Molecular Cell

Short Review
The Coming of Age for Piwi Proteins
Anita G. Seto,1,2,3 Robert E. Kingston,1,* and Nelson C. Lau1,2
1

Department of Molecular Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA
These authors contributed equally to this work.
3
Present address: Thermo Fisher Scientific, Dharmacon Products, Lafayette, CO 80026, USA.
*Correspondence: kingston@molbio.mgh.harvard.edu
DOI 10.1016/j.molcel.2007.05.021
2

Piwi proteins, a subfamily of Argonaute (Ago) proteins, have recently been shown to bind endogenous small RNAs. However, differences between Ago proteins (which bind microRNAs and smallinterfering RNAs) and Piwi proteins and Piwi-interacting RNAs (piRNAs) suggest novel functions
for Piwi proteins. Here, we highlight the recent progress in understanding Piwi function and theimplications for germline and stem cell development.
Argonaute (Ago) proteins comprise the central protein
component of RNA-mediated gene silencing complexes
and utilize short guide RNAs to direct function towarda specific nucleic acid target. The best-characterized
small RNA partners of Ago proteins are microRNAs(miRNAs), which predominantly silence genes posttranscriptionally, and small interfering RNAs (siRNAs) thatparticipate in transcriptional and posttranscriptional
mechanisms. Agos are defined by two major protein
motifs: the PAZ and PIWI domains. These motifs point toAgos’ function as an RNA interactor: the PAZ domain isa single-stranded nucleic acid binding motif and thePIWI domain has an RNase H fold (reviewed in Tolia and
Joshua-Tor [2007]).
Phylogenetic analysis indicates that known Agos can bedivided into two subclades: the Ago subclade based on
Arabidopsis thaliana Ago1 and the Piwi subclade basedon Drosophila melanogaster Piwi (see Figure 1). Much
progress has been made to elucidate the biological function and mechanism of action of the Ago subclade in RNA
interference pathways. The accompanying review in thisissue of Molecular Cell covers recent advances in thisarea (Peters and Meister, 2007). Meanwhile, functional
analysis of the Piwi subclade has lagged behind that of
the Ago subclade, in part due to the lack of definition concerning RNAs that interact with Piwi. This has changed
recently, as Piwi proteins have now been found to interact
with a new class of small RNAs that has intriguing differences from miRNAs and siRNAs. Recent reviews haveaddressed advances concerning the role for Piwi proteinsin the biogenesis of piRNAs (Lin, 2007; O’Donnell and
Boeke, 2007). The goal here is to summarize Piwi andpiRNA biology and to focus on their possible roles intranscriptional gene silencing and germline/stem cellmaintenance.
Piwi Proteins as Small RNA Interactors
Given the conservation of the major protein domains
between the Ago and Piwi subclades, it may not havebeen surprising to expect Piwi proteins to bind RNA. Indeed, the genetic studies of abrogated transposon andrepeat-element silencing observed in fly Piwi-familymutants were linked to RNA-dependent mechanisms.
Transgene silencing that was affected by mutations in
the piwi gene also seemed to depend on small RNAs
that are complementary to the transgenes (Pal-Bhadra
et al., 2004). Additionally, mouse Piwi proteins exhibited
RNA binding activity and appeared responsible for thestability of a subset of mRNAs (Deng and Lin, 2002; Kuramochi-Miyagawa et al., 2004). The surprise, however,came from the unusual qualities of endogenous RNAs
that interact with Piwi proteins.
In 2006, six groups converged upon the landmark finding that Piwi proteins in flies and mammals bind a class of
small RNAs distinct from miRNAs and typical siRNAs. Twogroups (Saito et al., 2006; Vagin et al., 2006) determinedthat Piwi and Aubergine bind repeat-associated siRNAs(rasiRNAs). The rasiRNAs were first characterized in small
RNA cloning studies from flies at different stages of development, which revealed that the rasiRNAs were longer inlength than canonical small RNAs (24–27 nucleotides, as
opposed to 21–22), were enriched in the testes and earlyin development, and derived from retrotransposons andother repetitive elements (Aravin et al., 2003). In addition
to their longer length, rasiRNAs can be distinguishedfrom miRNAs by a modification to the 30 terminal nucleotide (Vagin et al., 2006). rasiRNAs copurify with Piwi, but
not with DmAgo1, which binds miRNAs. Consistent with
the Piwi protein and the rasiRNAs working together, an
endonuclease activity on a target RNA complementary
to a short guide RNA in the protein could be detected(Gunawardane et al., 2007; Saito et al., 2006).
Working in mammals, four other groups revealed that
Piwi proteins interact with novel RNAs derived from intergenic regions but were not as dominated with matches torepeat elements as the rasiRNAs from flies (Aravin et al.,2006; Girard et al., 2006; Grivna et al., 2006a; Lau et al.,2006). Mammalian Piwi-interacting RNAs (piRNAs), likethe rasiRNAs, were longer than miRNAs, and the pool ofpiRNA sequences was extremely complex. These piRNAs
coimmunoprecipitated with two mouse Piwi subclade
members, Miwi and Mili (Aravin et al., 2006; Girard et al.,

