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Characterization of the miRNA-RISC loading complex andmiRNA-RISC formed in the Drosophila miRNA pathway
Keita Miyoshi, Tomoko N. Okada, Haruhiko Siomi, et al.
RNA published online May 18, 2009
Access the most recent version at doi:10.1261/rna.1541209

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Characterization of the miRNA-RISC loading complex
and miRNA-RISC formed in the Drosophila
miRNA pathway
KEITA MIYOSHI,1 TOMOKO N. OKADA,1 HARUHIKO SIOMI,1 and MIKIKO C. SIOMI1,2
1

School of Medicine, Keio University, Tokyo 160-8582, Japan
Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Saitama 332-0012, Japan

2

ABSTRACTIn Drosophila, miRNA is processed by Dicer-1 (DCR-1) from its precursor and loaded onto Argonaute1 (AGO1). AGO1
recognizes target mRNAs based on the miRNA sequence and suppresses the expression at post-transcriptional levels. GW182, aP-body component, localizes the AGO1 complex to processing bodies (P-bodies) where mRNA targets are decayed or stored.
However, the details of the pathway remain elusive. In this study, two distinct types of AGO1-containing complexes from
Drosophila Schneider2 (S2) cells were isolated and compared at the molecular level. The AGO1 complex with DCR-1 contained
neither mature miRNA nor GW182 but exhibited pre-miRNA processing activity. The resultant mature RNA was loaded onto
AGO1 within the complex. The AGO1 complex with GW182 excluded DCR-1, but possessed mature miRNA and showed no
pre-miRNA processing activity. Thus, the AGO1-DCR-1 and AGO1-GW182 complexes correspond to miRLC (miRISC loading
complex) and miRISC, respectively. The requirement for various domains of AGO1 in miRNA-loading and DCR-1/GW182
interaction was also examined. The Mid domain mutant (F2V2) interacted with DCR-1 but not with mature miRNA and GW182.
The AGO1-PAZ mutant lacks the mature miRNA-binding ability but associates with either DCR-1 or GW182. The AGO1-PIWI
mutant showed no Slicer activity but associates with mature miRNA. These results indicate that these domains are required
differently for miRLC and miRISC formation in the miRNA pathway.
Keywords: Argonaute1; GW182; Dicer-1; miRLC; miRISC; Drosophila

INTRODUCTION
RNA silencing is an evolutionarily conserved physiologicalprocess regulating gene expression (Zaratiegui et al. 2007).
RNA interference (RNAi), the prototype of RNA silencing,negatively regulates gene expression by cleaving target
mRNAs in a sequence-dependent manner (Tolia and
Joshua-Tor 2007). A molecule that renders sequence specificity to RNAi is a short interfering RNA (siRNA) of z21
nucleotides (nt) (Bernstein et al. 2001). siRNAs are produced from long double-stranded (ds) RNA precursors thatare exogenously introduced into, or expressed within, cells.
The RNase III enzyme required for dicing long dsRNA
precursors into siRNAs in Drosophila is Dicer-2 (DCR-2)(Bernstein et al. 2001; Lee et al. 2004a). DCR-2 associates

Reprint requests to: Mikiko C. Siomi, School of Medicine, Keio
University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan;
e-mail: siomim@sc.itc.keio.sc.jp; fax: 81-3-53633266.
Article published online ahead of print. Article and publication date areat http://www.rnajournal.org/cgi/doi/10.1261/rna.1541209.

with R2D2, a dsRNA-binding domain protein during theprocessing step (Liu et al. 2003). DCR-2-R2D2 is also
necessary for the siRNA loading step onto the RNAinduced silencing complex (RISC) (Liu et al. 2003); thus,
it is considered as the RISC-loading complex (RLC) (Leeet al. 2004a; Tomari et al. 2004a). Other components of the
RLC remain unclear. The siRNA duplex processed by DCR2-R2D2 is passed onto Argonaute2 (AGO2), an endonuclease required for cleaving target mRNAs in RNAi
(Tolia and Joshua-Tor 2007). AGO2 cleaves a particularstrand of the siRNA duplex (called ‘‘passenger’’; this strand
is not loaded onto RISC), which then disappears in theenvironment (Matranga et al. 2005; Miyoshi et al. 2005;
Rand et al. 2005; Leuschner et al. 2006; Kim et al. 2007).
Through these processes, RISC is finally activated.
miRNAs are an endogenous subset of small RNAs of 21–
23 nt that function in RNA silencing by repressing translation or by facilitating degradation of target mRNAs(Bushati and Cohen 2007; Farazi et al. 2008). miRNAtarget genes are implicated in vital biological processes suchas development, apoptosis, neural patterning, and viral

RNA (2009), 15:00–00. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2009 The Protein Society.

