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Interactions between transposable elements and Argonautes
have (probably) been shaping the Drosophila genome throughout
evolution
Haruhiko Siomi1 and Mikiko C Siomi
Transposable elements (TEs) are powerful mutagenic agents
responsible for generating variation in the host genome. As TEs
can be overtly deleterious, a variety of different mechanisms
have evolved to keep their activities in check. In plants, fungi,
and animals, RNA silencing has been implicated as a major
defense against repetitive element transposition. This nucleic
acid-based defense mechanism also appears to be directed atinherited silencing of TEs without altering the underlying DNA
sequence. Complex interactions between TEs and RNA
silencing machineries have been co-opted to regulate cellular
genes.
Addresses
Institute for Genome Research, The University of Tokushima, 3-18-15
Kuramoto, Tokushima 770-8503, Japan
Corresponding author: Siomi, Haruhiko(siomi@genome.tokushima-u.ac.jp)
1
Present address: Department of Molecular Biology, Keio University
School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582,
Japan.

Current Opinion in Genetics & Development 2008, 18:181–187
This review comes from a themed issue on
Chromosomes and expression mechanisms
Edited by Sarah Elgin and Moshe Yaniv
Available online 4th March 2008
0959-437X/$ – see front matter# 2008 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.gde.2008.01.002

relics [2]. Retroelements were long thought of as either‘selfish’ or ‘parasitic’ DNA elements that were there notfor the sake of the host organism, but for their own sake inan evolutionary sense; thus they were considered to beeither neutral or deleterious to their hosts. However, it is
becoming increasingly clear that there are more complex
interactions between retroelements and their hosts thanstrict parasitism; these elements produce changes thathave a broad range of fitness values at an organismal level.
Recent evidence indicates that these elements confer afitness benefit to the host more frequently than previously
recognized [3–5].
Retroelements, including retrotransposons (also called‘LTR retrotransposons’), retroposons (also called ‘nonLTR retrotransposons’), mobile introns and related
elements replicate through obligatory RNA intermediates
using reverse transcriptase-dependent mechanisms [3].
Transposition of these elements may not only shape
genomic landscapes by insertions, deletions and generearrangements but also create functional diversificationof the host’s genes on which natural selection can act. They
modulate host gene expression in several ways. In somecases, a new insertion can disrupt an existing host regulatory element. In other cases, the retroelement may contribute its own cis-regulatory sequences (e.g. potential splicesites, polyadenylation signals, transcriptional promoters orenhancers) to a host gene, providing the potential for
creating greater functional diversity of genes. Transcribed
retroelements may also have a particular function in translation regulation [6]. As well, inserted sequences may becandidates for co-option or ‘exaptation’ as (or future evolution of) regulatory sequences. Thus retroelements could
serve as dynamic reservoirs for new cellular functions [3–5].

Introduction‘Selfish DNA – the large amounts of DNA in our chromosomes with no obvious function – may turn out to be partof another [tricks that are used to make for smoother andmore rapid evolution]. It is entirely possible that thisselfish DNA may play an essential role in the rapid
evolution of some of the complex genetic control mechanisms essential for higher organisms.’
Francis Crick What Mad Pursuit (1988) [1].
It is remarkable to consider that more than 40% of thehuman genome is comprised of retroelements and theirwww.sciencedirect.com

It is clear that the unbridled activity of retroelements has
the potential to cause deleterious insertions and re-

Glossary
AUB: Aubergine
dsRNA: double-stranded RNA
HP1: heterochromatin protein 1
miRNA: microRNAs
piRNA: Piwi-interacting RNAPIWI: P element-induced wimpy testis
rasiRNA: repeat-associated small interfering RNA
RNAi: RNA interferencesiRNA: small interfering RNA
TEs: transposable elements

