drosophila gonadal soma, Piwi is expressed andassociates with primary piRNAs. However, the expression levels of Aub and Ago3 are too low for them tooperate in the amplification loop. Thus, the situation in
ovarian somatic cells, such as follicle cells, is different
from that in germ cells.
Like Piwi in Drosophila, in mice, MIWI2 loaded withsecondary piRNAs is localized to the nucleus upon
piRNA loading (Fig. 1; Carmell et al. 2007). Thus, MIWI2
likely does not contribute to the bearing of secondary
piRNAs via the Ping-Pong cycle. The pathway for producing MIWI2–piRNAs is thus considered to be the ‘‘oneway’’ secondary piRNA biogenesis pathway (Aravin et al.
2008; De Fazio et al. 2011; Reuter et al. 2011), although
there is evidence supporting the notion that the MILI–
piRNA complex may operate a homotypic Ping-Pongcycle in mouse testes (De Fazio et al. 2011). MIWI is
associated with pachytene piRNAs (piRNAs expressed
starting at the pachytene stage of meiosis in mouse
spermatogenesis), which are also barely involved in theamplification loop (Beyret et al. 2012). Based on theseobservations, it is highly likely that a heterotypic PingPong cycle does not operate in mice, unlike in Drosophila(Fig. 1). Although the details of the Ping-Pong cycle
differ slightly between flies and mice, secondary piRNAs
are 29-O-methylated by Hen1/Pimet in both species(Fig. 1).
Because the amplification loop depends on the Slicer
activity of PIWI proteins, secondary piRNAs on Aub inFigure 1. piRNA biogenesis in Drosophila and mice consists of the primary piRNA processing pathway and the amplification loop. The left side shows a model of the primaryprocessing pathway in flies. The primary transcripts of piRNA clusters are shortened into piRNA-like small RNAs. The factors necessary for this process largely remain to be
determined. Recent studies have suggested that Zucchini might be an endonuclease forming the 59 ends of piRNAs. The mouse Zucchini homolog (MitoPLD) might also function asa nuclease to produce primary piRNAs. piRNA-like small RNAs are loaded onto PIWI proteins (shown in pink) and then trimmed from the 39 end to the size of mature piRNAs by an
unknown nuclease. Hsp83/Shu may play a role in the Piwi loading step. piRNAs are 29-O-methylated by HEN1/Pimet. In flies, the PIWI proteins that associate with primarypiRNAs are Piwi (green) and Aub (blue). Piwi associated with piRNAs is translocated to the nucleus and thus likely does not contribute to the amplification loop. Aub associatedwith piRNAs now triggers the Ping-Pong cycle by cleaving transposon transcripts. Ago3 (orange) loaded with secondary piRNAs in turn produces piRNAs that associate with Aub.
Transposon transcripts are cleaved through this amplification cycle, resulting in cytoplasmic transposon silencing. In mice, primary piRNAs are loaded onto MILI (yellow) and MIWI
(red). MIWI associated with pachytene piRNAs functions in cytoplasmic silencing. The targets remain largely unknown. MILI associated with primary piRNAs contributes to thePing-Pong cycle to produce piRNAs that associate with MIWI2 (purple), upon which MIWI2 is localized to the nucleus to accomplish nuclear silencing. The contribution of MIWI2
to the Ping-Pong cycle may be negligible. MILI might operate a homotypic Ping-Pong cycle as indicated. HSP90/FKBP6 plays a role in producing secondary piRNAs that associatewith MIWI2. HEN1/Pimet 29-O-methylates secondary piRNAs, the products of the Ping-Pong cycle.

