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FEBS Letters xxx (2008) xxx–xxx

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How selﬁsh retrotransposons are silenced in Drosophila germline
and somatic cells
Mikiko C. Siomia,b,*, Kuniaki Saitoa, Haruhiko SiomiaaKeio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japanb
Japan Science and Technology Agency (JST), CREST, Saitama 332-0012, Japan
Received 21 May 2008; revised 12 June 2008; accepted 12 June 2008
Available online
Edited by Shou-Wei Ding

Abstract Transposable elements (TEs) are DNA elements
found in the genomes of various organisms. TEs have been highly
conserved during evolution, suggesting that they confer advantageous eﬀects to their hosts. However, due to their ability totranspose into virtually any locus, TEs have the ability to generate deleterious mutations in the host genome. In response, a variety of diﬀerent mechanisms have evolved to mitigate theiractivities. A main defense mechanism is RNA silencing, which
is a gene silencing mechanism triggered by small RNAs. In thisreview, we address RNA silencing mechanisms that silence retrotransposons, a subset of TEs, and discuss how germline andsomatic cells are equipped with diﬀerent retrotransposon silencing mechanisms.
Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Argonaute; Retrotransposon; siRNA; piRNA;RNA silencing; Drosophila

1. RNA silencing involving PIWI proteins
Biochemical and mutation analyses have revealed the existence of Dicer and Argonaute multigene families, membersof which are the key factors in RNA silencing in Drosophila[1–4]. Dicer functions in maturing small RNAs [20–30 nucleotides (nt)] that trigger RNA silencing, whereas Argonaute
functions in down-regulating gene expression post-transcriptionally by directly targeting particular mRNAs [5]. mRNAtargets are selected by base-pairing with small RNAs, whichhave been processed by Dicer and loaded onto the Argonautes.
Extensive studies at the molecular level have revealed that two
members of the Drosophila Argonaute family, Argonaute1(AGO1) and Argonaute2 (AGO2), speciﬁcally associate with
miRNA and siRNA, respectively, and function in gene silencing mechanisms mediated by miRNAs and RNAi, respectively[3]. We have analyzed gene silencing in living cells using monoclonal antibodies against each of the Argonaute proteins toimmunopurify endogenous antigens and speciﬁcally associated*Corresponding author. Address: Keio University School of Medicine,
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
E-mail address: Siomim@sc.itc.keio.ac.jp (M.C. Siomi).

small RNAs from living cells [6]. AGO1 and AGO2 have been
well studied, therefore, we focused on identifying small RNA
binding partners, as well as investigating RNA silencing pathways of other Drosophila Argonaute members, namely AGO3,
Aubergine (Aub), and Piwi. These three Argonautes are most
likely expressed only in the germline [7], and are collectively
called the PIWI proteins to distinguish them from ubiquitously
expressed AGO1 and AGO2 [8].
Forward genetic approaches have shown that mutations
introduced into the Piwi gene cause disruption to the germline
and that Piwi is an essential factor in germline stem cell (GSC)
self-renewal in both males and females [9–11]. It was also demonstrated that Piwi mutations impact retrotransposon mobility[12,13]; without functional Piwi, retrotransposons become
abnormally active. These were the ﬁrst studies to show the connection between Piwi function and regulation of retrotransposon activity. However, the molecular function of Piwi protein
had yet to be determined.
Genetic studies have shown that Aub is required for pole cell
formation [14] and for activating RNAi during Drosophila oocyte maturation [15]. Aub is also involved in silencing retrotransposons in the germline [16–18], and in silencing Stellate
genes in the testis by targeting the Suppressor of Stellate[Su(Ste)] repeats on the Y chromosome, which are highly
homologous to Stellate.
As mentioned above, AGO1 and AGO2 speciﬁc monoclonal
antibodies were key reagents for the molecular investigation of
Argonaute functions in RNA silencing. Thus, we endeavored
to produce monoclonal antibodies against the PIWI proteins
and were indeed successful in producing speciﬁc monoclonal
antibodies that recognize each of the antigens and that do
not cross-react with other Argonaute members [19–21].

