Distinct roles for Argonaute proteinsin small RNA-directed RNA
cleavage pathways
Katsutomo Okamura, Akira Ishizuka, Haruhiko Siomi,1 and Mikiko C. Siomi
Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan

In mammalian cells, both microRNAs (miRNAs) and small interfering RNAs (siRNAs) are thought to beloaded into the same RNA-induced silencing complex (RISC), where they guide mRNA degradation ortranslation silencing depending on the complementarity of the target. In Drosophila, Argonaute2 (AGO2) was
identified as part of the RISC complex. Here we show that AGO2 is an essential component forsiRNA-directed RNA interference (RNAi) response and is required for the unwinding of siRNA duplex and inconsequence assembly of siRNA into RISC in Drosophila embryos. However, Drosophila embryos lacking
AGO2, which are siRNA-directed RNAi-defective, are still capable of miRNA-directed target RNA cleavage. In
contrast, Argonaute1 (AGO1), another Argonaute protein in fly, which is dispensable for siRNA-directed target
RNA cleavage, is required for mature miRNA production that impacts on miRNA-directed RNA cleavage. The
association of AGO1 with Dicer-1 and pre-miRNA also suggests that AGO1 is involved in miRNA biogenesis.
Our findings show that distinct Argonaute proteins act at different steps of the small RNA silencing
mechanism and suggest that there are inherent differences between siRNA-initiated RISCs andmiRNA-initiated RISCs in Drosophila.
[Keywords: RNAi; siRNA; miRNA; AGO1; AGO2]
Received April 7, 2004; revised version accepted May 20, 2004.

Double-stranded (ds) RNA induces the sequence-specificposttranscriptional gene silencing of cognate genes innumerous organisms (Cogoni and Macino 2000; Hutvagner and Zamore 2002a; Denli and Hannon 2003). The
multidomain ribonuclease III enzyme Dicer excises longdsRNA into duplexes of 21–23 nucleotides (nt) termed
short interfering RNAs (siRNAs; Bernstein et al. 2001;
Ketting et al. 2001; Knight and Bass 2001), which direct
the cleavage of complementary mRNA targets, a process
known as RNA interference (RNAi; Fire et al. 1998).
Prior to target mRNA recognition, an siRNA duplex goes
through an ATP-dependent unwinding process and one
strand over the other is often preferentially loaded onto
the RNA-induced silencing complex (RISC), the multiple-turnover enzyme complex that mediates endonucleolytic cleavage in the RNAi pathway. The RISC is
guided to cleave target mRNAs sharing perfect complementarity across the center of the complementarysiRNA strand in the absence of high-energy cofactors(Hammond et al. 2001; Nykanen et al. 2001; Hutvagner
and Zamore 2002b; Martinez et al. 2002). siRNAs are notthe only products of Dicer. Natural dsRNA-encoding
genes, named microRNA (miRNA) genes, encode RNA
1

Corresponding author.
E-MAIL siomi@genome.tokushima-u.ac.jp; FAX 81-88-633-9451.
Article published online ahead of print. Article and publication date areat http://www.genesdev.org/cgi/doi/10.1101/gad.1210204.

products of ∼70 nt that are predicted to form imperfecthairpin structures and are processed by Dicer to mature
21–23-nt miRNAs (Grishok et al. 2001; Hutvagner et al.
2001). Only one Dicer enzyme is found in Caenorhabditis elegans and humans, therefore indicating that thesame Dicer is required for both RNAi and for the processing of miRNA precursors in these organisms(Grishok et al. 2001; Hutvagner et al. 2001; Knight andBass 2001). The expression of miRNAs is often developmentally regulated, suggesting an important role for
miRNAs in the regulation of endogenous gene expression (Lagos-Quintana et al. 2001; Lau et al. 2001; Lee andAmbros 2001; Reinhart et al. 2002; Brennecke et al.
2003; Xu et al. 2003). Target mRNAs containing sequences imperfectly complementary to the miRNA canbe subject to translational repression without alteringmRNA stability (Lee et al. 1993; Wightman et al. 1993;
Olsen and Ambros 1999; Reinhart et al. 2000; Bartel
2004).
Recent findings point to a tight connection between
miRNA and RNAi molecular machineries. Both miRNAs
and siRNAs have been shown to be capable of target
mRNA degradation or translation silencing in mammalian cells and plants (Hutvagner and Zamore 2002b;
Llave et al. 2002; Doench et al. 2003; Zeng et al. 2003);
additionally, it has been shown that both siRNAs andmiRNAs can be loaded onto the same RISC complex

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(Hutvagner and Zamore 2002b; Martinez et al. 2002).
These findings imply that, regardless of the maturation
process, once the small RNA is loaded, the RISC uses it
to degrade or inhibit translation depending on the degreeof complementarity between the small RNA and its
mRNA target.
In C. elegans, genetic analyses suggested that RDE-1, amember of the Argonaute family of proteins (Carmell et
al. 2002), is required for the initiation of RNAi with injected dsRNAs (Grishok et al. 2000), whereas Alg-1 andAlg-2, other Argonaute family members, are required forthe accumulation of stable mature miRNAs in C. elegans, but not for RNAi driven by dsRNA (Grishok et al.
2001). These results suggest that distinct members of the
Argonaute family of proteins may provide specificity totheir respective pathways, RNAi and translation inhibition, in C. elegans. However, the underlying molecularmechanisms are not well understood.
In Drosophila, it has been shown that Argonaute2(AGO2) protein, a member of the Argonaute family ofproteins (Carmell et al. 2002), is essential for RNAi
driven by exogenously introduced dsRNA and is the firstprotein component to be identified as part of the RISC
complex in cultured Drosophila S2 cells (Hammond etal. 2001). Although AGO2 is known to associate withseveral proteins such as the Drosophila homolog(dFMR1) of the human fragile X mental retardation protein and VIG (Caudy et al. 2002; Ishizuka et al. 2002), theprecise role that AGO2 plays in RNAi is not well understood. In this paper, we have produced AGO2 deletionmutant flies and found that embryos lacking AGO2 aresiRNA-directed RNAi defective but are still capable ofmiRNA-directed target RNA cleavage. We also showthat AGO2 mutant embryos are impaired in the assembly of siRNA into RISC. In contrast, Argonaute1(AGO1), another Argonaute protein in fly (Kataoka et al.
2001), is dispensable for siRNA-directed RNA cleavage
but is necessary for the accumulation of stable mature
miRNAs, and thus impacts on miRNA-directed target
RNA cleavage. Our findings suggest that distinct Argonaute proteins act at different steps of the small RNA
silencing mechanism, and provide specificity to their respective pathways in Drosophila.
Results