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Miyoshi et al.

infection (Bushati and Cohen 2007). To date, about 150
miRNAs have been identified in Drosophila. Each miRNA isthought to target multiple genes, suggesting that the vast
majority of protein-coding genes are regulated by miRNAs
in vivo (Bushati and Cohen 2007).
miRNAs are mostly transcribed by RNA polymerase II(Cai et al. 2004; Lee et al. 2004b). The primary transcripts,
pri-miRNAs, are processed to pre-miRNAs (miRNA precursors) in the nucleus by an RNase III enzyme, Drosha, in
Drosophila (Lee et al. 2003). Upon export to the cytoplasm,
pre-miRNAs are further processed by another RNase III
enzyme, DCR-1 (Lee et al. 2004a). Loquacious (LOQS;
also known as R3D1), a cytoplasmic double-stranded (ds)RNA-binding domain protein, stimulates and directs the
specific pre-miRNA processing activity of DCR-1 by tightly
associating with DCR-1 (Forstemann et al. 2005; Jiang et al.
2005; Saito et al. 2005). Upon processing, miRNAs are stillin a duplex form (miRNA/miRNA*) and thus should be
unwound prior to miRISC (miRNA-containing RISC) formation. In contrast to siRNA duplexes, strand rejection of
miRNA* is not accompanied by miRNA* strand cleavage
by AGO1 (Matranga et al. 2005). Factor(s) responsible for
miRNA/miRNA* unwinding remain unknown. Within
miRISC, AGO1, another member of the Argonaute family
in Drosophila, directly associates with mature, singlestranded miRNA (Miyoshi et al. 2005).
Human GW182 was shown to be involved in posttranscriptional regulation of a subset of mRNAs by localizing them to cytoplasmic foci termed P-bodies (Eystathioy
et al. 2002, 2003; Liu et al. 2005; Meister et al. 2005).
P-bodies contain enzymes required for mRNA decay, forexample, CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. In Caenorhabditis elegans, AIN-1, a homologof human GW182, interacts with ALG-1, one of thenematode Argonautes, and targets ALG-1 to P-bodies(Ding et al. 2005). GW182 in Drosophila (also known as
Gawky) (Schneider et al. 2006) was also shown to interact
with AGO1 and accumulate in P-bodies (Behm-Ansmant
et al. 2006; Schneider et al. 2006). Recent studies suggested
a functional involvement of GW182 in the miRNA-mediated silencing pathway. Loss of GW182 changes mRNA
expression profiles in a similar way to those observed in
AGO1 depletion (Behm-Ansmant et al. 2006). It was also
demonstrated that GW182 interaction with AGO1 is essential for miRNA-mediated translational repression andmRNA decay (Liu et al. 2005; Jakymiw et al. 2007; Eulalio
et al. 2008). Taken together, it is thought that DCR-1LOQS, AGO1, and GW182 are indispensable in the
Drosophila miRNA pathway. However, in contrast to theRNAi pathway, the RLC-like complex and miRISC, whichshould be formed in the miRNA pathway, have not yet
been isolated and characterized.
To gain further insights into the miRNA-mediated genesilencing mechanism, we immunopurified AGO1-containing
complexes from S2 cells and found two such complexes. The
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RNA, Vol. 15, No. 7

AGO1 complex that involves DCR-1 excludes GW182 andmature miRNAs but exhibits pre-miRNA activity. miRNAs
processed by the complex were loaded onto AGO1 within
the complex. The other AGO1 complex contained GW182
and mature miRNAs but excluded DCR-1 and pre-miRNA
activity. These results suggest that the association of AGO1
with DCR-1 and GW182 occurs in a mutually exclusive
manner and that the AGO1-DCR-1 and the AGO1-GW182
complexes correspond to miRLC and miRISC, respectively.
The requirements of the various domains in AGO1 forbinding with miRNA, DCR-1, and GW182 were also
examined. The Mid domain mutant (F2V2) interacted with
DCR-1. However, it does not associate with mature miRNA
and GW182; thus, it did not localize to P-bodies (Eulalio
et al. 2008; this study). The PAZ mutant lacks the mature
miRNA-binding activity, but associates with either DCR-1
or GW182. The Piwi mutant showed no Slicer activity but
still associates with mature miRNA. These results indicatethat these domains are required for different AGO1 functions and for miRLC and miRISC formation in the miRNA
pathway.
RESULTSAGO1 association with DCR-1 and GW182
is mutually exclusive
The identification of AGO1-associating proteins in S2 cells
by immunoprecipitation with anti-AGO1 antibodies revealed that an z175-kDa protein is a tight binder (Fig.
1A, ‘‘*’’). Mass spectrometric analysis identified the protein as GW182, a factor known to localize the AGO1
complex to P-bodies (Behm-Ansmant et al. 2006). Western
blotting using a newly produced anti-GW182 antibody(Fig. 1B) on anti-AGO1 immunoprecipitates confirmed
the association (Fig. 1C). Anti-GW182 immunoprecipitates were reciprocally probed with anti-AGO1. Bands
corresponding to AGO1 were detected (Fig. 1D; Supplemental Fig. 1A). However, under these circumstances,
DCR-1, which was previously shown to interact with
AGO1, was not detected (Fig. 1D; the specificity of antiDCR-1 monoclonal antibody is shown in Supplemental
Fig. 1B). These results led us to hypothesize that AGO1
associates with DCR-1 and GW182 in a mutually exclusive
manner. AGO2 did not interact with GW182 (Fig. 1D) as
reported previously (Behm-Ansmant et al. 2006). DCR-2
was also undetected in the complex (Fig. 1D; the specificity
of anti-DCR-2 monoclonal antibody is indicated in Supplemental Fig. 1B). Western blotting on anti-GW182
immunoprecipitates from ovaries revealed no associationof GW182 with Aub, Piwi (Fig. 1E), or AGO3 (datanot shown), the germline-specific Argonaute. Thus, inDrosophila, GW182 is dedicated to the function of AGO1
in the miRNA pathway and is most likely unrelated to thepiRNA pathway.