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Figure 1. A Piwi-Focused Phylogenetic
Tree of Argonaute-Family Proteins
Alignments, tree construction, and bootstrap
analysis were performed with the MEGA v3.1
software. Only bootstrap values greater than
60 are shown for this neighbor-joining tree.
Protein sequences and accession numbers
are available upon request. Abbreviations: At:
Arabidopsis thaliana; Ce: Caenorhabditis
elegans; Dd: Dictyostelium discodium; Dj:
Dugesia japonica; Dm: Drosophila melanogaster; Dr: Danio rerio; Gl: Giardia lambia;
Gg: Gallus gallus; Hs: Homo sapiens; Mm:
Mus musculus; Nc: Neurospora crassa; Pc: Podocoryne carnea; Pt: Paramecium tetraurelia;
Scp: Schizosaccaromyces pombe; Sm:
Schmidtea mediterranea; Sp: Strongylocentrotus purpuratus; Tb: Trypanosoma brucei; Tt:
Tetrahymena thermophila; and Xt: Xenopus
tropicalis.

2006; Grivna et al., 2006a), whereas a conventional
chromatographic purification of proteins associated with
piRNAs from rat testes followed by protein sequencing
identified piRC (piRNA complex), containing the rat Piwi
protein, Riwi, and a RecQ1 helicase that has genetic links
to known RNA silencing pathways (Lau et al., 2006). Most
recently, a new subset of prepachytene piRNAs was discovered to be bound by Mili (Aravin et al., 2007). These
are less abundant and are expressed earlier than the
major complement of adult piRNAs, which are expressed
starting at the pachytene stage of meiosis. Although prepachytene piRNAs are more enriched in repetitive
sequences than pachytene piRNAs, the majority of bothprepachytene and pachytene piRNAs each map as a single copy within the mouse genome (Aravin et al., 2007;
Girard et al., 2006; Lau et al., 2006).

604 Molecular Cell 26, June 8, 2007 ª2007 Elsevier Inc.

Genomic analysis of mammalian piRNAs indicates thatthey cluster in primarily unannotated regions. Pachytene
piRNA clusters range in size from 1 to 100 kb in length(for example see Figure 2A), comprise less than 0.1% ofrodent genomes, and distribute nonuniformly as about
100 different loci across mainly autosomes (Girard et al.,2006; Lau et al., 2006). Prepachytene piRNAs, however,are more broadly distributed, with over 900 smaller clusters present, and appear not to overlap with pachytene
piRNA clusters (Aravin et al., 2007). A distinguishing feature of clusters of uniquely mapping piRNAs is the pronounced strand bias of the sequences, thereby leading
to the proposal that the biogenesis of piRNAs might
involve a long single-stranded precursor. However, detectable secondary structures or pronounced doublestranded RNA precursors have not been systematically

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Short Review

Figure 2. Potential Sites and Modes of Action for piRC
(A) Clusters of piRNAs derive from discrete 20–100 kb loci, several of which are bidirectionally transcribed (i.e., piRNA cluster #1, which is 80 kb). Bars
indicate number of individual piRNAs identified in each interval (red, top strand; blue, bottom strand.).
(B) piRC may serve a function in the nucleus, such as marking important genomic loci through an epigenetic mechanism (similar to that in ciliates) or
through an RNA cleavage event (similar to S. pombe). piRC could also act in the cytoplasm, to suppress transposon activity (similar to the rasiRNAcomplex in Drosophila), or it might act at the chromatoid body, an organelle postulated as an RNA-processing center.