Figure 1. Characterization of the AGO2 deletion mutant
AGO2414. (A) Structure of the AGO2 locus. Exons are indicatedwith open boxes; the closed portion is the protein-coding region.
The translation initiation start site (Met) is located in the firstexon. The position of EP(3)3417 is represented as triangle withan arrow pointing in the direction of GAL4-induced transcription. The deleted genomic region of the AGO2414 chromosome
is shown as a dotted line. In this mutant, imprecise excision of
the EP element generated a 2.3-kb deletion of genomic DNA,
which included exons 1 and 2 of the AGO2 gene. Sequence
information from the Berkeley Drosophila Genome Project reveals that the AGO2 locus is positioned in a small region on thecytological location 71E1, on the left arm of the third chromosome. (B) Western blotting on protein extracts from adult ovaries. Western blotting was carried out using anti-AGO2 (4D2),
anti-dFMR1 (5A11), and antiribosomal protein P0 antibodies.
[WT(yw)] Yellow-white wild type; (AGO2414) AGO2414 homozygote; (AGO2414;WTrescue) AGO2414 homozygote with twocopies of wild-type rescue. The genomic rescue construct contained a 10.1-kb XhoI–FspI fragment of the AGO2 locus in A in
which no other known genes are included.

AGO2 is essential for RNAi in embryos
The Drosophila AGO2 protein is an essential factor for
RNAi as a component of the RISC complex in cultured
S2 cells (Hammond et al. 2001). However, the preciserole that AGO2 plays in RNAi is not well understood. To
gain an insight into the molecular functions of AGO2 in
RNAi, we produced fly strains that lack AGO2. To obtain fly strains bearing deletions in AGO2, we mobilizeda P{EP}element (Rorth et al. 1998) inserted in the firstexon of the AGO2 gene on the EP(3)3417 chromosome to
produce several partial deletions of AGO2. One suchmutation named AGO2414 was selected for further characterization (Fig. 1A). Western blot analysis with anti-

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in homozygous AGO2414 flies (Fig. 1B). We also confirmed the absence of AGO2 mRNA by Northern blot
analysis in homozygous AGO2414 flies (data not shown).
These results demonstrated that AGO2414 are AGO2
null flies. We found that homozygous AGO2414 flies proceed into adulthood, are fertile, and appear outwardly
normal. Because it is not known whether AGO2 is required for RNAi in embryos, we tested for RNAi in vivo
by assaying the ability of long dsRNA corresponding to
the fushi tarazu (ftz) gene to produce a ftz phenotype(Kennerdell and Carthew 1998) when injected into wild-

Distinct roles for AGO proteins in RNAi

type and AGO2 mutant embryos. We used AGO2 mutant adult females to make mutant eggs to remove thematernal contribution of AGO2 (simply called the“AGO2 mutant embryo” or AGO2414 embryos, hereafter). Wild-type embryos injected with ftz dsRNA exhibit
segmentation defects in their cuticle (Fig. 2B; Table 1). In
contrast, AGO2 mutant embryos showed a complete absence of interference in response to ftz dsRNA, indicating that they are RNAi defective (Fig. 2C). This is consistent with previous findings made with cultured S2cells (Hammond et al. 2001; Caudy et al. 2002; Ishizuka
et al. 2002). To confirm that disruption of the AGO2
gene is directly responsible for the RNAi-defective
phenotype, we transformed mutant flies with a P element containing wild-type AGO2 genomic sequences(Fig. 1A,B). The RNAi-defective phenotype was ameliorated by the introduction of this AGO2 minigene(Fig. 2D; Table 1), demonstrating that the RNAi-defective phenotype is caused by the AGO2 mutation rather
than a second site mutation elsewhere on the chromosome.
AGO2 is required for the assembly of siRNA
into RISC in embryos
To determine whether AGO2 is required for the formation of the siRNA duplex or for interference thereafter,
we injected synthetic ftz siRNA into AGO2414 embryos.
siRNA produced a ftz phenotype in wild-type embryos(Fig. 3A; Table 1). However, AGO2414 embryos were notresponsive to the ftz siRNA (Fig. 3B; Table 1). Similarly,
in vitro, AGO2414 embryo lysate processed long ftz
dsRNA into short ∼21-nt fragments, similar to lysatesfrom wild-type embryos (Fig. 3C). These findings suggestthat AGO2 is necessary for RNAi after the formation of
the siRNA duplex.
Before target mRNA recognition, the siRNA duplex isunwound (Nykanen et al. 2001), and each RISC contains
only one of the two strands of the siRNA duplex (Martinez et al. 2002). We tested whether AGO2 activity isnecessary for this unwinding process. Unwound siRNA

