Leading Edge

Review
Mighty Piwis Defend the Germline against
Genome Intruders
Kathryn A. O’Donnell1,2 and Jef D. Boeke1,2,*

Department of Molecular Biology and Genetics
The High Throughput Biology Center
The Johns Hopkins University School of Medicine, 733 N. Broadway, Baltimore, MD 21205, USA*Correspondence: jboeke@bs.jhmi.edu
DOI 10.1016/j.cell.2007.03.028
1
2

Piwis are a germline-specific subclass of the Argonaute family of RNA interference (RNAi)
effector proteins that are associated with a recently discovered group of small RNAs
(piRNAs). Recent studies in Drosophila and zebrafish directly implicate Piwi proteins in
piRNA biogenesis to maintain transposon silencing in the germline genome (Brennecke etal., 2007; Gunawardane et al., 2007; Houwing et al., 2007). This function may be conservedin mice as loss of Miwi2, a mouse Piwi homolog, leads to germline stem cell and meiotic
defects correlated with increased transposon activity (Carmell et al., 2007).
Mobile elements can insert themselves at new locations in host genomes to modify gene structure andalter gene expression. Rampant mobility of these elements would endanger both the host and, thereby, theelement. Thus a strong selective pressure exists tolimit their transposition. Mobile elements are classified
into two categories based on the mechanism of theirtransposition. DNA transposons, such as Drosophila P
elements, generally utilize a cut-and-paste mechanism
in which the transposon is excised from a donor site
and inserted into a new genomic location. Retrotransposons and endogenous retroviruses such as gypsy
elements represent a distinct class of mobile genetic
sequences that insert into new genomic locations byreverse transcription of an RNA intermediate. Expansion of these selfish elements can occur when noveltransposition events are transmitted to subsequent
generations after germline hopping; indeed metazoantransposons often show germline-restricted expression. Therefore, it seems likely that metazoan genomes
have evolved mechanisms to regulate germline mobilization of transposable elements. DNA methylation is
one important mechanism involved in the silencing oftransposons in plant, mammalian, and fungal germlines(Yoder et al., 1997; Martienssen and Colot, 2001; Selker,
2004). Additionally, APOBECs (a class of RNA/DNAediting enzymes) have been found to be potent genome
defense proteins against retroelements (Takaori-Kondo,
2006). RNAi is widely believed to control retrotransposition (Robert et al., 2004); however, this system hasa surprisingly modest effect on silencing mammalian
retrotransposons in somatic cells (Yang and Kazazian,2006). With the recent characterization of the molecularfunction of Piwi (P element-induced wimpy testes) proteins, a novel form of control for mobile elements has
emerged involving small RNAs in germ cells.

The founding member of this class of proteins, Piwi,
was first identified 10 years ago in a genetic screen formutants that affect asymmetric division of stem cells in
the Drosophila germline (Lin and Spradling, 1997). Early
studies demonstrated that Drosophila Piwi is essential
for spermatogenesis and is a key regulator of female
germline stem cells (Cox et al., 2000). It was also appreciated that Piwi proteins are an ancient subset of thelarger Argonaute protein family (Carmell et al., 2002;
Cerutti and Casas-Mollano, 2006), other members ofwhich associate with short-interfering (si)RNAs andmicro (mi)RNAs. These small RNAs serve as guides thatlead to degradation and/or reduced translation of target
mRNAs. Membership in the Argonaute family suggested
that Piwi proteins and their associated RNAs mightalso mediate RNA silencing. The recent identificationand characterization of the small Piwi-interacting RNAs
(dubbed piRNAs) has indicated that Piwi proteins mediate RNA-mediated silencing of mobile elements, thereby
defending the germline genome.
Identification of piRNAs that Bind Mammalian Piwi
Proteins
The murine Piwi orthologs Miwi and Mili are essential for
mammalian spermatogenesis (Deng and Lin, 2002; Kuramochi-Miyagawa et al., 2004). Mice with targeted mutations in either gene are sterile and have distinct defects
in gametogenesis, but unlike the Drosophila piwi mutant,neither loses germline stem cells. To investigate the roleof the third mouse Piwi family member in gametogenesis, the gene encoding Miwi2 has now been disrupted.
In a report described in Developmental Cell, Carmell etal. (2007) demonstrate that Miwi2 mutants are uniquein their loss of germline stem cells. These observations
suggest that the stem cell maintenance functions exhibited by Drosophila Piwi are conserved in mice throughCell 129, April 6, 2007 ©2007 Elsevier Inc. 37