2. The PIWI proteins associate with rasiRNAs in the
germline
Not only Piwi and Aub proteins but also AGO3 was successfully puriﬁed from Drosophila ovary lysates. Small RNAs contained in the immunoprecipitates from about 200 ovaries werevisible by silver staining ([21] and unpublished data), indicating
their abundance. Identiﬁcation and analysis of these small
RNAs revealed that all three PIWI proteins preferentiallyassociate with a particular set of small RNAs, previously
termed as rasiRNAs (repeat-associated small interferingRNAs) [22]. This indicates that the PIWI proteins most likely

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function in pathways silencing retrotransposons in the
germline since rasiRNAs are mainly derived from retrotransposons, remnants of ancient retrotransposons or from otherrepetitive sequences found in the genome [22]. Particular characteristics of small RNAs associated with the PIWI proteins
are that (1) Piwi and Aub show a strong preference to bindto rasiRNAs originating from the antisense transcripts of retrotransposons, whereas AGO3 prefers to bind rasiRNAs fromthe sense strand and (2) the preferred lengths of rasiRNAs thatassociate with each PIWI protein diﬀer. Piwi prefers to associate with longer rasiRNAs (24–30 nt), while AGO3 prefers tobind to relatively smaller rasiRNAs of approximately 23 nt[20]. rasiRNAs are longer in length than miRNAs and siRNAs, which function in gene silencing in association with
AGO1 and AGO2 [22]. Currently, small RNAs binding to
the PIWI proteins in other organisms, including mouse andﬁsh, are collectively referred to as piRNAs (PIWI-interactingRNAs) [23]; thus, we will follow this terminology.

3. piRNAs are methylated
In Drosophila piRNAs are chemically diﬀerent from miRNAs [18,20,24,25]. As opposed to miRNAs expressed inplants, miRNAs in animals show sensitivity to peridate(NaIO4) oxidation and beta-elimination treatments since they
contain a 2 0 , 3 0 -cis-diol at their 3 0 ends [26]. On the other hand,piRNAs are resistant to these chemical treatments, indicatingthat piRNAs lack one of the terminal hydroxyl groups[18,20,24,25]. Further investigation using the mutant ﬂy, piggyBacf00810, in which CG12367 is disrupted by an element called
piggyBac, revealed that piRNAs expressed in ﬂy ovaries andthose associated with the PIWI proteins are 2 0 -O-methylated
at their 3 0 ends [24,25]. The gene responsible for this modiﬁcation is DmHen1/Pimet (piRNA methyltransferase) (dubbed
CG12367 in FlyBase), which is the Drosophila orthologue ofthe A. thaliana gene, Hen1, identiﬁed as a source of miRNA
methyltransferase activity by Yu et al. [27]. Under conditions
where the PIWI proteins physically associate with DmHen1/
Pimet, the miRNA binding partner, AGO1, is not able to associate with the enzyme [24]. This might explain why miRNAs
are not methylated in Drosophila. Phil Zamore and his colleagues showed that siRNAs exogenously introduced in S2
cells are also methylated [25]. Recently, we performed periodate oxidation and beta-elimination on siRNAs associatedwith AGO2 in S2 cell lysates that had been pre-incubated withsiRNA duplexes, and we conﬁrmed that exogenous siRNAs
could indeed serve as substrates for DmHen1/Pimet (unpublished observations).