Current Opinion in Genetics & Development 2008, 18:181–187

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182 Chromosomes and expression mechanisms

arrangements in the host genome. Thus it is not surprising that multiple pathways have evolved to inhibit
retrotransposition [3]. Since new insertions and rearrangements that happen in germ cells are likely to be passed to
the next generation, these mechanisms are particularly
important for genome surveillance in the germline.
Conversely, these mechanisms are presumably also
important for retroelements to modulate the extent ofdamage to the host genome upon which they depend.
Recent evidence has linked RNA silencing with inhibition of expression and transposition of retrotransposons[4]. Here, we focus on recent work with the model animal
Drosophila melanogaster, one that has contributed significantly to our understanding of RNA silencing and howinteractions between retrotransposons and RNA silencing
machineries have been co-opted to influence gene
expression.

RNA silencing
RNA silencing is an evolutionarily conserved mechanism
in which small RNAs trigger various forms of sequencespecific gene silencing by guiding effecter complexes totarget RNAs via base-pairing [7,8]. Biochemical andgenetic analyses reveal the existence of multigene
families encoding the two key proteins – Dicer and
Argonaute – in RNA silencing. In RNA interference(RNAi) (see Glossary), small interfering RNAs (siRNAs)
are produced from long exogenous double-stranded RNA(dsRNA) (see Glossary), whereas microRNAs (miRNAs)
are excised in a stepwise process from RNA hairpins
encoded by the genome. In flies, DICER-1 generates
miRNAs while DICER-2 produces siRNAs [7,8]. These
small RNAs presumably are complementary to target
mRNA by strict Watson–Crick base pairing or according
to miRNA (see Glossary) targeting ‘seed’ rules [7,8].
Single-stranded siRNAs or miRNAs are loaded onto
RISCs (RNA induced silencing complexes) that participate in subsequent steps such as mRNA cleavage ormRNA translational repression.
Argonaute proteins are a defining component of RISC
complexes [9]. They are a protein family conserved fromfission yeast to humans; its name comes from its founding
member in Arabidopsis [9]. Argonaute proteins are100 kD basic proteins and are composed of two principal domains; an amino-terminal PAZ domain, a singlestranded nucleic acid binding domain; and an carboxylterminal PIWI domain that is responsible for the RNaseH-like catalytic or ‘Slicer’ activity [10]. The PIWI
domain contains two aspartates and a histidine that forma ‘DDH’ catalytic motif similar to the DDE catalytic triad(aspartate/aspartate/glutamate) motif seen in RNase H.
Duplex formation between the guide small RNA and thetarget places the scissile phosphate of the target strand(located between the 10th and 11th nucleotides asmeasured from the 50 end of the guide) adjacent to the
Slicer catalytic site in the PIWI domain. If the catalytic
Current Opinion in Genetics & Development 2008, 18:181–187