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flies and MILI in mice show complete complementarity
to the piRNAs on Ago3 (in flies) and MIWI2 (in mice),
respectively, through the first 10 bases from their 59 ends
(Brennecke et al. 2007; Gunawardane et al. 2007). In
addition, Aub-piRNAs show a strong bias for uracil atthe 59 end (1-U), and, accordingly, Ago3-piRNAs tend
to have adenosine at the 10th nucleotide from the 59 end(10-A). These are typical signatures of piRNAs made via
the Ping-Pong cycle or one-way secondary piRNA biogenesis, which is highly conserved in animal species(Grimson et al. 2008).
In the Ping-Pong cycle, transposon transcripts arecleaved by piRISCs. This means that the Ping-Pong cycle
accomplishes dual tasks simultaneously, producing secondary piRNAs and silencing transposons by cleaving
their transcripts. In mice, primary piRNAs on MILI are
predominantly ‘‘sense’’ to transposon transcripts and are
unable to target transposon transcripts. Nuclear MIWI2
associates with antisense piRNAs and thus is capable ofinducing transposon silencing, regardless of whether
nuclear silencing targets transposon loci directly or via
nascent RNAs transcribed from transposon loci. MIWI2(and also MILI) has been linked to DNA methylationof target gene loci in the genome (Aravin et al. 2008).
However, the molecular mechanisms underlying this action remain unclear. The functions of pachytene piRNAs
loaded onto MIWI are unclear.
piRNA biogenesis factors: Tudor domain-containing(TDRD) proteinspiRNA biogenesis requires several factors other than
PIWI proteins, involving Tudor superfamily members or
TDRD proteins (Fig. 2; Siomi et al. 2010). TDRD proteinsspecifically associate with particular protein substrates
through symmetrical dimethyl arginines (sDMAs) or
asymmetrical dimethyl arginines (aDMAs) in the substrates (Chen et al. 2011). This protein–protein interaction has been shown to play a role in aspects of RNA
metabolism such as splicing (Neuenkirchen et al. 2008).
RNA silencing is no exception. PIWI proteins containsDMAs toward their N-terminal ends (Chen et al. 2009;
Kirino et al. 2009; Nishida et al. 2009; Reuter et al. 2009;
Vagin et al. 2009). An enzyme responsible for the sDMA
modification is PRMT5/DART5/Capsleen (Kirino et al.
2009). To date, 11 TDRD proteins in flies (Tudor [Tud],
Partner of piwis [Papi], Qin/Kumo, Tejas, Spindle-E [Spn-E],
Yb, Brother of Yb [BoYb], Sister of Yb [SoYb], Krimper[Krimp], dSETDB1, and Vret) (Fig. 2; Anand and Kai 2011;
Handler et al. 2011; Liu et al. 2011; Rangan et al. 2011;
Zamparini et al. 2011; Zhang et al. 2011) and seven inmice (TDRD1, TDRD2, TDRD4, TDRD5, TDRD6,
TDRD7, and TDRD9) (Siomi et al. 2010) have been
known to be involved in the piRNA pathway.
Earlier genetic studies in flies showed that Tud mutants phenocopy Aub mutants (Arkov et al. 2006). Morerecent studies have revealed that Tud, consisting of 11
Tud domains (Fig. 2), associates with both Aub and Ago3
in an sDMA-dependent manner to act as a ‘‘platform’’ for
the Ping-Pong cycle (Nishida et al. 2009). In tud mutants,

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Aub and Ago3 associate with piRNAs more abundantly
than in wild-type flies; however, these associations arelikely predominantly aberrant. Thus, Tud is necessary forthe quality control of piRNAs in the germline (Nishida
et al. 2009). TDRD6 in mice, containing seven Tud
domains, is thought to be the nearest homolog of Drosophila
Tud. TDRD6 binds mouse Vasa homolog (MVH) (seebelow), MIWI, MILI, and TDRD1 (Kirino et al. 2010),
although the function of TDRD6 in piRNA biogenesis is
not yet clear.
Qin/Kumo (CG14303) (Anand and Kai 2011; Zhang
et al. 2011) and Vret (CG4771) (Handler et al. 2011;
Zamparini et al. 2011) are newly characterized Drosophila
TDRD genes (Fig. 2). Qin/Kumo encodes a protein containing one RING domain and two B-box domains in the
N-terminal region, followed by five Tud domains. The
nearest homolog of Qin/Kumo in mice is TDRD4/RNF17(Pan et al. 2005). Qin/Kumo localizes to the nuage,a nonmembranous perinuclear structure found in animal
germ cells, which is implicated as the site of piRNAbiogenesis (Anand and Kai 2011). Qin was named after the
famous ancient Chinese dynasty (Zhang et al. 2011),
while Kumo in Japanese means ‘‘cloud,’’ the French forwhich is nuage (Anand and Kai 2011). Mutations in Qin/
Kumo cause most nuage components, including Aub andAgo3, not to accumulate to the nuage; thus, Qin/Kumo is
considered to be the core of the nuage (Anand and Kai
2011). Loss of function of Qin/Kumo induces homotypicPing-Pong among Aub proteins, instead of natural heterotopic Ping-Pong between Aub and Ago3, although theexpression level of Ago3 in qin/kumo mutants is greaterthan that in ago3 heterozygotes (Zhang et al. 2011).
Mutations in Qin/Kumo weaken the association between
Aub and Ago3; this might be the cause of the weakeningof heterotopic Ping-Pong between Aub and Ago3. A comprehensive bioinformatic analysis of piRNAs revealed
that the homotypic Ping-Pong in qin/kumo mutants is,however, futile, resulting in derepression of transposons
and DNA damage in the ovaries. Thus, Qin/Kumo enforces the heterotypic amplification loop between Aub
and Ago3 to quality-control piRNAs to ensure transposon
silencing. It is of interest to note that the functions of Tud
and Qin/Kumo are identical to each other; namely, bothproteins function in the quality control of piRNAs.
However, tud and qin/kumo mutants show different
phenotypes. One could infer that Tud and Qin/Kumo
have additional functionalities and/or specificities that
contribute to successful Ping-Pong in the germ cells.
Qin/Kumo is localized to the nucleus in the germarium.
A potential nuclear function of Qin/Kumo was suggested;
namely, maintaining the transcription level of the bidirectional piRNA cluster 42AB, as does the HP1 homolog Rhino (Klattenhoff et al. 2009; Anand and Kai
2011). However, Qin/Kumo associates with HP1 but notwith Rhino. Findings to date suggest that Qin/Kumo
regulates the level of bidirectional piRNA clusters by
restricting HP1 binding to the clusters by physicallyassociating with HP1. However, the important questionof how Qin/Kumo/HP1 selectively regulates piRNA
clusters remains to be addressed. The roles of Qin/Kumo