the function of Miwi2. After initial characterization of the
MILI/MIWI proteins in mice, the next challenge was to
identify their small RNA-binding partners.
Last year, five independent laboratories reported theidentification of mammalian piRNAs from mouse and rat
testes (Aravin et al., 2006; Girard et al., 2006; Grivna etal., 2006; Lau et al., 2006; Watanabe et al., 2006). Twoof these groups purified ribonucleoprotein complexes
with a MILI- or MIWI-specific antibody from adult mousetestes and then cloned and sequenced the associatedsmall RNAs. These MILI- and MIWI-interacting RNAs
were termed piRNAs based on their interaction with themouse Piwi proteins.
piRNAs have several interesting characteristics. First,
these small RNAs were longer than miRNAs and siRNAs
and similar in size to a previously described class of
Drosophila RNAs corresponding to repeat sequences,“rasiRNAs” (repeat-associated siRNAs; Aravin et al.,2003). Second, the majority of piRNAs mapped to asmall number of genomic loci. Individual clusters range
between 1 and 100 kb in size and contain between 10
and 4500 piRNAs, demonstrating that thousands ofpiRNAs may be generated from one particular locus.
Third, many of these clusters exhibit remarkable asymmetry, meaning that within a given cluster all piRNAs arederived from the same strand. This asymmetric orientation suggests that piRNAs might be processed from longprimary transcripts. When two adjacent clusters werelocated in close proximity to each other, strand switching was also commonly observed. Aravin et al. (2006)
postulated that these neighboring clusters with opposite
strand polarity might be transcribed divergently fromone bidirectional promoter. Sequence analysis of the
MILI- and MIWI-associated piRNAs revealed a strong
bias for uridine residues at their 5′ termini. This 5′ uridine
bias is characteristic of siRNAs and miRNAs processedfrom double-stranded precursors by RNase III enzymes.
However, a computational search for stem loops similar to pre-miRNAs failed to identify any secondary
structures in regions flanking piRNAs, suggesting thatpiRNA processing is distinct from miRNA biogenesis.
Finally, ?17% of mammalian piRNAs mapped to repeat
sequences, including LINEs, SINEs, and several classesof DNA transposons. Although this is consistent with apossible role in mobile element defense, considering that
?40% of the mouse genome is composed of repetitiveelements, this is actually less than expected by chance.
However, a conserved role for Miwi2 in mobile element
control is suggested by the observation of increased L1
retrotransposon expression in the Miwi2 mutant testes(Carmell et al., 2007). Interestingly, this increase in L1
transcription was accompanied by decreased L1 DNA
methylation, suggesting a possible interplay between
Piwi (and perhaps piRNAs) and methylation machinery, reminiscent of the interaction between the siRNA
posttranscriptional silencing machinery and chromatinlevel transcriptional regulation in Schizosaccharomyces
pombe (Verdel et al., 2004). However, this analogy not38 Cell 129, April 6, 2007 ©2007 Elsevier Inc.

