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First published online 21 November 2007
doi: 10.1242/dev.006486
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Review |
Program in Molecular Medicine, Program in Cell Dynamics, University of Massachusetts Medical School, Worcester, MA 01605, USA.
* Author for correspondence (e-mail: william.theurkauf{at}umassmed.edu)
Accepted 15 October 2007
SUMMARY
Small interfering RNAs and microRNAs are generated from double-stranded RNA precursors by the Dicer endonucleases, and function with Argonaute-family proteins to target transcript destruction or to silence translation. A distinct class of 24- to 30-nucleotide-long RNAs, produced by a Dicer-independent mechanism, associates with Piwi-class Argonaute proteins. Studies in flies, fish and mice implicate these Piwi-associated RNAs (piRNAs) in germline development, silencing of selfish DNA elements, and in maintaining germline DNA integrity. However, whether piRNAs primarily control chromatin organization, gene transcription, RNA stability or RNA translation is not well understood, neither is piRNA biogenesis. Here, we review recent studies of piRNA production and function, and discuss unanswered questions about this intriguing new class of small RNAs.
Introduction
In 1993, Ambros and colleagues showed that the C. elegans lin-4
gene encodes a small regulatory RNA with complementarity to the
lin-14 transcription unit, which it negatively regulates
(Lee et al., 1993
). These
pioneering studies thus identified the first microRNA (miRNA). Small
non-coding RNAs have subsequently emerged as powerful experimental tools and
as crucial developmental regulators in animals and plants
(Baulcombe, 2004;
Hannon, 2002;
Kloosterman and Plasterk,
2006; Mello and Conte,
2004). 21-nucleotide (nt) small interfering RNAs (siRNAs), now
ubiquitously used to experimentally manipulate gene expression, are processed
from long, double-stranded RNA (dsRNA) precursors by the Dicer endonucleases.
The resulting 21-nt dsRNAs are incorporated into an intermediate RNA-protein
complex. Displacement of one of the RNA strands (referred to as the `passenger
strand') then produces the mature RNA-induced silencing complex (RISC), which
contains a single `guide strand' bound to a member of the Argonaute protein
family. When the guide-strand siRNA is perfectly complementary to a target
RNA, the Argonaute protein catalyzes sequence-specific endonucleolytic
cleavage (for reviews, see Hannon,
2002; Meister and Tuschl,
2004). In vivo, the siRNA pathway destabilizes RNA intermediates
generated during the viral life cycle, and thus plays an important role in
limiting virus infectivity (Wang et al.,
2006). By contrast, miRNAs are derived from stem-loop transcripts
encoded by chromosomal genes. Primary stem-loop RNAs (priRNA) are processed in
the nucleus by the ribonuclease Drosha, producing pre-miRNAs that are exported
from the nucleus and cleaved in the cytoplasm by a Dicer endonuclease to yield
22-nt mature miRNAs. These miRNAs associate with Argonaute proteins and
induce the homology-dependent downregulation of target gene activity.
Imperfect miRNA base-pairing to target transcripts appears to induce
translational silencing, whereas perfect base-pairing triggers RNA
destruction. Mutations in the miRNA pathway disrupt development and often lead
to embryonic lethality (reviewed by Du and
Zamore, 2005; Kloosterman and
Plasterk, 2006).
Recent studies have revealed a new class of 24- to 30-nt RNAs that are
generated by a Dicer-independent mechanism and that interact with a subset of
Argonaute proteins related to Piwi (Aravin
et al., 2006; Brennecke et
al., 2007; Girard et al.,
2006; Grivna et al.,
2006a; Gunawardane et al.,
2007; Houwing et al.,
2007; Lau et al.,
2006; Saito et al.,
2006; Vagin et al.,
2006; Watanabe et al.,
2006), which is required for female and male fertility in
Drosophila (Lin and Spradling,
1997). In some systems, these Piwi-interacting RNAs (piRNAs) are
primarily derived from transposons and other repeated sequence elements
(Brennecke et al., 2007;
Gunawardane et al., 2007;
Saito et al., 2006), leading
to their alternative designation as repeat-associated small interfering RNAs
(rasiRNAs) (Aravin et al.,
2003). It is now clear that piRNAs can be derived from either
repeated or complex DNA sequence elements
(Aravin et al., 2007;
Brennecke et al., 2007;
Houwing et al., 2007), and
that rasiRNAs are a subset of piRNAs. We therefore use the more generic term
piRNA in the following discussions. Genetic studies in mice,
Drosophila and zebrafish indicate that piRNAs are crucial to germline
development (Carmell et al.,
2007; Chen et al.,
2007; Cook et al.,
2004; Cox et al.,
1998; Cox et al.,
2000; Deng and Lin,
2002; Gillespie and Berg,
1995; Houwing et al.,
2007; Kuramochi-Miyagawa et
al., 2004; Pane et al.,
2007; Schupbach and Wieschaus,
1991). However, proteins involved in piRNA production have also
been implicated in the control of gene expression in somatic cells
(Grimaud et al., 2006;
Pal-Bhadra et al., 2002;
Pal-Bhadra et al., 2004) and
in learning and memory (Ashraf et al.,
2006), suggesting that piRNAs might have an impact on a broad
range of biological processes.