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miRLC and miRISC in Drosophilathat the AGO1-DCR-1 and AGO1GW182 complexes are distinct in S2
cells (Supplemental Fig. 2). Note that
GW182 and DCR-1 were detected
simultaneously in anti-AGO1 immunoprecipitates, although the DCR-1 and
LOQS signals were very faint comparedwith those of GW182 (Fig. 2A). Thissuggests that the AGO1-GW182 complex is more abundant than the AGO1DCR-1-LOQS complex in S2 cells.
Thus, to equalize the amount of AGO1
in the immunoprecipitated samples, weemployed 10-fold more (by volume; theprotein concentration was the same) S2
lysate for anti-LOQS immunoprecipitation (see Fig. 2A).
To compare miRNAs present in the
AGO1 complexes, RNAs isolated fromthe complexes were probed with DNA
oligos for bantam, an miRNA expressedin S2 cells, and its antisense strand,bantam* (Fig. 2B). The AGO1-GW182
complex contained abundant maturebantam (Fig. 2B; see anti-AGO1 andanti-GW182 lanes). In contrast, bantam*
and pre-bantam were barely detected.
The AGO1-DCR-1-LOQS complex (antiLOQS lane) contained both bantam andbantam* at very low levels. Faint signals
for pre-bantam also appeared. Both bantam and bantam* signals are z2.5-foldstronger compared with pre-bantam
FIGURE 1. AGO1 interacts in a mutually exclusive manner with DCR-1 and GW182. (A)AGO1 coimmunoprecipitates with an z175-kDa protein, GW182, from S2 cells. The signal on each gel. These results suggest
immunoprecipitated proteins were stained with silver. (n.i.) Non-immune IgG used as that miRNAs contained in the complex
negative control; (h.c. and l.c.) heavy and light chains of the antibodies, respectively. (B) are mostly in either duplex or precursor
Western blotting analyses show that anti-GW182 antibodies produced in our laboratory
forms and that AGO1 associated withspecifically recognize GW182 in S2 cells. (C) Anti-AGO1 immunoprecipitates from S2 cells
contain both AGO1 and GW182. (D) AGO1, but not AGO2, DCR-1, and DCR-2, is detected DCR-1 might be mostly devoid of main the immunoprecipitated complex with anti-GW182 from S2 cells. (E) The PIWI proteins, ture miRNA in vivo.
Aub and Piwi, are not detected in anti-GW182 immunoprecipitates obtained from fly ovaries.
In vitro target RNA cleavage assays
were performed using a target RNA
harboring a sequence completely complementary to banAGO1 with GW182, but not with DCR-1,
tam miRNA (bantam-130). The AGO1-GW182 comis associated with endogenous mature miRNAplex, which contains mature bantam, was able to cleave
To confirm the exclusion of GW182 from the AGO1the target, whereas the AGO1-DCR-1-LOQS complex
DCR-1 complex, immunoprecipitates with anti-LOQS were
failed (Fig. 2C). These results confirm that AGO1 associprobed with anti-GW182. The immunoprecipitation effiated with DCR-1-LOQS is unloaded with mature, active
ciency of anti-DCR-1 was very low (data not shown); thus,
miRNA.
hereinafter, we decided to use anti-LOQS to obtain the
AGO1-DCR-1 complexes. As expected, GW182 was undeThe AGO1-DCR-1 complex shows activities
tected in the AGO1-DCR-1-LOQS complex (Fig. 2A).
to process pre-miRNA and load miRNA onto AGO1
These results further suggest that two distinct AGO1containing complexes exist in S2 cells; one with DCRAGO1 associated with DCR-1-LOQS is not loaded with
1-LOQS and the other with GW182. Glycerol gradientmature miRNA (Fig. 2B,C). To examine if the miRNA
sedimentation experiments further supported the modelloading step induces the dissociation of AGO1 fromwww.rnajournal.org

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Miyoshi et al.

FIGURE 2. Characterization of two AGO1-containing complexes immunoprecipitated from
S2 cells. (A) GW182 is not detected in the AGO1-DCR-1 complex immunoprecipitated from
S2 cells using anti-LOQS. It should be noted that 10-fold more S2 lysate was used for n.i. (nonimmune IgG; negative control) and anti-LOQS immunoprecipitations compared to those foranti-AGO1 and anti-GW182 immunoprecipitations to equalize the amounts of AGO1 in the
immunoprecipitates. (Lowest panel, *) Bands corresponding to antibodies (heavy chains). (B)RNAs were extracted from the immunoprecipitated complexes (shown in A) and subjected to
Northern blotting with DNA oligos for bantam and the opposite strand, bantam*. Anti-AGO1
and anti-GW182 immunoprecipitates almost exclusively contain single-stranded maturebantam. On the other hand, anti-LOQS immunoprecipitates show very weak signals forbantam precursor and bantam/bantam* duplexes. (C) Immunoprecipitated complexes (shownin A) were subjected to in vitro target RNA cleavage assays using a target RNA (bantam-130)
showing full complementarity to bantam. The AGO1-GW182 complex, but not the AGO1DCR-1 complex, exhibits bantam-dependent Slicer activity.

