Slicer function of Drosophila Argonautes
and its involvement in RISC formation
Keita Miyoshi,1 Hiroko Tsukumo,1 Tomoko Nagami, Haruhiko Siomi, and Mikiko C. Siomi2
Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan

Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was
shown to be responsible for target RNA cleavage (“Slicer”) activity in RNA interference (RNAi), whereas other
Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown
to possess the Slicer activity. Here we show that AGO1, another member of the Drosophila Argonaute
subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target
RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length
AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further,reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that otherregions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage
activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwoundand consequently an RNA-induced silencing complex (RISC) is not formed. We show that upon addition of ansiRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, andnuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a
new model in which AGO2 is directly involved in RISC formation as “Slicer” of the passenger strand of thesiRNA duplex.
[Keywords: RNAi; Argonaute; Slicer, RISC; Drosophila]
Supplemental material is available at http://www.genesdev.org.
Received August 31, 2005; revised version accepted October 5, 2005.

RNA interference (RNAi) is a sequence-specific RNA
cleavage process triggered by double-stranded RNA(dsRNA) (for review, see Sontheimer 2005; Tomari andZmore 2005). Long dsRNA is first converted to 21–23nucleotide (nt) small interfering RNA (siRNA) by Dicer(Bernstein et al. 2001; Elbashir et al. 2001a; Ketting et al.
2001; Knight and Bass 2001). Subsequently, the siRNA
duplex goes through an ATP-dependent unwinding process and one strand over the other is often preferentiallyloaded onto the RNA-induced silencing complex (RISC)
that endonucleolytically cleaves target RNA at sites
completely complementary to the siRNA (Hammond etal. 2000; Nykanen et al. 2001; Hutvagner and Zamore
2002a; Martinez et al. 2002). Cleavage on the target RNA
sequence in RNAi is known to occur between +10 and+11 positions of the guide siRNA (the 5⬘-end of the guidesiRNA being assigned +1) (Elbashir et al. 2001b; Schwarz
et al. 2004).
microRNAs (miRNAs) are a large family of 21–22-nt
noncoding RNAs that interact with target mRNAs at

1

These authors contributed equally to this work.
Corresonding author.
E-MAIL siomim@genome.tokushima-u.ac.jp; FAX 81-88-6339451.
Article published online ahead of print. Article and publication date areat http://www.genesdev.org/cgi/doi/10.1101/gad.1370605.

2

specific sites to induce cleavage of the message or inhibittranslation (Olsen and Ambros 1999; Reinhart et al.
2000; Ambros 2004; Bartel 2004; Bagga et al. 2005). The
expression of miRNA itself is often developmentally
regulated in a spacious- and/or timing-specific manner,
implying an important developmental role for miRNAs
in the regulation of endogenous gene expression (LagosQuintana et al. 2001; Lau et al. 2001; Lee and Ambros
2001; Reinhart et al. 2002; Brennecke et al. 2003; Leaman et al. 2005; Pillai et al. 2005). Lately, direct relations
between miRNA and human diseases such as cancer
have been gradually elucidated (Garraway et al. 2005; Heet al. 2005; Johnson et al. 2005; Mattick and Makunin2005). miRNAs are excised in a stepwise process fromprimary miRNA transcripts (pri-miRNAs) (Bartel 2004;
Kim 2005). Then, mature miRNA is loaded onto RISC
(miRISC) as siRNA in RNAi (Hutvagner and Zamore
2002b).
A biochemical approach to identifying protein components involved in RISC in the Drosophila system led tothe identification of a member of the Argonaute familyof proteins as an essential factor in RNAi (Hammond etal. 2001). Members of the Argonaute family, defined by
the presence of PAZ and PIWI domains, are known toplay essential roles, while residing in RISC, in gene silencing triggered by small RNAs (Carmell et al. 2002).

GENES & DEVELOPMENT 19:2837–2848 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org

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Argonaute members can be divided into two groups, theArgonaute and the Piwi subfamilies (Sasaki et al. 2003).
Even within a subfamily, each member seems functionally distinct. Fly embryos lacking AGO2, a member ofthe Argonaute subfamily in fly, are siRNA-directed
RNAi defective, but still capable of miRNA-directed target RNA cleavage (Okamura et al. 2004). AGO1, another
Argonaute protein in fly (Kataoka et al. 2001), is dispensable for siRNA-directed RNA cleavage, but is necessary
for miRNA-directed target RNA cleavage (Okamura etal. 2004). Depletion of AGO2, but not AGO1, from Drosophila Schneider2 (S2) cells results in transgene mRNAstabilization with concomitant shortening of the mRNApoly(A) tail (Siomi et al. 2005). In humans, Ago2 (hAgo2)
is associated with both siRNA and miRNA, and mediates RNA cleavage targeted by small RNAs; however,other Argonaute subfamily members, such as hAgo1,
hAgo3, and hAgo4, do not mediate such RNA cleavage,
although all can associate with siRNA and miRNA (Liuet al. 2004; Meister et al. 2004a) and show high similarities to hAgo2 at peptide sequence levels (Sasaki et al.
2003). In plants, specific members of the Argonaute subfamily also show distinct biochemical activities (Qi et al.
2005).
Structural analysis of full-length Pyrococcus furiosus
AGO-related protein revealed that the PIWI domain, oneof the common domain structures in the Argonautes,
shows an RNaseH-like structure, and that the essential
residues for the endonucleolytic activity of RNaseH areconserved in the Argonaute-PIWI (AGO-PIWI) domain(Song et al. 2004). Mutagenizing the conserved residues
in hAgo2 abolished the RNA cleavage activity targetedby small RNAs (Liu et al. 2004). Furthermore, Slicer, theendonucleolytic enzyme residing in RISC activity canbe reconstituted with recombinant hAgo2 and singlestranded siRNA (Rivas et al. 2005). These observations
indicate that hAgo2 acts as Slicer in RNAi.
The crystal structure of Archaeoglobus fulgidus Piwi
protein (AfPiwi) associated with an siRNA-like small
RNA has been determined (Ma et al. 2005; Parker et al.
2005). AfPiwi, utilized as a model for understanding the
eukaryotic AGO-PIWI domain, was shown to bind to the5⬘-end of the guide strand of the siRNA duplex, althoughprevious structural and biochemical studies implied thatthe conserved PAZ domain in Argonaute proteins was
the guide siRNA-binding module through its 3⬘-end (Yanet al. 2003; Lingel et al. 2004; Ma et al. 2004; Song et al.
2004). The crystal structure of Aquifex aeolicus Argonaute (Aa-Ago), a site-specific DNA-guided RNA endonuclease, was recently solved (Yuan et al. 2005) and an
Ago-mediated target RNA cleavage cycle model proposed. According to this model, the Ago-PAZ domain
binds to the 3⬘-end of siRNA at the beginning, but once
target RNA comes and starts to anneal to the siRNA, the3⬘-end of the guide strand is released from the PAZ domain. This action consequently allows target RNA toform a full-length guide siRNA-target RNA duplex before the target RNA cleavage really occurs. However,crystallographic characterization of Argonaute from organisms furnishing genuine RNAi machinery will be