Table 1. Effects of ftz dsRNA and ftz siRNA injected into
Drosophila embryos
Genotype

Injected RNA

Wild
type

ftz

WT(yw)
WT(yw)AGO2414
P[w+: AGO2];AGO2414
WT(yw)AGO2414

mock (water)
ftz dsRNA
ftz dsRNA
ftz dsRNA
ftz siRNAftz siRNA

29
5
43
1
2
21

0 (0%)
30 (86%)0 (0%)
22 (96%)30 (94%)0 (0%)

Numbers indicate embryos with wild-type or ftz phenotype after injection. Percentages are given in parentheses. Embryos
were scored as ftz if they had six or fewer ventral denticle belts.
P[w+: AGO2];AGO2414 is the genotype of “rescued’ flies(AGO2414;WT rescue).

was detected in wild-type embryo lysate, whereas nosuch activity was detected in AGO2414 embryo lysate(Fig. 3D), suggesting that AGO2 is required for the unwinding of the siRNA duplex. An alternative explanation of the results could be that the absence of AGO2
leaves the unwound single-stranded RNA exposed to nucleolytic degradation and therefore the single-stranded
RNA could not be detected. However, this is unlikely
because the amount of input siRNA duplex left in
AGO2414 embryo lysate was not significantly reduced
even after 3 h incubation as opposed to that seen in wildtype embryo lysate (Fig. 3D, lower panel), indicating that
input siRNA duplex is indeed not unwound in AGO2
mutant embryo lysate. Using a recently developed RISC
assembly assay (Tomari et al. 2004), we next analyzed
RISC formation in AGO2 mutant embryo lysates.
ds-siRNA was incubated with embryo lysates in a standard RNAi reaction, and then RISC complexes were resolved by electrophoresis through a native gel. As shownin Figure 3E, three major complexes (B, A, and RISC)
were detected in wild-type embryo lysates as reported
previously (Tomari et al. 2004). Complexes B and A are
thought to be intermediate precursors to active RISC and

Figure 2. RNA interference of ftz activity
in embryos. Representative embryonic phenotypes of wild-type and AGO2414 mutant
embryos injected with ftz dsRNA are
shown. (A) A wild-type embryo injectedwith water. (B) A wild-type embryo injectedwith ftz dsRNA has a phenotype similar tothat of ftz. (C) An AGO2414 mutant embryo
injected with ftz dsRNA has a wild-type
phenotype. (D) An AGO2414;WTrescue embryo has a phenotype similar to that of ftz.

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Figure 3. AGO2 is required for interference after the formation of the siRNA duplex. (A) Wild-type embryo injected with synthetic
ftz siRNA has a phenotype similar to ftz. (B) AGO2414 embryo injected with synthetic ftz siRNA has a wild-type phenotype. (C) AGO2
is not required for the production of the siRNA duplex. Uniformly 32P-labeled dsRNA corresponding to the ftz gene, incubated in
extracts from yw and AGO2414 embryos. RNA products of the reaction were analyzed on a polyacrylamide gel. Incubation of lysateswith labeled dsRNA generates RNA fragments ∼22 nucleotides (nt) long. [WT(yw)] Yellow-white wild type; (AGO2414) AGO2414
homozygote. (D) Analysis of siRNA unwinding in AGO2414 embryo lysate. The siRNA duplex was incubated in the extracts fromwild-type (yw) and AGO2414 embryos. RNA products of the reaction were analyzed on a native acrylamide gel. The migration patternsof the siRNA duplex, the starting material, and the single-stranded siRNA, the expected product after unwinding, are shown on theleft. (Lower panel) The amounts of input siRNA duplex and unwound single-stranded (ss) siRNA at 0 and 3 h incubation are shown.
The average of five independent experiments is shown here. (E) Native gel analysis of labeled siRNA–protein complexes from wild-type
and AGO2414 embryo lysates. B and A complexes are observed in both lanes from wild-type and AGO2414 reactions. These twocomplexes correspond to intermediate precursors to RISC (Tomari et al. 2004). The complex corresponding to RISC (Tomari et al. 2004)
is not detected in the lane from an AGO2414 reaction.

contain ds-siRNA (Tomari et al. 2004). Both complexes B
and A were readily detected in AGO2 mutant embryo
lysates. However, RISC formation was impaired in
AGO2 mutant embryo lysates (Fig. 3E). Together, theseresults suggest that AGO2 is required in a step(s) in RISC
assembly after binding of the siRNA duplex to RISC precursors (complexes B and A).

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miRNA-initiated RNA cleavage does not need AGO2
but siRNA-initiated RNA cleavage does
Recently, it has been demonstrated that both siRNAs
and miRNAs are associated with the same RISC and thatmiRNAs can even direct cleavage of perfectly basepaired substrates in human cells (Hutvagner and Zamore

Distinct roles for AGO proteins in RNAi

2002b; Martinez et al. 2002). To see if this is also the casein Drosophila, we investigated whether the lack of
AGO2 affects the miRNA-mediated cleavage of target RNAs. 32P-radiolabeled target mRNA, containing
a sequence fully complementary to an endogenous
miRNA, miR-2b, as well as a sequence exactly complementary to one strand of a ftz siRNA duplex (Fig. 4A),
was incubated with AGO2414 embryo lysate in an invitro RNAi reaction. Both siRNA-directed and miRNAdirected cleavage products are seen in the wild-type lysate (Fig. 4B) as shown previously (Schwarz et al. 2002).
However, ftz siRNA did not direct cleavage in AGO2414
embryo lysate (Fig. 4B); in contrast, miR-2b still directed cleavage of the target RNA in AGO2414 embryo lysates (Fig. 4B). These results demonstrate that although
AGO2 is an essential factor in siRNA-directed RNAi,