4. Association of AGO2 with endogenous siRNAs
Flies utilize the RNAi mechanisms to defend against viral
infection [28]. siRNAs originating from infecting viruses that
produce double-stranded RNAs (dsRNAs) as part of their replication cycle associate with AGO2 [28]. Mutations in Ago2
and Dicer2 caused loss of viral infection resistance, whichstrengthens the idea that the RNAi system is involved in virus
defense.
A question we then raised was whether AGO2 could exist
simply to lie in wait for exogenous small RNAs, such as siR-

NAs originating from the viral dsRNAs, or does AGO2 haveits own endogenous partner(s) in naı̈ve cells and organs. To address this question, we speciﬁcally immunoprecipitated endogenous AGO2 from naı̈ve S2 cells that were grown undernormal conditions, and visualized small RNAs associated withthe protein [29]. We found that in these cells AGO2 existed inassociation with endogenous small RNAs of around 21 nt.
Interestingly, the size peak was 1 nt shorter than that of miRNAs associated with AGO1 in S2 cells. Since this was the ﬁrst
demonstration that AGO2 has its own endogenous small RNA
partners, as do other Argonautes in Drosophila, we referred tothem as endogenous short interfering RNA, esiRNA. Identiﬁcation and analysis of esiRNAs revealed that they are mainly
derived from retrotransposons and other genomic repetitiveelements. This property of esiRNAs resembled that of piRNAs. However, piRNAs and esiRNAs are clearly distinct classes of small RNAs in respect to their sizes and their protein
partners, as piRNAs bind to the PIWI proteins and their sizes
are about 24–30 nt in length. In contrast, esiRNAs are approximately 21 nt and associate speciﬁcally with AGO2. The
expression proﬁles of piRNAs and esiRNAs through development are also diﬀerent; piRNAs are found in principle only in
the germline, while esiRNAs are likely to be expressed ubiquitously, based on the observation that esiRNAs could be detected, not only in S2 cells, but also in adult bodies devoidof germline, as well as in embryos (unpublished data) wheremost of the cells are somatic.

5. piRNA and esiRNA biogenesis
piRNAs associated with Piwi and Aub in ovaries, show astrong preference for uracil (U) at the 5 0 ends, while AGO3associated piRNAs show a strong preference for adenosine(A) at the 10th nucleotide from the 5 0 ends. By contrast, esiRNAs show little or no bias for nucleotides at any position, indicating that the processing mechanisms of piRNAs andesiRNAs might be diﬀerent. In early 2007, the Hannon groupand ourselves proposed a model for ‘‘piRNA biogenesis’’[20,30]. In agreement with the observation of Vagin et al.
[18], in which piRNAs were produced Dicer-independently,
our model also excludes Dicer activities. Although it is not
yet entirely understood, it is generally accepted that the Slicer
activities of PIWI proteins [19–21] contribute to the generationof piRNAs, at least in determining and producing their 5 0
ends.
How, then, are esiRNAs produced in vivo? Using bioinformatic analyses of esiRNAs, we summed the number of uniquesmall RNAs in a 5 kb sliding window and plotted this against
the Drosophila draft genome assembly. Clusters of small RNA
production were observed, from which we estimated that mostesiRNAs could be divided into two types: those that matchedretrotransposons and those that arose from long stem–loop
structures from repetitive sequences located on the X chromosome. Plotting of the esiRNAs derived from retrotransposons
against the Drosophila draft genome assembly revealed a distinct ‘‘hotspot’’ that produces a number of esiRNAs and thatthe hotspot expresses esiRNAs from both sense and antisense
strands. By looking at the plotting pattern, we postulated that
esiRNAs must be largely derived from dsRNAs arising fromthe bi-directional transcripts of retrotransposons in a Dicer2dependent manner. By contrast, esiRNAs arising from stem–

