Vol 453 | 5 June 2008 | doi:10.1038/nature06938

LETTERS
Drosophila endogenous small RNAs bind to
Argonaute 2 in somatic cells
Yoshinori Kawamura1*, Kuniaki Saito1,2*, Taishin Kin3, Yukiteru Ono4, Kiyoshi Asai3,5, Takafumi Sunohara1,
Tomoko N. Okada1,2, Mikiko C. Siomi1,2,6 & Haruhiko Siomi1,2

RNA silencing is a conserved mechanism in which small RNAs
trigger various forms of sequence-specific gene silencing by
guiding Argonaute complexes to target RNAs by means of basepairing1,2. RNA silencing is thought to have evolved as a form of
nucleic-acid-based immunity to inactivate viruses and transposable elements. Although the activity of transposable elements
in animals has been thought largely to be restricted to the germline, recent studies have shown that they may also actively transpose in somatic cells, creating somatic mosaicism in animals3. In
the Drosophila germ line, Piwi-interacting RNAs arise from repetitive intergenic elements including retrotransposons by a Dicerindependent pathway and function through the Piwi subfamily of
Argonautes to ensure silencing of retrotransposons4–9. Here weshow that, in cultured Drosophila S2 cells, Argonaute 2 (AGO2),
an AGO subfamily member of Argonautes, associates with endogenous small RNAs of 20–22 nucleotides in length, which we havecollectively named endogenous short interfering RNAs (esiRNAs).
esiRNAs can be divided into two groups: one that mainly corresponds to a subset of retrotransposons, and the other that arises
from stem–loop structures. esiRNAs are produced in a Dicer-2dependent manner from distinctive genomic loci, are modified
at their 39 ends and can direct AGO2 to cleave target RNAs.
Mutations in Dicer-2 caused an increase in retrotransposon transcripts. Together, our findings indicate that different types ofsmall RNAs and Argonautes are used to repress retrotransposons
in germline and somatic cells in Drosophila.
Biochemical and mutation analyses have revealed the existenceof multigene families encoding two key proteins—Dicer and
Argonaute—in RNA silencing in Drosophila10–13. Dicer-1 generates
Drosophila microRNAs (miRNAs) whereas Dicer-2 creates short
Perfect matchinterfering RNAs (siRNAs). Argonaute proteins directly bindsmall guide RNAs and either display endonucleolytic (‘Slicer’) activity or have a platform role for the assembly of silencing complexes2.
The five Drosophila Argonaute proteins can be subdivided into
two subfamilies: the ubiquitous AGO (AGO1 and AGO2) and thegermline-specific Piwi (AGO3, Aubergine (Aub) and Piwi) subfamilies2,14. AGO1 is involved in the miRNA-dependent pathwaythat silences messenger RNA, whereas AGO2 functions in RNA
interference (RNAi)12 directed by exogenous siRNAs. Piwi proteins are involved in silencing retrotransposons through directinteraction with Piwi-interacting RNAs (piRNAs) in the germ line4–9
by forming a Dicer-independent cycle that amplifies piRNAs6,7. In
contrast, how RNA silencing might operate to repress retrotransposons in Drosophila somatic cells, and to what extent, remain
unknown.
Unlike for the other Argonaute proteins in Drosophila, endogenous small RNA partners for AGO2 have not been identified. AGO2
could exist simply to lie in wait for small RNAs that exogenously
come into contact with it, including those originating from infecting
viruses that produce double-stranded RNAs (dsRNAs) as part oftheir replication cycle15. However, we surmised that AGO2 mightalso interact with endogenous siRNAs that are products of Dicer
processing of dsRNAs, akin to endogenous siRNAs in Arabidopsis
thaliana1. In animals, endogenous siRNAs have only been identified
in Caenorhabditis elegans16. To test whether AGO2 exists in a complex
with endogenous siRNAs, we immunopurified AGO2 with a specificmonoclonal antibody from a cultured Drosophila somatic S2 cell line
(Supplementary Fig. 1a, b) and examined its associated RNAs. AGO2
in S2 cells was predominantly associated with small RNAs of about 21
nucleotides in length (Supplementary Fig. 1c).