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necessary to create a fully elaborated biochemical modelof the small RNA-directed target RNA cleavage pathway.
In Drosophila, AGO2 was reported to be the sole protein required for target RNA cleavage activity in RNAi
(Rand et al. 2004). While AGO2 may indeed function as
the Slicer, AGO1 is also thought responsible for cleaving
target RNA since it is more similar to hAgo2 at the peptide sequence level than is AGO2; in addition, AGO1 isalso required for efficient miRNA-directed target RNA
cleavage in fly embryo (Okamura et al. 2004). In humans,
hAgo1 does not show Slicer activity as opposed tohAgo2. This is quite striking since hAgo1 and hAgo2
show strong similarities at both the peptide sequencelevel and in their biochemical properties, such that bothproteins bind siRNA and miRNA (Liu et al. 2004; Meister et al. 2004a). They have even been shown to coimmunoprecipitate from human culture cells (Sen and Blau
2005). Although the function of hAgo1 has yet to be elucidated, it seems quite unlikely that Drosophila AGO1
is not simply the counterpart of hAgo1.
In this study, we show that in Drosophila, unlike inhumans, not only AGO2, but also AGO1 has the capacity to show Slicer activity. Recombinant full-length
AGO1 and the PIWI domains of AGO1 (AGO1-PIWI) andAGO2 (AGO2-PIWI) were successfully produced in Escherichia coli. All recombinants were found capable ofcleaving RNA targets when associated with small guideRNA. Our data indicate that the PAZ domain is notstrictly necessary for the Argonautes to exhibit Slicer
activity. It has been reported that recombinant RISC
does not require ATP for cleaving target RNA and releasing the RNA products from itself (Rivas et al. 2005). In
this study, using a Drosophila system, we showed thateach step indeed occurs without ATP and that they are
not stimulated by the addition of ATP. Of the siRNA
duplex programmed in S2 lysate, the passenger strand
was cleaved in an AGO2-dependent manner and the
AGO2 cleavage-resistant passenger strand clearly impaired RISC formation in S2 cell lysate. These findings
give rise to a new model in which AGO2 is directly involved in RISC formation as “Slicer” of the passenger
strand of the siRNA duplex. Finally, reconstitution of
Slicer activity was accomplished with recombinant fulllength AGO1 together with the siRNA duplex and its
target RNA.

Results
Immunopurified AGO2 from siRNA-programmed
S2 lysate shows target RNA cleavage activity
We assessed whether Drosophila AGO2 immunopurified from S2 cells could show target RNA cleavage activity. S2 lysate was incubated with the luc siRNA duplex, from which AGO2 was immunoprecipitated undera high-salt condition using an anti-AGO2 antibody
raised specifically for the N terminus of the protein. Silver-staining of the protein contents in the immunoprecipitate showed that AGO2 was nearly uniform in theFly Argonautes as Slicer

fraction (Fig. 1A). Western blot analysis confirmed thatthe immunoprecipitate contains AGO2, but not AGO1,
as an undesired contaminant (Supplementary Fig. S1).
Northern blotting revealed that the immunopurified
AGO2 associates with luc guide siRNA (Fig. 1B). Thisdata agreed well with previous reports showing that Argonaute proteins are very tightly bound to singlestranded siRNA within RISC (Martinez et al. 2002; Rand
et al. 2004). luc target RNA radiolabeled with 32P at the5⬘-end was then incubated with the AGO2-luc guidesiRNA complex immobilized on beads. Figure 1C showsthat the complex is able to cleave luc target RNA. AGO2
immunopurified from naïve S2 lysate under the same
conditions did not show such activity (data not shown).
These data demonstrate that after incubation in S2 lysate, the siRNA duplex was properly processed andsingle-stranded guide siRNA was loaded onto AGO2,
and further, that AGO2 associated with guide siRNA iscapable of cleaving the RNA target harboring a sequence
completely complementary to the siRNA. Previously it
was revealed in a biochemical approach that the sole
protein required for RISC activity in S2 cells was AGO2
(Rand et al. 2004). Our current data confirm that AGO2
is indeed an element required for the target RNA cleavage activity in Drosophila.
AGO1 also functions as Slicer in Drosophila

Figure 1. AGO2 immunopurified from siRNA-programmed S2cells is associated with guide siRNA and exhibits target RNA
cleavage activity. (A) Silver-staining of the immunoprecipitateusing a specific monoclonal antibody against AGO2. The lane
indicated as −lysate shows the proteins that originated from theantibody. The lane indicated as +lysate contains a band corresponding to AGO2 (shown with an asterisk). Other protein
bands were not observed in the +lysate lane compared with the−lysate lane, indicating that the AGO2 immunoprecipitated
was nearly uniformly purified. (h.c.) Heavy chains of the antibody; (l.c.) light chains of the antibody. (B) Northern blot showsthat luc guide siRNA is specifically associated with the AGO2
immunopurified in A. The lane indicated as n.i.-IP shows that
luc guide siRNA is not in the immunoprecipitate with nonimmune IgG used as a negative control. (C) In vitro target RNA
cleavage assay using luc target RNA. Immunopurified AGO2, asshown in A, shows ability for cleaving the target RNA (luc180;
5⬘-radiolabeled with 32P), whereas the control (n.i.-IP) does not.