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loop structures showed a strong strand bias, indicating that
their production must be Dicer2-independent. However, it
was subsequently found that in the latter case the precursor
presumably folds into a long stem–loop structure, and theesiRNAs must arise from the long stem region. This further
supported the idea that esiRNAs are Dicer2-dependently produced.
We then assessed whether or not the production and normal
accumulation of esiRNAs require Dicer2. We depleted Dicer2
and other small RNA processing factors from S2 cells one by
one by RNAi and monitored the abundance of esiRNAs. A
marked reduction of esiRNAs in Dicer2-depleted S2 cells
was observed, whereas Drosha and Dicer1 depletion did notaﬀect esiRNA accumulation. esiRNAs were hardly detectablein Dicer2 mutant ovaries. There results indicated that esiRNAs
are produced in a Dicer2-dependent manner.
As mentioned above, exogenous siRNAs processed fromlong dsRNAs by Dicer2 and loaded onto AGO2 resisted periodate oxidation and beta-elimination reactions because they
are methylated at their 3 0 ends. We, therefore, wondered if
esiRNAs, associated with AGO2 in S2 cells, are also methylated in vivo. To address this, esiRNAs, associated with
AGO2 in S2 cells, were subjected to periodate oxidation andbeta-elimination. esiRNAs in DmHen1/Pimet mutant ovaries
were also subjected to these reactions. The results clearly indicated that esiRNAs are also methylated. Furthermore, thegene responsible for the esiRNA modiﬁcation was DmHen1/
Pimet. These data strongly support the idea that esiRNAs
are processed by Dicer2, in the same manner as siRNAs originating from exogenous dsRNAs.

6. Speciﬁc association of esiRNAs with AGO2
AGO2 in Drosophila is known to exhibit Slicer activity [6].
In our recent study, we showed that the AGO2-esiRNA complexes, immunopuriﬁed from S2 cells, had activity for cleaving
an RNA target (esiRNA-sl-1 target) harboring a sequence
completely matching one found in the most abundant esiRNA(esiRNA-sl-1) in the complexes. However, another target witha sequence complementary to bantammiRNA (bantam target),one of miRNAs expressed in S2 cells, was not cleaved by thecomplexes, indicting that at least bantam is not loaded onto
AGO2.bantam was previously shown to be associated with
AGO1 in S2 cells and AGO1 complexes immunopuriﬁed from
S2 cells were able to cleave the bantam target [3], indicatingthat AGO1 also has Slicer activity, depending on the sequenceof the small RNA with which AGO1 is associated. Recently,
we found that the AGO1 complexes isolated from S2 cells were
not able to cleave the esiRNA-sl-1 target, suggesting that esiRNAs are not loaded onto AGO1 in vivo.
One might then ask if all Drosophila miRNAs are exclusively
loaded onto AGO1, or are some sorted onto AGO2 as has
been suggested by the Forstemann et al. [31]. We characterized
miRNAs in an AGO2-associated small RNA library. It was
found that, for example, miR-20071, which was one of the
newly identiﬁed miRNAs in the study, did indeed bind to
AGO2, as assayed by northern blotting. However, the association was not exclusive to AGO2. Rather, it was more strongly
associated with AGO1. Respective sorting of miRNAs andsiRNAs into association with AGO1 and AGO2 was again
strongly supported.

3

7. Retrotransposon silencing by the AGO2–esiRNA complex
It has been previously shown that loss of PIWI proteins in
the germline caused abnormally high expression of retrotransposon transcripts. Does loss of esiRNAs, or rather, loss of the
AGO2–esiRNA-mediated silencing pathway, cause the same
phenomenon in somatic cells? To address this question, we
have performed qRT-PCR to detect expression of retrotransposons from total RNAs isolated from S2 cells, as well as fromadult male ﬂies devoid of testes. It was clearly indicated thatloss of Dicer2, the esiRNA processing factor, caused higher
expression of 297, 1731 and other retrotransposons but notof rp49 (also known as RpL32), a ribosomal protein gene used
as a control, which suggests that somatic expression of retrotransposons is suppressed by the AGO2–esiRNA pathway.
All these results in somatic cells, together with previous datafrom the germline [18], indicate that retrotransposon silencing
occurs in both these Drosophila cell types but that they utilize
diﬀerent molecules/pathways for silencing the selﬁsh elements.
Since the Piwi proteins and piRNAs are not detectable in S2
cells, at least in our laboratory, we assume that the retrotransposon silencing pathway mediated by piRNAs and the PIWI
proteins does not function in S2 cells. But, retrotransposons
are, to some extent, active even in somatic cells, potentiallycausing lethal mutations. For this reason, somatic cells may
have evolved a pathway for silencing transposable genes(Fig. 1). Subsequently, Drosophila somatic cells may havemodiﬁed this pathway to confer protection from invasive
viruses that produce dsRNAs during the replication cycle.
We wonder if somatic cells in ovaries and testes use the
AGO2–esiRNA pathway, as in S2 cells. AGO2 is expressed
throughout ovary development, but in early-stage oocytes
RNAi seems to be inactive [15]. Thus, even though somatic
cells in the germline are equipped with the AGO2–esiRNA
pathways, they may not be active. In the germline, retrotransposons are thought to be very active and thus there is a large
chance for the genome to be invaded by the mobile genes. To
eﬃciently repress this deleterious activity, the germline may
have acquired the additional system(s) mediated by the PIWI
proteins and piRNAs (Fig. 1).
We experimentally showed that the AGO2–esiRNA complexes exhibit Slicer activity. This suggests that the complexes
might silence retrotransposon genes by transcript cleavage.
However, studies in the ﬁssion yeast Schizosaccharomyces
pombe have shown that RNAi contributes to the formationof heterochromatin [32]. Similarly, a loss of Dicer2 or Ago2
activity is correlated with defects in heterochromatin formation in Drosophila [33,34]. Thus, we propose that the AGO2–
esiRNA complex induces heterochromatin formation at speciﬁc chromosomal regions containing a high density of retrotransposon genes.