DCR-1-LOQS, pre-miRNA loading assays were performed.
Both the AGO1 complexes with DCR-1-LOQS and GW182
were immunopurified from S2 cells (as in Fig. 2) and32
P-labeled pre-let-7 was added to the complexes andincubated. The AGO complex that contains DCR-1-LOQS
was able to show processive activity of pre-let-7 to mature
let-7, as expected (Fig. 3A; anti-AGO1, anti-LOQS). In
contrast, the AGO1-GW182 complex failed to show
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the activity, confirming the earlier
observation that the AGO1-GW182
complex is devoid of DCR-1 (Fig. 1D).
Immunoprecipitates with anti-LOQS
and anti-AGO1, after being exposed to
pre-let-7 processing in vitro, were able
to cleave the let-7 target (Fig. 3B). Thus,
let-7 loading onto AGO1 that was originally contained in the complex was
confirmed. When RNA cleavage assays
were performed using the supernatantand the bead fractions separated after
the miRNA loading, both fractions ofanti-LOQS showed the Slicer activity(Fig. 3C). The same fractions were subjected to Western blotting using antiAGO1, which showed the displacementof AGO1 from DCR-1. Under the same
conditions, DCR-1 remained mostly on
the beads (Fig. 3C). However, we
noticed that under conditions in which
pre-let-7 was not added, AGO1 was also
detected in the supernatant (Fig. 3C),
suggesting that some population of
AGO1 might be displaced from DCR-1
independently of miRNA loading.
The AGO1-DCR-1 complex showedstrong miRNA processing activity,
although it excludes GW182 (Fig. 3A).
To address the question of whether
GW182 is unnecessary for the process,
GW182 was depleted from S2 cells by
RNAi, and Northern blotting was performed. In contrast to DCR-1 depletion, where pre-bantam accumulated,
GW182 depletion did not cause suchan effect (Fig. 3D). Neither did GW182
depletion abolish the pre-miRNA processing activity (Fig. 3E). Thus, GW182
is unnecessary for miRNA processing.
The above data imply that the two
distinct AGO1 complexes found in Drosophila S2 cells, one with DCR-1-LOQS
and the other with GW182, correspondto miRLC and miRISC, respectively.

Migration of miRLC and miRISC on native gels differsfrom that of siRLC and siRISC
Gel shift assays have shown that siRLC and siRISC formed
in RNAi migrated differently on native gel systems (Phamet al. 2004; Tomari et al. 2004b). We examined how miRLC
and miRISC that are formed in the miRNA pathway
migrate on the gel systems. 32P-labeled pre-let-7 was added

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miRLC and miRISC in Drosophila

into the S2 cell lysate and incubated for 30 min in thepresence of EDTA, which should inhibit DCR-1 (Saito
et al. 2005). During this time period, we observed that a
particular complex, indicated by an asterisk in Figure 4A,
accumulated. However, upon DCR-1 activation by theaddition of Mg2+ (Saito et al. 2005), the RNP band
gradually disappeared, but another complex, which shiftedslightly up the gel, appeared (shown with a double asterisk
in Fig. 4A). Examination of RNAs contained in bothcomplexes on native acrylamide gels revealed that thelower (*) and upper (**) complexes hold predominantly
pre-miRNA and single-stranded mature miRNA, respectively (Fig. 4B). Considering these data, we determined thelower and upper complexes as miRLC and miRISC,
respectively.
On the gel system, we noticed thatsiRLC (Tomari et al. 2004b) and siRISC
migrated much slower than miRLC andmiRISC, respectively (Fig. 4A), indicating that the components in siRLC andmiRLC, as well as siRISC and miRISC,
must be distinct from each other.

could it associate with GW182 (Fig. 5A), as shown previously. The F2V2 mutant does not accumulate in P-bodies
but instead formed larger aggregations in the cytoplasm(Fig. 5B; Supplemental Fig. 2B), which correlates well withthe fact that the mutant lacks the GW182-binding activity.
In contrast, the PAZ mutant, in which four amino acids(F369, Y386, F387, and Y391) that are supposedly necessary
for association with small RNAs (Song et al. 2003) werealtered to alanines, associated with DCR-1 and GW182 to asimilar extent as the WT, although the PAZ mutant was notloaded with mature miRNA (Fig. 5A; Supplemental Fig.
2A). These data suggest that miRNA loading is not aprerequisite for association with GW182 and P-body
localization.

The requirement for various
domains of AGO1 in miRNA,
DCR-1, and GW182 binding
Argonautes are characterized by two
domains, PAZ and PIWI (Farazi et al.
2008; Hutvagner and Simard 2008). The
PAZ domain has the ability to bind tosingle-stranded RNA, whereas the PIWI
domain endows Slicer activity to Argonautes by folding a three-dimensional
structure similar to that of RNase H(Song et al. 2003, 2004; Ma et al. 2005).
Another functional domain in Argonautes, the Mid domain, is locatedbetween the PAZ and PIWI domains,and is known to function as a 59phosphate binding module (Ma et al.
2005; Parker et al. 2005; Rivas et al.
2005). When two conserved phenylalanines in the Mid domain of AGO1,
F560 and F595, were mutated to valines(F2V2), its binding to miRNAs and
GW182 was abolished, suggesting that
the Mid domain is important for AGO1
effector activity (Eulalio et al. 2008). We
also produced the F2V2 mutant andfound that it could associate with
DCR-1 just as well as wild-type (WT)AGO1 (Fig. 5A); however, the mutant
was not loaded with mature miRNA(Fig. 5A; Supplemental Fig. 2A), nor

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Miyoshi et al.