Figure 2. piRNA biogenesis factors in Drosophila. Drosophila piRNA factors can be subgrouped into PIWI proteins, TDRD proteins, and others. The cellular localizations in
ovaries and protein–protein interaction partners of individual factors are summarized on the right. (N.D.) Not determined. Interaction of Tral, Me31B, and TER94 with Papi wasreported by Liu et al. (2011). HP1 interaction with Qin/Kumo was reported by Anand and Kai (2011). In addition to perinuclear localization in the nurse cells, Kumo also appears asfoci in the nuclei of the germ cells in the germarium (indicated by an asterisk). Interaction of MTOC proteins with Mael was reported by Sato et al. (2011).

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in transcription of piRNA clusters and piRNA biogenesis are not yet known. However, the Qin/Kumo studies
by Anand and Kai (2011) and Zhang et al. (2011) raised
the interesting idea that ubiquitination might be involved in the piRNA pathway because the RING domain
is known to specify the activity of E3 ligases, which
specifically modify their substrates with ubiquitins. It isof great interest to address how ubiquitination is molecularly linked to piRNA biogenesis, if ubiquitination isindeed involved in the pathway.
Drosophila Vret consists of one RNA recognition motif(RRM), one MYND domain, and two Tud domains and isnecessary for piRNA-based transposon regulation in bothgerm cells and gonadal somas (Fig. 2; Handler et al. 2011;
Zamparini et al. 2011). Loss of function of Vret abolishes
the primary piRNA processing activity, whereas the PingPong pathway is not greatly affected by vret mutations.
Thus, Vret plays a role exclusively in primary piRNA
production, although the expression of Vret can also be
observed in germ cells, where the Ping-Pong amplification takes place. Vret interacts with the somatic primarypiRNA factors Armitage (Armi), Yb, and Zuc (Fig. 3; seebelow). The transcriptional levels of piRNA clusters are
not changed in vret mutants. Thus, the requirement for
Vret in the piRNA pathway is distinct from that for Qin/
Kumo, although both Vret and Qin/Kumo are TDRD
members and bind PIWI proteins. Vret contains a RRM,
suggesting a direct association of Vret with RNA molecules. Investigations to identify the RNA-binding substrates are awaited. It is noted that no obvious homolog ofVret has been found in mice.

piRNA biogenesis factors: other than PIWI and TDRD
proteins
Several non-PIWI, non-TDRD proteins are also involvedin piRNA biogenesis: These include Vasa (Vas), Maelstrom (Mael), Armi, Zuc, Squash (Squ), and Shu in
Drosophila (Fig. 2). All of these factors except Squ areconserved in mice: The mouse homologs are MVH, Mael,
MOV10/MOV10l1, MitoPLD/PLD6, and FKBP6, respectively (Soper et al. 2008; Frost et al. 2010; Huang et al.
2011; Watanabe et al. 2011; Xiol et al. 2012). Most of thesefactors play a role in the amplification loop. The representative is Vas, a germ cell-specific DEAD-box-type
RNA helicase required for pole cell development in
Drosophila (Lasko and Ashburner 1990).
Vas was implied to regulate translation of target genes,
such as Gurken, because it associates with the translation
initiation factor eIF5B, and Gurken does not accumulate
in oocytes in vas mutants (Carrera et al. 2000). Translation of oskar and nanos may also be activated by Vas in
the pole plasm. MVH also has pivotal roles specificallyin male germ cells in mice (Tanaka et al. 2000). MVHdeficient mouse testes phenocopy MILI- and MIWI2deficient testes; IAP and Line-1 retrotransposons are derepressed, and de novo DNA methylation of their regulatory regions is impaired (Kuramochi-Miyagawa et al.
2010). Without MVH function, MIWI2 becomes almost
free from secondary piRNAs, although MILI’s association
with primary piRNAs is maintained. Thus, MVH isconsidered to be necessary for the Ping-Pong cycle.
Mutations in Vas in Drosophila also impair piRNA
accumulation in the gonads and derepress transposons