detected for the putative piRNA precursors, thus thebiogenesis of mammalian piRNAs remains a mystery.
Nevertheless, recent characterization of a third Piwi
member in flies, Ago3, has yielded insight into the biogenesis of rasiRNAs. Piwi, Aub, and Ago3 each bind a different
set of rasiRNAs, and all three proteins produced in E. coli
exhibit RNA cleavage activity in vitro (Brennecke et al.,2007; Gunawardane et al., 2007). Aub- and Piwi- associated rasiRNAs derive mainly from the antisense strand of
retrotransposons, whereas Ago3 rasiRNAs derive mainly
from the sense strand. The finding that many Aub-associated rasiRNAs are complementary to Ago3-bound rasiRNAs
at only the first ten base pairs led to a model that Ago3
is involved in rasiRNA biogenesis by cleaving acrossa dsRNA precursor, thus specifying the 50 end of rasiRNAs

that are bound to Aub (Brennecke et al., 2007; Gunawardane et al., 2007). The base-paired configuration is supported by a strong preference for adenine at position 10
of Ago3 rasiRNAs, which would be opposite the biased
presence of uridine at the 50 end of Aub rasiRNAs. Thus,
the endonuclease activities of Piwi proteins might servea dual capacity: to degrade sense transposon transcripts
and to produce the antisense rasiRNAs.
This biogenesis mechanism might extend to mammals.
The population of repetitive piRNAs that bind to Mili during
the prepachytene stage of spermatogenesis contains
sense and antisense piRNAs derived from transposable
elements that have the same base-paired configuration
as the Ago3/Aub rasiRNA pairing (Aravin et al., 2007).
The biochemically purified endogenous piRC exhibits

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RNA cleavage activity, which presumably comes from theRiwi protein (Lau et al., 2006). However, whether recombinant mammalian Piwi proteins have the same biochemical
attributes as the Drosophila proteins remains to be seen.
In addition, piRNAs associated with Miwi/Riwi appear toderive from a single strand, not both strands as most
simply expected if this biogenesis model is general.
A model invoking Piwi-family proteins as the sole cleavage agents would explain how rasiRNA biogenesis canproceed without either Dcr-1 or Dcr-2, the RNase III-type
enzymes that produce miRNAs and siRNAs, respectively,in flies (Vagin et al., 2006). However, this model does notaddress the mechanism for the 30 end modification ofrasiRNAs and piRNAs that is not seen for miRNAs (Houwing et al., 2007; Vagin et al., 2006). Thus, there are open
questions. How are Drosophila Ago3, Piwi, and Aub able
to specifically recognize the respective sense andantisense strands of repetitive elements? Which Piwi proteins are the mammalian counterparts to the Drosophila
proteins?
The discovery of piRNAs and rasiRNAs in mammals andflies implied that other organisms that express Piwi
proteins might express piRNAs. In fact, the first Piwi protein shown to bind small RNAs was TWI1, a Tetrahymena
thermophila homolog. TWI1 binds 30 nt long scan RNAs
(scnRNAs), which derive from the micronucleus (reviewed
in Mochizuki and Gorovsky [2004]). During conjugative
replication, the complex of TWI1 and scnRNAs serves tointerrogate the genome of the daughter macronucleus,
where the complex directs elimination of DNA sequencesthat are homologous to the scnRNAs. In other vertebrates,
the zebrafish Piwi protein, Ziwi, has recently been shownto bind fish piRNAs that derive from single-stranded clusters as well as repetitive elements (Houwing et al., 2007).
Because the interaction of Piwi proteins with small RNAsis clearly a conserved feature, key aspects of the molecular function for Piwi proteins might also be conserved.
A Function for Piwi in Transcriptional Gene
Silencing Processes?
It is apparent from their conservation and distinct characteristics that elucidating Piwi and piRNA function in
mammals will be important; however, at present, wemust look toward model genetic organisms for clues
about function. Work in flies indicated a role for Piwi
proteins and rasiRNAs in silencing of repetitive elements.
In the fission yeast Schizosaccharomyces pombe, small
RNAs and the RNAi pathway are involved in transcriptional
gene silencing (TGS). By analogy, mammalian piRNAs
might also play a role in TGS (Figure 2B).
The genetics of Piwi proteins in flies have suggested
interplay with TGS mechanisms. Piwi, aubergine, andspindle-E (a helicase) mutants, which have compromisedlevels of fly rasiRNAs (Vagin et al., 2006), can suppress
position effect variegation of a white reporter and display
loss of histone H3-lysine 9 methyl marks (H3K9-Me) andheterochromatin protein 1 (HP1) binding at normallysilenced loci (Pal-Bhadra et al., 2004). Additionally, the606 Molecular Cell 26, June 8, 2007 ª2007 Elsevier Inc.