Drosophila AGO1 shows a higher similarity than Drosophila AGO2 to hAgo2 at the amino acid sequence level(Supplementary Fig. S2). We previously showed thateven in AGO2-lacking embryo lysate, target RNA cleavage takes place in a miRNA-dependent manner (Okamura et al. 2004); therefore, it was assumed that AGO1
in Drosophila should also have the ability to cleave the
RNA target as well as AGO2. Immunoprecipitation was
carried out from luc siRNA-programmed S2 lysate withan anti-AGO1 antibody under a high-salt condition (Fig.
2A), and cleavage assay was performed using luc target
RNA as in Figure 1C. The fact that the immunoprecipitate contains AGO1 but not AGO2 was determined byWestern blotting (Supplementary Fig. S1). Unlike AGO2
(Fig. 1C), immunopurified AGO1 did not show luc target
RNA cleavage activity (data not shown). Northern blotting revealed that immunopurified AGO1 was not associated with luc guide siRNA (Fig. 2B, top). We then assessed whether the immunopurified AGO1 was associated with miRNAs by performing Northern blotting
using an oligo DNA probe for mi-RNA bantam (miRban), one of the miRNAs known to be expressed in S2
cells (Okamura et al. 2004). Immunopurified AGO1, but
not AGO2, associates with miR-ban (Fig. 2B, bottom). In
vitro target RNA cleavage assay was then performed using an RNA containing a sequence completely matched
to miR-ban (bantam38). We observed the expected cleavage product when bantam38 was incubated with immunopurified AGO1 (Fig. 2C), indicating that AGO1
also shows Slicer activity depending on the miRNA withwhich AGO1 is associated. Under the same conditions,immunopurified AGO2 not associating with miR-ban(Fig. 2B) was unable to cleave bantam38 (Fig. 2C). In thenext experiment, the same assay as in Figure 2C was
performed, but after incubation the supernatant was
separated from the beads on which AGO1-miR-ban was
immobilized. RNAs were then isolated from both fractions and analyzed. About 65% of the cleavage productwas observed in the supernatant fraction under the condition where ATP was not added exogenously to the reaction mixture. Depletion of ATP (treatment of AGO1miR-ban complex with hexokinase and glucose prior tothe cleavage reaction; see Materials and Methods) (Ny-GENES & DEVELOPMENT

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kanen et al. 2001) or addition of ATP did not affect theresult (data not shown), indicating that the target RNAcleaved products were released from AGO1 in solution
in an ATP-independent manner. This is not a uniqueproperty of Drosophila AGO1, since the same phenomenon was also observed with Drosophila AGO2-guidesiRNA (data not shown) and recombinant hAGO2-guidesiRNA (Rivas et al. 2005) complexes. Nonetheless, ATPhydrolysis may accelerate the product release in RNAi
occurring in vivo (Haley and Zamore 2004).
Reconstitution of Slicer activity with recombinant
full-length AGO1 and the PIWI domains of AGO1
and AGO2

Figure 2. AGO1 immunopurified from naïve S2 cells is associated with endogenous miRNA and cleaves target RNA harboring a sequence completely complementary to the miRNA.
(A) Silver-staining of the immunoprecipitate with anti-AGO1
monoclonal antibody. The lane indicated as anti-AGO1 shows
the protein components in the immunoprecipitate with antiAGO1 antibody. The n.i. lane contains the protein components
in the immunoprecipitate with negative control nonimmune
IgG. A doublet corresponding to AGO1 is shown with an asterisk. The same protein band is not observed in the n.i. lane. (h.c.)
Heavy chains of the antibody; (l.c.) light chains of the antibody.
(B, top) Northern blot shows that luc guide siRNA is not associated with AGO1 immunopurified from luc siRNA-programmed S2 lysate. (Bottom) miR-ban, a miRNA known to be
expressed in S2 cells, is associated with immunopurified AGO1.
(Bottom) Under the same conditions, the AGO2 immunoprecipitated was not associated with miR-ban. (C) In vitro target
RNA cleavage assay using miR-ban target RNA (bantam38),
which was radiolabeled with 32P at the 5⬘-end. Immunopurified
AGO1 (AGO1-IP) (A) and AGO2 (AGO2-IP) (Fig. 1A) was incubated with bantam38, and then the reaction RNAs were prepared and analyzed on a gel. AGO1-IP shows the ability tocleave bantam38, whereas the control (n.i.) and AGO2-IP
showed no such activity for cleaving bantam38. (D) The in vitro
target RNA cleavage assay shown in C was repeated. The supernatant and the bead fractions were separated after reaction
and RNAs were prepared and analyzed. The cleaved product isobserved in the supernatant fraction, meaning that the productwas released in buffer after cleavage. The same was observed
even under conditions without ATP (data not shown); indicating that the releasing of cleaved target RNA from AGO1 occurs
in an ATP-independent manner.

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Even though immunoprecipitation was performed under
stringent conditions, in which almost all protein–protein
interaction was presumably abolished, AGO1 and AGO2
immunopurified from S2 lysate may still contain tiny
amounts of contaminants that could participate to somedegree in target RNA cleavage activity. To verify
whether Argonaute was indeed the Slicer, recombinant
Argonaute expressed in a system that does not furnish
the RNAi machinery (i.e., bacteria) was required. In ourstudy, recombinant Drosophila AGO1 fused to glutathione S-transferase (GST-AGO1) was successfully produced in E. coli (Supplementary Fig. S3). This prompted
us to question whether the Slicer activity can be reconstituted with GST-AGO1. Purified GST-AGO1 was incubated with luc guide siRNA, and then luc target RNA
radiolabeled at the 5⬘-end was added to the reaction mixture. The target RNA was efficiently cleaved by GSTAGO1 (Fig. 3A). Addition of EDTA at 10 mM to thereaction abolished the activity (Fig. 3A). Without luc
guide siRNA, target RNA cleavage was not observed (Fig.
3A). A similar experiment was carried out using miRNAlet-7 (let-7) and let-7 target RNA, where the let-7 target
RNA was cleaved in an AGO1-dependent manner (Fig.
3B). In both cases, GST itself did not show target RNA
cleavage activity. These results clearly demonstrate that
AGO1 does indeed function as Slicer. In these experiments, recombinant AGO1 did not distinguish siRNA or
miRNA to associate with. siRNA was not loaded onto
AGO1 when the siRNA duplex was programmed into S2
lysate (Fig. 2B). These results suggest that a well-ordered,programmed mechanism distinguishing siRNA frommiRNA being loaded onto AGO2 exists in the lysate. It
has been already shown that R2D2 bridges the initiationand effector steps of the Drosophila RNAi pathway byfacilitating siRNA passage from Dicer2 to AGO2 (Liu etal. 2003; Tomari et al. 2004b). Similarly, mature miRNAloading onto AGO1 is likely restricted by specific, consecutive interactions of pre-miRNA with the processing
complex including Dicer1/Loquacious (R3D1), and thenof the complex with AGO1 (Forstemann et al. 2005;
Jiang et al. 2005; Saito et al. 2005).
The PIWI domain conserved in Argonaute proteins
adopts an RNaseH fold (Liu et al. 2004; Song et al. 2004)
and provides the binding pocket for the 5⬘-end of guide
RNA (Ma et al. 2005; Parker et al. 2005). We then sought