site is functional and the guide-target duplex is complementary in the region of the scissile phosphate, thetarget strand can be cleaved. If the guide contains mismatches to the target sequences in the region of thescissile bond or the Argonaute does not contain the
Slicer catalytic residues, slicing does not occur but
the Argonaute-guide complex remains associated withthe target and can induce translational repression or
cleavage-independent mRNA decay.
Argonaute proteins have proliferated and evolved a rangeof functions for endonucleolytic cleavage, translational
repression, recruitment of chromatin-modification factors,
and biogenesis of small RNAs (see below). Argonaute
proteins fall into two classes [9]; the AGO subfamily
based on Arabidopsis thaliana Ago1 and the PIWI subfamily based on Drosophila melanogaster PIWI. Drosophila
has two AGO proteins (AGO1 and AGO2), that are
ubiquitously expressed, and three PIWI proteins[AGO3, AUB (Aubergine), PIWI (P-element induced
wimpy testis)], (see Glossary) whose expression is primarily observed in the germline [11]. Each has specialized
functions. For instance, AGO1 is involved in the miRNAdependent mRNA silencing pathway (mostly translation
repression), whereas AGO2 functions in a pathway for
siRNA-directed RNAi (see Glossary) [8]. AGO2 also
promotes RISC assembly by slicing siRNA duplex precursors [7].
Genetic studies have indicated that PIWI is an essential
factor in germline stem cell self-renewal while AUB is
required for pole cell formation and spermatogenesis[12,13]. These PIWI proteins are also involved in silencing retrotransposons and repetitive sequences in themale and female germlines of Drosophila. PIWI proteinsbind repeat-associated small interfering RNAs (rasiRNAs) [12,14,15,16]. rasiRNAs are longer in lengththan siRNAs and miRNAs (24–27 nucleotides, as opposed
to 20–22) and are mainly derived from transposons, theirremnants or other repetitive elements in the genome. As
well as their longer length, rasiRNAs are chemicallydifferent in that they lack one of the 30 -terminal hydroxyl
groups characteristic of animal miRNAs; instead they are
20 -O-methylated at their 30 ends [17,18]. Currently, small
RNA binding partners for PIWI proteins (including rasiRNAs) are collectively referred to as PIWI-interacting
RNAs (piRNAs); we will use the term hereafter. PIWI
proteins are able to exhibit Slicer activity in vitro[12,14,15]. Thus, it can be postulated that these PIWI
proteins function, at least in part, as a Slicer, to cleavetranscripts from repetitive TEs (see Glossary) throughtheir association with piRNAs. In this way, they silence
active transposons, thereby protecting the genome frominvasive elements. piRNAs map to discrete genomic
clusters, suggesting that a limited number of master
piRNA (see Glossary) loci might control germline transposon activity [16].
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Interactions between transposable elements and Argonautes Siomi and Siomi 183