Piwi gene interacts genetically with the regulation of
a Polycomb response element and Polycomb proteinfunction, suggesting a role in influencing cellular identity
during embryonic development, through a chromatindependent mechanism (Grimaud et al., 2006).
Because yeast and plants lack Piwi subfamily members,
small RNA-dependent mechanisms in these organisms
rely entirely on Argonaute subclade proteins. Certain
Ago proteins in yeast and plants function in TGS (reviewed
in Zaratiegui et al. [2007]). S. pombe Ago1 binds heterochromatic siRNAs and constitutes part of the RNAinitiated transcriptional silencing (RITS) complex, which
directs the establishment of H3K9-Me marks and the subsequent recruitment of the repressive S. pombe HP1 protein. Genetic studies suggested that RITS acts in cis bybase pairing to nascent transcripts formed at these lociand is necessary for proper chromosome segregation. In
A. thaliana, Ago4 is involved in the silencing of inverted repeats, whereby heterochromatic siRNAs guide Ago4 to
specify H3K9-Me marks and DNA methylation to silent
repetitive regions of the genome. Like piRNAs andrasiRNAs, plant heterochromatic siRNAs (R24 nt) arelonger than miRNAs and siRNAs. Additionally, both fungal
and plant siRNAs map as clusters at heterochromatic lociand lack the sequence conservation seen for plant andanimal miRNAs.
Despite the precedents established in yeast and plants,
a direct mechanistic link for fly and mammalian Piwi proteins acting on chromatin remains to be demonstrated.
There is some support for this notion: piRNA features
are clearly distinct from those of miRNAs (piRNAs havepoor primary sequence conservation and immense complexity), and piRNA sequences cluster to discrete originating loci. Thus, piRNA clusters resemble plant and fungalloci that produce clusters of heterochromatic siRNAs
and the scnRNA elements in ciliates. A major proportionof fly and fish piRNAs also map to transposons, repetitiveelements, and heterochromatic regions, which parallels
the repetitive nature of heterochromatic siRNA loci inlower eukaryotes (Aravin et al., 2003; Brennecke et al.,2007; Houwing et al., 2007). Various epigenetic mechanisms have been described for controlling repetitive elements (Zaratiegui et al., 2007). Although the biogenesis
model proposed above for fly piRNAs suggests a PTGS
mechanism for transposon control, it does not rule out
TGS processes.
The link between repetitive elements and piRNAs is lessobvious in mammals, because repeat element sequences
are underrepresented in pachytene piRNA sequences(20%), compared to the 40% total repetitive elementsin the genome (Girard et al., 2006; Lau et al., 2006). However, prepachytene piRNAs are more enriched in repeats(35%), and Mili mutant testis exhibit substantial DNA
demethylation of the L1 retrotransposon (Aravin et al.,2007). In addition, the knockout of Miwi2, a third mouse
Piwi member, results in transposon expression and lossof DNA methylation at some transposon loci (Carmell
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Short Review
Piwi proteins in TGS by regulating transposable elements(TEs) via an RNA-dependent DNA methylation mechanism. The mammalian germline is known to undergo extensive genome-wide reprogramming events during development—perhaps a portion of these events involve
Piwi and piRNAs, given the abundant, germline-specificexpression of this ribonucleoprotein. However, it remains
to be determined whether piRNA complexes target transposable elements throughout the genome, or whether
they target the piRNA clusters in cis. Determining the chromatin structure at piRNA loci will be important for assessing their role in TGS.
Piwi’s Roles in Germline and Stem
Cell Maintenance
An epigenetic role for Piwi proteins in animals is an attractive hypothesis, because epigenetic regulation has
emerged as an important paradigm for how stem cells
and germ cells maintain pluripotency. Such a consideration puts into new perspective the rich biology of germline and stem cell regulation that was previously characterized for animal Piwi proteins. Fly piwi and aubergine
mutants have been known to have defects in gaemetogenesis (reviewed in Carmell et al. [2002]). Female aubergine flies produce mostly deformed oocytes, whereas
male aubergine mutants are sterile because sperm areunable to silence the deleterious and repetitive Stellateelements. Male and female piwi mutants begin with normal numbers of germ cells at the onset of gametogenesis,but the renewal of the germ stem cells are disrupted andadults become completely depleted of gametes. Knockdowns of both Caenorhabditis elegans Piwi homologs,
prg-1 and prg-2, produces various penetrance of reduced
gonads (Cox et al., 1998); however, only a knockout of
prg-2 results in a temperature-sensitive sperm defect(C.C. Mello and P.J. Batista, personal communication).
Because C. elegans has an expanded class of wormspecific Ago members that segregate from both the Ago
and Piwi subfamilies (Tolia and Joshua-Tor, 2007), assessing function and redundancy of prg-1 and prg-2 iscomplicated.
The germline role for Piwi proteins is conserved in
mammals. Homozygous male knockout mice of Miwi,
Mili, and Miwi2 become sterile as a result of arrested spermatogenesis and eventual apoptosis of the germ cells,
which is evident in the smaller size of the testis (Carmell
et al., 2002, 2007; Deng and Lin, 2002; KuramochiMiyagawa et al., 2004). Despite the similar phenotypic
outcomes of apoptotic sperm and depleted germ cells inthese knockouts, there may be subtle functional differences between the three proteins because of differential
timings of expression and different aspects of spermatogenic arrest. In contrast, homozygous knockout femalesof Miwi, Mili, and Miwi2 are fertile and produce normal
broods, suggesting either a lack of function or redundancy
for these proteins in mammalian oogenesis.
Amongst vertebrates, the male-specific germline function of Piwi proteins may be unique to mammals, because