Fly Argonautes as Slicer

Figure 3. Recombinant Drosophila full-length AGO1
and the PIWI domains of AGO1 and AGO2 cleave target
RNA, depending on the small RNA sequences withwhich they are associated. (A) luc target RNA was efficiently cleaved by GST-AGO1 associated with luc guidesiRNA. Purified GST-AGO1 (Supplementary Fig. S3)
was first incubated with luc guide siRNA and then luc
target RNA (luc180) radiolabeled at the 5⬘-end wasadded. Addition to the reaction of EDTA at 10 mM abolished the activity. Without siRNA addition, the target
RNA cleavage is not observed. GST itself does not show
the activity. (B) A similar experiment to A was carriedout using miRNA let-7 (miR-let-7) and the miR-let-7
target RNA (Okamura et al. 2004). Target RNA iscleaved in an AGO1-miR-let-7-dependent manner. (C)Reconstitution of Slicer activity with the PIWI domain
of AGO2. All of the recombinants used were first incubated with luc guide siRNA and then luc target RNA(luc180) radiolabeled at the 5⬘-end was added and incubated. GST-AGO2-PIWI-I and GST-AGO2-PIWI-G
cover the PIWI domain of AGO2, but GST-AGO2PIWI-D contains only a portion of the PIWI domain as
indicated below. (D) The PIWI domain of AGO1 preassociated with guide siRNA also shows activity to cleavetarget RNA.

to test whether the PIWI domain alone of Drosophila
Argonautes would be sufficient for binding to guide RNA
and for cleaving its RNA target. Recombinant peptides
harboring the PIWI domains of both Drosophila AGO1
and AGO2 tagged with GST were purified from E. coli
(Supplementary Fig. S4), and target RNA cleavage assays
were performed. luc target RNA was cleaved by GSTAGO2-PIWI-G, GST-AGO2-PIWI-I (Fig. 3C), and GSTAGO1-PIWI-C (Fig. 3D) preassociated with luc guidesiRNA. A small portion of the PIWI domain of AGO2
tagged with GST (GST-AGO2-PIWI-D) did not exhibit
the activity (Fig. 3C). These data demonstrate that thePIWI domain is itself sufficient, and that the PAZ domain is dispensable for initiating guide siRNA-dependent target RNA cleavage in vitro. GST-AGO1-PIWI-B
showed much less Slicer activity compared with GSTAGO1-PIWI-C (Fig. 3D), suggesting that the amino acidresidues adjacent to the PIWI domain are necessary for,for instance, supporting the domain structure.
AGO1/AGO2 associated with guide siRNA is able
to cleave passenger siRNAEndonucleolytically cleaved target RNAs were released
from AGO1 and AGO2 in a manner independent of ATP(Fig. 2D; data not shown). It has previously been shownthat without AGO2, the siRNA duplex was not unwound even after 1 h incubation in fly embryo lysate,
and as a result, RISC formation did not occur (Okamura
et al. 2004; Tomari et al. 2004b). We hypothesized that
AGO2 might be involved in the siRNA duplex unwinding process as an enzyme cleaving the passenger strand
while it is still in a duplex form with guide siRNA. After
this process, the cleaved passenger could be thrown away
in buffer and, consequently, AGO2 would be able to bind
only with the remaining single-stranded guide siRNA.
To test this hypothesis, we first examined whether
AGO2 associated with guide siRNA is capable of cleaving passenger siRNA. AGO2 immunopurified fromsiRNA-programmed S2 lysate under a high-salt condition was incubated with luc passenger siRNA radiolabeled at the 5⬘-end. A band corresponding to the cleavage
product (9 nt) was clearly observed, indicating that thepassenger siRNA was cleaved as expected by AGO2 associated with guide siRNA (Fig. 4A). This result is consistent with the previous observation that RISC is able torecognize a target RNA molecule as short as 15 nt (Martinez and Tuschl 2004). We next questioned whether thecleaved form of the passenger strand (9 nt in length)
could be detected in lysates programmed with siRNA
duplex. In this experiment, two kinds of siRNA duplex(the nucleotide sequences are the same, but only the pas-

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Figure 4. Cleavage of the passenger strand of siRNA duplex by
AGO2. (A) AGO2 immunopurified from siRNA-programmed S2lysate under high-salt conditions was incubated with luc passenger siRNA radiolabeled at the 5⬘-end. The passenger strand iscleaved by AGO2 preloaded with luc guide siRNA. luc guideand passenger strand sequences are shown on the right. The
cleavage site on the passenger is indicated with an arrow. (B)Detection of the cleaved passenger strand of siRNA duplex programmed in S2 lysates. Two sets of luc siRNA duplex (thenucleotide sequences are the same, but only passenger or guidestrand of the duplex was radiolabeled at the 5⬘-end) were used.
The passenger strand, but not guide siRNA being cleaved (9 nt)
is detected. (C) In AGO2-lacking embryo lysate (AGO2414 lysate) (Okamura et al. 2004), the cleaved passenger is not detected, as opposed to the wild-type embryo lysate, which indicates that cleavage of the passenger strand of the siRNA duplex
occurs in an AGO2-dependent manner. The luc siRNA duplex(the passenger was radiolabeled at the 5⬘-end) was used.

senger or guide strand of the duplex was radiolabeled at
the 5⬘-end) were prepared. We could detect the passenger
strand but not the guide siRNA being cleaved in S2 lysate (Fig. 4B). In AGO2-lacking embryo lysate (AGO2414
lysate) (Okamura et al. 2004), the cleaved passenger was
not detected as opposed to the wild-type embryo lysate(Fig. 4C), indicating that cleavage of the passenger strandof the siRNA duplex in lysates occurs in an AGO2-dependent manner.