7.1. Additional proteins of the AGO2–esiRNA pathway
It should be noted that we are not the sole group to haveidentiﬁed esiRNAs in Drosophila somatic cells [35–37]. Threeother groups in the United States also reported identiﬁcationand characterization of Drosophila somatic esiRNAs (in theirstudies, the small RNAs are referred to as endo-siRNAs). In
agreement with us, Ghildiyal et al. reported that endo-siRNAs
show high homology to retrotransposons [35]. It was also concluded that endo-siRNAs are produced by Dicer2, although

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Fig. 1. Predicted actions of retrotransposon silencing in Drosophila germline and somatic cells. Although the extent may diﬀer, retrotransposons areactive and transposable in both germline and somatic cells. In the germline, Aub, Piwi, and AGO3, the germline-speciﬁc Argonautes, function insilencing selﬁsh DNA elements by associating with piRNAs that originate from retrotransposon transcripts from both sense and antisense directions.
It has previously been demonstrated that piRNA biogenesis occurs Dicer-independently and the Slicer activities of the germline-speciﬁc Argonautes
are involved in the process. By contrast, in somatic cells, where Aub, Piwi, AGO3, and piRNAs are hardly detectable, AGO2 and esiRNAs function
in an equivalent manner. It should be noted that esiRNAs are processed in a Dicer2-dependent manner, unlike piRNAs. It is speculated that
Drosophila has evolved two distinct machineries/pathways to make selﬁsh DNA elements silent, one for the germline and the other for somatic cells.
The AGO2–esiRNA complexes may also function in the germline. In the germline, retrotransposons might be so active that additional mechanisms
are required to eﬀectively silence these selﬁsh genes.

some endo-siRNAs were shown to persist in dicer2 mutants.
One of the peculiar observations made by both Czech et al.
and Okamura et al. is that Loquacious, a factor known to be
involved in miRNA processing, along with Dicer1 [38–40], isalso required for endo-siRNA processing, along with Dicer2[36,37]. An additional observation made by both groups is thatsome endo-siRNAs target transcripts of regular protein-coding
genes. It is most likely that Ago2–endo-siRNA complexes are
not solely dedicated to silencing retrotransposons and otherrepetitive genes.
Our ﬁndings, together with related studies, potentially havean important implication in human disease; AGO2 is known toform a complex with the Drosophila homolog of fragile X mental retardation protein (FMRP) [41,42]. This provides a link

between transposon and protein-coding gene silencing by
AGO2 and post-transcriptional gene control by FMRP [43].
7.2. Are similar mechanisms found in other species?
Two independent groups simultaneously reported the identiﬁcation and characterization of endogenous siRNAs (endosiRNAs) expressed in mouse oocytes [44,45]. Unlike mouse
piRNAs, endo-siRNAs are about 21 nt and originate frommany diﬀerent retrotransposons in a Dicer-dependent manner.
Loss of Dicer activity caused higher expression of particular
retrotransposons, such as RLTR10 and IAP, in mouse ovaries.
By these criteria, we understand that mouse endo-siRNAs areequivalent to or are involved in the same group as Drosophila
esiRNAs. However, which mouse Argonautes, PIWI proteins