Figure 3. Zucchini in primary piRNA biogenesis in Drosophila ovarian somas. (A) Long, single-stranded piRNA precursors aretranscribed from piRNA clusters in the genome and are processed into piRNA intermediates through an unknown mechanism. (B)An enlarged cartoon of the area shown by a dotted square in A: The RNA helicase Armi and the Tud domain-containing RNA
helicase Yb localize to Yb bodies, which are often located adjacent to mitochondria. Armi and Yb form a complex that contains
piRNA intermediates with 59-hydroxyl and 39-cyclic phosphate ends. Nascent, piRNA-free Piwi transiently localizes to Yb bodies
to interact with the Armi–Yb complex. Zuc is anchored on the outer surfaces of mitochondria with the catalytic site facing thecytosol and processes piRNA intermediates into piRNA fragments, which are loaded onto Piwi. Primary piRNAs show a strong
bias for 1-U, although Zuc endonuclease showed little sequence specificity in vitro. These findings lead to the possibility that
cofactors may be involved in 1-U determination in vivo (‘‘cofactors?’’). Alternatively, Piwi may preferentially bind 1-U piRNAs
among all Zuc cleavage products (‘‘selection?’’). Vret and Shu are newly characterized piRNA factors that localize to Yb-bodies.
The functions of Vret and Shu have not been fully elucidated. The enzymes participating in 39 end formation of piRNAs remain tobe identified.