Figure 4. AGO2 is not required for miRNA-initiated RNA cleavage. (A) Schematic drawing of the target RNA and its pairing withftz siRNA and miRNA (miR-2b or let-7). (Magenta) miR-2b, or let-7 and their complementary sequences (below); (green) ftz siRNA andits complementary sequence. An asterisk indicates the 5⬘-32P radiolabel. (B) miRNA-directed target RNA cleavage is catalyzed byembryo lysates with and without AGO2. In vitro RNAi assays were carried out with 14–16-h embryo lysates of yw and AGO2414.
Target RNA contains the ftz siRNA and the miR-2b target sequences as shown in A. (C) In vitro processing of pre-let-7 RNAs internallylabeled with ␣-32P-GTP. Mature let-7-generating activities were compared between lysates from 0- to 2-h yw and AGO2414 embryos.
Both synthetic 21-nt let-7 RNA (not shown) and in vitro processed let-7 migrate at ∼25 nt relative to the markers. The discrepancy
between molecular mass and mobility in polyacrylamide gel electrophoresis is frequently observed for small RNAs (Hutvagner et al.
2001; Lee et al. 2003). (D) In vitro RNAi assays with lysates prepared from 0- to 2-h yw and AGO2414 embryos. The RNA target
contains let-7 and ftz siRNA target sequences as shown in A.

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AGO2 is dispensable for miRNA-directed RNA cleavage
in RNAi.
In this experiment, however, the timings of miRNAloading and siRNA loading into RISC are different. miR2b is already incorporated into the RISC by the embryo
before the lysate is made, whereas siRNA is added to thereaction mixture. We therefore added synthetic Drosophila melanogaster let-7 precursor RNA to an in vitro
RNAi reaction mixture containing the let-7 complementary target RNA. It should be noted that the sequence ofthe synthetic let-7 precursor used here (Fig. 4C, bottom
panel) is slightly longer than that of naturally processed
pre-let-7, which contains short 3⬘ overhangs (Basyuk et
al. 2003; Lee et al. 2003). However, the synthetic let-7
precursor has been previously shown to be processed andproduce mature let-7 with precisely the same 5⬘ end as
authentic let-7 in the Drosophila embryo lysates(Hutvagner et al. 2001). In this experiment, the synthetic
let-7 precursor RNA was also converted to mature let-7
in vitro, not only in the wild-type embryo lysate, but alsoin AGO2414 embryo lysate (Fig. 4C; data not shown). In
the in vitro RNAi assay, target RNA was cleaved within
the let-7 complementary sequence in AGO2414 embryo
lysate as efficiently as in wild type (Fig. 4D). Thus,
miRNA that is not preloaded by the embryo is still capable of entering into the RNAi pathway without AGO2
activity. These results demonstrate that AGO2 is not
required for miRNA production or the loading of
miRNAs into the RISC and suggest that AGO2 has a
specific role for siRNA activity, likely as a specific unwinding and loading factor of siRNA into RISC as shown
earlier, and possibly as a necessary component of ansiRNA-initiated RISC.
AGO1 is necessary for efficient miRNA-directed
RNA cleavage
We next tested whether siRNA- and miRNA-directed
RNA cleavage pathways have differential requirements
for AGO1. Mutations in AGO1 result in late embryonic/
early larval lethality with developmental defects(Kataoka et al. 2001). Although AGO1 has been shown
necessary for efficient RNAi in embryos (Williams andRubin 2002), AGO1 appears to have little or no effect on
the efficiency of RNAi in cultured S2 cells (Fig. 5A; seealso Caudy et al. 2002). AGO1k08121 is a strong allele(Kataoka et al. 2001). AGO1k08121 was balanced over a
CyO chromosome carrying a kruppel (Kr)-Gal4 and a
UAS–GFP transgene. Homozygous embryos were separated from heterozygous and CyO-Kr-Gal4-UAS–GFP
homozygous embryos on the basis of GFP expression.
Because there is strong reduction of AGO1 transcripts in
AGO1k08121 embryos (Kataoka et al. 2001; data notshown) and AGO1 protein was not detected in lysate
from 14- to 16-h staged AGO1k08121 embryos (Fig. 5B),
we tested 14–16-h staged AGO1k08121 embryo lysate for
its ability to direct cleavage of siRNA or miRNA
complementary RNA targets. ftz siRNA directed its
cleavage in AGO1k08121 embryo lysate as effectively as
in wild type (Fig. 5C). AGO1k08121 embryo lysate also

