A Slicer-Mediated Mechanism for
Repeat-Associated siRNA 5' End Formation in
Drosophila
Lalith S. Gunawardane, et al.
Science 315, 1587 (2007);
DOI: 10.1126/science.1140494
The following resources related to this article are available online atwww.sciencemag.org (this information is current as of March 16, 2007 ):Supporting Online Material can be found at:
http://www.sciencemag.org/cgi/content/full/1140494/DC1
This article cites 18 articles, 14 of which can be accessed for free:
http://www.sciencemag.org/cgi/content/full/315/5818/1587#otherarticles
This article appears in the following subject collections:
Molecular Biology
http://www.sciencemag.org/cgi/collection/molec_biol
Information about obtaining reprints of this article or about obtaining permission to reproduce
this article in whole or in part can be found at:
http://www.sciencemag.org/about/permissions.dtl

Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyrightc 2007 by the American Association for the Advancement of Science; all rights reserved. The title SCIENCE is a
registered trademark of AAAS.

Downloaded from www.sciencemag.org on March 16, 2007

Updated information and services, including high-resolution figures, can be found in the online
version of this article at:
http://www.sciencemag.org/cgi/content/full/315/5818/1587

A Slicer-Mediated Mechanism for
Repeat-Associated siRNA 5´ End
Formation in Drosophila
Lalith S. Gunawardane,* Kuniaki Saito,* Kazumichi M. Nishida,* Keita Miyoshi,
Yoshinori Kawamura, Tomoko Nagami, Haruhiko Siomi,† Mikiko C. Siomi†
In Drosophila, repeat-associated small interfering RNAs (rasiRNAs) are produced in thegerm line by a Dicer-independent pathway and function through the PIWI subfamily of
Argonautes to ensure silencing of retrotransposons. We sequenced small RNAs associatedwith the PIWI subfamily member AGO3. Although other members of PIWI, Aubergine (Aub) andPiwi, associated with rasiRNAs derived mainly from the antisense strand of retrotransposons,AGO3-associated rasiRNAs arose mainly from the sense strand. Aub- and Piwi-associatedrasiRNAs showed a strong preference for uracil at their 5′ ends, and AGO3-associatedrasiRNAs showed a strong preference for adenine at nucleotide 10. Comparisons between
AGO3- and Aub-associated rasiRNAs revealed pairs of rasiRNAs showing complementarities
in their first 10 nucleotides. Aub and AGO3 exhibited Slicer activity in vitro. These data
support a model in which formation of a 5′ terminus within rasiRNA precursors is
guided by rasiRNAs originating from transcripts of the other strand in concert with the
Slicer activity of PIWI.
mall noncoding RNAs trigger various
forms of sequence specific gene silencing, including RNA interference (RNAi),
translational repression, and heterochromatin
formation in a variety of eukaryotic organisms,
commonly referred to as RNA silencing (1–3).
Members of the Argonaute family of proteinsare essential components of RNA silencing(4, 5). In Drosophila, five genes encode distinct
members of the Argonaute family: AGO1,
AGO2, Aubergine (Aub), Piwi, and AGO3.
AGO1 and AGO2 constitute the Argonaute(AGO) subfamily and bind microRNA (miRNA)and small interfering RNA (siRNA), respectively (6–8). Aub, Piwi, and AGO3 belong to
the PIWI subfamily of the Argonaute family(4, 5) and are enriched in germline cells (9),and Aub and Piwi have been shown to play
important roles in germline cell formation(10, 11). They are involved in silencing retrotransposons and other repetitive elements(12–15) and exhibit target RNA cleavage(slicing) activity in vitro (16). Both Aub andPiwi associate with repeat-associated siRNAs(rasiRNAs) (15, 16). Aub- and Piwi-associatedrasiRNAs are derived mainly from the antisense strand of retrotransposons, with little orno phasing, and have a strong preference foruracil (U) at the 5′ end (15, 16). Small RNA
processing factors such as Dicer and Drosha
are known to cleave preferentially at the 5′ sideof U (17); however, rasiRNAs are thought to be