The D-D-H triad necessary for exhibiting Slicer activity(Rivas et al. 2005) was subsequently mutated to either A-DH (D687A) or D-A-H (D760A) in AGO1 to examine if
such mutations could affect interactions with DCR-1 and
GW182, as well as miRNA loading. That both the mutations abolished Slicer activity was confirmed by target RNA
cleavage assays (Fig. 5C). It was revealed that both mutants
were loaded with mature bantam (but not with bantam*)and interacted with DCR-1 and GW182 as did the WT(Fig. 5C). We further examined miRNA loading onto the
AGO1 mutants, which revealed that even miRNA with afully paired seed sequence (‘‘seed’’ sequence corresponds tothe second to seventh nucleotides of mature miRNAs fromthe 59-ends) (Bushati and Cohen 2007), such as miR-277,
was loaded onto the Slicer-deficient AGO1 mutants asefficiently as onto WT AGO1 (Supplemental Fig. 3A).
These results indicate that Slicer activity is not necessary,in most cases, for efficient miRNA loading in vivo. The
Dpiwi mutant, where the PIWI domain of AGO1 wasdeleted, did not interact with GW182 (Fig. 5C), as reported
previously (Behm-Ansmant et al. 2006). This mutant
interacted with DCR-1 but was not loaded with miRNA(Fig. 5C). It seems that the PAZ and Mid domains, which
are thought to be necessary for small RNA association(Farazi et al. 2008), are not sufficient to receive mature
miRNA upon processing by DCR-1. All these data indicatethat DCR-1 and GW182 require different domains of
AGO1 for their association and that active miRISC formation requires not only both the functional PAZ and MID

domains but also the existence of the Piwi domain even
without the slicing activity.
DISCUSSION

In this study, we determined that there are two distinct
types of AGO1-containing RNP complexes in Drosophila S2cells. One such complex, immunoprecipitated with antiLOQS, contained DCR-1, but no GW182 (Fig. 2A). This
complex contained little or no endogenous, mature miRNAand showed no target RNA cleavage activity in an endogenous miRNA-dependent manner (Fig. 2B,C). Pre-miRNAand miRNA/miRNA* duplexes were detected in the complex, but at negligible levels (Fig. 2B), suggesting that the
majority of AGO1 in the complex are ‘‘empty’’ and thus
ready to be loaded with mature miRNA. In fact, mature
miRNA processed by the complex from the precursor wasable to be loaded onto AGO1 that was originally containedin the complex (Fig. 3B). Earlier studies showed that theAgo2-Dicer-TRBP complex isolated from mammalian cells
is able to process pre-miRNAs to mature miRNAs and theresultant miRNAs are loaded onto Ago2, the homolog of
Drosophila AGO1, residing in the complex (Gregory et al.
2005; Maniataki and Mourelatos 2005). The Ago2-DicerTRBP complex was referred to as miRLC (Maniataki andMourelatos 2005). Thus, we consider the AGO1-DCR-1LOQS complex as miRLC in Drosophila.
In the other AGO1-containing complex, GW182 wasdetected, but DCR-1 and LOQS were absent (Fig. 2A).
Mature bantam existed abundantlyin the complex, but it excluded preFIGURE 3. The AGO1-DCR-1 complex exhibits pre-miRNA processing activity, but the AGO1- bantam and bantam* (Fig. 2B). The
GW182 does not. (A) Immunoprecipitated complexes (Fig. 2A) were subjected to in vitro pre- complex showed strong activity inmiRNA processing assays using pre-let-7 labeled with 32P. The AGO1-GW182 complex does notcleaving bantam target (Fig. 2C),
show the activity because the complex contains little or no DCR-1. (B) Immunoprecipitatedcomplexes (Fig. 2A) were first subjected to in vitro pre-miRNA processing using pre-let-7 (cold), although no pre-miRNA processing
and then target RNA cleavage assays were performed. The AGO1-GW182 complex, which did not activity was observed (Fig. 3A). Thisshow pre-let-7 processing activity in A, does not cleave the target (let-7 target; L7Pv), whereas the correlated well with the fact that
AGO1-DCR-1 complex shows the activity, indicating that the AGO1 that was originally containedin the complex was loaded with mature let-7 processed by the immunoprecipitated complex. (C) the AGO1-GW182 complex lacks
pre-let-7 processing was performed using let-7-130 as a target. After the reaction, the supernatant DCR-1. Thus, we consider this parand bead fractions were separated by centrifugation, and then target RNA cleavage assays were ticular complex as miRISC in Droperformed as in B. The fractions were also subjected to Western blotting using anti-DCR-1 and sophila. It should be noted thatanti-AGO1. AGO1 is detected both in the supernatant (S) and bead (B) fractions of anti-LOQS
immunoprecipitates, whereas DCR-1 is only detected in the bead fraction, indicating that AGO1 AGO1 and GW182 might only bedissociates from DCR-1 upon miRNA loading. Such dissociation was confirmed by performing the core components of miRISC, andtarget RNA cleavage assays. The cleavage products are observed in both the supernatant and bead other unknown proteins might also
fractions of anti-LOQS immunoprecipitates. The supernatant fraction of anti-AGO1 immuno- be involved. The determination of
precipitates shows no activity to cleave the target RNA, because AGO1 stayed on the beads during
the assays. The bead fraction of anti-LOQS shows stronger cleavage activity than expected. This such components is currently under
was because pre-miRNA processing continuously occurred even after the target RNA cleavage investigation in our laboratory.
reaction was started. The lowest panel shows that even under conditions where pre-let-7 was not
siRLC and siRISC were visualized
added, AGO1 was released to the supernatant, indicating that AGO1 displacement from DCR-1
inthe native gel systems by incubatoccurs in an miRNA-loading-independent manner. (D) GW182 and DCR-1 were depleted from32
S2 cells by RNAi, and RNAs isolated from individual cells were subjected to Northern blotting ing P-labeled siRNA duplex withusing a DNA oligo for bantam. When DCR-1 was depleted, pre-bantam was aberrantly Drosophila cell lysates (Pham et al.
accumulated, whereas GW182 depletion did not cause such an effect. The lower panel (Western 2004; Tomari et al. 2004b). In thisblot) shows the high efficiency of DCR-1 and GW182 depletion in each sample. (E) GW182 andstudy, we visualized miRLC and
DCR-1 are depleted from S2 cells as in D, and the resultant cell lysates were subjected to premiRNA processing assays. Only DCR-1 depletion causes a severe decrease in pre-miRNA miRISC on native agarose gels byprocessing activity.
incubating 32P-labeled pre-miRNA
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miRLC and miRISC in Drosophila