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processed long ftz dsRNA into short ∼21-nt fragments(Fig. 5D). Furthermore, RISC formation directed bysiRNA duplex was not impaired in AGO1k08121 embryo
lysate as judged by the native gel shift assay (Fig. 5E).
These findings suggest that AGO1 is not necessary forthe production of the siRNA duplex, siRNA-initiated
RISC formation, or siRNA-directed cleavage. However,cleavage of the target RNA directed by miR-2b was suppressed in AGO1k08121 embryo lysate by a factor of fivefold compared with that in wild type (Fig. 5C), suggestingthat AGO1 is necessary for efficient target RNA cleavage
mediated by miRNAs. Residual miRNA activity seen in
AGO1k08121 could be due to some degree of functional
complementation among Argonaute family proteins expressed in embryos (Williams and Rubin 2002). Our invitro experiments with AGO1k08121 mutant embryo lysates argue against AGO1 being required for siRNA-mediated RNAi in embryos when injected with either longdsRNA or siRNA (Williams and Rubin 2002). Although
we currently do not understand the discrepancy, ourfindings are at least consistent with the findings using
cultured S2 cells (Fig. 5A; Caudy et al. 2002).
AGO1 is involved in the stable maturationof miRNAs
We hypothesized that AGO1 might be involved in thematuration and/or function of miRNAs. To directly determine the contribution of AGO1 and AGO2 to miRNA
production in Drosophila cells, we depleted each from S2cells by dsRNA soaking (Fig. 6A) and performed Northern blot analyses to monitor the abundance of bantam
miRNA (miR-ban; Brennecke et al. 2003). A marked reduction of mature miR-ban was observed in AGO1-depleted S2 cells but not in AGO2-depleted S2 cells (Fig.
6A). We also confirmed that the miRNA level was significantly reduced in the AGO1k08121 embryos (Fig. 6B).
The accumulation of pre-miR-ban was not observed inboth cases (data not shown; see also Fig. 6C). Similar
results were obtained when the same RNA preparations
were probed for the expression of another miRNA, miR-2b,
which is also known to be expressed both in S2 cells andembryos (data not shown). These results suggest that
AGO1 is involved in the maturation and/or stability ofmiRNAs.
Production of both siRNAs and miRNAs require
Dicers, which interact with RISC components (Hammond et al. 2001; Tabara et al. 2002), suggesting that
Dicer action is coupled to loading small RNAs onto the
RISC. Two Dicers, Dicer-1 and Dicer-2, have been identified in Drosophila. siRNA production is associatedwith Dicer-2, but not Dicer-1 (Liu et al. 2003). Dicer-2,
together with the dsRNA-binding protein R2D2, facilitates siRNA loading onto RISC (Liu et al. 2003). We
found that the targeted destruction of only one of thetwo Dicers, Dicer-1, leads to an accumulation of themiR-ban precursor in S2 cells (Fig. 6C), consistent withthe recent genetic studies showing that Dicer-1 is required for miRNA processing (Lee et al. 2004). The
amount of pre-miR-ban was further increased after pro-

Distinct roles for AGO proteins in RNAi

Figure 5. AGO1 is required for efficient miRNA-initiated RNA cleavage. (A) When AGO2 is suppressed by introducing specificdsRNA in S2 cells expressing EGFP, the ability of the cells to silence EGFP by RNAi is severely reduced. In contrast, when AGO1 is
suppressed, the EGFP silencing effect is unaffected, indicating that AGO1 is not essential for the RNAi pathway in S2 cells. (B) Lysates
were prepared from 14- to 16-h yw, AGO2414 mutant, and AGO1k08121 mutant embryos. Western blots were performed using theantibodies against AGO2 (upper), AGO1 (middle), and ribosomal protein P0 (lower) as a loading control. (C) In vitro RNAi assays withlysates prepared from 14- to 16-h yw, AGO2414, and AGO1k08121 embryos. The RNA target was the same as that used in Figure 4B.
The amount of specifically cleaved bands was quantified and normalized to that of bands in wild-type embryos. (D) AGO1 is not
required for the production of the siRNA duplex. Uniformly 32P-labeled dsRNA corresponding to the ftz gene, incubated in extracts
from yw and AGO1k08121 embryos. RNA products of the reaction were analyzed on a polyacrylamide gel. Incubation of lysates withlabeled dsRNA generates RNA fragments ∼22 nt long. [WT(yw)] Yellow-white wild type; (AGO1k08121) AGO1k08121 embryos. (E) RISC
formation is not impaired in AGO1k08121 embryo lysates.

longed Dicer-1 knockdown for 8 d. In contrast, prolonged
suppression of AGO1 expression did not result in theaccumulation of pre-miR-ban (Fig. 6C). These findings
suggest that mature miRNAs are processed from pre-miRNAs by Dicer-1 and are stabilized by AGO1. We
next asked whether AGO1 is physically associated with
Dicer-1 by purifying AGO1-associated complexes from
S2 cells using a TAP method (Rigaut et al. 1999). It was

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Figure 6. AGO1 is involved in miRNA biogenesis. (A)AGO1 is required for stable, mature miRNA production. Expression of AGO1, AGO2, or EGFP (control)
was suppressed by RNAi in S2 cells. Western blots(lower panels) show that the Argonaute proteins are indeed down-regulated. RNAs were obtained from eachcell and subjected to Northern blots to analyze theamounts of miR-ban using a specific probe. (B) The
miRNA level in the wild-type, AGO1, and AGO2 mutant embryos. Northern blot analysis on the AGO1 mutant (AGO1k08121) shows a marked reduction of maturemiR-ban. (C) Dicer-1 and AGO1 are related to miRNAprocessing and stabilization, respectively. Expression ofeither Dicer-1 (Dcr-1), Dicer-2 (Dcr-2), AGO1, AGO2,
or EGFP (control) was reduced by RNAi in S2 cells forindicated days, then total RNAs were obtained fromeach cell and subjected to Northern blots to visualize
mature and pre-miR-ban. (D) AGO1 associates with
Dicer-1. TEV extract prepared from S2 cells expressing
AGO1-TAP, AGO2-TAP, or dFMR1-TAP, or the parental S2 cells (control), were subjected to Western blots
using antibodies against Dicer-1, AGO1, dFMR1, andAGO2. Protein bands with asterisks indicate AGO1,
AGO2, and dFMR1 with a CBP tag, a converted form ofa TAP tag after TEV cleavage. (E) AGO1 associates withboth pre-miRNA and mature miRNA. Northern blots
show that all AGO1-TAP, AGO2-TAP, and dFMR1TAP complexes contain mature miR-ban, but the precursor is associated only with the AGO1-TAP complex.