withstanding, it is important to note that no Miwi2-specific piRNAs have yet been described, so it is formallypossible that this pathway is piRNA independent. This
raises the question, how pervasive is the Piwi-piRNAgenome defense association?
In several of the earlier piRNA sequence studies, themajority of piRNAs were identified only once, suggesting a high degree of complexity in piRNA populations.
Comparative genomics further revealed that the piRNAloci, but not their sequences, are conserved throughout
evolution. As Girard et al. (2006) point out, this may indicate that the sequence of a piRNA does not necessarily specify its function. Rather, its true function may be
determined by the abundance of piRNAs produced fromany individual locus.
Despite these interesting and confounding discoveries, several important questions remained. Do piRNAs
exist in invertebrates and other vertebrate species?
What are their mRNA targets? Are piRNAs similar to Drosophila rasiRNAs? Is there more compelling evidence
that piRNAs provide defense against genome intruders
like mobile elements? Two new papers, one in this issue
of Cell (Brennecke et al., 2007; Houwing et al., 2007),shed light on some of these questions and provide us
with more food for thought.
piRNAs and Mobile Element Defense in Drosophila
Although piRNAs were first identified in mammals,
analogous studies in flies revealed that this class of
small RNAs also exists in invertebrates. Recently, two
Piwi family members in Drosophila, Aubergine andPiwi, were found to bind small RNAs (Saito et al., 2006;
Vagin et al., 2006). In a study reporting a few hundred
piRNA sequences, Saito et al. demonstrated that Piwi
complex immunopurified from Drosophila ovaries contained a class of small RNAs distinct in size from siRNAs
and miRNAs. Sequencing revealed that most of these­piRNAs corresponded to repetitive elements and heterochromatic genome regions. Tuschl and colleagues had
previously identified about 4000 Drosophila germline
rasiRNAs (Aravin et al., 2003), which also corresponded
to repetitive elements, suggesting that they might regulate chromatin structure and transposon activity. Basedon current evidence, it appears that most rasiRNAs inflies are simply a (very important) subclass of piRNAs.
In recent years, Piwi proteins were recognized as having potential anti-mobile element activity. Transpositionof telomeric retroelements and P elements is enhanced
in aubergine mutants whereas piwi mutants mobilized
the endogenous retrovirus gypsy (Sarot et al., 2004)
and showed increased expression of copia and mdg1
elements (Kalmykova et al., 2005). Vagin et al. (2006)
also demonstrated that expression of retrotransposons
was derepressed in the germline of piwi and aubergine
mutants. Importantly, silencing of these retroelements
did not require RNAi or miRNA proteins. These findings
suggested that Piwi proteins and their associated small
RNAs might silence mobile elements in the germline.

Figure 1. piRNAs Control Mobile­Elements in the Drosophila Germline

piRNAs (Piwi-interacting RNAs) are generated
from specific loci throughout the Drosophila
germline genome. Two examples are depicted. The Flamenco and X-TAS loci are locatedin heterochromatic regions on the X chromosome. Hundreds of distinct piRNAs are produced from each of these loci and correspondto mobile element repeats dispersed throughout the genome. According to the current
model, piRNAs associate with Piwi proteinsin the germline and serve as guides that leadto cleavage of transposon targets. Flamenco
is known to control expression of the gypsy,
Idefix, and ZAM retroelements, and X-TAS has
been linked to the control of P elements.