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New insights into piRNA biology(Lim and Kai 2007; Nagao et al. 2010), although theunderlying mechanism remains to be elucidated. Ovariansomas in Drosophila are devoid of Vas expression, suggesting that the somatic primary processing does not
require Vas.
Many non-PIWI, non-TDRD piRNA factors also playa role in primary processing. Drosophila ovarian somatic
cells use the primary processing pathway but not thePing-Pong cycle (Lau et al. 2009; Li et al. 2009; Malone
et al. 2009; Saito et al. 2009) simply because they do not
express Aub and Ago3. In these cells, primary processing
occurs in perinuclear Yb bodies. The core component Yb andother piRNA factors—Armi, Shu, and Vret—accumulate
in Yb bodies, where they exert their functions (Fig. 3).
Upon processing, mature primary piRNAs are loaded
onto Piwi to assemble active piRISCs. This assembly
allows piRISCs to be transported to the nucleus, where
they function in transposon silencing (Saito et al. 2009;
Ishizu et al. 2011). Piwi Slicer activity is dispensable fornuclear silencing (Saito et al. 2009), although it is still
unclear whether the silencing occurs transcriptionally or
post-transcriptionally.
Zuc, a member of the phospholipase D (PLD) superfamily, is required for primary piRNA biogenesis in
Drosophila ovarian somatic cells. Zuc has been considered a candidate ribonuclease for piRNA maturation(Pane et al. 2007; Saito et al. 2009, 2010; Haase et al.
2010; Olivieri et al. 2010). This is because a bacterial
protein, Nuc, another member of the PLD superfamily,
has endonuclease activity (Pohlman et al. 1993). MitoPLD,
the mammalian homolog of Zuc, also plays a crucial rolein piRNA biogenesis (Huang et al. 2011; Watanabe et al.
2011). Previous reports showed that MitoPLD generates
a lipid signal molecule, phosphatidic acid, using mitochondrial lipid cardiolipin as a substrate (Choi et al. 2006;
Huang et al. 2011), although MitoPLD showed no nuclease activity toward DNA and RNA substrates in in vitro
assays (Watanabe et al. 2011). These observations suggested that mitochondrial morphology regulated by the
mitochondrial lipid signaling pathway might be related
to piRNA production. Like MitoPLD, Zuc localizes tomitochondria in Drosophila cells (Saito et al. 2010). However, it was unclear whether Drosophila and mammalian
Zuc have conserved enzymatic functions. Furthermore,these studies could not rule out the possibility that Zuc/
MitoPLD might exhibit a nuclease activity in intracellularenvironments.
Recent crystal structural analyses revealed that, similar to other PLD enzymes, Zuc/MitoPLD forms dimers
with a single active site at the subunit interface (Ipsaro
et al. 2012; Nishimasu et al. 2012). The active site contains
two conserved catalytic HKD (His–Lys–Asp) motifs, one
from each subunit. Zuc was found by in vitro nucleaseassays to have single-strand-specific endonuclease activity.
Mutational analyses showed that Zuc cleaves the target
phosphodiester bond in a manner similar to other PLD
family nucleases, by leaving products with a 59-phosphateterminus. Since the 59-phosphate terminus is one of thehallmarks of mature piRNAs, it was postulated that Zuccleaves piRNA intermediates to generate the 59 ends ofpiRNAs (Fig. 3). Zuc showed little sequence specificity invitro. These findings appear to contradict the finding thatprimary piRNAs show a strong bias for 1-U. One possibility might be that cofactors—for example, TDRD
proteins—are involved in 1-U determination in vivo. An
alternative possibility is that Piwi selects 1-U piRNAs
among all Zuc cleavage products to bind. In any case,these findings suggest that Zuc plays a role equivalent tothat of Aub/Ago3 in the Ping-Pong cycle; that is, to form
the 59 end of piRNAs.
Shu is a female sterile gene in Drosophila (Schüpbachand Wieschaus 1991) encoding an evolutionarily conserved cochaperone of the immunophilin class that harbors
a peptidyl-propyl-cis/trans-isomerase domain and a tetratricopeptide repeat (TPR) domain (Munn and Steward2000). Recent studies identified Shu as a new piRNA
factor necessary for both primary processing and theamplification loop (Olivieri et al. 2012; Preall et al.
2012). Indeed, Shu was found to be a component of boththe nuage and Yb bodies in germ cells and ovarian somas,
respectively, both of which are considered to be centers ofpiRNA biogenesis. Mutations in Shu in ovarian somas do
not affect the localization of Yb, Armi, and Vret, meaningthat Shu is located downstream from these molecules.
However, the Yb body localization of Shu depends on
Piwi. Shu may be unnecessary for somatic primarypiRNA biogenesis but rather may be required for Piwi
loading. In mice, FKBP6 (a Shu homolog in mice), whichis essential for male fertility (Crackower et al. 2003), wasdetermined to be unnecessary for primary processing,unlike Shu in flies, but required for the amplification asa remover of 16-nt slicing products (Xiol et al. 2012).
FKBP6 associates with Hsp90 through its TPR domain
and plays a role in producing secondary piRNAs thatassociate with MIWI2. Hsp90 has been implicated inpiRNA biogenesis (Specchia et al. 2010). FKBP6 also binds
with PIWI proteins (Vagin et al. 2009). Inhibition of
HSP90 results in the accumulation of ;16-nt by-productsof piRNA amplification specifically on BmAgo3, one of
two PIWI proteins expressed in cultured Bombyx germ
cells. One proposal is that the chaperone machinery
consisting of HSP90 and FKBP6 removes the 16-nt byproducts of piRNA amplification from BmAgo3, facilitating its turnover in the Ping-Pong pathway to enforce thehigh expression of piRNAs in germ cells. How, then, arethe by-products on SIWI, another PIWI in Bombyx, removed from SIWI? One suggestion is that other chaperone machinery similar to HSP90/FKBP6 might exist for
SIWI, although currently this remains unclear.
piRNA functions outside of the germline cells
A unique, fascinating study using the Aplysia CNS
revealed that piRNAs can play roles in regulating memorystorage in the brain by silencing CREB2, the major inhibitory constraint of memory formation (Rajasethupathy
et al. 2012).
piRNAs in the Aplysia CNS cluster in the genome (372clusters were found in the study), similar to the piRNAs
in germline cells. Neuronal piRNAs preferably contain