miR-2 induces dense complexes, heavier
than the 80S complex, termed pseudopolysomes, in Drosophila embryo lysates(Thermann and Hentze 2007). Pseudopolysomes were formed in the environment to which mRNA targets were
exogenously added, hence the large
apparent size. miRISC observed in ourstudy was rather small, which may be
due to the unavailability or accessibilityof the targets for let-7 in S2 lysates
utilized in the assays.
Upon miRNA loading, AGO1 wasdetected in a fraction separate from the
DCR-1-containing fraction (Fig. 3C),
indicating that the loading of mature
miRNA frees AGO1 from the miRLC, as
has been reported in mammalian systems (Maniataki et al. 2005). However,
the amounts of DCR-1 associated withWT AGO1 and the PAZ mutant wereapproximately equal (Fig. 5A), indicating that DCR-1 can be displaced from
FIGURE 4. Visualization of miRLC and miRISC on native agarose gels. (A) pre-let-7 was AGO1 independently of miRNA loadlabeled with 32P and incubated in S2 cell lysates in the presence of EDTA for 3 min and 30 min. ing. It is possible that the substitution of
After 30 min of incubation, Mg2+ was added to the reaction mixture to induce DCR-1 activity. amino acids may have caused considerFurther incubation was carried out as indicated. The resultant samples at each time point were able conformational changes in AGO1,
loaded onto an agarose gel, and complexes containing either pre-let-7 or mature let-7 werevisualized after running the gel. Simultaneously, siRNA duplex labeled with 32P was incubated and thus DCR-1 was then easily dissoin the S2 lysates in the presence of EDTA or Mg2+. After 30 min of incubation, both the lysates ciated; however, it also suggests thatwere loaded on the same gel system. The input lanes on the far left and right sides show pre- miRNA loading onto AGO1 might only
miRNA and siRNA duplexes by themselves, respectively. The bands with an asterisk and twopartially contribute to such displaceasterisks correspond to miRLC and miRISC, respectively. The bands corresponding to siRLC
and siRISC are as indicated on the gel. The band shown with a black triangle is an unknown ment and other systems might also
product, which did not appear in previous equivalent experiments (Miyoshi et al. 2005) but operate in vivo.
appeared in this study. (B) The bands indicated as miRLC and miRISC in A were isolated fromThe PAZ mutants of mammalian
the gel, and RNAs isolated were subjected to a native acrylamide gel. The miRLC contained
Ago2(Ago2-PAZ9 and Ago2-PAZ10)
pre-let-7, whereas miRISC contained mostly single-stranded let-7. (Input lane) Contains predid
notlocalize to P-bodies (Liu et al.
let-7 as the starting material. Pre-let-7 incubated in S2 lysates was also subjected to the same gel(S2 lysate + pre-let-7), which shows the migration patterns of pre-let-7, let-7/let-7*, and 2005). In contrast, the PAZ mutant of
mature let-7.
Drosophila AGO1 was associated with
GW182 and was localized to P-bodies asin the case of the WT (Fig. 5A), meanwith S2 lysates. It was found that both miRLC and miRISC
ing that, at least in Drosophila, GW182 does not discrimmigrated differently from siRLC and siRISC, respectively
inate between AGO1 with and without miRNA. Thus, it is(Fig. 4A). The migration distances between miRLC andspeculated that even WT AGO1 could be localized to thesiRLC were obvious, but not large. However, the situation
P-bodies if it encounters GW182 prior to miRNA loading.
observed for siRISC and miRISC clearly differs. siRISC is
However, P-bodies are not the place for the dead-ending of
known to be associated with ribosomes (Pham et al. 2004);
the AGO1 function. It is generally thought that once target
thus, it is massive and thus hardly runs on the native gels,
mRNAs are degraded in P-bodies, AGO1 is released fromeven under conditions in which the obvious target RNAs
P-bodies to the cytosol for subsequent silencing cycles. This
were absent (Fig. 4A). However, miRISC migrated much
step might displace GW182 from AGO1, although solid
faster, suggesting that miRISC does not seem to associate
evidence for this scenario is still missing. Such displacementwith polysomes, even under the same conditions. Althoughof GW182, upon release from P-bodies, might happen to
the Argonautes involved in miRISC and siRISC, AGO1 andAGO1 that was somehow accidentally localized to P-bodies
AGO2, are similar in size and sequence, their behavior
without being associated with miRNA, which provides
in terms of protein–protein interaction should be very
another chance for AGO1 to encounter DCR-1 and become
different from one another. It was previously reported that
activated.
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7