S

Institute for Genome Research, University of Tokushima,
Tokushima 770-8503, Japan.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:siomi@genome.tokushima-u.ac.jp (H.S.); siomim@genome.
tokushima-u.ac.jp (M.C.S.)

produced by a Dicer-independent pathway(15). The mechanisms governing rasiRNA
production remain to be elucidated.
Very little is known about the function of
AGO3 (9), the third member of the Drosophila PIWI subfamily. We isolated a full-length
cDNA of AGO3, revealing that the AGO3 geneis ~83 kb in length (fig. S1). Peptide sequencealignments among Drosophila Argonaute proteins revealed that AGO3 is most similar to
Piwi (fig. S2A). The Asp-Asp-His motif in the
PIWI domain, originally identified as the catalytic center for Slicer activity in human AGO2
(5, 18), is conserved in AGO3 (fig. S2B).
Embryonic RNA expression patterns of
AGO3 are very similar to those of Piwi andAub; they are expressed maternally, but theirexpression disappears by embryonic stages
10 to 12 (9). To confirm these results, we produced a monoclonal antibody (mAb) to AGO3
(Fig. 1A), which revealed that AGO3 is strongly expressed in earlier embryonic stages but
decreases as development proceeds (Fig. 1B).
AGO3 accumulated in the cytoplasm of germline cells including germline stem cells (GSCs),
germline cyst cells, nurse cells, and oocytes at
earlier stages (Fig. 1C and fig. S3). In testes,
AGO3 is expressed in GSC, primary gonial
cells, and early spermatocytes (Fig. 1D). Unlike Piwi (16), AGO3 expression was undetected in the hub (fig. S3), a tiny cluster of
postmitotic somatic cells localized at the apical tip of the testis that functions as a niche for
GSC (19). Thus, with respect to expression in
germline cells, AGO3 is more similar to Aub
than to Piwi (10, 16).
All of the other members of the fly
Argonautes are specifically associated with asubset of small RNAs: siRNAs, miRNAs, orwww.sciencemag.org

SCIENCEVOL 315

rasiRNAs (6–8, 15, 16). We therefore investigated whether AGO3 also associates withsmall RNAs produced in the fly ovary. Immunoprecipitation with AGO3 mAb fromovary lysate revealed small RNAs ~23 to 26
nucleotides (nt) long (Fig. 2A). The size distribution of AGO3-associated small RNAsis similar to that of Aub-associated small
RNAs (Fig. 2B); in both cases, the peak is
24 nt and the longest is 27 nt. Small RNAs
associated with AGO3 are likely to lack eithera 2′ or 3′ hydroxyl group, because they do not
migrate faster after b-elimination as opposed
to a synthetic siRNA that has 2′ and 3′hydroxyl groups at the 3′ end, the latter being
the hallmarks of Dicer cleavage (Fig. 2C).
These results suggest that AGO3-associatedsmall RNAs in the ovary are produced by apathway similar to those involved in production of rasiRNAs that associate with Aub
and Piwi.
We constructed a cDNA library of small
RNAs associated with AGO3 in the ovary. Of
420 clones sequenced, 410 matched Drosophila genomic sequences in a database search(table S1), and most were rasiRNAs (~86%;
353 of 410), as in the case of Aub and Piwi
(table S2). Like rasiRNAs associated with
Aub or Piwi (15, 16), rasiRNAs associatedwith AGO3 included various kinds of transposable elements, both LTR (long terminal
repeat) retrotransposons and LINE (long interspersed nuclear element)–like elements(tables S1 and S2). rasiRNAs associated with
Aub or Piwi in ovaries are derived mainly
from the antisense strand of retrotransposons,and the 5′ end is predominantly U (15, 16).
These characteristics were not found for
rasiRNAs associated with AGO3. However,AGO3-associated rasiRNAs were derived
mainly from the sense strand of retrotransposons (~82%; table S3), and they showed astrong preference for adenine (A) at nucleotide 10, but no preference for U at the 5′ end(Fig. 3A). These results suggest that AGO3associated rasiRNAs belong to a subset ofrasiRNAs that are distinct from Aub- andPiwi-associated rasiRNAs.
Some Argonaute proteins exhibit Slicer activity that directs cleavage of its cognate mRNAtarget across from nucleotides 10 and 11, measured from the 5′ end of the small RNA guidestrand (20). Thus, our findings suggest a model
for rasiRNA biogenesis, in which the 5′ end of
Aub- and Piwi-associated rasiRNAs is determined and cleaved by AGO3-rasiRNA complexes, and the 5′ end of AGO3-associatedrasiRNAs is determined by Aub- and PiwirasiRNA complexes through a similar rasiRNAguided cleavage event (Fig. 3B). For instance,
AGO3 associated with a rasiRNA with A atnucleotide 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 5′
end. Similarly, when Aub or Piwi associated with