In a recent issue of Cell, Brennecke et al. (2007) investigate the small RNA-binding partners of Piwi, Aubergine, and Ago3 in the Drosophila female germline athigh resolution. After purifying RNP complexes using
antibodies specific to each of the three proteins, cDNA
libraries were prepared from each of the piRNA populations. 454 sequencing yielded more than 60,000 piRNA
reads, providing a much larger sequence population to
analyze than in the earlier fly studies. Similar to mammalian piRNAs, Drosophila piRNAs are longer than ­miRNAs
and siRNAs and map to discrete genomic clusters. For
example, the largest 15 clusters account for 70% of allpiRNAs, suggesting that a limited number of master
piRNA loci might control germline mobile element activity. Unlike their mammalian counterparts, most piRNAs
in flies (?80%) are present in pericentromeric and telomeric heterochromatin and correspond to truncated or
defective repeat elements.
How do these findings square with earlier studies oftransposon control mechanisms? One model of transposon control proposes that transposon resistance isdue to discrete genomic loci and is supported by studies of the gypsy element, the first endogenous retrovirus
discovered in invertebrates. The mobility of gypsy andtwo other retroelements, Idefix and ZAM, is controlled
by flamenco, a specific heterochromatic locus in the X
chromosome (Bucheton, 1995). Despite intensive studyof flamenco, no “transposon repressor locus” could
be identified in the sequence. Rather, it contained ajumble of different types of transposable elements,
but exactly how these elements might be involved ina transposon defense system remained unclear. Sarotet al. (2004) provided one connection by showing that­flamenco-mediated silencing of gypsy depends on Piwi.
Now, Brennecke and colleagues (2007) provide direct
sequence evidence that a large piRNA locus spanningmore than 150 kb corresponds to the flamenco locus.
The depth of the sequencing allowed them to find many
instances of mobile element-derived piRNAs mapping
uniquely to flamenco. Further supporting the notionthat piRNA clusters are control loci that regulate transposon activity through the Piwi pathway, Brennecke etal. performed several functional tests using flamenco
mutants. In agreement with their hypothesis, maturepiRNA expression levels decreased in flamenco mutants
whereas gypsy mRNA expression increased.
Brennecke et al. also demonstrate that the subtelomeric TAS repeat on the X chromosome (X-TAS) corresponds to yet another piRNA cluster. Previous studies
have linked specific alleles of this locus, here designated
X-TASP, to the global control of P elements (see references in Brennecke et al., 2007). Those alleles are distinguished by containing P element insertions in X-TAS.
The sites from which piRNAs (not complementary to P
elements) emanate in the Oregon R fly strain analyzed
by Brennecke et al. (2007) correspond to the insertion
positions of three P elements found in a series of XTASP strains. The Oregon R fly strain does not contain
P sequences at X-TAS. Thus it seems likely that the XTASP loci will produce P element-derived piRNAs. Thisis truly remarkable because P elements invaded the D.
melanogaster genome only within the last 50 years, presumably sweeping in through contact with a sibling species (Kidwell, 1983). The implication is that the resistance locus was born when P elements inserted into
X-TAS, within very recent history, showing how dynamic
the interplay between host and genome parasite can beeven on a short time scale.
A model for piRNA-mediated suppression of transposons is shown in Figure 1. Using flamenco and X-TAS
as examples, these heterochromatic loci generate hundreds of distinct piRNAs that correspond to transposon
repeats dispersed throughout the Drosophila genome.
These piRNAs associate with Piwi proteins and serve as
guides that lead to cleavage of expressed transposon
targets.
By examining the strand bias of piRNAs derived fromeach of the three Piwi complexes, these authors, as well
as Gunawardane et al. (2007) who performed a smaller
piRNA sequencing study, made several other important
observations consistent with a genome defense mechanism. Piwi and Aubergine preferentially bind piRNAs
corresponding to the antisense strand of transposons.
In contrast, Ago3 complexes are biased for the sense
strand of transposons. Perhaps one of the most intriguing findings is the observation of a unique complementary relationship between these sense and antisense­piRNAs. Assuming that piRNAs are ?25 nucleotides long,
Cell 129, April 6, 2007 ©2007 Elsevier Inc. 39

Figure 2. An Amplification Loop for piRNA Biogenesis
Piwi-mediated cleavage events generate new piRNAs, thereby setting up a self-reinforcing amplification cycle. The cycle begins with processing ofprimary piRNAs, which are derived from defective transposon copies in regions of heterochromatin (labeled A–E). Piwi proteins cleave targets between nucleotides 10 and 11 from the 5′ end of piRNAs. An unidentified endonuclease cleaves the 3′ end of piRNA precursors. Primary piRNAs are
antisense to expressed transposons and bind either Piwi or Aubergine (Aub) proteins. Together, the Piwi/Aub-piRNA complexes identify and cleave
their transposon target transcripts, generating new sense piRNAs that bind the Ago3 protein. This secondary piRNA-Ago3 complex directs a second
cleavage event of another piRNA cluster transcript, which creates a new antisense piRNA capable of binding to Piwi or Aubergine. Amplification canoccur whenever transcription of transposons and/or pre-piRNA transcripts pumps additional unprocessed RNAs into the system (green arrows).The
cycle continues as long as secondary piRNAs are able to recognize and cleave their target transposon elements, generating new piRNAs. This cycle
has the potential to generate an uncontrolled positive feedback loop and thus must be regulated. Potentially, accumulation of unprocessed piRNA
precursors (i.e., in response to diminished transposon RNA production) might dampen the piRNA response.