piRNAs are distinct from both siRNAs and miRNAs not
only in their length but also in their biogenesis. piRNA
production appears not to require DICER-1 or DICER-2[12]. Rather, piRNA biogenesis involves a Dicer-independent cycle that amplifies piRNAs [15,16]. piRNAs
associated with PIWI proteins show a strong sequencebias. AUB and PIWI preferentially bind piRNAs derived
from the antisense strand of reterotransposons, and the 50
end is predominantly uridine (U), while AGO3 associated piRNAs arise mainly from the sense strand andshow a strong preference for adenine (A) at nucleotide
10, but no preference for U at the 50 end. As describedabove, Argonaute proteins exhibit Slicer activity that
directs cleavage of its cognate RNA target across from
nucleotides 10 and 11, measured from the 50 end of thesmall RNA guide strand. These results together suggestthat AGO3 associated with a rasiRNA (see Glossary)with A at nucleotide 10, can target a long RNA molecule
by Watson–Crick base pairing and cleave the target
RNA, resulting in sliced RNAs with U at the 50 end.
Similarly, when AUB or PIWI associated with rasiRNAs
with U at the 5 end, slices its cognate RNA target, theresulting cleaved RNA will have an A at nucleotide 10.
In this model, the Slicer activities of PIWI proteins servea dual capacity; they degrade sense transposon transcripts and produce the antisense piRNAs. Thus, inpiRNA biogenesis, sense and antisense transcripts fromretrotransposons appear to interact through the action of
PIWI proteins in a cycle that amplifies piRNAs(Figure 1). Therefore, retrotransposons are both a source
gene for piRNAs and a target of piRNA-mediated silencing. Changes in the activity of the piRNA pathway inresponse to environmental stimuli or stress may allow awindow of opportunity for expression or even transposition of some retrotransposons to occur. By creating newnodes in the piRNA biogenesis cycle or new inheritable
insertions of retrotransposons, such transposons could
influence end points of gene expression regulated by thepiRNA pathway.
Generation of Drosophila piRNAs is also distinct frombiogenesis of plant siRNAs and Caenorhabditis elegans
secondary siRNAs, both of which also involve amplification steps [7]. Plant siRNAs might prime dsRNA synthesis by RNA-dependent RNA polymerase (RdRP),
leading to dicing and amplification of siRNAs as longas the target RNA continues to be produced. By contrast,
each secondary siRNA in C. elegans is synthesized individually by RdRP [19,20]. However, the Drosophila genome lacks genes encoding RdRP. The biogenesis
mechanism of piRNAs appears conserved in fish andmammals [21,22]. A key difference between transposon
management in Drosophila and in mammals is the role ofcytosine methylation in maintaining stable repression.
Recent evidence indicates that mammalian PIWI–
piRNA complexes may function in an RNA-dependent
DNA methylation pathway [22].
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How then is the piRNA pathway connected with germline maintenance? One possibility is that loss of thepiRNA pathway affects germline integrity throughderepression of transposones, whose activity can leadto DNA damage [23]. Another possibility, not mutually
exclusively, is that loss of the piRNA pathway affects
expression of specific genes that might be important for
germline development. Since the biogenesis of piRNAs
appears to involve a long single-stranded precursor[15,16], transcripts from cellular genes (whose product benefits the cell) in the germline, in principal,
could participate in an amplification cycle as nodes ifthey have perfect or near perfect piRNA target sites.
Cellular transcripts containing a stretch of sequencesthat are sufficiently complementary to a piRNA could
also be targets of piRNA. In the latter case, piRNAs
could guide PIWI proteins either to cleave the target
with low turnover rates or to repress translation of thetarget in a fashion similar to miRNA mediated control.
In this regard, it is interesting that many AUB-associated piRNAs in testis arise from the Suppressor of Stellate[Su(Ste)] repeats on chromosome Y which are required
to silence Stellate genes on the X chromosome [14]. In
addition, some AUB-associated piRNAs in testis also
show significant complementarity to part of a vasa
transcript, a germline specific transcript involved inoocyte differentiation and cyst development. An immunopurified AUB-piRNA complex from testes displays
Slicer activity to cleave target RNA containing theStellate sequences or the vasa sequence [14]. Therefore, piRNAs have the potential to trigger RNA silencing in cis (at the locus from which they are derived) andin trans (at a locus from which they do not arise). It isalso interesting to note that a fraction of mouse PIWI
(Miwi) comigrates with polysomes on density gradients[24]. A recent computer-based pattern discovery search
revealed that short blocks (16 nucleotides) termed‘pyknons’ are dispersed at multiple sites in eukaryotic
genomes; the great majority of them are found both
inside repeat TEs and in repeat-free regions [25,26],
suggesting that many cellular genes may have piRNAtarget sites. In support of the view that piRNAs could
regulate expression of cellular genes as well, we notethat many miRNAs appear to have evolved from transposable elements, and their involvement in gene regulation appears to be an outcome of the antagonistic
relationship between transposons and the host genome[5,27].

Epigenetic silencing
Retroelements have been estimated to constitute 27%
(retrotransposons: 22%; retroposons; 5%) of the Drosophilamelanogaster genome [3]. These repetitive sequences
account for the majority of all heterochromatic DNA in
D. melanogaster, whose genome is 30% heterochromatic[28], suggesting that they play a central role in theassembly of heterochromatin.
Current Opinion in Genetics & Development 2008, 18:181–187

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184 Chromosomes and expression mechanisms