Nuclease-resistant modification of the passenger
strand blocks RISC formation
However, it could be possible that the siRNA duplex
programmed in lysates comes apart right after being
added to the lysate; then, single-stranded guide siRNA is2842

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loaded onto RISC, which then captures the passenger asa target and cleaves it. Thus, a similar assay as in Figure
4B was performed using a nuclease-resistant-modified
oligonucleotide. The presence of a single-stranded passenger modified with 2⬘-O-methyl groups, which is
known to act as competitive inhibitors of RISC by binding to guide siRNA within RISC (Haley and Zamore
2004; Hutvagner et al. 2004; Meister et al. 2004b), did not
block the production of 9 nt (Fig. 5A). The result demonstrates that the cleavage of the passenger occurs while
it is still in the initial duplex form with guide siRNA. In
the next experiment, we used the siRNA duplex, in
which only one nucleotide of the passenger strand (theninth nucleotide from the 5⬘-end, see Fig. 4A) was modified by the 2⬘-O-methyl group (OMe-9). This single
modification is known to block target RNA cleavage in
RNAi (Martinez and Tuschl 2004). The modified passenger strand, but not the guide strand, was radiolabeledwith 32P at the 5⬘-end for visualization. After incubationin the lysate, we did not observe cleaved passenger ofsi-duplex (OMe-9), as expected (Fig. 5B). Native agarose
gel electrophoresis showed that RISC formation was
clearly impaired when si-duplex (OMe-9) (in this particular experiment the unmodified guide strand was labeledwith 32P at the 5⬘-end) was added to S2 lysate (Fig. 5C,
left panel). The target RNA cleavage activity of S2 lysate
was also markedly impaired when si-duplex (OMe-9) wasused in the target RNA cleavage assay (Fig. 5C, rightpanel). These results indicate that the cleavage of thepassenger strand of the siRNA duplex is required for efficient formation of active RISC. These findings give rise
to a new model, in which AGO2 is directly involved in
RISC formation as Slicer of the passenger strand of thesiRNA duplex.
RISC formation was clearly impaired but not completely blocked with the 2⬘-O-methyl-modified passenger (Fig. 5C), implying that activities other than AGO2
Slicer function might be cooperatively involved in thesiRNA duplex unwinding process and concomitant RISC
formation with AGO2. A most likely candidate for suchactivities is the helicase activity of, for example, a
DEAD-box RNA helicase. To distinguish whether or not
such an RNA helicase is indeed involved in RISC formation, as has been presumed for a long time, we re-examined ATP requirements in RISC formation by native agarose gel electrophoresis. S2 lysate was prepared and divided in half. One-half was treated with hexokinase in
the presence of glucose as previously reported (Nykanen
et al. 2001). The other half of the lysate was also incubated, but hexokinase and glucose were not added prior
to incubation. Both portions of the lysate were then used
for the RISC formation with the luc siRNA duplex, in
which the guide strand was radiolabeled with 32P at the5⬘-end and the passenger was phosphorylated with cold
ATP. After annealing, the siRNA duplex was gel-purified
to avoid any contamination of residual ATP from thephosphorylation reaction. ATP, GTP, creatine phosphate, and creatine kinase were omitted for −ATP reaction. Even in ATP-depleted lysate, RISC was formed aswell with ATP (Fig. 5D). Furthermore, addition of a com-

Fly Argonautes as Slicer

petitive inhibitor of ATP, AMP-PNP, at 1µM to ATPdepleted lysate did not affect the result (data not shown).
RISC formed in ATP-depleted lysate is active, since target RNA cleavage occurred quite similarly in both lysate
portions (Fig. 5E). Taken together, it is suggested thatenzyme(s) requiring ATP as a cofactor for their functions, such as a typical ATP-dependent DEAD-box RNA
helicase, are not strictly required for the siRNA duplex
unwinding process in concert with AGO2.

Reconstitution of Slicer activity using recombinant
full-length AGO1 together with siRNA duplex
and its target RNA
Reconstitution of Slicer activity has so far been demonstrated using single-stranded siRNA or miRNA (Fig.
3A,B; Rivas et al. 2005). Next, we asked whether Slicer
activity could be reconstituted with a sole protein, recombinant Argonaute, and the siRNA duplex. We firstchecked whether GST-AGO1 has the capacity to cleave
passenger strands still in a duplex form with guidesiRNA. The siRNA duplex (both strands phosphorylated,but only the passenger radiolabeled with 32P) was incubated in buffer containing GST-AGO1 or GST. Cleaved
passenger was observed with GST-AGO1, but not with
GST (Fig. 6A), indicating that the passenger was cleaved

by AGO1 even in a duplex form. Then, a similar reaction
was set up using the cold luc siRNA duplex. After incubation to allow GST-AGO1 to process the siRNA duplex, the target RNA (luc target radiolabeled with 32P atthe 5⬘-end; 130 nt in length) was added to the reaction
mixture. As a control, GST-AGO1 preassociated withsingle-stranded luc guide siRNA was also used. Although it was not as robust compared with GST-AGO1
preassociated with guide siRNA, GST-AGO1 preincubated with the siRNA duplex clearly showed Slicer activity (Fig. 6B). These results demonstrate that Slicer activity was reconstituted with recombinant full-length
AGO1 together with siRNA duplex and its target RNA.

Discussion
In this study, we showed that endogenous AGO1 andAGO2 immunopurified with specific monoclonal antibodies under harsh conditions in which almost all protein–protein interactions were disrupted exhibited target
RNA cleavage activity. We proceeded to reconstitute
Slicer activity with recombinant full-length AGO1 andthe PIWI domains of both AGO1 and AGO2 purified
from E. coli. These experiments made a compelling argument that both AGO1 and AGO2 function as Slicer in
Drosophila.