one would expect corresponding sense and antisense
piRNAs to overlap by 23 nucleotides with a 2 nucleotide
3′ overhang at each end if processed in an siRNA- or
miRNA-like manner. In fact, this was not observed withcomplementary piRNAs. Instead, the 5′ ends of complementary piRNAs were separated by 10 nucleotides,with the strongest complementarity observed between
Ago3- and Aubergine-associated piRNAs.
Yet another surprise was the enrichment of 5′-terminal uridine residues in Piwi- and Aubergine-boundpiRNAs, which correspond to the antisense strand oftransposons. As might be expected for sense strand
piRNAs bound to Ago3, these show a dramatic enrichment for adenine at position 10, and they complement
the 5′-terminal uridine of an antisense piRNA bound to
40 Cell 129, April 6, 2007 ©2007 Elsevier Inc.

Piwi or Aubergine. Notably, the same strand bias wasobserved for piRNAs bound to Drosophila Piwi proteins(Gunawardane et al., 2007). These findings suggest that
Piwi-mediated cleavage events generate new piRNAs.
In light of these findings, both groups propose a selfreinforcing amplification cycle for piRNA generation that
may be analogous to secondary siRNA generation by
RNA-dependent RNA polymerases (Figure 2). According
to this model, initiation of the cycle begins with processing of primary piRNAs, which are derived from defective
transposon copies in regions of heterochromatin. ThesepiRNAs are antisense to expressed transposons andbind either Piwi or Aubergine. Together, the Piwi/Aubergine-piRNA complexes identify and cleave their active
transposon targets, generating new sense piRNAs thatbind Ago3. Next, a sense piRNA-Ago3 complex directs
another cleavage event of a piRNA cluster transcript, creating a new antisense piRNA capable of binding to Piwi
or Aubergine. Amplification of the response is dependent on the interaction between piRNA sequences in different clusters. As long as secondary antisense piRNAcomplexes are able to recognize and silence their target
transposable elements, the cycle is reinforced throughthe production of additional sense piRNAs. Althoughseveral aspects of the model have yet to be validated,
this amplification loop has important implications formobile element control in the germline.
The proposed model raises several important questions. How is the amplification cycle initiated with primary antisense piRNAs loaded into Piwi or Aubergine?
Although it is logical that Ago3-bound piRNAs would bein the sense orientation if they were generated solely by
piRNA-mediated cleavage of transposon sequences,
the origin of the strict antisense strand bias of Piwi- andAubergine-bound piRNAs is not intuitive. Brennecke etal. (2007) demonstrate that there are special loci suchas flamenco from which piRNAs are generated fromonly one strand and specifically loaded onto Piwi. Yet
most piRNA-producing loci have the potential to produce both sense and antisense piRNAs. Knockout
studies of individual piRNA clusters will be necessary
in order to better understand their function.
What prevents constitutive autoamplification, whereby
sequences in different piRNA clusters interact to amplify
the response? Given that the majority of transposon
sequences are present in different clusters in both orientations, a transposon challenge is not really required
to amplify the response. Therefore, a mechanism must
be in place to prevent rampant, uncontrolled generationof piRNAs.
Is it possible that the subcellular localization of the different Piwi proteins in Drosophila may reflect how they areloaded with piRNAs? The nuclear localization of Piwi may
indicate that this protein is uniquely loaded with primarypiRNAs at sites of transcription, for example at the flamenco
locus. In contrast, Aubergine and Ago3 may be specificallyloaded with secondary piRNAs generated by target RNA
cleavage in the cytoplasm or perhaps P bodies.
In summary, the Brennecke et al. (2007) study extends
our understanding of the role of Piwi proteins in mobile
element silencing in the Drosophila germline. The discovery that piRNAs are generated from previously identified transposon control loci such as X-TAS and flamenco
illuminates previous findings from the transposon andRNA-silencing communities. Furthermore, this work
highlights the power of deep sequencing and reveals an
unexpected and exciting role for Piwi proteins in the biogenesis pathway of their small RNA-binding partners.
Vertebrate piRNAs
Although there are distinct differences in Piwi function between Drosophila and mice, it has remained
unclear to what extent Piwi function is conserved