16 MARCH 2007

Downloaded from www.sciencemag.org on March 16, 2007

REPORTS

1587

Fig. 1. AGO3 expression. (A) Western blotting was
performed on S2 and fly ovary lysates with AGO3
mAb. AGO3 expression is detected only in the ovary.
(B) AGO3 expression pattern through development.
Expression is high in early embryos but graduallydiminishes through development. (C) Immunostaining pattern of AGO3 in fly ovary and testis. All
images shown represent one confocal section. In a
germarium region, AGO3 is strongly accumulated in
the cytoplasm of germline stem cells (GSC) andgermline cyst cells (GCC) (upper panel). In an egg chamber at stage 7 (lower panel), AGO3 was found in bothoocytes (OO) and nurse cells (NC). A weak signal of AGO3 was also observed in follicle cells (FC). AGO3 isclearly cytoplasmic. (D) Fluorescent image of AGO3 in testis. AGO3 expression is detected in germline
stem cells, primary gonial cells, and early spermatocytes.
Fig. 2. Small RNAs associated with AGO3 in fly
ovary. (A) Small RNAs associated with AGO3 immunopurified from ovary are
visualized by [32P]adenosine triphosphate labeling. (B) Sizes of small RNAs
associated with AGO1, Piwi,
Aub, and AGO3 are compared. The peak of small
RNAs associated with Aub
and AGO3 is about 24 nt,
whereas that of Piwi isabout 26 nt; rasiRNAs with
AGO3 contain shorter ones
(23 nt) relative to those of
Aub. The ~30-nt bands are
2S rRNA. (C) rasiRNAs associated with AGO3 show resistance to b-elimination treatment, which made 21-nt siRNA (control RNA)
without 3′ end modification shorter, indicating that rasiRNAs associated with AGO3 are modified atthe 3′ end.