piRNA production
The 24- to 30-nt length of piRNAs is an indication that they are not
generated by a Dicer, which produce 21- to 22-nt products from double-stranded
precursors (Bernstein et al.,
2001). Recent genetic studies are consistent with the conclusion
that piRNA production is a Dicer-independent process
(Houwing et al., 2007;
Vagin et al., 2006). Insight
into the mechanism of piRNA production has come from studies of their genomic
origin and of Argonaute binding in Drosophila
(Aravin et al., 2006;
Brennecke et al., 2007;
Girard et al., 2006;
Grivna et al., 2006a;
Gunawardane et al., 2007;
Houwing et al., 2007;
Lau et al., 2006;
Saito et al., 2006;
Vagin et al., 2006). In
Drosophila ovaries, the vast majority of piRNAs appear to be derived
from a limited number of pericentromeric and telomeric sites that are enriched
for retrotransposon sequences (Brennecke
et al., 2007). The most abundant piRNAs derive from the antisense
strand of retrotransposon sequences, and these RNAs preferentially associate
with the Argonaute proteins Piwi and Aubergine (Aub). Sense-strand piRNAs, by
contrast, preferentially associate with Argonaute 3 (Ago3)
(Brennecke et al., 2007;
Gunawardane et al., 2007).
Piwi, Aub and Ago3, in complex with piRNAs, can cleave target RNAs between
positions 10 and 11 of the guide strand
(Gunawardane et al., 2007;
Saito et al., 2006).
Significantly, Drosophila piRNAs from opposite strands tend to have a
10-nt overlap. Furthermore, antisense piRNAs bound to Piwi and Aub show a
strong bias toward a U at the 5' end, whereas sense-strand piRNAs bound
to Ago3 tend to have an A residue at position 10
(Brennecke et al., 2007;
Gunawardane et al., 2007).
Based on these observations, two groups concurrently proposed a `ping-pong'
model of piRNA production, in which Ago3 bound to sense-strand piRNAs
catalyzes antisense-strand cleavage at an A:U base-pair that generates the
5' end of antisense piRNAs (Fig.
1) (Brennecke et al.,
2007; Gunawardane et al.,
2007). The 5' ends of the resulting cleavage products are
proposed to associate with Aub or Piwi, with nucleolytic processing of the
3' overhangs generating mature 23- to 30-nt antisense piRNAs
(Fig. 1B). The mature antisense
piRNA-Argonaute complexes are then proposed to bind and cleave sense-strand
RNAs, silencing gene expression and generating the 5' end of
sense-strand piRNA precursors that associate with Ago3
(Fig. 1D). Processing of the
3' overhang produces mature sense-strand piRNAs, completing the cycle
(Fig. 1E). This model is based
on studies in Drosophila, but recent findings suggest that a similar
mechanism might operate in mouse (Aravin et
al., 2007).
|
). Zucchini and Squash may therefore process the 3'
end extensions to generate mature piRNAs
(Fig. 1). In addition, the
3' end of mature piRNAs are methylated
(Kirino and Mourelatos, 2007;
Ohara et al., 2007). In
Drosophila, this reaction is carried out by the Hen1 RNA
methyltransferase (also known as DmHen1 and Pimet), and methylation appears to
take place after piRNAs bind to Argonautes
(Horwich et al., 2007;
Saito et al., 2007). A
mutation in Drosophila hen1 reduces the length and steady-state level
of piRNAs, suggesting that methylation limits the extent of 3'
processing and increases the stability of piRNAs
(Horwich et al., 2007).