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1-U, are 29-O-methylated, and induce DNA methylationof target genes, including CREB2, a transcriptional repressor of memory. Notably, all of these features arehallmarks of piRNAs in germline cells. The associationof acaPiwi (the single PIWI in Aplysia) with piRNAs was
also confirmed experimentally.
One or a few individual piRNAs were frequently cloned
from the same cluster, suggesting that some piRNAs are
selectively enriched in the CNS. In this regard, Aplysia
neuronal piRNAs may be similar to Suppressor of Stellate[Su(Ste)]-derived piRNAs in the Drosophila testis (Nishida
et al. 2007). Su(Ste) piRNAs were suggested to be theproducts of primary processing (Nagao et al. 2010), andneuronal piRNAs in Aplysia may be produced by anequivalent pathway. Aplysia expresses only PIWI (acaPiwi);
thus, the animal lacks the amplification loop. As a result,
all Aplysia piRNAs are considered to be primary piRNAs.
It remains unclear whether primary piRNA biogenesis
factors are conserved in Aplysia. acaPiwi is localized to
the nucleus, as is MIWI2 in mouse testis and Piwi in
fly gonads. Nuclear localization of MIWI2 and Piwi isachieved by piRNA loading onto the proteins. It would
be of interest to determine whether piRNA loading isnecessary for the nuclear localization of acaPiwi. Whether
nuclear localization of acaPiwi can also be observed in the
ovotestes remains to be determined.
Knockdown of acaPiwi induces up-regulation of CREB2
but does not affect the related genes C/EBP and CPEB.
Rajasethupathy et al. (2012) suggested that acaPiwi hastarget genes other than CREB2, although these are
currently unknown. Inhibition of acaPiwi completely
abolished the serotonin-dependent increase in methylation at the promoter of CREB2 (Rajasethupathy et al.
2012); however, the details of the mechanism underlying
this effect are also unknown.
The following questions remain to be addressed: (1)What is the benefit for Aplysia in using miRNA andpiRNA to silence CREB1 and CREB2, respectively? (2) Do
mammals also use piRNAs in brains to establish longterm memory storage? The answers to these questions
are eagerly awaited.
A recent study found piRNAs in the mouse hippocampus (Lee et al. 2011). These piRNAs associate with MIWI
to form an effector piRISC, which is localized to thecytoplasm in mouse hippocampal neurons. piRNA function seems to be required for dendritic spine development
because inhibition of piRNA function led to a decrease indendrite spine area. Computational analyses have suggested target genes of these hippocampal piRNAs. However, it remains to be determined whether expression ofthese genes is regulated by piRNAs.
piRNAs in Caenorhabditis elegans
One of the outstanding questions in piRNA research is how
the piRNA system selects transposable elements—and notother cellular genes—as targets to silence. In other words,
how does the system discriminate between foreign
nucleic acid elements and cellular mRNAs? This question
has conceptual similarities to questions about immune

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systems, which achieve ‘‘self’’ versus ‘‘nonself’’ recognition. Recent studies in C. elegans have provided important insights into piRNA-mediated genome-wide surveillance of germline transcripts, leading to long-termsilencing of transcripts recognized as nonself and tolicensing to protect cellular protein-coding genes as selffrom piRNA-mediated silencing (Fig. 4; Ashe et al. 2012;
Bagijn et al. 2012; Lee et al. 2012; Luteijn et al. 2012;
Shirayama et al. 2012).
The piRNAs in C. elegans are 21-U RNAs, a populationof 21-nt small RNAs characterized by a 1-U bias anda characteristic sequence motif ;42 nt upstream of thestart of the small RNA (Ruby et al. 2006; Batista et al.
2008). 21-U RNAs appear to be derived from thousands ofindividual, autonomously expressed loci broadly scattered in two large clusters on chromosome IV (Rubyet al. 2006; Batista et al. 2008). The most conserved 8-mersequence (CTGTTTCA) in the upstream region is required for their individual expression and is specificallyrecognized by Forkhead family transcription factors(Cecere et al. 2012). piRNAs are expressed in germline
cells, where they interact with the PIWI protein PRG-1.
Mutations in prg-1 result in a reduced brood size and atemperature-sensitive sterile phenotype, consistent with
the notion that PIWI proteins are linked to germline
maintenance. Like the abundant pachytene piRNAs in
mammals (Aravin et al. 2006; Lau et al. 2006), C. eleganspiRNAs encode remarkable sequence diversity and yetlack obvious targets. Although the piRNA pathway isimplicated in the silencing of only a single transposon,
Tc3, by acting upstream of secondary siRNA production
via RNA-dependent RNA polymerase (RdRP) activity(Das et al. 2008), the lack of perfect complementarity
to Tc3 and other transposable elements displayed by;16,000 different piRNAs encoded in the C. elegans
genome has been a long-standing conundrum.
A combination of C. elegans genetics together withbioinformatics for small RNAs answered a very important question about how 21-U RNAs recognize andsilence target genes (Ashe et al. 2012; Bagijn et al. 2012;
Lee et al. 2012; Shirayama et al. 2012). Compelling
evidence supported the concept that PRG-1–piRNA complexes scan foreign RNA sequences and drive the production and initial amplification of RdRP-dependentsiRNAs known as 22G-RNAs, which are loaded onto
worm-specific Argonaute members, WAGO proteins. An
important recurring theme that emerged is that RNA
silencing in C. elegans occurs in a two-step pathway, andmultiple C. elegans small RNA pathways converge on
WAGO proteins, including the RDE-1 exogenous siRNA
pathway (RNAi) and the ERGO-1 endogenous siRNA
pathway (Yigit et al. 2006; Pak and Fire 2007; Gu et al.
2009). Amplification of the silencing signal in the twostep system occurs via the production of secondarysiRNAs (22G-RNAs) by RdRPs that are then discriminately bound to WAGO proteins, which mediate downstream silencing; WAGOs that localize to the cytoplasm
mediate mRNA turnover, whereas WAGOs that localize
to the nucleus mediate transcriptional silencing (Guanget al. 2008; Gu et al. 2009). Once a piRNA initiates theDownloaded from genesdev.cshlp.org on November 1, 2012 - Published by Cold Spring Harbor Laboratory Press