Single mismatch
1.4%

58.2%

84.9%
18.9%

2.6%
1.4%
0.3%
0.5%
6.5%

Transposons
Repeats
rRNAs
tRNAs/ncRNAs
miRNAsSingle mismatch
Coding genes
No annotation

Figure 1 | Identification of endogenous small
RNAs that bind AGO2. The left chart shows thatthe contents of AGO2-associated small RNAs
perfectly match the Drosophila genome sequence(perfect match). Clones with single mismatchescomprise 12,240 from a total of 64,588; these
populations are shown on the right (single
mismatch).

3.7%

9.8%

3.0%
1.3%
1.4%
5.9%

1
Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan. 2Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan. 3Computational Biology Research Center, Advanced Industrial Science and Technology, Tokyo 135-0664, Japan. 4Information and Mathematical
Science Laboratory, Inc., Tokyo 112-0012, Japan. 5Graduate School of Frontier Science, University of Tokyo, Chiba 277-8561, Japan. 6Japan Science and Technology Agency (JST), Core
Research for Evolutional Science and Technology (CREST), Saitama 332-0012, Japan.
*These authors contributed equally to this work.

793©2008 Nature Publishing GroupLETTERS

NATURE | Vol 453 | 5 June 2008

d

a
Chr X

5 Mbp

Antisense

Sense

1,000

2,000

′

* **

5284000

5285000

5286000

5287000

IR elements

AGO2

AGO3

Aub

Piwi

AGO2

AGO3

Aub

Piwi

Figure 2 | esiRNA clusters. a, Frequency map of AGO2-associated small
RNAs on chromosome X. The x and y axes represent the chromosomal
position and the number of reads, respectively. The strand is represented by
the colour of the bars: plus (red) and minus (blue). Two major hotspots
indicated by the yellow box are enlarged in the rectangle below; one, indicatedwith the asterisk, expresses only from the minus strand, where 20 similar
sequence patterns (,274 nt) are repeatedly encoded (Supplementary Fig. 5).
Each fragment has a distinct stem–loop structure, as shown in b. The mostabundant small RNA (esiRNA-sl-1) derived from this fragment is indicated bya red bar. The other hotspot (indicated with two asterisks) expresses esiRNAs

1360
Bari1
HB
hobo
hopperhopper2
looper1
mariner2
NOF
P-elementpogo
S2
S-element
Tc1
Tc1-2
transib1
transib2transib3transib4

IR elements

1360
Bari1
HB
hobo
hopperhopper2
looper1
mariner2
NOF
P-elementpogo
S2
S-element
Tc1
Tc1-2
transib1
transib2transib3transib4

LINE elements

esiRNA-sl-1

cbaggins
BS
BS3
Cr1a
Doc
Doc2-element
Doc3-element
Doc4-element
F-element
Fw2
Fw3
G2
G3
G4
G5
G5A
G6
G-element
HelenaHeT-A
I-element
Ivk
jockeyjockey2Juan
Penelope
Porto1
R1-2
R2-element
Rt1a
Rt1b
Rt1c
TAHRE
X-element

17.5
297
412
1731
3S18
accordaccord2
aurora-element
blood
Burdock
Circe
copia
diver
diver 2
Dm88
flea
froggerGATE
gt wingypsy
gypsy2
gypsy3
gypsy4
gypsy5
gypsy6
gypsy7
gypsy8
gypsy9gypsy10
gypsy11
gyspy12HMS-Beagle
HMS-Beagle2
idefix
invader1
invader2
invader3
invader4
invader5
invader6
Max-element
McClintock
mdg1
mgd3
micropia
opus
OsvaldoQuasimodo
roo
rooA
rover
springer
Stalker
Stalker2
Stalker4
Tabor
Tirnt
TrnspacZAM
baggins
BS
BS3
Cr1a
Doc
Doc2-element
Doc3-element
Doc4-element
F-element
Fw2
Fw3
G2
G3
G4
G5
G5A
G6
G-element
HelenaHeT-A
I-element
Ivk
jockeyjockey2Juan
Penelope
Porto1
R1-2
R2-element
Rt1a
Rt1b
Rt1c
TAHRE
X-element

LTR elementsbLINE elements

0

17.5
297
412
1731
3S18
accordaccord 2
aurora-element
blood
Burdock
Circe
copia
diver
diver 2
Dm88
flea
froggerGATE
gt wingypsy
gypsy2
gypsy3
gypsy4
gypsy5
gypsy6
gypsy7
gypsy8
gypsy9gypsy10
gypsy11
gyspy12HMS-Beagle
HMS-Beagle2
idefix
invader1
invader2
invader3
invader4
invader5
invader6
Max-element
McClintock
mdg1
mgd3
micropia
opus
OsvaldoQuasimodo
roo
rooA
rover
springer
Stalker
Stalker 2
Stalker 4
Tabor
Tirnt
Trnspac
ZAM