1588

16 MARCH 2007

VOL 315SCIENCE

rasiRNAs with U at the 5′ end slices its cognate
RNA target, the resulting cleaved RNA will
have A at nucleotide 10.
To test this model, we examined AGO3
for Slicer activity by performing in vitro target RNA cleavage assays with glutathione
S-transferase (GST)–AGO3 fusions (Fig. 4A).
The target RNA, luc passenger siRNA (21 nt
long, 5′ end labeled with 32P) (8), was efficiently cleaved by GST-AGO3, as was thecase for GST-AGO1 and GST-Aub. The sizeof the cleaved products (9 nt) indicated thatthey direct cleavage of target RNA acrossfrom nucleotides 10 and 11 as measured fromthe 5′ end of the small RNA guide strand (Fig.
4A). Both GST-Aub and GST-AGO3 with alonger guide RNA (26 nt) were also able tocleave a long transcript (180 nt) (fig. S4).
Long precursors of rasiRNAs both in sense
and antisense orientations appear to exist in
fly ovaries (fig. S5). These results corroborate
the model in which the 5′ end of rasiRNAs
within the precursors is determined by rasiRNAs
and cleaved by members of PIWI that associatewith these rasiRNAs.
Our model predicts that some AGO3associated rasiRNAs should be complementary
to the first 10 nt of Aub- and Piwi-associatedrasiRNAs. Sequence comparison between AGO3and Aub-associated rasiRNAs indeed revealed
pairs of rasiRNAs that show complementarities at their first 10 nt (fig. S6). Sixteen of
353 AGO3-associated rasiRNAs had such pairs
with 11 of 676 Aub-associated rasiRNAs (fig.
S6). However, such pairings were only foundbetween AGO3- and Aub-associated rasiRNAs,
and no pairs were observed between AGO3and Piwi-associated rasiRNAs (353 versus
330). Like Aub-associated rasiRNAs (tableS3), Piwi-associated rasiRNAs arise mainly
from the antisense strand and their 5′ ends
show a strong preference for U (16); thus, it
is difficult to argue that Piwi is not involvedin this type of rasiRNA biogenesis. One possible reason is that Piwi is nuclear, whereas
AGO3 and Aub are cytoplasmic (16). This
type of rasiRNA biogenesis may operate in
the cytoplasm. Alternatively, formation of
5′ ends of Piwi-associated rasiRNAs may
occur only at an earlier time during germline
development.
RasiRNAs are involved in genome surveillance by silencing repetitive elements andcontrolling their mobilization in the Drosophila germ line. It was recently shown that
rasiRNAs are produced by a mechanism that
requires neither Dicer-1 nor Dicer-2 in flies(15). Our data suggest that rasiRNAs in asense orientation guide formation of the 5′
end of rasiRNAs in an antisense orientation,and vice versa; as well, this cycle of mutual
dependency elaborates optimal rasiRNA production (Fig. 4B). In this model, proteins of
the PIWI subfamily function as Slicer forformation of the 5′ end during rasiRNA

www.sciencemag.org

Downloaded from www.sciencemag.org on March 16, 2007

REPORTSREPORTS
biogenesis. This model requires that sliced
rasiRNA precursors then be cleaved again at
the 3′ end by an as yet unidentified endo-

ber of the PIWI. Once “primary” complexesof rasiRNAs with proteins of PIWI are
produced, these complexes will in turn function

A
( )

80

A=73%

UGC A

70
60

5'

3'

50
40
30
20
10
0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

B
rasiRNAs associated with Aub and Piwi
5'-

-3'

3'-

-5'

rasiRNAs associated with AGO3

10 th

Fig. 4. (A) Target RNA
cleavage assay using GSTAGO1, GST-AGO3, and
GST-Aub produced in
Escherichia coli. GST-fusedproteins were first boundwith a single-stranded luc
guide siRNA; then, target
RNA (single-stranded luc
passenger siRNA labeledwith 32P at the 5′ end)
was added to the reaction
mixture and further incubated. All the proteins,
but not GST itself, wereable to cleave target RNA
across from nucleotides
10 and 11, measured
from the 5′ end of theguide RNA. The asterisk
indicates cleaved productswith the expected size
(9 nt); the cross marks byproducts of 8 nt in in vitro
assays with GST fusions(8). (B) A model for arasiRNA biogenesis cycle.
AGO3-associated sense
rasiRNAs (upper left) guide
Slicer-mediated cleavageof primary antisense transcripts, yielding the antisense rasiRNA precursors
with U at the 5′ end. Reciprocally, Aub-associatedantisense rasiRNAs (lower
right) guide Slicer-mediatedcleavage of sense transcripts, yielding the sense
rasiRNA precursors with A at nucleotide 10. Formation of rasiRNAs at the 3′ end may occur (a) while the precursors still remain in the complexes, or (b) after they areloaded onto the other PIWI by an as-yet-unidentified nuclease.
www.sciencemag.org

SCIENCEVOL 31516 MARCH 2007

Downloaded from www.sciencemag.org on March 16, 2007

Fig. 3. Characteristics ofrasiRNAs associated with
AGO3 in the ovary. (A)rasiRNAs associated with
AGO3 contain A predominantly at nucleotide 10
from the 5′ end. (B) A
predicted model indicatingthat the first 10 nt of
AGO3-associated rasiRNAs
show complementarities tothe first 10 nt of Aub- or
Piwi-associated rasiRNAs.