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Miyoshi et al.

strand would occur; therefore, unwinding
should not be mediated by Slicer. In fact,
Matranga et al. (2005) have previously
shown that strand rejection of miRNA* is
not accompanied by miRNA* strand
cleavage by hAgo. In contrast, in mammals, Slicer activity of Ago2 (the homologof Drosophila AGO1) was shown to be
involved in let-7 maturation (Diederichsand Haber 2007). It seems that the requirements of Argonaute-Slicer activity inmiRNA maturation in mammals and flies
are different from each other. ATP depletion from S2 cell lysates caused lessefficient miRNA maturation, althoughsuch treatment did not affect the siRNA
unwinding activity (MC Siomi, unpubl.).
Thus, we can speculate that there are yetto-be identified factor(s) that require ATPhydrolysis in the miRNA/miRNA* unwinding process, as opposed to the mammalian systems, where ATP is unnecessary
for miRNA maturation (Gregory et al.
2005).
MATERIALS AND METHODS
Plasmid constructs

FIGURE 5. The requirement for various domains of AGO1 in binding with miRNA, DCR-1,
and GW182. (A) Two mutants of AGO1, the PAZ and F2V2 mutants, were produced bymutagenesis and employed for binding assays with miRNA, DCR-1 and GW182. Proteinsassociated with Flag-AGO1-WT and Flag-AGO1-PAZ and Flag-AGO1-F2V2 mutants were
blotted with anti-DCR-1, anti-GW182, and anti-Flag antibodies. All interacted with DCR-1,
but the F2V2 mutant failed to associate with GW182, unlike the wild type (WT) and the PAZ
mutant. Northern blotting revealed that neither of the mutants associates with mature bantam.
(B) The F2V2 mutant (red), which lacks binding capacity to GW182, fails to accumulate in
P-bodies, whereas the PAZ mutant (red) co-localizes with DCP1 (green), a marker for
P-bodies. (C) The D-D-H motif in the PIWI domain was mutated to A-D-H or D-A-H. Bothmutants were expressed in S2 cells in a Flag-tagged form. Both mutants interacted with DCR-1
and GW182 and also mature single-stranded miRNA. In vitro target RNA cleavage assays showthat both mutants lack Slicer activity, indicating that Slicer activity of AGO1 is dispensable for
miRNA loading. The Dpiwi mutant interacts with DCR-1 but not with GW182, indicating thatthe domains required for binding with DCR-1 and GW182 are different.

The PIWI mutants of AGO1, D687A and D706A, showed no
Slicer activity, but mature miRNAs were loaded onto themutants as efficiently as onto WT AGO1 (Fig. 5C; Supplemental Fig. 3A). This means that the miRNA unwinding step
occurs, in most cases, in a Slicer-independent manner. It waspreviously shown that in the Drosophila RNAi system, the Slicer
activity of AGO2 largely functions in the siRNA unwinding
process (Matranga et al. 2005; Miyoshi et al. 2005; Rand et al.
2005). The miRNA/miRNA* duplex tends to contain a bulge inthe middle of the molecule, where the slicing of the passenger
8RNA, Vol. 15, No. 7

The Flag-AGO1 (WT) expression plasmid
was generated by inserting the coding regionof AGO1 from pGEX-AGO1-PB (Miyoshi
et al. 2005) into the pRH3xflag vector(Saito et al. 2005). The expression plasmids
for AGO1 mutants, Flag-AGO1-PAZ (F369A,
Y386A, F387A, Y391A), Flag-AGO1-F2V2(F560V, F595V), Flag-AGO1-D687A, andFlag-AGO1-D760A were produced by sitedirected mutagenesis using the QuikChange
Kit (Stratagene). The expression plasmid
for Flag-AGO1-Dpiwi (containing AGO1
peptide from the first methionine to P691;
D687 is altered to alanine) was obtained as aby-product during the construction of the
Flag-AGO1-D687A plasmid.

Immunoprecipitation, silver staining,and Western blotting
Monoclonal antibodies against GW182 were produced against
full-length GW182 (CG31992) tagged with GST. Anti-AGO1,
anti-Aub, and anti-Piwi antibodies were described previously(Miyoshi et al. 2005; Saito et al. 2006; Nishida et al. 2007).
Monoclonal antibodies against LOQS were produced against fulllength LOQS (CG6866) tagged with GST. Monoclonal antibodies
against DCR-1 were produced against the N terminus of DCR-1(750 amino acids). Monoclonal antibodies against DCR-2 were

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miRLC and miRISC in Drosophila

produced against the C terminus of DCR-2 (680 amino acids)
tagged with GST. Immunoprecipitation from S2 cell lysates orovary lysates were performed as described previously (Miyoshi
et al. 2005). After immunoprecipitation, the beads were washedextensively with a buffer containing 30 mM HEPES (pH 7.4), 150
mM potassium acetate, 5 mM magnesium acetate, 5 mM DTT,
0.1% NP-40, 2 mg/mL pepstatin, 2 mg/mL leupeptin, and 0.5%
aprotinin. The immunoprecipitated proteins were eluted with
SDS sample buffer without DTT. After elution, DTT was added tothe eluates to 100 mM. The eluates were then boiled and loaded
onto SDS-acrylamide gels. After electrophoresis, protein bands
were visualized using Silver Quest (Invitrogen). Western blotting
was performed as described previously (Miyoshi et al. 2005).