between invertebrates and vertebrates. In this issue,
Houwing et al. (2007) present a study on one of thePiwi orthologs in zebrafish, Ziwi, and its associatedpiRNAs. The characterization of Ziwi mutant phenotypes reveals some common features but also someinteresting differences between fish and mammalian Piwi proteins. Expression of Ziwi is not required
for early specification of germ cells, yet loss of Ziwi
function results in a progressive loss of germ cells
and elevated levels of apoptosis in premeiotic cells inzebrafish after 3 weeks of age. However, it remains
uncertain whether increased apoptosis is a direct orindirect consequence of Ziwi loss.
In order to determine whether piRNAs are expressedin zebrafish germline cells, the investigators firstdetected a population of small RNAs 26–30 nucleotides long. Fractionation experiments demonstrated
that these small RNAs coelute with Ziwi, strongly suggesting that they are zebrafish piRNAs. In contrast to
mammalian piRNAs, which have only been identified
in testes to date, zebrafish piRNAs are expressedin male and female germ cells. In general, zebrafish
piRNAs have a strong 5′-terminal uridine bias, and as
in the fly, transposon repeats are modestly overrepresented (40% of piRNAs versus ?30%–40% of the
genome). But there was also an intriguing strand bias
observed for piRNAs derived from retroelements notseen with DNA elements. Ziwi piRNAs corresponding
to long terminal repeat (LTR) retrotransposons corresponded to the antisense strand of these repeats. In
fewer cases, when LTR-derived piRNAs matched thesense strand, they lacked a 5′-terminal uridine andinstead were enriched for an adenine at position 10,
reminiscent of the Ago3-bound piRNAs in Drosophila
and implying that a piRNA-based amplification loop
might be conserved in zebrafish.
Several lines of evidence suggest that zebrafish
piRNA biogenesis might be distinct from the mammalian piRNA-processing pathway. First, there is a striking periodicity of piRNAs within transposons occurring every 200–300 nucleotides. Second, zebrafish
strand bias can switch back and forth within a given
piRNA cluster. This was not seen in mammals, suggesting that transcription of piRNAs in zebrafish andmammals might differ.
Previous studies have suggested that piRNAs havea modified 3′ end structure (Vagin et al., 2006). Using
mass spectrometry, Houwing and colleagues present
evidence suggesting that 3′ ends of fish and mammalianpiRNAs may be 2′O-methyl modified. The significanceof this modification is unknown but is reminiscent of the
3′ ends of plant miRNAs, which have the same modification (Yu et al., 2005).
Normal levels of piRNAs are seen in zebrafish Dicer
mutants, demonstrating that just as in flies, Dicer is dispensable for the production of mature piRNAs. In contrast, piRNAs are not detected in morpholino-induced
Ziwi mutant testes. So what happens to transposon
Cell 129, April 6, 2007 ©2007 Elsevier Inc. 41