LTR elements

1,000

30%
20%
15%
10%
3%

100%
75%
50%
75%
100%

2,000

′

Cloning frequency

from both strands, with details shown in c. d, The heat map (left panel)indicates the strand bias of cloned esiRNAs and piRNAs7 with respect tocanonical transposon sequences (indicated at the right). Transposons aregrouped into long terminal repeat (LTR), long interspersed nuclear (LINE)and inverted repeat (IR) elements. The colour intensities indicate the degreeof strand bias (green, sense; red, antisense; yellow, unbiased). Note that sometransposons in the AGO2 lane are coloured either green or red, but thecloning frequency of these transposons was very low (see the right panel). The
cloning frequencies of individual transposons in all four complexes (thisstudy and ref. 7) are indicated as a heat map (right panel).

794©2008 Nature Publishing GroupLETTERS

NATURE | Vol 453 | 5 June 2008

IP from
S2 cells
Anti- AntiAGO1 AGO2

– + – + βdS2 totalRNA– +βesiRNAsl-1

–

+

–

Pimet–/–

c

Wild type

Target onlyn.i.
Anti-AGO1
Anti-AGO2

esiRNA-sl-1 target siteb
IP from
S2 cells

bantam target siteaclearly different. Thus, esiRNAs and piRNAs are distinct classes ofendogenous small RNAs. Some retrotransposons including 1731 and297 elements are overrepresented in the esiRNA populations.
Drosophila piRNAs show a strong strand bias for sequences; Auband Piwi mainly bind antisense-strand piRNAs, whereas AGO3 binds
sense-strand piRNAs6,7. esiRNAs, however, seems to be unbiased(Fig. 2d and Supplementary Fig. 4). For example, one hotspot forsmall RNA production within a 7,600-nucleotide intergenic region
on chromosome X was almost entirely covered by small RNAs on
both strands (Fig. 2c). Thus, they must be derived from dsRNAs.
In contrast, esiRNAs arising from stem–loop structures (Fig. 2b)
showed strand bias with exceptionally high cloning frequency(Supplementary Fig. 5). The stem–loop-generating region contains
twenty ,274-nucleotide repeats and appeared to extend to theCG4068 gene (Supplementary Fig. 5). All of the esiRNAs from thisregion were found in a sense orientation with respect to the orientation of CG4068 (Supplementary Fig. 5). Northern blotting revealed
that both types of esiRNAs specifically co-purified with AGO2, but
not with AGO1 (Supplementary Fig. 6a–c). An in vitro Slicer assay
also confirmed that AGO2–esiRNA complexes were capable of specifically cleaving target RNAs containing sequences perfectly complementary to esiRNAs (Fig. 3a). This also implied that esiRNAs canguide AGO2 to cleave complementary RNAs including retrotransposon transcripts in cells.
piRNAs and exogenous siRNAs in Drosophila are modified with
29-O-methylation at their 39 terminus, requiring an RNA methyltransferase, Pimet (also known as DmHen1 and CG12367) (refs 21
and 22). esiRNAs in S2 cells were resistant to periodate oxidation(NaIO4) and b-elimination reactions (Fig. 3b, c), but esiRNAs from
DmHen1/Pimet mutant ovaries could be b-eliminated (Fig. 3d).
Thus, esiRNAs are likely to have the same 39 modification, as is thecase for piRNAs and exogenous siRNAs. These findings suggested
that esiRNAs are produced by a pathway similar to that of exogenous
siRNAs that bind AGO2 in S2 cells. Because exogenous siRNA
could not displace esiRNAs from AGO2 (Supplementary Fig. 7a,b), ‘empty’ AGO2 (apo-AGO2) seems to exit to accommodate small
RNAs from exogenous sources.
To test whether the production and normal accumulation of
esiRNAs require Dicers and other known small-RNA-processing
factors, we depleted each of these proteins from S2 cells by RNAiand monitored the abundance of small RNAs (Fig. 4a). A marked
reduction of esiRNA-sl-1, one of the very abundant esiRNAs (Fig. 2b),
was observed in Dicer-2-depleted S2 cells; this was observed lessefficiently in AGO2-depleted S2 cells, and was not observed in
Drosha-, Dicer-1- or AGO1-depleted S2 cells. We also confirmed