other vertebrates like zebrafish depend on Piwi proteins for
germ-cell development in both the testis and ovary (Houwing et al., 2007). Perhaps Piwi proteins exert a pronouncedrole in mammalian males because only the male germ cells
undergo continuous mitosis and meiosis during adulthood, like the oocytes of flies and fish. Alternatively, Ziwi
may resemble fly Piwi proteins more closely than mammalian Piwi proteins, as reflected in not only the genetic
requirement in oogenesis but also the enrichment of repeat-associated piRNAs. This parallel might be extended
by the maternal contribution of Piwi proteins and piRNAs
to the oocyte, which has also been observed for the sea
urchin Piwi homolog, Seawi (Rodriguez et al., 2005).
Maternal contribution of Piwi proteins and piRNAs, aswell as the sequences of repetitive piRNA themselves,supports the hypothesis that they have roles in transposon
control in the Drosophila and zebrafish germline. In fact,
inheritance of transposon control may be a necessarycomponent of the female germline. For example, in thegenetic phenomenon of hybrid dysgenesis, the cross ofa male carrying a repressed transposon to a female lacking the transposon, results in offspring containing theactive transposon, which ultimately results in sterility.
The reciprocal case—a cross between a female carrying
a repressed transposon with a male lacking it—does not
result in transmission of the transposon to the offspring.
In Drosophila, the inheritance of transposon repression
is mediated by rasiRNAs and Piwi proteins. rasiRNAs corresponding to the Penelope transposon mediate female
germline fertility in D.virilis (Blumenstiel and Hartl, 2005),
whereas the flamenco locus, a master controller of theactivity of the retroviral gypsy element, is a rasiRNA cluster
that, when mutated, results in severe reductions inflamenco-derived rasiRNAs (Brennecke et al., 2007).
Additionally, a piwi mutant results in elevated gypsy transcription (Brennecke et al., 2007; Pelisson et al., 2007). In
mice, the prepachytene piRNAs map to loci that resemble
the master transposon control loci in Drosophila, andtogether with Mili, may have a function in transposon suppression (Aravin et al., 2007).
There is both supporting and confounding evidence forthe hypothesis that Piwi proteins serve an epigenetic rolein nuclear surveillance. Piwi is observed in the nucleus aswell as the cytoplasm of germline cells (Cox et al., 2000;
Megosh et al., 2006; Saito et al., 2006), whereas Ago3and Aub are mostly detected in the cytoplasm and in
germline granules called nuage (Brennecke et al., 2007;
Gunawardane et al., 2007; Harris and Macdonald, 2001).
Miwi and Ziwi also appear to localize intensely in perinuclear germline granules (Grivna et al., 2006b; Houwing
et al., 2007), and in particular, Miwi is a component of
the chromatoid body, a dynamic cytoplasmic structurethat repeatedly contacts the nuclear periphery (Kotaja
and Sassone-Corsi, 2007). Finally, a fraction of Miwi comigrates with polysomes on density gradients, suggestive ofa large cytoplasmic piRNA complex (Grivna et al., 2006b).
However, the cytology of Piwi will not necessarily reflect
the dynamic functions of this protein. For example, Ago1