Historically, heterochromatin has been regarded as transcriptionally inert. Recent evidence in fission yeast S.
pombe indicates that components of the RNAi system are
involved in heterochromatin formation. While it seems
counterintuitive, the data suggest that transcription ofsilenced loci by RNA pol II and processing into siRNA are
essential for the maintenance of the heterochromatic state[29]. Thus, heterochromatin must be expressed to maintain ‘silence.’
The first indication that components of RNA silencing
pathways function in assembly of heterochromatin-likedomains came from studies of gene silencing in Drosophila. Genetic studies revealed that repeat-induced genesilencing in Drosophila requires components of the Polycomb group proteins and PIWI [30,31]. The chromatincontext of a gene can determine its expression, as illustrated by position-effect variegation (PEV) in Drosophila,
in which the mosaic silencing of a normally active geneoccurs when it is located in or near heterochromatin. piwi
and aub mutants can suppress PEV of a white reporter, andappear to result in some delocalization of the heterochromatin proteins HP1 (see Glossary) and HP2, proteinsthat bind normally at silenced loci [32]. Collectively,
these observations suggest that PIWI proteins (and presumably their associated piRNAs) are involved in setting
chromatin states. This also implies that the factors which
confer silencing status in heterochromatin can spread
into nearby genes that would otherwise be active. Therefore, insertion of transposons into or near genic regions
could affect expression of nearby genes, on which natural
selection can act. A recent study indicates that a new
insertion of a P transposable element carrying a 1360
repetitious element can indeed suppress expression of an
adjacent reporter gene in the same element, given an
appropriate chromosomal environment [33]. The piwi
gene also interacts genetically with the regulation of a
Polycomb response element and Polycomb protein function, suggesting a role influencing cellular identity during
embryonic development through a chromatin-dependent mechanism [34]. Furthermore, recent studies haveshown that PIWI appears to interact directly with HP1,
which also implicates PIWI in heterochromatin formation [35]. Surprisingly, the protein is also implicatedin activation of transcription in some cases, notably attelomeres [36].
Given the biogenesis model of piRNAs, the potential for
RNA silencing machineries in Drosophila to have an
epigenetic characteristic appears to involve both heterochromatin formation and a second mechanism at the RNAlevel. In the former mechanism, piRNAs may home in on
homologous nascent RNAs traversing the silencing lociand recruit, together with PIWI proteins, chromatinmodification factors to these regions. In the latter mechanism, RNA transcripts from silenced loci would be
expressed continuously [37] and enter into a piRNA
Current Opinion in Genetics & Development 2008, 18:181–187

amplification cycle in concert with the Slicer activity of
PIWI proteins to initiate epigenetic silencing. In thiscycle, piRNAs would have the potential to target further‘maintenance’ rounds of piRNA production on theoriginal transcripts and on homologous transcripts. This
would result in the degradation of transposon transcripts,
thereby leading to the suppression of transposon activity.
In effect, these maintenance rounds of silencing provide
both a genetic memory of transposons to which the
population has been exposed and a molecular memoryof primary piRNAs.

Implications of RNA mediated inheritance
Since expression of PIWI proteins is largely restricted to
the germline, the manifestation of heterochromatic states,
PEV and in some cases the control of gene expression insomatic cells, mediated by PIWI proteins [30,31,34,38]
should be preprogrammed during very early ontogeny
and therefore must involve chromatin memory. How
could this be achieved?
In C. elegans, silencing induced by RNAi can be transmitted for multiple generations [39], and chromatin-modifying factors appear to play a role in this inheritancemechanism [40]. In Drosophila, misregulation of normallysilenced sequences occurs when males carrying certaintransposable elements are crossed with females lacking
them [4,12]. This results in transposon activation withdetrimental consequences that induce hybrid sterility.
The reciprocal cross of a female carrying a repressedtransposon with a male lacking it does not result insterility associated with high rates of germline transposition in the offspring. These phenotypes are collectively
referred to as hybrid dysgenesis. The maternal suppression of hybrid dysgenesis suggests the involvement of acytoplasmic factor, termed cytotype, that suppresses theactivity of transposable elements, and that the repressive
signal is passed from females to their offspring. Recent
evidence indicates that maternal suppression of hybrid
dysgenesis depends on PIWI proteins and piRNAs [4,41].
AUB and PIWI proteins are deposited in the developing
germline during oogenesis through germline transmission[12,42]. Thus, the maternal contribution of PIWI proteinsand presumably their associated piRNAs, as well as thesequences of piRNA themselves, provide a physical basis
for the hypothesis that the transfer of silencing signals andchromatin memory to off-springs is mediated in the formof PIWI–piRNA complexes. This also implies that Drosophila may possess the capacity to store information atnumerous genetic loci in the form of PIWI–piRNA complexes and so pass them on from generation to generation.
These genetic loci not only include transposons and otherrepetitive elements but may also include their homologous sequences that can be part of cellular genes. The
maternal transmission of PIWI–piRNA complexes may
also imply that primary piRNAs that initiate an amplification cycle of piRNA biogenesis are supplied throughwww.sciencedirect.com