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or non-PIWI proteins, the endo-siRNAs are associated with isstill unknown. Raising antibodies against each mouse Argonaute protein (mouse possesses eight Argonaute genes) would
provide very helpful reagents to address these questions.
Acknowledgements: We are grateful to members of the Siomi laboratory for discussion and comments on the manuscript. This work was
supported by Ministry of Education, Culture, Sports, Science andTechnology (MEXT) grants to H.S., Japan Society for the Promotionof Science (JSPS) grants to K.S., New Energy and Industrial Technology Development Organization (NEDO) grants to M.C.S. M.C.S. is
supported by Core Research for Evolutional Science and Technology(CREST) from Japan Science and Technology Agency (JST). H.S. isa member of the Genome Network Project by MEXT. M.C.S. is Associate Professor of Global Centers of Excellence (GCOE) for Human
Metabolomics Systems Biology by MEXT.

References[1] Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R. and
Hannon, G.J. (2001) Argonaute2, a link between genetic andbiochemical analyses of RNAi. Science 293, 1146–1150.
[2] Bernstein, E., Caudy, A.A., Hammond, S.M. and Hannon, G.J.
(2001) Role for a bidentate ribonuclease in the initiation step of
RNA interference. Nature 409, 363–366.
[3] Okamura, K., Ishizuka, A., Siomi, H. and Siomi, M.C. (2004)
Distinct roles for Argonaute proteins in small RNA-directed
RNA cleavage pathways. Genes Dev. 18, 1655–1666.
[4] Lee, Y.S., Nakahara, K., Pham, J.W., Kim, K., He, Z.,
Sontheimer, E.J. and Carthew, R.W. (2004) Distinct roles for
Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing
pathways. Cell 117, 69–78.
[5] Hammond, S.M. (2005) Dicing and slicing: the core machinery of
the RNA interference pathway. FEBS Lett. 579, 5822–5829.
[6] Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H. and Siomi,
M.C. (2005) Slicer function of Drosophila Argonautes and its
involvement in RISC formation. Genes Dev. 19, 2837–2848.
[7] Williams, R.W. and Rubin, G.M. (2002) ARGONAUTE1 isrequired for eﬃcient RNA interference in Drosophila embryos.
Proc. Natl. Acad. Sci. USA 99, 6889–6894.
[8] Peters, L. and Meister, G. (2007) Argonaute Proteins: mediators
of RNA silencing. Mol. Cell 26, 611–623.
[9] Cox, D.N., Chao, A., Baker, J., Chang, L., Qiao, D. and Lin, H.
(1998) A novel class of evolutionarily conserved genes deﬁned bypiwi are essential for stem cell self-renewal. Genes Dev. 12, 3715–
3727.
[10] Cox, D.N., Chao, A. and Lin, H. (2000) piwi encodes anucleoplasmic factor whose activity modulates the number anddivision rate of germline stem cells. Development 127, 503–514.
[11] Szakmary, A., Cox, D.N., Wang, Z. and Lin, H. (2005)
Regulatory relationship among piwi, pumilio, and bag-of-marbles
in Drosophila germline stem cell self-renewal and diﬀerentiation.
Curr. Biol. 15, 171–178.
[12] Sarot, E., Payen-Groschene, G., Bucheton, A. and Pelisson, A.
(2004) Evidence for a piwi-dependent RNA silencing of the gypsy
endogenous retrovirus by the Drosophila melanogaster ﬂamenco
gene. Genetics 166, 1313–1321.
[13] Kalmykova, A.I., Klenov, M.S. and Gvozdev, V.A. (2005)
Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline. Nucleic Acids Res. 33,
2052–2059.
[14] Harris, A.N. and Macdonald, P.M. (2001) Aubergine encodes a
Drosophila polar granule component required for pole cell
formation and related to eIF2C. Development 128, 2823–2832.
[15] Kennerdell, J.R., Yamaguchi, S. and Carthew, R.W. (2002) RNAi
is activated during Drosophila oocyte maturation in a mannerdependent on aubergine and spindle-E. Genes Dev. 16, 1884–1889.
[16] Vagin, V.V., Klenov, M.S., Kalmykova, A.I., Stolyarenko, A.D.,
Kotelnikov, R.N. and Gvozdev, V.A. (2004) The RNA Interference proteins and Vasa locus are involved in the silencing ofretrotransposons in the female germline of Drosophila melanogaster. RNA Biol. 1, 54–58.