nuclease (or nibbled by exonuclease) to produce
mature rasiRNAs before or after loading of theresulting cleavage products onto another mem-

1589

REPORTS

References and Notes
1. Y. Tomari, P. D. Zamore, Genes Dev. 19, 517(2005).
2. H. H. Kavi, H. R. Fernandez, W. Xie, J. A. Birchler,
FEBS Lett. 579, 5940 (2005).
3. W. P. Kloosterman, R. H. Plasterk, Dev. Cell 11, 441(2006).
4. M. A. Carmell, Z. Xuan, M. Q. Zhang, G. J. Hannon,
Genes Dev. 16, 2733 (2002).
5. J. S. Parker, D. Barford, Trends Biochem. Sci. 31, 622(2006).

6. K. Okamura, A. Ishizuka, H. Siomi, M. C. Siomi,
Genes Dev. 18, 1655 (2004).
7. Y. Tomari, C. Matranga, B. Haley, N. Martinez,
P. D. Zamore, Science 306, 1377 (2004).
8. K. Miyoshi, H. Tsukumo, T. Nagami, H. Siomi,
M. C. Siomi, Genes Dev. 19, 2837 (2005).
9. R. W. Williams, G. M. Rubin, Proc. Natl. Acad. Sci. U.S.A.
99, 6889 (2002).
10. A. N. Harris, P. M. Macdonald, Development 128, 2823
(2001).
11. D. N. Cox et al., Genes Dev. 12, 3715 (1998).
12. A. I. Kalmykova, M. S. Klenov, V. A. Gvozdev,
Nucleic Acids Res. 33, 2052 (2005).
13. A. A. Aravin et al., Mol. Cell. Biol. 24, 6742 (2004).
14. V. V. Vagin et al., RNA Biol. 1, 54 (2004).
15. V. V. Vagin et al., Science 313, 320 (2006); published
online 28 June 2006 (10.1126/science.1129333).
16. K. Saito et al., Genes Dev. 20, 2214 (2006).
17. A. A. Aravin et al., Dev. Cell 5, 337 (2003).
18. J. Liu et al., Science 305, 1437 (2004); published online
29 July 2004 (10.1126/science.1102513).
19. Y. M. Yamashita, M. T. Fuller, D. L. Jones, J. Cell Sci. 118,665 (2005).
20. S. M. Elbashir, W. Lendeckel, T. Tuschl, Genes Dev. 15,
188 (2001).

Attention-Like Processes in
Drosophila Require Short-Term
Memory Genes
Bruno van Swinderen
Although there is much behavioral evidence for complex brain functions in insects, it is notknown whether insects have selective attention. In humans, selective attention is a dynamic
process restricting perception to a succession of salient stimuli, while less relevant competingstimuli are suppressed. Local field potential recordings in the brains of flies responding tovisual novelty revealed attention-like processes with stereotypical temporal properties. These
processes were modulated by genes involved in short-term memory formation, namely dunce andrutabaga. Attention defects in these mutants were associated with distinct optomotor effects inbehavioral assays.
tudies of visual discrimination in flies
have revealed sophisticated perceptual
effects that are relevant to selective attention (1, 2), such as associative learning (3),context generalization (4, 5), cross-modal binding (6), and position invariance (7). Visual choice
behavior in Drosophila is correlated with local
field potential (LFP) activity in the brain, centered around 20 to 30 Hz (8). This activity istransiently increased in amplitude by classicalconditioning (8), is suppressed during sleep (8)or light anesthesia (9), and is modulated bydopamine (10). Electrophysiological and behavioral measures of visual attention in flies
were developed to test whether these shortterm processes depend on the effect of genes
involved in memory formation and plasticity(11–13).
LFP responses to two distinct visual objects(a cross or a box, 180° apart, each moving aroundS

The Neurosciences Institute, 10640 John Jay Hopkins Drive,
San Diego, CA 92121, USA. E-mail: van@nsi.edu