found that AGO1 was associated with Dicer-1 (Fig. 6D).
This interaction of AGO1 with Dicer-1 seems to be quite
specific for AGO1 because Dicer-1 was not detected in
AGO2- and in dFMR1-associated complexes under theconditions used for the purification (Fig. 6D). RNA
preparations from AGO1-, AGO2-, and dFMR1-associated complexes were then probed for the presence ofmiR-ban. AGO2- and dFMR1-associated complexes contained the mature form of miR-ban (Fig. 6E). Only the
AGO1-associated complex contained the pre-miR-ban
and mature forms (Fig. 6E). The interaction of AGO1
with Dicer-1 and pre-miRNA further suggests that
AGO1 is involved in miRNA biogenesis.
Discussion
In mammals, there appear to be no real distinctions between the siRNA and miRNA pathways downstream ofDicer (Hutvagner and Zamore 2002b). In contrast, genetic studies in C. elegans have shown that distinct Argonaute homologs appear to be dedicated to distinct
RNAi and translation repression pathways (Grishok et
al. 2001). Using target RNA cleavage assays, we showthat siRNA-directed RNA cleavage depends on AGO2

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but does not require AGO1, whereas miRNA-directed
RNA cleavage depends on AGO1 but does not need
AGO2 in Drosophila embryos. Our results suggest thatmaturation and the function of siRNAs and miRNAs
have differential requirements for Argonaute proteins in
Drosophila. These findings also suggest that Argonaute
proteins regulate entry points of small RNAs to RISC but
may not act as determinants for target RNA cleavage ortranslation repression. AGO2 is an essential componentof RNAi and part of the RISC complex (Hammond et al.
2001). We show that the particular function of AGO2lies downstream of siRNA duplex production in theRNAi pathway (Fig. 3). It has been shown that duplex
siRNAs are incorporated in RISC precursors and ATPdependent unwinding of siRNAs converts RISC precursors into active RISC that then degrades the specific target mRNAs (Hammond et al. 2000; Nykanen et al. 2001;
Tomari et al. 2004). Thus, AGO2 is thought to function
at some or all of these steps in RNAi. In this study, wedemonstrate that both the unwinding of siRNA duplex
and the assembly of siRNA into functional RISC are impaired in AGO2 mutant embryos (Fig. 3). Therefore,
these results indicate that AGO2 functions at a step(s) in
RISC assembly after binding of the siRNA duplex toDistinct roles for AGO proteins in RNAi

RISC precursors. Recently, Liu et al. (2003) demonstrated that Dicer-2 not only associates with siRNA production, but also, together with R2D2, facilitates siRNAloading onto RISC in Drosophila. These data suggestthat AGO2, together with Dicer-2 and R2D2, assembles
siRNA into functional RISC in Drosophila embryos.
AGO2414 flies that we produced are developmentallynormal, which provides circumstantial evidence that
AGO2 is not important for development and by inference not essential for utilization and function of miRNA
that often regulates the developmental pathways (Bartel
2004). In contrast, our findings that AGO1 is required forthe stable production of mature miRNAs might explain
the fact that AGO1 is essential for normal development,
particularly in the nervous system (Kataoka et al. 2001).
The physical association of AGO1 with Dicer-1 and premiRNA suggests that AGO1 is involved in miRNA biogenesis. In fact, recent genetic studies of Dicers in Drosophila have shown that mutations in Dicer-1 block processing of miRNA precursors (Lee et al. 2004). Together,
these results suggest that in Drosophila, distinct pathways exist for siRNA and miRNA production and theirconcomitant assembly into RISC complexes. Do AGO1
and AGO2 functional specificities reflect physically distinct miRNA-associated RISC and siRNA-associatedRISC, or differential processing and/or loading of small
RNAs into generic RISC? Although there is no definitive
evidence that there is a generic RISC for both smallRNAs, recent studies have shown that the RISC containing active miRNAs and the RISC involved in siRNAdirected RNAi are very similar, if not identical, as endogenous miRNAs can cleave mRNAs with perfectcomplementarity (Hutvagner and Zamore 2002b; Llave
et al. 2002), and exogenously introduced siRNAs cantranslationally repress mRNAs bearing imperfectly
complementary binding sites (Doench et al. 2003; Zenget al. 2003). Our findings show that the miRNA-associated RISC that cleaves RNA does not need AGO2,
whereas siRNA-associated RISC does. This argues that
there are inherent differences between siRNA-initiated
RISC and miRNA-initiated RISC in Drosophila. Physically, they might be the same generic complex (for instance, both containing AGO2) but one does not need
AGO2 activity. It is also possible that they might bephysically distinct with respect to Argonaute proteins.
AGO1 is not present in AGO2-associated complexes(Fig. 6D) and can be biochemically separated fromsiRNA-loaded RISCs (Caudy et al. 2002). Therefore,
AGO1 and AGO2, conceivably, are not incorporated into
the same RISC. However, miRNAs and siRNAs arefound, to some extent, in both AGO1 and AGO2 complexes (Fig. 6E; see also Caudy et al. 2002), suggestingthat association of Argonaute proteins with small RNAsis preferential rather than absolutely specific. Alternatively, a fraction of small RNAs might be exchangeable
between the two complexes, probably during recycling of
their silencing function.
The recent study has shown that miRNA-associatedRISCs and siRNA-associated RISC are different with respect to Dicers in Drosophila (Lee et al. 2004). Our re-sults clearly suggest that in conjunction with Dicers,
Argonaute proteins regulate the formation of siRNA-associated RISC or miRNA-associated RISC, as their particular functions lie downstream of Dicers in a step(s) in
the assembly of small RNAs into RISC.
Materials and methods
Drosophila strains
Flies were maintained under standard procedures. P element
insertion in AGO2 EP(3)3417 was out-crossed to yw to clean up
the genetic background. AGO1k08121 (Kataoka et al. 2001) wasbalanced over a CyO chromosome carrying a Kruppel-Gal4 anda UAS–GFP transgene (CyO-Kr-Gal4-UAS–GFP chromosome).
Generation of AGO2 mutants
EP(3)3417 contains the EP transposon (Rorth et al. 1998) in the5⬘ UTR region of the AGO2 locus. To isolate new AGO2 alleles,
we generated 400 independent lines from the cross of virgin
EP(3)3417 females with males possessing ⌬2–3 transposase. Genomic DNA from these lines were prepared and used as PCR
templates to screen lines that carried a deletion within the
AGO2 locus. The breakpoints of the deletion were determinedby sequencing the PCR products. AGO1k08121 was maintained
as a heterozygous stock balanced over a CyO-Kr-Gal4-UAS–GFP chromosome. One-quarter of the embryos produced in thisstock were AGO1k08121 homozygous mutants, as identified by
the absence of zygotic GFP expression. The remaining threequarters expressed GFP and represented both heterozygous and
CyO-Kr-Gal4-UAS–GFP homozygous embryos.
RNAi in embryos
For dsRNA production, a region of the fushi tarazu (ftz) gene
was amplified with oligonucleotides 5⬘-ATCCGATATGGCCACCAC-3⬘ and 5⬘-CGCTCTGGGGAAGAGAGTAA-3⬘ by using an embryonic cDNA pool as a template. The PCR productwas cloned into the pGEM-T vector (pGEM-T-ftz) and the insert
was amplified with T7 and SP6 primers. The resulting PCR
product was used as a template for in vitro transcription reactions using MEGAScript T7 and SP6 kits (Ambion) according to
the manufacturer’s instructions. Equimolar concentrations ofsense and antisense RNAs were boiled and allowed to anneal>12 h at room temperature. siRNA (21 bp) with 2 base 3⬘ overhangs corresponding to the ftz gene (siFTZ 5⬘-UGCCUACUAUCAGAACACC-3⬘) was purchased from Dharmacon. The
siRNA was 5⬘-phosphorylated with T4 polynucleotide kinase(PNK; TaKaRa). dsRNA or siRNA (1 mg/mL in H2O) was injected into syncytial blastoderm embryos (Kennerdell andCarthew 1998). After 20–24 h at room temperature, the surviving embryos were scored for the number of ventral denticle
belts. For consistency, any embryos with four, five, or six belts
were scored as ftz. For cuticle analysis, embryos were incubatedat room temperature under oil for 20–24 h. After dissection fromtheir vitelline membranes, embryos were washed and fixed, andtheir cuticles were prepared as described (Kennerdell andCarthew 1998).
RNAi in S2 cells
dsRNAs (∼700 bp long) of EGFP, AGO2 and AGO1, Dicer-1, andDicer-2 were produced by in vitro T7 transcription, denatured