Figure 5. Nuclease-resistant passenger of the siRNA duplex
blocks RISC formation, but ATP depletion does not. (A) The
reaction shown in Figure 4B was repeated. Either EDTA (to 10mM) or 2⬘-OMe-modified passenger was added when the reactions were initiated. Total incubation time was 15 min. Addition of EDTA blocks the cleavage of the passenger; in contrast,
2⬘-O-methyl-modified passenger does not. (B) The siRNA duplex, of which the passenger strand was modified with the 2⬘O-methyl group at the ninth nucleotide (OMe-9) and radiolabeled with 32P at the 5⬘-end, was programmed in S2 lysate. In
si-duplex (OMe-9), the cleaved passenger is not observed, unlikewith nonmodified si-duplex. (C, left) Native agarose gel electrophoresis shows that even after 30 min incubation, RISC formation is strongly impaired with si-duplex (OMe-9) (the passenger
was modified with the 2⬘-O-methyl group at the ninth nucleotide, and the guide was labeled with 32P at the 5⬘-end). (Right)
The target RNA cleavage activity of S2 lysate was also markedly
impaired when the si-duplex (OMe-9) was preprogrammed to
the lysate. These results suggest that cleavage of the passenger
strand of the siRNA duplex by AGO2 is required for efficient
formation of active RISC. A target RNA cleavage assay was
performed as described previously (Okamura et al. 2004). luc180
target RNA was used as in Figure 1C. (D) Even in ATP-depleted
lysate (indicated as −ATP), RISC is formed as well as that with
ATP (+ATP). To create −ATP conditions, S2 lysate was treatedwith hexokinase in the presence of glucose as previously reported (Nykanen et al. 2001). RISC formation was performed
with gel-purified luc siRNA duplex (the guide strand was radiolabeled with 32P at the 5⬘-end and the passenger was phosphorylated with cold ATP). ATP, GTP, creatine phosphate, and creatine kinase were omitted for the −ATP reaction. (E) RISC
formed in ATP-depleted lysate is active. ATP-depleted S2 lysate
was prepared as in D. Target RNA cleavage assay was performed
with luc180 target RNA.

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Miyoshi et al.

Figure 6. (A) Recombinant GST-AGO1 was incubated with luc
siRNA duplex. A 9-nt RNA product is observed in the GSTAGO1 lane, but not in the GST-only lane. (B) Reconstitution of
Slicer activity with recombinant GST-AGO1, the siRNA duplex, and its target RNA in vitro. GST-AGO1 preprogrammedwith the siRNA duplex shows Slicer activity, although it is not
as robust compared with GST-AGO1 preassociated with guidesiRNA.

Structural and mutagenesis analyses on hAgo2 revealed that an Asp–Asp–His (DDH) motif is the catalytic
triad coordinating Mg2+ ions at the active site of theprotein (Rivas et al. 2005). At a peptide sequence level,
the DDH motif is well conserved in AGO1 and AGO2 in
Drosophila (Supplementary Fig. S2). In contrast, theGln–His (Q-H) sequence that has been shown to be required in hAgo2 Slicer function (Liu et al. 2004) is only
conserved in AGO1, while in AGO2, Arg–Gly (R-G) corresponds to it. The hAgo2 mutant (H634A; His in the
Q-H sequence is changed to Ala) shows no Slicer activity(Liu et al. 2004). The peptide sequence of the entire PIWI
domain in AGO1 is highly similar to that in hAgo2(∼88% identity), but AGO2 only shows ∼43% identity(Supplementary Fig. S2). Taken together, in Drosophila,
AGO1 is more closely related to hAgo2 than AGO2;
thus, it is reasonable that AGO1 also has Slicer activity
in Drosophila.
Drosophila AGO1 functions in miRNA processing(Okamura et al. 2004; Saito et al. 2005) and potentially ina translational repression pathway mediated by miRNAs
without altering levels of the messenger. Why then does
AGO1 have to contain the PIWI domain that can exhibit
Slicer activity? There is as yet no clear answer to this,but it can be speculated that there may be unidentified,but endogenous miRNAs existing in flies that can leadtarget mRNA degradation as siRNA in the RNAi path-

2844

GENES & DEVELOPMENT

way. Recently, it was found that ALG-1, a Caenorhabditis elegans Argonaute family protein that is part of
miRISC (Grishok et al. 2001), interacts with AIN-1 (Dinget al. 2005). AIN-1 shares homology with GW182 (Eystathioy et al. 2002), a protein recruited in cytoplasmic locicalled processing bodies (P-bodies) that are known to besites of mRNA decapping and degradation (Sheth andParker 2003). It was also shown that human Argonautes(hAgo1 and hAgo2) are localized in P-bodies in cultured
cells and the localization is linked to siRNA/miRNA
function (Liu et al. 2005; Sen and Blau 2005). We foundthat when Flag-tagged AGO1 is expressed in S2 cells, it
localizes in P-bodies (K. Miyoshi and M.C. Siomi, unpubl.), suggesting that even in Drosophila, AGO1 may
function in degrading mRNA targeted by miRISC after
being sequestered in P-bodies.
We produced recombinant AGO1-PIWI and AGO2PIWI domains and showed that they are active in RISC
assays, indicating that other Argonaute domains, including the PAZ domain, are not strictly necessary for siRNAbinding and target RNA cleavage. However, cleavage of
AGO1-PIWI (GST-AGO1-PIWI-C) was less efficientcompared with that of recombinant full-length AGO1
(GST-AGO1) (Fig. 3C), although the concentrations ofproteins used in assays were equal to each other. Thus,
we cannot exclude the possibility that other regions of
AGO1, including the PAZ domain, contribute to Slicer
activity to some extent. According to the target RNA
cleavage cycle model recently proposed by Yuan et al.
(2005), 5⬘- and 3⬘-ends of siRNA are first bound to the
PIWI and the PAZ, respectively, domains of Argonautes.
Once the cycle moves forward, the 3⬘-end is released
from the PAZ domain and a siRNA/target RNA duplex isformed, while the 5⬘-end of siRNA is still fixed on the
Argonaute-PIWI domain. The action of binding and releasing the 3⬘-end of guide siRNA by the PAZ domain
itself, and/or protein conformational change madethrough such action, may accelerate Argonaute Slicer
activity.
One of the crucial issues for elucidating the molecularmechanisms underlying the RNAi pathway is to identify
the siRNA unwinding enzyme(s). We previously showedthat without AGO2, the siRNA duplex is not unwound
and RISC is not formed in fly embryo lysate (Okamura etal. 2004). As in the case of embryo lysate, S2 lysate without AGO2 also has no ability to form RISC (H. Tsukumo
and M.C. Siomi, unpubl.). In this study, we found that
AGO2 associated with guide siRNA is able to cleave
passenger siRNA (Fig. 4A) and that cleaved passenger
strands originating from the siRNA duplex programmed
into the lysates can be detected as 9 nt in an AGO2dependent manner (Fig. 4B,C). A 2⬘-O-methyl modification of passenger strands of the siRNA duplex, whichshows resistance to AGO2 Slicer activity, did not completely abolish, but clearly impaired RISC formation inS2 lysate (Fig. 5C, left panel). The target RNA cleavage
activity of S2 lysate was also markedly impaired when
si-duplex (OMe-9) was preprogramed to the lysate (Fig.
5C, right panel). Taken together, it can be speculated
that AGO2 is directly involved in the siRNA duplex un-Fly Argonautes as Slicer