Target RNA cleavage assay
The reactions were performed as previously described (Miyoshi
et al. 2008). To make a let-7 target RNA (L7Pv in Fig. 3B), pBSlet-7-L7Pv was digested with PvuII, and in vitro transcription was
performed using MEGAscript T3 (Ambion). To make a shorter
let-7 target RNA (let-7-130 in Fig. 3C), a PCR fragment was
amplified with T7 and T3 primers from pBS-let-7-130 and in vitrotranscription was performed using MEGAscript T7 (Ambion).
The pBS-let-7-130 and pBS-let-7-L7Pv plasmids were constructed
as follows. pBS-let-7-130, a KpnI-EcoRI fragment containing asequence completely complementary to mature let-7 was produced by annealing a set of DNA oligonucleotides (59-CACTATACAACCTACTACCTCAG-39 and 59-AATTCTGAGGTAGTAGGTTGTATAGTGGTAC-39) and inserting the resultant DNA fragment into pBluescript digested with KpnI and EcoRI. pBS-let-7L7Pv; a KpnI-EcoRI fragment was produced by annealing two
DNA oligonucleotides (59-CTGAGGTAGTAGGTTGTATAGTG-39
and 59-AATTCACTATACAACCTACTACCTCAGGTAC-39) andinserted into pBluescript digested with KpnI and EcoRI. To makemiR-bantam target RNA (bantam-130), a PCR fragment was
amplified with T7 and T3 primers from pBS-miR-ban-target(Miyoshi et al. 2005), and in vitro transcription was performed
using MEGAscript T7. All target RNAs were 32P-labeled at the
59-G cap by guanylyltransferase (Ambion) as previously described(Miyoshi et al. 2005).

Northern blot analysis
Northern blot analysis was performed as described previously(Miyoshi et al. 2005). The probe used for detecting miR-bantam*
was 59-GTCAAACCAAATCGAAAACC-39.

RNAi
RNAi in S2 cells was performed as described previously (Saito et al.
2005). dsRNA utilized for GW182 depletion corresponded tonucleotides 1–698 of the GW182 coding sequence. The dsRNAs usedfor DCR-1 and EGFP were described previously (Saito et al. 2005).

Immunofluorescence
Immunofluorescence was performed by fixing S2 cells with 2%
formaldehyde for 15 min. Cells were permeabilized using 0.1%
Triton X-100. To visualize Flag-tagged AGO1 proteins, cells were
stained with anti-Flag M2 antibody (Sigma). Alexa-546 antimouse IgG was used as a secondary antibody. After extensive
washing, the cells were further stained with anti-DCP1 antibody
coupled to fluorescein using a Fluorescein Labeling kit-NH2(Dojindo). Monoclonal antibodies against DCP1 were produced
against full-length DCP1 (CG11183) tagged with GST. Cells werealso mounted with SlowFade Gold Antifade Reagents with DAPI
(Invitrogen). All images were collected using a Zeiss LSM510 laser
scanning microscope.

Native gel electrophoresis
Native gel electrophoresis was performed as described previously(Miyoshi et al. 2005). 32P-labeled pre-let-7 or luc siRNA duplex
was first incubated in S2 lysates in the presence of 5 mM EDTA at
26°C, and then 30 mM magnesium acetate was added to the
mixture. After incubation, samples were adjusted to 6% (w/v)
glycerol and resolved by submarine native 1.5% agarose gelelectrophoresis. The RNP bands were visualized by exposure to
an imaging plate of a BAS 2500. To purify RNAs from the RNP
complexes, corresponding bands were excised from the gels andRNAs were purified using SuprecEZ (Takara). Purified RNAs weresolubilized in loading buffer (5 mM HEPES-KOH at pH 7.4,
5 mM EDTA, 5% glycerol, and 0.5% SDS) and resolved by 15%
native acrylamide gel electrophoresis. As a control, the reaction
mixture was incubated in the presence of magnesium acetate.
After reaction, the sample was mixed with the same volume of thestop solution (5 mM HEPES-KOH at pH 7.4, 5 mM EDTA, 5%
glycerol, 0.5% SDS, and 0.5 mg/mL proteinase K) and incubatedfor 10 min 37°C before loading.

SUPPLEMENTAL MATERIAL
Supplemental material can be found at http://www.rnajournal.org.

ACKNOWLEDGMENTS
We thank K. Saito, K.M. Nishida, T. Sunohara, and A. Ishizuka fortechnical assistance, and other members of the Siomi laboratoryfor discussions and comments on the manuscript. This work was
supported by MEXT grants to H.S., the New Energy and Industrial
Technology Development Organization (NEDO) grants to M.C.S.,
and JSPS Research Fellowships for Young Scientists to K.M. M.C.S.
is supported by CREST from JST. H.S. is a member of the Genome
Network Project (MEXT). M.C.S. is Associate Professor of Global
COE for Human Metabolomics Systems Biology by MEXT.
Received January 4, 2009; accepted April 14, 2009.

Pre-miRNA processing assay
The pre-miRNA processing assays were performed as described
previously (Saito et al. 2005). Pre-let7 (UGAGGUAGUAGGUUGUAUAGUAGUAAUUACACAUCAUACUAUACAAUGUGCUAGCUUUCU; Dharmacon) was labeled with 32P at the 59-end andself-annealed.

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