Pimet+/–To characterize the small RNAs associated with AGO2, we constructed a complementary DNA library and performed highthroughput pyrosequencing17 that generated 77,327 reads of thecDNA library. Consistent with the [c-32P]ATP-labelled result, thesizes of the small RNAs ranged from 20 to 22 nucleotides in length(Supplementary Fig. 1d), similar to the sizes of miRNAs but clearly
different from the sizes of piRNAs (24–29 nucleotides). Most of theseendogenous binding partners for AGO2 were a previously undescribed class of ,21-nucleotide small RNAs (see below), which wehave collectively referred to as esiRNAs (also known as endosiRNAs). Unlike many other small RNAs that bind Argonautes1,18,
esiRNAs do not display a nucleotide-bias at any particular position(data not shown). Among the 77,327 reads, 52,348 sequences
matched the Drosophila genome with 100% identity over their entire
length (Fig. 1). In addition, a large number of the AGO2-associatedsmall RNAs showed single mismatches (12,168; Fig. 1). Adenosineto-guanosine single mismatches were vastly overrepresented(Supplementary Fig. 1e), suggesting that a portion of the small
RNAs may be adenosine-to-inosine RNA edited by ADAR (adenosine deaminase acting on RNA) enzymes19.
Database searching revealed that a large number of the AGO2associated small RNAs corresponded to transposons and other repetitive elements in the genome (Fig. 1). Pieces of protein-coding sense
mRNAs (,6%) as well as ribosomal RNAs (,3%) were conspicuousin this screening, as is the case for piRNAs in fly9 and mouse20. We
also observed a small number of miRNAs (,1.4%) among the
AGO2-associated small RNAs sequenced (Supplementary Table 1),
including two previously unknown miRNAs (miR-20071 and miR20072) derived from introns of prp8 and cdc2, respectively(Supplementary Figs 2 and 3).
We summed the number of unique small RNAs in a 5-kilobase(kb) sliding window and plotted this against the Drosophila draft
assembly (Berkeley Drosophila Genome Project release 5). Clustersof small RNA production were observed (Fig. 2a, Supplementary Fig.
4 and Supplementary Table 2). Most esiRNAs could be divided into
two types: those that matched retrotransposons and those that arosefrom long stem–loop structures from repetitive sequences located in
an intergenic region on chromosome X (Fig. 2b, c and Supplementary Fig. 5). Populations of esiRNAs derived from retrotransposons
are quite complex, with most RNAs being cloned only once (57.2% in
perfect and single matches). Like Drosophila piRNAs4–9, esiRNAs arederived from a subset of retrotransposons (Fig. 2d). Consistent with
this, a number of esiRNAs (24.5%) overlap with registered piRNAsequences6–9; however, their sizes (20–22 nucleotides versus 24–27
nucleotides) and binding partners (AGO2 versus Piwi proteins) were

+

–

+ β
esiRNAsl-1

miR-279roo

miR-310Figure 3 | Specific association and modification of esiRNAs. a, Target
RNAs containing sequences complementary to bantam (red bar) andesiRNA-sl-1 (green bar) are cleaved at each position with AGO1–miRNA and
AGO2–esiRNA complexes, as expected. n.i.; non-immune IgG; IP,
immunoprecipitation. b, Unlike miRNAs, esiRNAs show resistance tob-elimination. c, Total RNAs of S2 cells were probed for esiRNA-sl-1 andmiR-279 before and after b-elimination. d, Loss of Pimet causes esiRNA to besensitive to b-elimination, suggesting that esiRNAs are methylated by Pimet.
roo, piRNA corresponding to the LTR retrotransposon roo.
795