New insights into piRNA biology

Figure 4. piRNAs in C. elegans. C. elegans piRNAs
are 21 nt in length with 1-U (21U-RNAs) and areloaded onto the PIWI protein PRG-1. piRNAs are
genomically encoded in two large clusters on chromosome IV. These clusters are depleted of proteincoding genes. piRNAs have a characteristic sequencemotif ;42 nt upstream of the start of the small RNA,
which may be required for RNA polymerase II (Pol II)transcription. piRNAs scan virtually all transcripts
expressed in the germline and guide PRG-1 to targets
by means of relaxed base-pairing with up to fourmismatches. PRG-1–piRNA complexes then recruit
RNA-dependent RNA polymerases (RdRPs) to thetarget site to produce 22G-RNAs (which are loaded
onto WAGOs) that silence foreign genetic elements(Exo). WAGOs that localize to the cytoplasm mediate mRNA turnover (PTGS), whereas WAGOs that
localize to the nucleus mediate transcriptional genesilencing (TGS). The CSR-1 22G RNA pathway may
provide a memory of self (Endo) and act as an antisilencing signal.

two-step pathway against nonself elements—a processthat is independent of PRG-1 Slicer activity (Bagijn et al.
2012)—the silencing is permanently heritable and nolonger requires piRNAs and PRG-1 (Ashe et al. 2012;
Luteijn et al. 2012; Shirayama et al. 2012). This phenomenon (which involves nuclear WAGO proteins and canonical chromatic modifications, including H3K9me3) isreferred to as RNA-induced epigenetic silencing or RNAe
(Shirayama et al. 2012). Thus, piRNAs represent theinitial triggers of siRNA amplification and heterochromatin formation, revealing intriguing parallels with theroles of fission yeast primal RNAs (priRNAs) that nucleate a positive feedback loop of siRNA production, whichthen promotes heterochromatin formation (Halic andMoazed 2010).
Mutagenesis studies and genome-wide computational
predictions revealed that piRNAs guide PRG-1 to target
transcripts by base-pairing with up to four mismatches,
initiating localized generation of 22G-RNAs withina 650-nt window around the site of piRNA complementarity on the target RNA (Ashe et al. 2012; Lee et al. 2012).
However, just like the target recognition of miRNAs(Bartel 2009) and similar to the target recognition ofmouse piRNAs (Reuter et al. 2011), the 59 end of theC. elegans piRNA, particularly the seed sequence (nucleotides 2–8), appears critical for targeting. Thus, C. eleganspiRNAs have the potential to silence transcripts in transthrough imperfectly complementary sites. However,such relaxed targeting requirements imply that piRNAs
with ;16,000 distinct sequences target not only multiple
transposable elements and exogenously introduced elements (transgenes), but also most cellular protein-coding
transcripts. How do piRNAs selectively target foreign
elements and avoid targeting endogenous protein-coding
genes? Perhaps piRNAs that silence protein-coding genes
are negatively selected, as depletion of mismatch-tolerantpiRNAs that base-pair with germline protein-coding
transcripts is observed (Bagijn et al. 2012; Lee et al.
2012). Instead, piRNA clusters tend to overlap transposonends, suggesting the occurrence of recent transposon
integrations downstream from the sequence motif
thought to be required for piRNA biogenesis. Thus, theC. elegans piRNA system bears many conceptual similarities to Drosophila piRNA clusters. These systems
must cope with a diverse range of foreign genetic elements that show little similarity at the primary sequencelevel. The strategies used to build a piRNA repertoire
seem to rely on transposition by chance into piRNA