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in S. pombe clearly regulates chromosome segregation,
yet cytology shows pronounced cytoplasmic staining
and modest nuclear localization (Buker et al., 2007).
Studies that catalog interacting partners of Piwi proteinsshould yield insight into these issues. For example,
evidence has suggested that Seawi can interact with thespindle apparatus (Rodriguez et al., 2005), whereas
aubergine ovarioles have disorganized microtubules(Klattenhoff et al., 2007). Thus, it is possible that animal
Piwi proteins in the cytoplasm are poised in machinerythat impacts chromatin during cell division.
The strongest evidence for a direct epigenetic role for
Piwi-like proteins has been described in ciliates, whichhave expanded the numbers of Piwi members in theirgenome (and notably do not contain Ago-subclade
members, Figure 1). TWI1 is essential for conjugative
replication and maturation of the macronucleus, or the‘‘somatic’’ genome that is actively transcribed and is separate from the micronucleus, or ‘‘germline’’ genome that istranscriptionally inert (Mochizuki and Gorovsky, 2004).
TWI1 triggers site-specific elimination of repetitive, transposon-like sequences within the macronucleus by using
scnRNAs to direct H3K9-Me marks to these loci. Thus,
in contrast to animals, TWI1 seems to directly impact
the somatic genome rather than the germline in the ciliate.
Do Piwi proteins have a broader function beyond regulating the germline? Although the data for mammalian Piwi
proteins impacting somatic stem cells is still unclear,a remarkable example of Piwi proteins regulating stem
cells is Smedwi-2 from the flatworm planaria Schmidtea
mediterranea. Smedwi-2’s expression pattern nearcompletely coincides with the patterns of the neoblasts,
totipotent stem cells that endow planarians the extraordinary capacity to regenerate entire organs and body structures (Reddien et al., 2005). Knockdown of Smedwi-2
significantly impairs planaria from restoring body parts
after wounding; however, this does not alter the expansion
and migration of neoblasts to the wound. Rather, properdifferentiation of the neoblast progeny is perturbed, andrenewal of neoblasts at the damage site is not maintained(Reddien et al., 2005). Although planarian piRNAs have notbeen described, it is tempting to speculate what genetic
elements are being regulated by Smedwi-2.
Resolving the function of Piwi proteins and piRNAs has
broad implications not only in understanding their essential role in fertility, germline, and stem cell development
but also in the basic control and evolution of animal
genomes. Because intergenic and repetitive elements
comprise the majority of animal genomes, cells must
effectively deal with this complex yet poorly understoodbulk sequence. Piwi proteins and piRNAs add to our comprehension of genomic elements that previously were notknown to transcribe functional gene products.

due to space constraints. N.C.L. is supported by a Whitney Foundation
Fellowship, and R.E.K. is funded by the National Institutes of Health.

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