©2008 Nature Publishing GroupLETTERS

NATURE | Vol 453 | 5 June 200810

bantam

1731

blood
roo

5

mdg1

Pre-bantam

1731
mdg1
297
blood
roo
HeT-A
HMS-Beagle

Relative mRNA levels

esiRNAsl-1

HeT-A
HMS-Beagle

297

dsAGO2dsDroshadsAGO1

b

RNAi

–

–

dsDcr1RNAi

dsDcr2a

1
0

U6snRNA

Dcr2+/–

Dcr2–/–

Figure 4 | esiRNAs are produced in a Dicer-2-dependent manner, and are
involved in transposon repression in somatic cells. a, Dicer-2 (Dcr2)
depletion causes a severe reduction of esiRNAs. b, Increase ofretrotransposon transcript abundance in homozygous Dcr2 mutants. The
steady-state levels of transcripts from retrotransposons were measured byquantitative RT–PCR in carcasses (flies without testes) of heterozygous or
homozygous Dcr2 mutant males, relative to rp49 (also known as RpL32).
The error bars report the average 6 s.d. for at least three independent
experiments.

that esiRNAs were almost undetectable in the ovaries of Dicer-2
mutants and were significantly reduced in AGO2 mutants (Supplementary Fig. 8). Although the normal accumulation of piRNAs
requires Spindle-E (Spn-E)4, a lack of the Spn-E activity does notaffect the levels of esiRNAs in the ovary (Supplementary Fig. 8).
These results demonstrated that esiRNAs are produced in a Dicer2-dependent manner.
We examined whether the somatic expression of retrotransposons
is suppressed by the esiRNA pathway. Depletion of Dicer-2 or AGO2
coincided with higher levels of retrotransposon transcripts (Supplementary Fig. 9a, b). Mutations in Dicer-2 also caused an increase in
retrotransposon transcripts (Fig. 4b). We therefore concluded thatthe expression of a subset of retrotransposons was reduced by the
esiRNA pathway in somatic cells.
Transposable elements are powerful mutator elements responsible
for generating variations in the host genome, and therefore have arole as crucial factors shaping the genome through evolution23.
Previous studies have suggested that expression of transposable elements in animal somatic cells is mainly silenced by DNA methylation
at transcriptional levels23. However, we found that the expression of asubset of retrotransposons was reduced by the esiRNA pathway that
requires Dicer-2 and AGO2 in Drosophila somatic cells. This wasclearly distinct from the silencing of retrotransposons by thepiRNA pathway that requires Piwi proteins but not Dicers in thegerm line4,6,7. Our findings, in turn, suggested that some retrotransposons are actively transcribed in somatic cells, which are thenprocessed into small RNAs by Dicer-2. Recent studies in thefission yeast Schizosaccharomyces pombe have shown that RNAimediated heterochromatin formation requires the production of
RNA from heterochromatic regions to generate small RNAs andserve as part of a feed-forward loop required to maintain theheterochromatic state24. Thus, it is tempting to speculate that
esiRNAs could not only guide AGO2 to cleave transcripts ofretrotransposons but are also able to direct silencing complexes tospecific chromosomal regions. Interestingly, a loss of Dicer-2 or
AGO2 activity is correlated with defects in the formation of heterochromatin in Drosophila25,26. These results together suggest thatchanges in the activity of Dicer-2 and/or AGO2 could allow theexpression or even transposition of retrotransposons that may inturn create quantitative genetic variation in gene expression withinsomatic cell populations.

METHODS SUMMARY
AGO1 and AGO2 were immunopurified from S2 cells using specific antibodies27.
Cloning of small RNAs associated with AGO2 in S2 cells was carried out asdescribed9. Genome mapping and annotation was performed as described in
the Methods section. Northern blotting5, the in vitro target-RNA cleavage assay9,
RNAi for S2 cells12, perioxidation/b-elimination6,21 and RT–PCR analysis4 were
performed as described.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 9 December 2007; accepted 2 April 2008.
Published online 7 May 2008.
1.
2.
3.
4.
5.

6.
7.
8.
9.
10.

11.

12.

13.
14.

15.