winding process as an enzyme-cleaving passenger strand
forming a duplex with guide siRNA and discarding thecleaved passenger in solution. However, it might not be
the only mechanism for conversion of siRNA duplex
into single-stranded siRNA. Although the unwinding of
the siRNA duplex is totally dependent upon the AGO2
activity, other factors likely function cooperatively with
AGO2 or act as a substitute, yet with low efficacy, for
AGO2 when AGO2 cannot cleave the passenger strandof the siRNA duplex for some reason, such as the passenger showing AGO2-cleavage resistance. miRNA/
miRNA* duplexes usually contain small bulge/internalloops in the middle of the duplexes (Bartel 2004; Kim
2005), which likely interfere with AGO Slicer activity.
Thus, supposedly our model can apply only to an siRNA
duplex but not to a miRNA/miRNA* duplex. How, then,can a miRNA duplex be converted to single-stranded
miRNA? We observed that the bantam/bantam* duplex
is unstable and easily becomes single-stranded in solution (K. Miyoshi and M.C. Siomi, unpubl.). Therefore,
one might imagine that the miRNA/miRNA* duplex
may come apart upon release from the precursor by
Dicer.
We depleted ATP from S2 cell lysate by treating itwith hexokinase in the presence of glucose, and performed RISC formation assay by adding siRNA duplex
with presumably no ATP contamination. Surprisingly,
RISC was formed even under such conditions, as in thecase with an energy-regenerating system and exogenous
ATP and GTP (Fig. 5D). Addition of a nonhydrolyzable
ATP analog (AMP-PNP) to ATP-depleted lysate did notaffect the result (data not shown). The simple implication is that RISC formation occurs ATP independently;
however, there is the possibility that a trace of ATP molecules remaining in the lysate, even after the hexokinase
treatment (∼1 nM) and even in the presence of AMP-PNP(1 µM), is just enough to form RISC in the lysate. If
Argonaute is indeed directly involved in the siRNA duplex unwinding process, and this process does not require ATP, where (in which step) in RISC formation is
ATP hydrolysis required? siRNA duplex programmed inlysate is promptly incorporated in RLC and then active
RISC is gradually formed (Tomari et al. 2004a). Although
RLC components other than R2D2 and Dicer2 are notyet fully understood, it can be imagined that the siRNA
duplex is entirely covered with protein components inthe complex, and if so, the siRNA duplex in RLC would
not be accessed by AGO2. ATP would stimulate the remodeling of the siRNA–RLC complex in vivo, by which
the siRNA duplex becomes accessible to AGO2.
Materials and methods
Immunoprecipitation and silver-staining
Monoclonal antibodies against Drosophila AGO1 and AGO2
were produced against both N termini of AGO1 (300 aminoacids) and AGO2 (300 amino acids). Immunoprecipitation fromS2 lysate was performed using anti-AGO1 or anti-AGO2 antibodies immobilized on Gamma-Bind beads (Amersham Biosci-ence). Sodium chloride was added to the lysates to 800 mM justbefore immunoprecipitation was started. The reaction mixtures
were rocked at 4°C for at least 1 h, and the beads washed extensively with a buffer containing 30 mM HEPES (pH 7.4), 800
mM sodium chloride, 2 mM magnesium acetate, 2 mM DTT,
0.1% NP40, 2 µg/mL pepstatin, 2 µg/mL leupeptin, and 0.5%
aprotinin. The final wash was carried out with a cleavage reaction buffer (30 mM HEPES at pH 7.4, 40 mM potassium acetate,
5 mM magnesium acetate, and 5 mM DTT). Figures 1A and 2A
show proteins eluted with SDS sample buffer without DTT.
After elution, DTT was added to the eluates to 100 mM, whichwere then boiled and loaded onto SDS–acrylamide gels. Afterelectrophoresis, protein bands were visualized using SilverQuest (Invitrogen).

Northern blot analysis
RNAs were isolated from the immunoprecipitates with ISOGEN (Nippon Gene). Northern blot was performed as described(Ishizuka et al. 2002). The probes used for detecting luc guidesiRNA and miR-ban were 5⬘-CGUACGCGGAAUACUUCGAAA-3⬘ and 5⬘-CAGCTTTCAAAATGATCTCAC-3⬘, respectively.