Methylation could also influence interactions between piRNAs and additional
components of the piRNA pathway. However, Drosophila hen1 mutants are
viable and fertile, indicating that this modification is not essential to
piRNA function (Saito et al.,
2007).
Genetic screens in Drosophila have identified several additional
factors that are required for piRNA production or function. For example, the
armitage and spindle E genes encode putative helicases that
are required for piRNA production and for retrotransposon silencing
(Aravin et al., 2004;
Vagin et al., 2006). These
proteins could unwind duplex intermediates formed during piRNA production,
target recognition, or cleavage. By contrast, the cutoff gene is
required for retrotransposon silencing, but is not needed for piRNA production
(Chen et al., 2007). Yeast
homologs of Cutoff have been implicated in RNA decay
(Kim et al., 2004;
Xue et al., 2000). Cutoff
might therefore facilitate gene silencing by enhancing the activity of
piRNA-Argonaute complexes.
Compartmentalization of the piRNA pathway
Recently, the Drosophila Tudor-domain protein Krimper has been
implicated in both retrotransposon repression and piRNA production
(Lim and Kai, 2007). This
protein is a component of nuage, a germline-specific perinuclear structure
that has been implicated in RNA processing, and krimper mutations
block nuage assembly. Intriguingly, many piRNA-pathway-related proteins
accumulate in nuage, which is prominent in nurse cells. The
Drosophila oocyte develops in a cyst with 15 nurse cells, which
synthesize RNAs and proteins that are transported through ring canals to the
oocyte (Spradling, 1993).
Nuage was first identified in electron micrographs as an amorphous
electron-dense cloud that surrounds the nurse cell nuclei
(Allis et al., 1979;
Mahowald, 1971). Nuage is
enriched for the Piwi-class Argonautes Aub and Ago3
(Fig. 2A)
(Brennecke et al., 2007;
Harris and Macdonald, 2001),
the helicases Armitage and Spindle E (Cook
et al., 2004; Lim and Kai,
2007), the nucleases Zucchini and Squash
(Pane et al., 2007), Maelstrom
and Cutoff (Chen et al., 2007;
Findley et al., 2003). In
contrast to most piRNA-pathway proteins, Drosophila Piwi localizes
almost exclusively to nurse cell nuclei
(Fig. 2A)
(Brennecke et al., 2007;
Cox et al., 2000;
Saito et al., 2006). These
observations suggest that piRNA production and function might be
compartmentalized (Lim and Kai,
2007).
piRNA-Argonaute complexes appear to be the catalytically active effectors of the pathway, and these localization studies thus suggest that Piwi mediates nuclear functions for the piRNA pathway, whereas Ago3 and Aub drive cytoplasmic functions (Fig. 2B). We speculate that piRNA biogenesis, which is proposed to require sense- and antisense-strand Argonaute complexes, takes place in the nuage. In this model, the sense-strand piRNA precursor transcripts are exported to the nuage, where they are cleaved by Aub-antisense piRNA complexes, silencing target gene expression and generating precursors of sense-strand piRNAs. These sense-strand precursors associate with Ago3 and are trimmed to mature length. Ago3-sense-strand complexes then catalyze the cleavage of the antisense transcripts, producing piRNA precursors that associate with Aub and Piwi. Mature Aub complexes then remain in the nuage and function in piRNA production and sense-strand transcript destruction, whereas mature Piwi complexes are imported into the nucleus and mediate heterochromatin assembly and transcriptional silencing, or co-transcriptional RNA destruction. This model is highly speculative, but makes a number of clear predictions and might therefore serve as a useful framework for further studies on piRNA biogenesis.