1590

the fly once every 3 s) were investigated (Fig. 1A).
When the objects were presented individually
to wild-type flies, they evoked brain responses
that were maximal when the single object
swept directly in front of the flies (Fig. 1B). In
contrast, dunce mutants (dnc1) (14), which aredefective in short-term memory (15, 16), displayed attenuated and delayed brain responses toeach visual object, as compared to wild-type flies(Fig. 1C).
To test for visual selection between theseobjects, I presented them together after having
increased the salience for one object specificallyin a recurrent-novelty paradigm (Fig. 1D) (17).
To measure visual selection, the 20- to 30-Hz
brain response (mapped onto the 3-s sequence)(Fig. 1B) was averaged for 10 s (about threerotations) after each transition to novelty, andthis was compared to the response for the 10 s
before novelty transitions (red versus blue lines,
respectively, in Fig. 1D). When wild-type flies
were trained with two identical boxes for 100 s(~33 rotations) before one of the boxes changed

16 MARCH 2007

VOL 315SCIENCE

21. We thank T. Suzuki and V. V. Vagin for technicaladvice; T. Mori, M. Shinomiya, and A. Kinouchi forassistance with fly husbandry and dissection; andmembers of the Siomi laboratory for discussions andcomments on the manuscript. Supported by the
Ministry of Education, Culture, Sports, Science, andTechnology of Japan (M.C.S., H.S.) and its 21st Century
Centers of Excellence Program (L.S.G., K.S.), the Japan
Society for the Promotion of Science (JSPS) (M.C.S. andH.S.), a JSPS research fellowship (K.M.), and the New
Energy and Industrial Technology Development
Organization (M.C.S).

Supporting Online Materialwww.sciencemag.org/cgi/content/full/1140494/DC1
Materials and Methods
Figs. S1 to S6Tables S1 to S3
References
29 January 2007; accepted 12 February 2007
Published online 22 February 2007;
10.1126/science.1140494
Include this information when citing this paper.

to a cross, the response mapped selectively tothe sectors of the rotation sequence associatedwith the (novel) cross (100 s, red line in Fig. 1D),
and the response for the competing box was
significantly suppressed. Converse experiments
attaching novelty salience to the alternate image(the box) after 100 s of cross training mapped
20- to 30-Hz responses to the novel box, showing that novelty selection was plastic (fig. S1).
Novelty selection was also found to be positioninvariant (7) in a subset of trials, suggesting a
cognitive effect rather than habituation (fig. S2).
By decreasing the time between transitions inotherwise identical experiments, this paradigm
provided a way to estimate the minimum exposure required for selection of recurrent novelty.
When the training time was decreased to 50 s(~16 rotations), significant selection of the novel
object and corresponding suppression of thecompeting object were still seen in wild-type
flies (fig. S3). However, when the training time
was further decreased to 25 s (about eight rotations), these novelty effects were lost (Fig. 1D).
To control for the effect of change alone
without novelty, I tested transitions from a crossand a box back to two boxes (Fig. 1E). In thiscase, an object changed to one that was alreadypresent during training. Such changes did not
produce any selective 20- to 30-Hz responses forany training time in wild-type flies (100 s and 25
s in Fig. 1E). The response is therefore unlikely
to emanate from a startle reflex or an electricalartifact.
Salience is a transient phenomenon. To investigate the extinction of novelty, I analyzed thetemporal sequence of selective brain responses
for successive rotations of a novel panorama
after a transition (Fig. 2A). In wild-type flies, 20to 30-Hz activity was strongly selective for thenovel object (the cross) for 9 s (three successive
panorama rotations) on average (red lines in Fig.
2A), and this was matched at a lower level forwww.sciencemag.org

Downloaded from www.sciencemag.org on March 16, 2007

as the “initiator” of secondary rasiRNA biogenesis, and so nascent rasiRNAs should becontinuously supplied in the ovary and testis.
Such a process may occur through rasiRNA
germline transmission. Of the PIWI members,
at least Aub is accumulated to the posteriorpole in oocytes and remains in polar granules
in early embryos. It is then incorporated inpole cells, the progenitor of the Drosophila
germ line (10).