Chapman, E. J. & Carrington, J. C. Specialization and evolution of endogenous
small RNA pathways. Nature Rev. Genet. 8, 884–896 (2007).
Peters, L. & Meister, G. Argonaute proteins: mediators of RNA silencing. Mol. Cell
26, 611–623 (2007).
Muotri, A. R. et al. Somatic mosaicism in neuronal precursor cells mediated by L1
reterotranspositioin. Nature 435, 903–910 (2005).
Vagin, V. V. et al. A distinct small RNA pathway silences selfish genetic elementsin the germline. Science 313, 320–324 (2006).
Saito, K. et al. Specific association of Piwi with rasiRNAs derived fromretrotransposon and heterochromatic regions in the Drosophila genome. Genes
Dev. 20, 2214–2222 (2006).
Gunawardane, L. S. et al. A Slicer-mediated mechanism for repeat-associatedsiRNA 59 end formation in Drosophila. Science 315, 1587–1590 (2007).
Brennecke, J. et al. Discrete small RNA-generating loci as master regulators oftransposon activity in Drosophila. Cell 128, 1089–1103 (2007).
Yin, H. & Lin, H. An epigenetic activation role of Piwi and a Piwi-associatedpiRNAs in Drosophila melanogaster. Nature 450, 304–308 (2007).
Nishida, K. M. et al. Gene silencing mechanisms mediated by Aubergine–piRNAcomplexes in Drosophila male gonad. RNA 13, 1911–1922 (2007).
Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. & Hannon, G. J.
Argonaute2, a link between genetic and biochemical analyses of RNAi. Science
293, 1146–1150 (2001).
Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate
ribonuclease in the initiation step of RNA interference. Nature 409, 363–366(2001).
Okamura, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Distinct roles for Argonaute
proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18,
1655–1666 (2004).
Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/
miRNA silencing pathways. Cell 117, 69–81 (2004).
Williams, R. W. & Rubin, G. M. ARGONAUTE1 is required for efficient RNAinterference in Drosophila embryos. Proc. Natl Acad. Sci. USA 99, 6889–6894(2002).
Wang, X. H. et al. RNA interference directs innate immunity against viruses inadult Drosophila. Science 312, 452–454 (2006).

796©2008 Nature Publishing GroupLETTERS

NATURE | Vol 453 | 5 June 2008

16. Ruby, J. G. et al. Large-scale sequencing reveals 21U-RNAs and additional
microRNAs and endogenous siRNAs in C. elegans. Cell 127, 1193–1207 (2006).
17. Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre
reactors. Nature 437, 376–380 (2005).
18. Kim, V. N. Small RNAs just got bigger: Piwi-interacting RNAs (piRNAs) inmammalian testes. Genes Dev. 20, 1993–1997 (2006).
19. Nishikura, K. Editor meets silencer: crosstalk between RNA editing and RNAinterference. Nature Rev. Mol. Cell Biol. 7, 919–931 (2006).
20. Girard, A., Sachidanandam, R., Hannon, G. J. & Carmell, M. A. A germline-specificclass of small RNAs binds mammalian Piwi proteins. Nature 442, 199–202(2006).
21. Saito, K. et al. Pimet, the Drosophila homolog of HEN1, mediates 29-Omethylation of Piwi-interacting RNAs at their 39 ends. Genes Dev. 21, 1603–1608(2007).
22. Horwich, M. D. et al. The Drosophila RNA methyltransferase, DmHen1, modifies
germline piRNAs and single-stranded siRNAs in RISC. Curr. Biol. 17, 1265–1272(2007).
23. Muotri, A. R., Marchetto, M. C. N., Coufal, N. G. & Gage, F. H. The necessary junk:
new functions for transposable elements. Hum. Mol. Genet. 16, R159–R167(2007).
24. Grewal, S. I. & Elgin, S. C. Transcription and RNA interference in the formation ofheterochromatin. Nature 447, 399–406 (2007).
25. Peng, J. C. & Karpen, G. H. H3K9 methylation and RNA interference regulate
nucleolar organization and repeated DNA stability. Nature Cell Biol. 9, 25–35(2007).
26. Deshpande, G., Calhoun, G. & Schedl, P. Drosophila argonute-2 is required early inembryogenesis for the assembly of centric/centromeric heterochromatin,
nuclear division, nuclear migration, and germ-cell formation. Genes Dev. 19,
1680–1685 (2005).

27. Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H. & Siomi, M. C. Slicer function of
Drosophila Argonautes and its involvement in RISC formation. Genes Dev. 19,
2837–2848 (2005).

Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We are grateful to T. Suzuki and members of the Siomi
laboratory for discussion and comments on this manuscript. We thank K. Yamada
and E. Hattori for expert assistance in AGO2-associated small RNA mapping andannotation, N. Iwanami and T. Hirose for help in qRT–PCR, and M. Itakura forcontinuous support and encouragement. This work was supported by MEXT grants
to M.C.S. and H.S., a MEXT (Ministry of Education, Culture, Sports, Science andTechnology, Japan) 21st COE (Centers of Excellence) postdoctoral fellowship to
K.S. and T.S., and NEDO (New Energy and Industrial Technology Development
Organization) grants to M.C.S., T.K. and K.A. M.C.S. is supported by CREST from
JST. H.S. is a member of the Genome Network Project (MEXT).
Author Contributions Y.K., K.S., T.N.O. and M.C.S. performed AGO2
immunoprecipitations, northern blotting, RNAi, the in vitro cleavage assay,b-elimination and qRT–PCR, and prepared the AGO2-associated small RNA
library. T.S. characterized and purified the AGO2 antibody. The bioinformatics
analyses of AGO2-associated small RNAs were designed and carried out by T.K.,
K.S., Y.O. and K.A. M.C.S., K.S., Y.K. and H.S. designed the experiments, discussed
the interpretation of the results and co-wrote the manuscript.
Author Information Small RNA sequences were deposited in the Gene Expression
Omnibus (www.ncbi.nlm.nih.gov/geo/) under the accession number GPL6452.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to M.C.S.
(siomim@sc.itc.keio.ac.jp) or H.S. (awa403@sc.itc.keio.ac.jp).

797©2008 Nature Publishing Groupdoi:10.1038/nature06938

METHODS
Drosophila strains. Yellow white (y w) was used as a wild-type strain. The strain
bearing the Spn-E100.37 mutation was provided by P. Zamore. The strain bearing
the Dcr-2L811fsX mutation13 was provided by K. Nakahara. The mutant fly(piggyBacf00810) was used as the DmHen1/Pimet mutant. Production of the
AGO2 mutant, AGO2414, was described previously12.
Cloning of small RNAs associated with AGO2 in S2 cells. AGO1 and AGO2
were immunopurified from S2 cells using specific antibodies against each of theproteins27. Cell lysis buffer was 13 PBS supplemented with 1 mM EDTA, 0.1 mM
DTT, 1% Empigen (Calbiochem), 2 mg ml21 pepstatin, 2 mg ml21 leupeptin
and 0.5% aprotinin. After immunoprecipitation, GammaBind beads (GEHealthcare) that were in a bound form with the immunoprecipitates were
washed five times with cell lysis buffer. Total RNAs were isolated from theimmunoprecipitates with phenol:chloroform and were precipitated withethanol. RNAs were dephosphorylated with calf intestinal alkaline phosphatase(CIP) (NEB) and labelled with [c-32P]ATP with T4 polynucleotide kinase(Takara) for visualization. Cloning of small RNAs associated with AGO2 in S2
cells was carried out as described5,6,9. The deep sequencing of the libraries was
performed on a GS20 system (Roche).
Genome mapping and annotation. Small RNA sequences were mapped to theD. melanogaster draft assembly28 (Berkeley Drosophila Genome Project release 5)
using the NCBI BLASTN program. The annotation of each small RNA wasdetermined by examining the overlap between its mapped regions and otherfeature track data of the UCSC Genome Browser (http://genome.ucsc.edu/). A
small RNA was assigned to a feature when the length of its overlap was longer
than 90% of the small RNA. We defined the priority of the feature assignment toavoid any conflict of assignment. The assignment to miRNA, rRNA, transfer
RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) andother non-coding RNAs (ncRNAs) was performed by using the FlyBase29 noncoding track. We used the FlyBase genes track for the annotation of coding genes.
We also used our earlier data of piRNAs22 for annotating piRNAs. RepeatMasker(http://www.repeatmasker.org) and the natural transposon tracks were used forannotating transposons. Repetitive elements other than transposons were used
to assign repeat elements.
Northern blot analysis. Northern blot analysis was carried out essentially as
described previously5. Total RNAs of S2 cells, fly embryos and adult male flies(carcasses, flies without testes) were isolated using ISOGEN (Invitrogen) according to the manufacturer’s instructions. Total RNAs from the immunoprecipitates were isolated with phenol:chloroform and precipitated with ethanol. A
DNA fragment for detecting retrotransposon transcripts was cloned into thepBS SK1 vector using primers, 1731-forward and 1731-reverse, for 1731
(GenBank accession number X07656). The sequences of the primers were as
follows: 1731-forward, 59-TATACGGCTTGAAGCAGTCAGGCAG-39; 1731reverse, 59-ACGCCAAGTACCGGAGGATGTGCTT-39. PCR products wereused as templates. DNA probes were synthesized in vitro using a random primelabelling kit (Takara) in the presence of [a-32P]dCTP. Probes used for miR-279,
miR-310, roo piRNA, bantam, esiRNA-1731-1 and esiRNA-sl-1 were as follows;
miR-279, 59-ATGAGTGTGGATCTAGTCA-39; miR-310, 59-AAAGGCCGGGAAGTGTGCAATA-39; roo piRNA, 59-TCGACTCAGTGGCACAATAAAT-39;
bantam, 59-CAGCTTTCAAAATGATCTCAC-39; esiRNA-1731-1, 59-AAGGT-