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Stem cell division and axis specification in Drosophila females
Mutations in piRNA-pathway genes were first identified in
Drosophila through screens for mutations that disrupt oogenesis and
embryonic axis specification (Gillespie
and Berg, 1995; Schupbach and
Wieschaus, 1991). Drosophila oogenesis begins with a
germline stem cell division that produces a cystoblast, which divides four
times with incomplete cytokinesis to produce 16 interconnected cells that form
a single oocyte and 15 nurse cells (for a review, see
Spradling, 1993). The nurse
cells provide most of the RNA and protein components of the oocyte, which
remains transcriptionally silent through most of oogenesis. Embryonic axis
specification in Drosophila depends on the asymmetric localization of
a small number of morphogenetic RNAs in the oocyte. These RNAs are transferred
from the nurse cells to the oocyte, where localization is driven by
interactions with a polarized microtubule cytoskeleton. Microtubule
polarization is controlled by a cascade of germline-to-soma and
soma-to-germline signaling events (reviewed by
Grunert and St Johnston,
1996). Mutations in piRNA-pathway genes disrupt both stem cell
maintenance and oocyte production, and the localization of morphogenetic RNAs
in the oocyte during axis specification
(Chen et al., 2007;
Cook et al., 2004;
Cox et al., 1998;
Cox et al., 2000;
Gillespie and Berg, 1995;
Lin and Spradling, 1997;
Pane et al., 2007;
Schupbach and Wieschaus,
1991).
The piwi gene encodes the founding member of the Piwi class of
Argonautes (Cox et al., 1998).
Mutations in piwi lead to severe defects in oogenesis, including loss
of germline stem cells (Fig.
3A) (Cox et al.,
1998; Cox et al.,
2000; Lin and Spradling,
1997). Clonal studies indicate that stem cell maintenance and
division require piwi expression in the somatic cells that form the
stem cell niche. Loss of piwi in the germline, by contrast, reduces
stem cell division rates, but does not lead to a loss of stem cells or to a
block in oogenesis (Cox et al.,
2000). Mutations in other piRNA-pathway genes, including
zucchini and squash, also lead to germline stem cell loss
(Chen et al., 2007;
Pane et al., 2007). It is
unclear whether these genes are required in the germline, soma, or both. The
molecular functions of Piwi and the piRNA pathway in germline stem cell
division and maintenance have not been defined.
Mutations in most piRNA genes in Drosophila, including
aubergine, spindle E, armitage, maelstrom, krimper, zucchini and
squash, disrupt the localization of dorsal and posterior RNAs
(Cook et al., 2004;
Gillespie and Berg, 1995;
Pane et al., 2007;
Schupbach and Wieschaus,
1991). These mutations do not disrupt anterior localization of
bicoid mRNA or block oocyte development. Therefore, these genes were
initially assumed to control the expression of a specific subset of genes
required for anterior-posterior and dorsal-ventral patterning
(Cook et al., 2004).
Subsequent studies, however, have demonstrated that the dramatic
axis-specification defects that are associated with piRNA mutations are a
secondary consequence of DNA damage signaling
(Chen et al., 2007;
Klattenhoff et al., 2007;
Pane et al., 2007). These
studies suggest that piRNAs have a primary function in maintaining germline
DNA integrity.
The link between piRNAs and DNA damage signaling was suggested by studies
of Drosophila meiotic DNA-repair genes. Meiotic recombination
requires DNA break formation by the Spo11 nuclease
(Cao et al., 1990), and
Schupbach and colleagues have shown that mutations that disrupt meiotic DNA
break repair also disrupt anterior-posterior and dorsal-ventral axis
specification (Abdu et al.,
2002; Ghabrial et al.,
1998). Significantly, the Drosophila embryonic patterning
defects that are linked to repair mutations are dramatically suppressed by
mutations in mei-41 and mnk (also known as loki -
FlyBase), which encode the ATR and Chk2 kinases, respectively, which function
in DNA damage signaling, and by mutations in mei-W68
(Abdu et al., 2002;
Ghabrial et al., 1998), which
encodes the fly Spo11 homolog (McKim and
Hayashi-Hagihara, 1998). Unrepaired meiotic breaks thus appear to
activate the ATR and Chk2 kinases, which in turn trigger the observed
axis-specification defects. Recent studies show that the axis-specification
defects in armitage, aubergine, cutoff and squash are also
suppressed by mei-41 and/or mnk
(Chen et al., 2007;
Klattenhoff et al., 2007;
Pane et al., 2007).
Furthermore, armitage, aubergine and spindle E mutations
lead to a dramatic accumulation of phosphorylated histone H2Av (
-H2Av)
foci in germline nuclei (Fig.
3B); these foci are generally linked to DNA double-strand breaks
(Modesti and Kanaar, 2001).