Author's personal copy

Interactions between transposable elements and Argonautes Siomi and Siomi 185

Figure 1

The biogenesis and functions of piRNAs in Drosophila. piRNAs bind the PIWI subfamily of Argonaute proteins – AGO3, Aubergine (AUB) and PIWI – andtarget homologous transposons scattered around the genome for silencing. PIWI–piRNA complex might also target cellular transcripts, if a part of thetranscript is homologous to piRNA. The nuclear localization of PIWI [14,16,42] may indicate that this protein directly participates in heterochromatin
formation. In piRNA biogenesis, the Slicer activities of PIWI proteins serve a dual capacity: cleaving sense transposon transcripts and producing
antisense piRNAs. PIWI-mediated cleavage events generate new piRNAs, thereby setting up a self-reinforcing cycle. It is not yet known how AGO3,
AUB, PIWI are able to specifically recognize the respective sense and antisense strands of repetitive elements.

germline transmission; thus the cycle could operate between generations.

Conclusions
RNA silencing is thought to have evolved as a form of
nucleic acid-based, and thus sequence-directed, immunity
to block the action of viruses and TEs [43]. Host–parasite
interactions are typically associated with rapid evolution
because of a permanent antagonistic relationship resembling an ‘arms race’ in which parasite adaptations arecountered by host adaptations [44]. Consistent with this,recent evidence indicates that components of RNA silencing pathways are among the fastest evolving immunerelated genes [45]. It is becoming increasingly clear thatthe ‘arms race’ between transposons and hosts leads topositive selection for cellular defense mechanisms, part ofwhich are co-opted or ‘borrowed’ for evolving new regulatory circuits, thus enabling the integration and networking of complex suites of gene activity. However, there are
numerous gaps in our understanding of the biochemical
events that transpire in this ‘arms race’, which has thepotential to create diversification of gene expression inhosts. Many questions remain unanswered; someexamples follow. How are Drosophila AGO3, PIWI,AUB able to specifically recognize the respective sense
and antisense strands of retrotransposones and repetitiveelements? What might be the mechanism that specifies the
30 ends of piRNAs? How are antisense transcripts of retrotransposons produced and how are they regulated? How
can a de novo piRNA biogenesis cycle be initiated? Prewww.sciencedirect.com

cisely how do piRNAs mediate epigenetic regulation ofgene expression? What are the developmental roles ofpiRNAs? As well as silencing transposons in the germline,
can piRNAs regulate germline development by affecting
cellular genes directly? Components of piRNA pathways
are also implicated in activation of transcription and translation in some cases [36,42,46]. So, what can turn normallyrepressors of gene expression into activators? Recent studies have shown that TEs may also actively transpose insome somatic cells, creating somatic mosaicism in animals[47]. Some retrotransposons are transcribed in Drosophila
somatic cells and they are not repressed by the piRNA
pathway [48]. Thus, how is transposon activity in somatic
cells controlled? Since small RNA triggers guide Argonaute proteins to ensure that they specifically silencetranscripts homologous to the small RNA, deciphering
how these partner Argonautes contribute to the functionof small RNAs is integral to understanding their biologicalroles. Studies that catalog interacting small RNA partnersof all of the Drosophila Argonaute proteins in the germline
and various somatic tissues by high-throughput sequencing should yield some insight into these issues. We lookforward to the day when these and other important mysteries are solved and when other, for now unexpected, roles
for Argonautes and transposons are uncovered.

Acknowledgements
We thank members of the Siomi laboratory for useful discussions. HS is amember of the Genome Network Project of MEXT. MCS is supported by
CREST from JST.
Current Opinion in Genetics & Development 2008, 18:181–187

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186 Chromosomes and expression mechanisms

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Current Opinion in Genetics & Development 2008, 18:181–187