GTCGTCGCTGGTCTAC-39; esiRNA-sl-1, 59-GGAGCGAACTTGTTGGAGTCAA-39; and U6snRNA, 59-GGGCCATGCTAATCTTCTCTGTA-39. The
DNA oligonucleotides were labelled with T4 polynucleotide kinase in the presence of [c-32P]ATP.
In vitro target-RNA cleavage assay. To make an RNA target harboring
sequences completely complementary to bantam miRNA and esiRNA-sl-1—a
short dsDNA fragment (a target sequence for bantam) produced from two
oligonucleotide DNAs (59- AGCTTGAGATCATTTTGAAAGCTGATT-39 and
59- AGCTAATCAGCTTTCAAAATGATCTCA-39) by annealing was first
inserted in a pBS SKII1 vector at the HindIII site (yielding pBS-ban).
Following this, another DNA fragment (a target sequence for esiRNA-sl-1) produced from two DNA oligonucleotides (59-GATCGGAGCGAACTTGTTGGAGTCAA-39 and 59-GGCCTTGACTCCAACAAGTTCGCTCC-39) was inserted
into a pBS-ban (between BamHI and NotI). PCR was again performed using
primers for the T7 and T3 promoter sequences, and the PCR products used as
templates for in vitro transcription using a MEGAscript T7 kit (Ambion). The
resultant RNAs were radiolabelled at their 59 ends with [c-32P]ATP using T4
polynucleotide kinase and were gel-purified. The target-RNA cleavage assay was
performed as described previously27 using AGO1 and AGO2 immunoprecipitates from S2 in a buffer containing Empigen.
RNA interference. RNAi in S2 cells was performed essentially as described12.
Production of dsRNAs was described previously12. dsRNAs used in RNAi were:dsDrosha, homologous to nucleotides 2701–3925 of the Drosha open reading
flame (ORF) sequence (accession number AE013599); dsDicer-1, 10–950 of the
Dicer-1 ORF sequence (accession number AE014297); dsDicer-2, 4091–4888 of
the Dicer-2 ORF sequence (accession number NM_079054); dsAGO1, 1035–
2042 of the AGO1 ORF sequence (accession number NM_166021); dsAGO2,
1304–2282 of the AGO2 ORF sequence (accession number NM_168626).
Periodate oxidation and b-elimination. Periodate oxidation and b-eliminationof RNAs were performed as described6,21. RNAs isolated from AGO1 and AGO2
immunoprecipitates were labelled with [c-32P]ATP after CIP treatment.
10,000 counts per min of 32P-labelled RNAs was used per reaction. Total
RNAs of S2 cells and of fly embryos were subjected to perioxidation andb-elimination without 32P labelling. Visualization of esiRNA-sl-1, miR-279, roo
piRNA and miR-310 was done by northern blotting.
Quantitative RT–PCR analysis. Total RNA (0.4 mg) was used to reverse transcribe target sequences using oligo(dT) primer and PrimeScript RT reagent Kit(Takara) according to the manufacturer’s instructions. The resulting cDNA was
analysed by quantitative RT–PCR in a LightCycler real-time PCR system (Roche
Diagnostics) using the SYBR Premix Ex Taq (Takara). Relative steady-statemRNA levels were determined from the threshold cycle for amplification.
Supplementary Table 3 lists the PCR primer sequences. Rp49 was used as aninternal control.
28. Celniker, S. E. et al. Finishing a whole genome shotgun: Release 3 of the Drosophilamelanogaster euchromatic genome sequence. Gen. Biol. 3,
research0079.1–0079.14 (2002).
29. Crosby, M. A., Coodman, J. L., Strelets, V. B., Zhang, P., Gelbart, W. M., the FlyBase
Consortium. FlyBase: genomes by the dozen. Nucleic Acids Res. 35 (Databaseissue), D486–D491 (2007).

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