5[17] Savitsky, M., Kwon, D., Georgiev, P., Kalmykova, A. and
Gvozdev, V. (2006) Telomere elongation is under the control of
the RNAi-based mechanism in the Drosophila germline. Genes
Dev. 20, 345–354.
[18] Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V. andZamore, P.D. (2006) A distinct small RNA pathway silences
selﬁsh genetic elements in the germline. Science 313, 320–324.
[19] Saito, K., Nishida, K.M., Mori, T., Kawamura, Y., Miyoshi, K.,
Nagami, T., Siomi, H. and Siomi, M.C. (2006) Speciﬁc association of Piwi with rasiRNAs derived from retrotransposon andheterochromatic regions in the Drosophila genome. Genes Dev.
20, 2214–2222.
[20] Gunawardane, L.S., Saito, K., Nishida, K.M., Miyoshi, K.,
Kawamura, Y., Nagami, T., Siomi, H. and Siomi, M.C. (2007) A
Slicer-mediated mechanism for repeat-associated siRNA 5 0 end
formation in Drosophila. Science 315, 1587–1590.
[21] Nishida, K.M., Saito, K., Mori, T., Kawamura, Y., NagamiOkada, T., Inagaki, S., Siomi, H. and Siomi, M.C. (2007) Genesilencing mechanisms mediated by Aubergine piRNA complexes
in Drosophila male gonad. RNA 13, 1911–1922.
[22] Aravin, A.A. et al. (2003) The small RNA proﬁle during
Drosophila melanogaster development. Dev. Cell 5, 337–350.
[23] OÕDonnell, K.A. and Boeke, J.D. (2007) Mighty Piwis defend thegermline against genome intruders. Cell 129, 37–44.
[24] Saito, K., Sakaguchi, Y., Suzuki, T., Suzuki, T., Siomi, H. andSiomi, M.C. (2007) Pimet, the Drosophila homolog of HEN1,
mediates 2 0 -O-methylation of Piwi-interacting RNAs at their 3 0
ends. Genes Dev. 21, 1603–1608.
[25] Horwich, M.D., Li, C., Matranga, C., Vagin, V., Farley, G.,
Wang, P. and Zamore, P.D. (2007) The Drosophila RNA
methyltransferase, DmHen1, modiﬁes germline piRNAs andsingle-stranded siRNAs in RISC. Curr. Biol. 17, 1265–1272.
[26] HutvaÕgner, G., McLachlan, J., Pasquinelli, A.E., Bálint, E.,
Tuschl, T. and Zamore, P.D. (2001) Science 293, 834–838.
[27] Yu, B., Yang, Z., Li, J., Minakhina, S., Yang, M., Padgett, R.W.,
Steward, R. and Chen, X. (2005) Methylation as a crucial step inplant microRNA biogenesis. Science 307, 932–935.
[28] Li, H., Li, W.X. and Ding, S.W. (2002) Induction and suppressionof RNA silencing by an animal virus. Science 296, 1319–1321.
[29] Kawamura, Y. et al. (2008) Drosophila endogenous small RNAsbind to Argonaute 2 in somatic cells. Nature, doi:10.1038/
nature06938.
[30] Brennecke, J., Aravin, A.A., Stark, A., Dus, M., Kellis, M.,
Sachidanandam, R. and Hannon, G.J. (2007) Discrete small
RNA-generating loci as master regulators of transposon activity
in Drosophila. Cell 128, 1089–1103.
[31] Förstemann, K., Horwich, M.D., Wee, L., Tomari, Y. andZamore, P.D. (2007) Drosophila microRNAs are sorted intofunctionally distinct argonaute complexes after production by
dicer-1. Cell 130, 287–297.
[32] Bühler, M. and Moazed, D. (2007) Transcription and RNAi inheterochromatic gene silencing. Nat. Struct. Mol. Biol. 14, 1041–
1048.
[33] Peng, J.C. and Karpen, G.H. (2007) H3K9 methylation and RNAinterference regulate nucleolar organization and repeated DNA
stability. Nature Cell. Biol. 9, 25–35.
[34] Deshpande, G., Calhoun, G. and Schedl, P. (2005) Drosophilaargonaute-2 is required early in embryogenesis for the assembly ofcentric/centromeric heterochromatin, nuclear division, nuclear
migration, and germ-cell formation. Genes Dev. 19, 1680–1685.
[35] Ghildiyal, M. et al. (2008) Endogenous siRNAs derived fromtransposons and mRNAs in Drosophila somatic cells. Science,
doi:10.1126/science.1157396.
[36] Czech, B. et al. (2008) An endogenous small interfering RNA
pathway in Drosophila. Nature, doi:10.1038/nature07007.
[37] Okamura, K., Chung, W., Ruby, J.G., Guo, H., Bartel, D.P. andLai, E.C. (2008) The Drosophila hairpin RNA pathway generates
endogenous short interfering RNAs. Nature, doi:10.1038/nature07015.
[38] Forstemann, K. et al. (2005) Normal microRNA maturation andgerm-line stem cell maintenance requires Loquacious, a doublestranded RNA-binding domain protein. PLoS Biol. 3, e236.
[39] Jiang, F., Ye, X., Liu, X., Fincher, L., McKearin, D. and Liu, Q.
(2005) Dicer-1 and R3D1-L catalyze microRNA maturation in
Drosophila. Genes Dev. 19, 1674–1679.