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by heating, and reannealed in water. The dsRNA of AGO1 or
AGO2 was introduced into S2 cells expressing EGFP by soaking(Clemens et al. 2000). After 3 d, cells were harvested and divided
into half. To one half, AGO1 or AGO2 dsRNA was again introduced. To the other half, AGO1 or AGO2 dsRNA plus EGFP
dsRNA were introduced into the cells. All cell pools were incubated for another 4 d and subjected to Western blot analyses
using anti-EGFP antibody. In Figure 6, to start RNAi for AGO1,
AGO2, or EGFP in S2 cells expressing EGFP, dsRNA for eachgene was introduced into cells by soaking. After 4 d incubation
or after double transfection of siRNA or dsRNA and prolonged
incubation for 8 d, total RNAs were prepared from each sample
and Northern blotting was performed using a bantam antisense
oligo labeled with gamma-32P-ATP.
In vitro RNAi assays
To prepare ftz RNA with miR-2b or let-7 target site, we amplified a region of the ftz gene from pGEM-T-ftz with the following
primer pairs: 5⬘-ACAACATGCATCACCCCCACAGCCT-3⬘
and 5⬘-AGAGCTCGAGTATCACAGCCAGCTTTGAGGAGCGTTGTAGTAGTAGCAGCTCTCCGAG-3⬘ (for the miR-2b target) or 5⬘-AGAGCTCGAGTGAGGTAGTAGGTTGTATAGTT
GTTGTAGTAGTAGCAGCTCTCCGAG -3⬘ (for the let-7 target). The resulting PCR products were cloned into the NsiI–SalIsite of pGEM-T-ftz as an NsiI–XhoI fragment (pGEM-T-ftz-2b or
pGEM-T-ftz-let-7). The target RNAs were transcribed with a
MEGAScript SP6 kit from PCR templates prepared with SP6
and 3⬘ (5⬘-CGCCGGGTGATGTATCTATT-3⬘) primers andpGEM-T-ftz-2b or pGEM-T-ftz-let-7. RNA was radiolabeled at
the 5⬘-G cap by guanylyltransferase (Ambion). In a 20-µL reaction, 500 ng RNA, 40 U RNasin (Promega), 0.2 U inorganic
pyrophosphatase (Sigma), and 5 µL of ␣-32P GTP (800 mCi/
mmole) were incubated in 50 mM Tris-HCl (pH 7.9), 12.5 mM
MgCl2, 2 mM EDTA, 60 mM KCl, 1 mg/mL BSA, and 25 mM
DTT. The reaction mixture was incubated for 2 h at 37°C, andthe radiolabeled RNA was then PAGE-gel purified. Pre-let-7
RNA was transcribed from a synthetic deoxynucleotide template with T7 RNA polymerase in the presence or absence of
␣-32P-GTP (4). Extract used for in vitro RNAi assays were prepared as described in Tuschl et al. (1999) and Hutvagner andZamore (2002b) with some modifications. In short, embryos
were collected for 2 h and immediately homogenized (0–2-hembryo extract), or then aged for 14 h at room temperatureand homogenized (14–16-h embryo extract). Homozygous
AGO1k08121 embryos were sorted from their AGO1k08121/CyOKr-Gal4-UAS–GFP and CyO-Kr-Gal4-UAS–GFP/CyO-Kr-Gal4UAS–GFP siblings under fluorescent microscopy. Approximately 100 mg embryos were homogenized in 100 µL of lysis
buffer (100 mM potassium acetate, 30 mM HEPES at pH 7.4, 2
mM magnesium acetate, 5 mM DTT, and 0.5 mg/mL Pefabloc
SC), centrifuged for 25 min at 14,600 × g at 4°C, and flash-frozen
in 10-µL aliquots. Total protein concentration in the extracts
was determined with a colorimetric Bio-Rad assay. Cleavage ofdsRNA or processing of pre-let-7 was assayed by incubating1000 cpm of labeled dsRNA or pre-let-7 in a 10-µL reaction
containing 15 µg of extract proteins for increasing times at room
temperature. Reactions were stopped by the addition of stopbuffer (100 mM Tris-HCl at pH 7.9, 50 mM NaCl, 50 mM
EDTA, 1% SDS, and 100 µg/mL proteinase K). For miR-2b target
RNA cleavage reactions, 100 nM ftz siRNA duplex was incubated with 2000 cpm of cap-labeled miR-2b target RNA in 14–
16-h (Figs. 4, 5) embryo lysate under standard conditions for 90
or 180 min at room temperature. For let-7 target RNA cleavage
reactions, 100nM ftz siRNA duplex and 100 nM in vitro transcribed pre-let-7 RNA were incubated with 2000 cpm of cap-