Significantly, mei-W68 (Spo11) does not suppress the patterning
defects associated with armitage, or the formation of -H2Av
foci (Klattenhoff et al.,
2007). piRNA mutations, like DNA-repair mutations, thus disrupt
axis specification through activation of the ATR/Chk2 pathway. However, unlike
DNA-repair-pathway mutations, meiotic breaks are not the source of damage.
|
;
Chen et al., 2007;
Kalmykova et al., 2005;
Pane et al., 2007;
Sarot et al., 2004;
Vagin et al., 2006), and
piwi mutants have been shown to mobilize at least one class of
transposon in the male germline (Kalmykova
et al., 2005). Retrotransposon mobilization can induce DNA damage
(Belgnaoui et al., 2006;
Gasior et al., 2006), and high
rates of transposon insertion in piRNA mutants could overwhelm the DNA-repair
machinery, leading to the breaks that activate ATR and Chk2. However, there is
no direct evidence for transposon mobilization in the female germline when
piRNA-pathway components are disrupted, and this pathway could have a more
direct role in DNA repair, or in establishing chromatin structures that resist
damage. Drosophila telomeres are composed of retrotransposon repeats,
and piRNA mutations increase the number of these repeats
(Savitsky et al., 2006). These
findings suggest that piRNA mutations could lead to a loss of telomere
protection, leading to the recognition of chromosome ends as DNA breaks.
However, it is currently unclear whether the break sites in piRNA-pathway
mutations are random, linked to transposon insertions, or restricted to
specific chromatin domains, and the precise functions of these RNAs in
maintaining germline genome integrity remain to be determined.
The Oskar protein is essential for pole plasm assembly and for embryonic
patterning, and piRNA-pathway mutations disrupt osk mRNA and protein
localization (Cook et al.,
2004). The translation of osk mRNA is tightly linked to
its posterior localization, which begins during stage 9 of oogenesis
(Kim-Ha et al., 1995;
Markussen et al., 1995;
Rongo et al., 1995). Mutations
in a number of piRNA-pathway mutations lead to Oskar protein expression during
earlier stages of oogenesis, and the mnk (Chk2) mutation does not
suppress premature osk mRNA translation
(Cook et al., 2004;
Pane et al., 2007). Therefore,
premature Osk protein accumulation is not a consequence of DNA damage
signaling. Piwi-class Argonaute-piRNA complexes, like siRNA or miRNA-Argonaute
complexes, can cleave perfectly matched RNA targets in vitro
(Gunawardane et al., 2007;
Lau et al., 2006;
Saito et al., 2006). As noted
above, miRNA-Argonaute complexes that imperfectly pair with target mRNAs
induce translational silencing
(Valencia-Sanchez et al.,
2006). It is therefore possible that piRNA-Piwi-class-Argonaute
complexes also trigger the translational silencing of imperfectly matched
targets, including mRNAs from single-copy genes such as oskar.
Consistent with this speculation, a subset of piRNAs associates with polysomes
in the mouse (Grivna et al.,
2006b).
Fertility and Stellate silencing in Drosophila males
Most Drosophila piRNA-pathway mutations reduce male fertility
(Cox et al., 1998;
Schmidt et al., 1999;
Stapleton et al., 2001;
Tomari et al., 2004), and this
is linked to the overexpression of Stellate protein. The function of Stellate
is not known, but it is encoded by repeated genes on the X chromosome that are
suppressed by the Y-linked Suppressor of Stellate [Su(Ste)]
locus (Aravin et al., 2001;
Livak, 1990). Su(Ste)
consists of bi-directionally transcribed repeats that are highly homologous to
Stellate, and deletion of the Su(Ste) locus leads to the
massive overexpression of Stellate protein, which assembles into crystals in
the testes (Bozzetti et al.,
1995; Livak, 1984;
Palumbo et al., 1994). piRNAs
of 25 to 27 nt are produced from the Su(Ste) locus, and
mutations in piRNA-pathway genes lead to Stellate crystal formation
(Aravin et al., 2001;
Pane et al., 2007;
Stapleton et al., 2001;
Tomari et al., 2004). piRNAs
from the Su(Ste) locus thus silence expression of Stellate
in trans. It is currently unclear whether this reflects transcriptional or
post-transcriptional silencing. Stellate overexpression alone could induce
sterility, but defects in silencing of other genes could also impact male
fertility. piRNA-pathway mutations lead to mobilization of at least a subset
of transposons in the male germline
(Kalmykova et al., 2005), and
insertional mutations associated with transposon mobilization might also
reduce male fertility.