expression in Ziwi mutants? Unfortunately, the authors
were unable to draw any conclusion because germ cells
died in the absence of Ziwi, confounding the analysis oftransposon activity. So the question remains: Do germ
cells die due to the unchecked activation of mobile element activity? A conditional Ziwi mutant might help
answer this question. The roles of the second zebrafish
Piwi ortholog, Zili, and its associated piRNAs are yet
unknown.
Other Functions for piRNAs?
The fact that mammalian piRNAs differ from Drosophila
and zebrafish piRNAs—the majority of the former are
not recognizably transposon related—suggests thepossibility that mammalian piRNA machinery may haveacquired additional germline-specific functions. Perhaps this opportunity arose because most mammalian genomes, unlike those of invertebrates and lower
vertebrates, appear to have successfully eradicated
all but a few major lineages of mobile elements in theirgenomes. Thus the piRNA machinery may have been
exapted (i.e., usurped evolutionarily for a new purpose
over time) to perform some other germline functions.
A surprising clue to this comes from the fungus Neurospora, which has evolved an extensive “genome
paranoia” perhaps due to its “single cytoplasm–manynuclei” lifestyle, leaving it particularly vulnerable togenome invaders (Borkovich et al., 2004; Galagan etal., 2003). Indeed, a connection has been observed
between piRNA-mediated silencing in germ cells andthe phenomenon of “meiotic silencing”—which damps
meiotic expression of all copies of transposon/genefamilies, even if most copies are paired (Aramayo
and Metzenberg, 1996). The “unpaired” region generates a diffusible signal (presumably aberrant RNA)
processed by the SAD-1, SMS-2, and SMS-3 proteins
corresponding to the RNA-directed RNA polymerase,
Argonaute, and Dicer proteins of the meiotic RNAsilencing pathway, respectively (Shiu et al., 2001, 2006;
Lee et al., 2003). The meiotic nucleus of Neurospora isremarkable in that it resembles a primordial chromatoid body. This perinuclear granular structure is foundin mammalian postmeiotic round spermatids and isthought to be equivalent to the germ cell specification
structure nuage in Drosophila and zebrafish. There,
components of the RNA-silencing machinery localize
to the meiotic perinuclear membrane, suggesting that
Neurospora genome defense occurs at this location.
Therefore Neurospora (and possibly other eukaryotic)
germline cells recognize unpaired (unsynapsed) chromatin and use an RNAi-like mechanism to silence it.
A Perimeter Defense for the Genome?
Recently, the mouse maelstrom protein, MAEL, wasfound to interact with mouse MIWI and MILI proteins in
the chromatoid body (Costa et al., 2006). Although theexact function of MAEL remains unknown, it is localized to unsynapsed chromosomes during male meio42 Cell 129, April 6, 2007 ©2007 Elsevier Inc.

sis. Thus, the interaction between MAEL and mouse
Piwi proteins suggests that the mechanisms controlling meiotic silencing are perhaps related to piRNAmediated silencing of transposons during mammalianmeiosis. Like the mysterious P body found in mitotic
cells, the meiotic chromatoid body contains many proteins involved in the siRNA and miRNA pathways andis thought to be a site of RNA storage and processing.
Given that, MILI and MIWI in mouse, Aubergine andAgo3 in Drosophila, and Ziwi in zebrafish all localize tochromatoid bodies/nuage (Kotaja and Sassone-Corsi,2007); this structure may well be where the piRNA
pathway actually defends the genome from intrusionof mobile elements. The argument against this is thatin mouse, chromatoid body formation occurs after
retrotransposon methylation/silencing (Deng and Lin,
2002; Kuramochi-Miyagawa et al., 2004; Bourc’hisand Bestor, 2004; Lees-Murdock et al., 2005). However, evidence supporting this hypothesis comes fromrecent work from Lim and Kai (T. Kai, personal communication). They demonstrated that mutations in several proteins that localize to the nuage in Drosophilacaused a reduction in piRNA levels and derepressionof some transposons. Nevertheless, future studies
are necessary to determine the spatial and temporalsequence of events involved in the regulation of transposon silencing in the germline.
Perspective
Although major advances have been made in theidentification of piRNAs, we are at the very earliest stages of understanding their functions. One of
the most exciting challenges ahead will be to dissect the spectrum of piRNA function in meiosis, inmobile element control, and perhaps in other germline functions. There are many open questions in thepiRNA field ripe for further study. For example, whatis the nature of primary piRNA transcripts and how
are mature piRNAs processed? What happens when
specific piRNA loci are knocked out? How might bulk
production of piRNAs from individual loci dictate
function? In addition to suppressing repetitive elements in the germline, can piRNAs regulate spermatogenesis by affecting meiosis directly? It is tempting
to speculate that piRNAs have acquired additional
functions in mammals. This might explain the observation that far fewer piRNAs correspond to repetitiveelements in mammals as compared to Drosophila
and zebrafish piRNAs. Certainly, addressing theseand other questions will be essential if the functionof piRNAs is to be fully appreciated.
Acknowledgments
We thank R. Aramayo, G. Seydoux, J. Mendell, and especially W.
Engels for insightful comments and K. Howe at the Wellcome Trust
Sanger Institute for assistance in estimating transposon content inzebrafish. K.A.O. is a fellow of the Damon Runyon Cancer Research
Foundation.

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