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labeled let-7 target RNA in 0–2-h embryo lysate under standard
conditions for 90 or 180 min at room temperature.
siRNA unwinding assay
siRNA unwinding assay was performed as described in Nykanen et al. (2001) with some modifications. The 5⬘ end of
the antisense strand siRNA was 32P-labeled with PNK. RNAi
reactions (10 µL) were quenched with 40 µL of stop buffer and a
100 molar excess of Pp-luc-antisense RNA; incubated for 15
min at 25°C; and immediately ethanol precipitated, redissolved
in 4 µL of 1× loading buffer (Takara), and immediately analyzed
by electrophoresis at 4°C through 15% native polyacrylamidegel. siRNA sequences were 5⬘-UCGAAGUAUUCCGCGUAC
GUG-3⬘ (Pp-luc-antisense) and 5⬘-CGUACGCGGAAUACUUCGAAA-3⬘ (Pp-luc-sense).
RISC assembly assay
RISC assembly assay was performed as described in Tomari etal. (2004). 32P-radiolabeled siRNA duplex was incubated withembryo lysates at room temperature. After incubation, thesamples were adjusted to 6% (w/v) glycerol and resolved bysubmarine native agarose gel electrophoresis. Gels were dried
under vacuum onto Hybond-N+ nylon membrane (Amersham
Bioscience).
Northern blot analysis
RNA was isolated from S2 cells or the IgG bound fractions with
ISOGEN (Nippon gene). Northern blot was performed as described (Ishizuka et al. 2002), except that the RNA was run on
12% acrylamide-denaturing gels and transferred onto HybondN+ membrane. The hybridizations were performed at 42°C in
0.2 M sodium phosphate (pH 7.2) and 7% SDS and washed at
42°C in 2× saline sodium citrate and 0.1% SDS.
TAP purification
The expression of AGO1-TAP, AGO2-TAP, or dFMR1-TAP in
S2 cells was induced by adding copper ion into the medium.
After overnight incubation, the cytoplasmic lysate was preparedin a buffer containing 10 mM HEPES (pH 7.0), 200 mM KOAc,
2 mM MgAc, 0.1% NP40, 2 µg/mL leupeptin, 2 µg/mL pepstatin, and 0.5% aprotinin. AGO1-TAP, AGO2-TAP or dFMR1TAP, and associated materials to the TAP-tagged fusion protein
were bound to IgG beads. After extensive washing, the boundfraction was eluted by treatment with TEV protease. The polyclonal antibodies against Dicer-1 and AGO1 were kind gifts
from G. Hannon and T. Uemura, respectively. The AGO2 anddFMR1 monoclonal antibodies (4D2 and 5A11, respectively)
were produced by injecting AGO2 or dFMR1 peptides into mice,followed by fusing the mouse spleen cells to myeloma cells to
obtain hybridomas producing the specific antibodies.

Acknowledgments
We thank the Bloomington Stock Center for fly stocks, T. Uemura for gifts of AGO1 antisera and AGO1 mutant fly stocks,
G. Hannon for anti-Dicer antibodies, S.B. Inoue for help producing AGO2 mutant fly strains, K. Mizumoto for purified capping
enzyme, J. Wilusz for an expression vector for capping enzyme,
Y. Kurihara for helping produce monoclonal antibodies, and T.
Uchiumi for anti-P0 antibody. Some EP fly strains came fromDistinct roles for AGO proteins in RNAi

Exelixis, Inc. We thank S. Nakielny, A. Nakamura, and members of the Siomi laboratory for discussions and comments on
the manuscript. K.O. and A.I. are predoctoral fellows of the 21st
century COE Program from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (MEXT). Supported inpart by grants to M.C.S. and H.S. from MEXT.
The publication costs of this article were defrayed in part by
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 USC section
1734 solely to indicate this fact.

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