Male germline development in the mouse
The mouse genome encodes three Piwi homologs, Miwi, Miwi2 and Mili (also
known as Piwil1, Piwil4 and Piwil2, respectively - Mouse Genome Informatics),
and all three are expressed at high levels in testes and are required for male
fertility (Aravin et al., 2006;
Deng and Lin, 2002;
Girard et al., 2006;
Grivna et al., 2006a;
Kuramochi-Miyagawa et al.,
2001; Sasaki et al.,
2003). Both Mili and Miwi bind piRNAs, and knockout mutations in
the Mili and Miwi genes block piRNA production
(Aravin et al., 2006;
Girard et al., 2006;
Grivna et al., 2006a). Single
null-mutations in each of the three genes lead to male sterility
(Carmell et al., 2007;
Deng and Lin, 2002;
Kuramochi-Miyagawa et al.,
2004). Spermatogenesis in the mouse is a coordinated process that
can be divided into three phases: mitosis, meiosis and spermiogenesis
(de Rooij and Grootegoed,
1998). In the first phase, stem cells localized in the basal layer
of the epithelium divide mitotically to self-renew and generate a population
of primary spermatocytes. In the second phase, the primary spermatocytes
progress through meiosis to generate haploid round-spermatids. During
leptotene of meiotic prophase I, duplicated chromosomes condense and begin to
pair. Pairing is completed and the synaptonemal complex forms during zygotene,
and crossing over occurs in pachytene. The homologs begin to separate in
diplotene and finally resolve in diakinesis. During the third phase, round
spermatids mature and elongate and are then released into the lumen of the
tubule. In Mili, Miwi and Miwi2 mutants, the testes appear
normal until about 2 weeks post-partum, which roughly corresponds with the
first round of meiosis. However, post-meiotic cells do not form
(Fig. 3C). Mutations in
Mili and Miwi2 block progression through pachytene, whereas
Miwi mutant spermatocytes develop to the round-spermatid stage but do
not complete spermiogenesis (Carmell et
al., 2007; Deng and Lin,
2002; Kuramochi-Miyagawa et
al., 2004). The timing of developmental arrest correlates with the
temporal expression of Mili and Miwi proteins. Mili is first detected in male
primordial germ cells and is present throughout pachytene, whereas Miwi is
expressed only from mid-pachytene to the round-spermatid stage
(Deng and Lin, 2002;
Kuramochi-Miyagawa et al.,
2004). The temporal expression pattern of Miwi2 during
spermatogenesis has not been reported.
Mutations in each of the three genes lead to the degeneration of the male
germline, whereas somatic cells appear to remain relatively unaffected
(Carmell et al., 2007;
Deng and Lin, 2002;
Kuramochi-Miyagawa et al.,
2004) (Fig. 3C).
Similar spermatogenesis-arrest phenotypes have been observed in mutants that
disrupt synapsis or DNA repair (Baarends et
al., 2001; Barchi et al.,
2005; Xu et al.,
2003). Additionally, high levels of -H2AX staining,
indicative of DNA break formation, have been observed in Miwi2
mutants (Carmell et al.,
2007). All of the above suggest that mutations in piwi
homologs in mice, as with piRNA-pathway mutations in Drosophila, lead
to DNA damage and to the activation of a DNA damage response, including
apoptotic degeneration of germline cells.
In piRNA-pathway Drosophila mutants, germline DNA damage is
associated with the massive overexpression of retrotransposons, and most of
the piRNAs are linked to retrotransposon and repeated sequences
(Brennecke et al., 2007;
Klattenhoff et al., 2007;
Vagin et al., 2006). These
observations suggest that germline DNA damage is caused by transposon
mobilization, although this has not been demonstrated
(Klattenhoff et al., 2007). By
contrast, the piRNAs from adult mouse testes are depleted of repeated and
retrotransposon sequences (Aravin et al.,
2006; Girard et al.,
2006; Grivna et al.,
2006a). However, a recent study has identified a pre-pachytene
cluster of Mili-interacting piRNAs that include a substantial number of repeat
and retrotransposon sequences (Aravin et
al., 2007). Moreover, Mili and Miwi2 mutations
in mice lead to the derepression of retrotransposon transcripts
(Aravin et al., 2007;
Carmell et al., 2007). The
piRNA pathway might therefore have a conserved function in silencing
retrotransposons and preventing DNA damage in the germline. However, in
contrast to flies, the female germline is not affected by single mutations in
mouse Piwi-class Argonaute genes (Carmell
et al., 2007; Deng and Lin,
2002; Kuramochi-Miyagawa et
al., 2004). This could indicate that a distinct pathway fulfils
this role in the mammalian female germline. However, the Piwi-class Argonautes
could also act redundantly during oogenesis, and double or triple mutants
might therefore reveal a role for piRNAs in the mouse female germline.