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clusters, which can then become fixed by evolutionary
selection. In both cases, piRNA clusters act as traps fortransposable elements.
In addition, germline mRNAs seem to be protectedfrom piRNA-induced silencing, and this is potentiallymediated by Argonaute CSR-1. CSR-1 lies in one of two
distinct germline 22G-RNA pathways, and the interacting 22G-RNAs are antisense to virtually all germline cellexpressed protein-coding genes (Claycomb et al. 2009).
However, endogenous CSR-1 22G-RNA targets do notappear to be down-regulated by CSR-1. CSR-1 22G-RNAsare expressed at low levels relative to WAGO 22G-RNAs,
perhaps below the threshold required to trigger mRNA
turnover. piRNAs that map to CSR-1 22G-RNA targets
trigger a less robust secondary siRNA response comparedwith those mapping to WAGO 22G-RNA targets. Perhaps
CSR-1 22G-RNA complexes can compete for bindingsites with PRG-1–piRNA and/or WAGO 22G-RNA complexes. This finding led to the hypothesis that CSR-1
22G-RNA complexes function as a memory of previous
gene expression in germline cells and that the CSR1-dependent licensing (‘‘anti-silencing’’) protects endogenous protein-coding genes from piRNA-mediated silencing(Shirayama et al. 2012). Thus, the balance between nonself
recognition by the PRG-1–piRNA pathway and self recognition by the CSR-1–22G-RNA pathway might determine
the outcome of gene expression in germline cells.
Although recent biochemical analyses revealed no
obvious targets of mouse pachytene piRNAs (Vourekas
et al. 2012), the findings in C. elegans may spur reexamination of the idea that pachytene piRNAs are theend products of RNA processing in mice using extensive
genome-wide computational analysis to identify targets
with relaxed base-pairing.
Perspective
The molecular mechanisms underlying piRNA biogenesis and functions are complex and diverse; therefore,
many questions still need to be answered. The firstquestion is related to the discrimination of piRNA precursors from non-piRNA precursors. As noted above, thetranscripts arising from piRNA clusters and a few specificprotein-coding mRNAs, including traffic jam, are recognized as piRNA precursors and give rise to piRNAs(Robine et al. 2009; Saito et al. 2009). piRNA clusters
normally span a wide region in the genome, and thus it isfeasible to think that they are transcribed by RNA polymerase II and later are capped and poly(A)-tailed at the
59 and 39 ends like normal mRNAs. Why and how are
regular mRNAs able to escape being victims of thepiRNA production system in vivo? Answering this question is crucial because the answer will enhance ourunderstanding of the mechanism of piRNA biogenesis
at the molecular level. The second question is related tothe identity and nature of piRNA biogenesis factors
required upstream of Zuc. Mutations in Zuc lead to theaccumulation of piRNA intermediates but impair piRNAmaturation. The intermediates are approximately several
hundred bases long, although piRNA precursors are much

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larger, meaning that other factors downsize the piRNA
primary precursors into their intermediates. Establishingan in vitro system to recapitulate piRNA processing frompiRNA cluster transcripts in combination with cellular
fractionation would be helpful for dissecting the piRNAbiology. Cultured cell lines established from ovaries and/or testes of various species or induced pluripotent stem
cells would also be useful for biochemical studies. Ultimately, we strive to understand the piRNA system inhumans.
Impairment of piRNA expression and mutations in
PIWI proteins result in sterility in animals. Are there
any human reproductive diseases, such as azoospermia,that involve piRNA clusters and PIWI loci? Wholegenome sequencing of patients may provide the answer
to this question. Interestingly, the human genome contains
one additional PIWI gene, PIWIL3 (HIWI3 in humans),
compared with mouse and fly genomes. Since the current
piRNA biogenesis model was based on insights gained
from investigations mainly involving mice and flies, it
obviously excludes PIWIL3. Thus, to understand thepiRNA pathway correctly, new insights into the molecular function of PIWIL3 should be incorporated. The roleof piRNAs in cancer is another area that warrants investigation, as there is evidence that PIWI proteins andpiRNAs may have important roles in epigenetic regulation in tumorigenesis (Siddiqi and Matushansky 2012),
as has been shown in gametogenesis. It will be of greatinterest to examine whether impairment of the functionsof piRNAs and/or PIWI proteins in cancer cells would
inhibit tumorigenesis.
Acknowledgments
We thank Kaoru Sato and other members of the Siomi laboratories at Keio University School of Medicine and at the Graduate
School of Science, The University of Tokyo, for useful discussion
and comments. H.I. is supported by JSPS (Japan Society for thepromotion of Science). H.S. and M.C.S. are supported by MEXT
(Ministry of Education, Culture, Sports, Science, and Technology). M.C.S. is supported by CREST (Core Research for Evolutional Science and Technology) from JST (Japanese Science andTechnology Agency).

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