Please cite this article in press as: Siomi, M.C. et al., How selﬁsh retrotransposons are silenced in Drosophila germline ..., FEBS Lett. (2008), doi:10.1016/
j.febslet.2008.06.018

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6

M.C. Siomi et al. / FEBS Letters xxx (2008) xxx–xxx[40] Saito, K., Ishizuka, A., Siomi, H. and Siomi, M.C. (2005)
Processing of pre-microRNAs by the Dicer-1–Loquacious complex in Drosophila cells. PLoS Biol. 3, e235.
[41] Caudy, A.A., Myers, M., Hannon, G.J. and Hammond, S.M.
(2002) Fragile X-related protein and VIG associate with the RNAinterference machinery. Genes Dev. 16, 2491–2496.
[42] Ishizuka, A., Siomi, M.C. and Siomi, H. (2002) A Drosophila
protein interacts with components of RNAi and ribosomal
proteins. Genes Dev. 16, 2497–2508.

[43] Laggerbauer, B. et al. (2001) Evidence that fragile X mental
retardation protein is a negative regulator of translation. Hum.
Mol. Genet. 10, 329–338.
[44] Tam, O.H. et al. (2008) Pseudogene-derived small interfering
RNAs regulate gene expression in mouse oocytes. Nature 1,
doi:10.1038/nature06904.
[45] Watanabe, T. et al. (2008) Endogenous siRNAs from naturally
formed dsRNAs regulate transcripts in mouse oocytes. Nature,
doi:10.1038/nature06908.

Please cite this article in press as: Siomi, M.C. et al., How selﬁsh retrotransposons are silenced in Drosophila germline ..., FEBS Lett. (2008), doi:10.1016/
j.febslet.2008.06.018