Sex determination and germline development in zebrafish
The zebrafish genome encodes two clear Piwi homologs, ziwi (also
known as piwil1 - ZFIN) and zili. Ziwi appears to be an
ortholog of the mouse Miwi protein, whereas Zili is more similar to mouse
Mili. Only Ziwi, which is expressed specifically in the male and female
germline cells, has been characterized
(Houwing et al., 2007). Ziwi,
like the Drosophila Ago3 and Aub proteins, is primarily cytoplasmic
and localizes to perinuclear nuage. Strikingly, ziwi-null mutations
also result in the apoptotic loss of germ cells from the testes
(Fig. 3D). Reduced levels of
ziwi function permit the survival of male germ cells to the adult
stage, but lead to elevated levels of apoptosis in adult germ cells and to
varying levels of infertility. piRNAs isolated from zebrafish testes and
ovaries show the same molecular properties as piRNAs from other organisms, and
many are derived from repetitive sequences. Mutations in ziwi also
affect sex determination, and all surviving mutant animals are male
(Houwing et al., 2007). As a
result, the role of ziwi in the female germline could not be
assessed. Other mutations that reduce germ cell number also lead to male
development, suggesting that the sex-determination phenotype is secondary to
the loss of germ cells (Slanchev et al.,
2005). However, a more direct role for piRNAs in sex determination
cannot be excluded.
Conclusions and open questions
In flies, fish and mice, piRNA-pathway mutations lead to germline-specific
defects, and studies in Drosophila indicate that some of these
defects result from DNA damage signaling. piRNAs might therefore have a
conserved function in preserving germline genome integrity. In flies, piRNA
mutations lead to the overexpression of retrotransposons, and retrotransposon
mobilization could cause the DNA lesions that lead to germline DNA damage.
However, piRNA-pathway mutations have been linked to the mobilization of a
single transposon in the Drosophila male germline
(Kalmykova et al., 2005), and
there is no direct evidence that the breaks that accumulate in piRNA-pathway
mutations in the female germline are associated with transposition events.
piRNAs could therefore directly promote repair, induce the assembly of
damage-resistant chromosome structures, or suppress the expression of
euchromatic genes that induce DNA breaks.
The mechanism of piRNA-based gene silencing also remains to be determined.
Mutations in genes involved in the piRNA pathway in Drosophila have
been reported to disrupt position-effect variegation (PEV), a form of
transcriptional silencing that is caused by the spreading of heterochromatin
from peri-centromeric and telomeric regions
(Pal-Bhadra et al., 2002;
Pal-Bhadra et al., 2004).
piRNAs could therefore silence gene expression by promoting heterochromatin
assembly, which could directly suppress transcription. Alternatively,
piRNA-Argonaute complexes could associate with heterochromatin and catalyze
the co-transcriptional destruction of nascent transcripts. The latter
possibility is suggested by studies in fission yeast that indicate that
siRNA-containing Argonaute proteins are recruited to heterochromatic regions,
where they degrade transcripts as they are produced
(Verdel and Moazed, 2005).
However, mouse and Drosophila Piwi-class Argonautes are also present
in the cytoplasm, and piRNA-Piwi-class-Argonaute complexes could silence gene
expression by targeting the destruction of mature mRNA following exit from the
nucleus. It is also possible that piRNA-Argonaute complexes function in both
the nucleus and cytoplasm during the development of complex multi-cellular
organisms.
ACKNOWLEDGMENTS
We thank Beatrice Benoit, Jaspreet Khurana, Hanne Varmark and other members of the Theurkauf laboratory for comments on the manuscript, and Phil Zamore and members of the Zamore laboratory for helpful discussions.
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