In vitro target RNA cleavage assays
Target RNA cleavage assays using the immunoprecipitates
were performed in a reaction buffer containing 30 mM HEPES(pH 7.4), 40 mM potassium acetate, 5 mM magnesium acetate,
5 mM DTT, 0.5 µg of yeast RNA (Ambion), and 40 U RNasin(Promega). After reaction at room temperature, RNAs were isolated from the whole-reaction mixtures, run on gels, and visualized on the BAS 2500. In Figure 2D, after the cleavage reaction, the supernatant and the beads were separated by centrifugation and RNAs isolated from each fraction. Target RNAs weretranscribed with a Megascript T7 kit (Ambion) and then radiolabeled at the 5⬘-G cap by guanylyltransferase (Ambion). In eachreaction 2000 to ∼5000 cpm of cap-labeled target RNA was used.
To prepare a template to make luc target RNA (luc180), PCR
was performed to amplify a portion of luc cDNA (160 nt fromthe 5⬘-end). Forward and reverse primers used in the PCR reaction contain T7 and T3 promotor sequences, respectively. To
make miR-ban target RNA harboring a sequence completely
complementary to miR-ban (bantam38), a PvuII–EcoRI fragment excised from the pBS-miR-ban-target plasmid was used asa template. The pBS-miR-ban-target plasmid was constructed asfollows: a KpnI–EcoRI fragment containing a sequence completely complementary to miR-ban was produced by annealing
a set of oligo DNAs (5⬘-CCAGCTTTCAAAATGATCTCACG-3⬘ and 3⬘-CATGGGTCGAAAGTTTTACTAGAGTGCTTAA5⬘), which was then inserted into pBluescript digested with KpnIand EcoRI. Production of target RNA for let-7 (let-7 target RNA)has been described previously (Okamura et al. 2004). To makeluc target RNA (luc130) harboring a sequence completely
complementary to luc siRNA (Fig. 6B), a PCR fragment was
amplified with T7 and T3 primers from the pBS-luc-target plasmid as a template, and in vitro transcription was performed.
The pBS-luc target plasmid was constructed as follows: a KpnI–
EcoRI fragment containing a sequence completely complementary to luc siRNA was produced by annealing a set of oligo
DNAs (5⬘-CCACGTACGCGGAATACTTCGAG-3⬘ and 5⬘AATTCTCGAAGTATTCCGCGTACGTGGGTAC-3⬘), which
was inserted into pBluescript digested with KpnI and EcoRI. luc
passenger strand (Fig. 4A) was labeled with T4 polynucleotide
kinase (PNK; TaKaRa) in the presence of [␥-32P]ATP.

GENES & DEVELOPMENT

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Miyoshi et al.

Recombinant protein concentrations used in each target RNA
cleavage assay was ∼100 nM. One microgram of BSA was also
added together with recombinant proteins in each reaction.
Single-stranded siRNA or miRNA was preincubated with GST
fusion proteins at ∼100 nM. In Figure 6B, GST-AGO1 or GST
itself was first incubated with a luc siRNA duplex (100 nM) for
1.5 h at room temperature and luc target RNA (luc130) was
added and then incubated for another 1.5 h at room temperature.
Production of recombinant Argonaute proteins
To obtain cDNA encoding AGO1, poly(A)+ RNAs were purified
from S2 cells and RT–PCR was carried out using a ProSTAR
Ultra-HF RT–PCR system (Stratagene). To produce a GSTAGO1 fusion protein, full-length AGO1 cDNA was subcloned
into a pGEX-5X expression vector (Amersham Bioscience).
GST-AGO1-PIWI-B and GST-AG01-PIWI-C were generated bycloning PCR fragments covering a portion of AGO1 that included the PIWI domain (as shown in Fig. 3D) into pGEX-5X.
GST-AGO2-PIWI-D, GST-AG02-PIWI-G, and GST-AGO2PIWI-I were generated by cloning PCR fragments covering a
portion of AGO2 (as shown in Fig. 3C) into pGEX-5X. All of the
GST-tagged proteins and GST itself were expressed in
BL21(DE3). After incubation to A600 of ∼0.8 at 37°C, IPTG wasadded to the medium to 1 mM and cells were grown overnightat 16°C. Cells were then lysed by sonication and GST and GST
fusion proteins were bound onto glutathione-Sepharose 4B resins according to the manufacturer’s instructions (Amersham
Bioscience). After extensive washing with PBS, GST, and GST
fusion proteins were eluted from the beads with PBS containing
10 mM glutathione diluted with PBS containing 5 mM magnesium acetate and then concentrated on spin columns.
Detection of cleaved passenger strand of siRNA duplex
in lysates
S2 and embryo lysates were prepared as described previously(Okamura et al. 2004). siRNA duplexes were gel-purified before
use. Then, siRNA duplex (∼20,000 cpm), 5× cleavage buffer (150mM HEPES at pH 7.4, 200 mM potassium acetate, 25 mM magnesium acetate, 25 mM DTT), 0.5 µg of yeast RNA, and 40 U
RNasin were added to lysates and incubated at room temperature. Reactions were stopped by the addition of stop buffer (50mM sodium chloride, 50 mM EDTA, 1% SDS, and 100 µg/mL
proteinase K). RNA was then extracted using phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with isopropanol,
and analyzed. luc passenger strands modified with 2⬘-O-methylgroup(s) at the ninth nucleotide (the 5⬘-end of the passenger
being assigned first) (OMe-9) and at all of the nucleotides (OMe)were purchased from Hokkaido System Science. In Figure 5A,
the modified passenger strand (OMe) was added to the reaction
mixture at 150 nM.
Native agarose gel electrophoresis
Native gel electrophoresis of RLC and RISC was performed as
described previously (Okamura et al. 2004; Tomari et al. 2004a).
32
P-radiolabeled luc siRNA duplex was incubated with S2 lysate
at room temperature. After incubation, samples were adjusted
to 6% (w/v) glycerol and resolved by submarine native agarose
gel electrophoresis. Gels were dried under a vacuum onto Hybond-N+ nylon membrane (Amersham Bioscience) and exposed
to an imaging plate of a BAS 2500. For depleting ATP from thelysate, hexokinase and glucose were added and incubated as
described previously (Nykanen et al. 2001). ATP concentration

2846

GENES & DEVELOPMENTof lysate was monitored using an ATP Bioluminescent Assay kit(Sigma). A nonhydrolyzable ATP analog (AMP-PNP) was purchased from CALBIOCHEM.

Acknowledgments
We thank members of the Siomi laboratory for discussions andcomments on the manuscript. K.M is a post-doctoral fellow ofthe 21st century COE Program from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (MEXT). This
work was supported in part by grants to M.C.S and H.S from
MEXT, JSPS (the Japan Society for the Promotion of Science),
and The Novartis Foundation (Japan) for the Promotion of Science.

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