Molecular Cell

ArticleRoles of R2D2, a Cytoplasmic D2 Body Component,in the Endogenous siRNA Pathway in Drosophila
Kazumichi M. Nishida,1,4 Keita Miyoshi,1,4,5 Akiyo Ogino,1,2,4 Tomohiro Miyoshi,1,6 Haruhiko Siomi,1and Mikiko C. Siomi1,3,7,*
1Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan
2Graduate School of Environment and Information Science, Yokohama National University, Yokohama 240-8501, Japan
3JST, CREST, Saitama 332-0012, Japan
4These authors contributed equally to this work
5Present address: School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA
6Present address: Center for Transdisciplinary Research, Niigata University, Niigata 950-2181, Japan
7Present address: Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan*Correspondence: siomim@biochem.s.u-tokyo.ac.jp
http://dx.doi.org/10.1016/j.molcel.2012.12.024

SUMMARY

Endogenous small interfering RNAs (endo-siRNAs) in
Drosophila are processed by Dicer-2 (Dcr-2) andloaded onto Ago2 by the Dcr-2/R2D2 heterodimer.
In r2d2 mutants, the level of endo-siRNAs is unchanged, but endo-siRNAs are misloaded onto
Ago1. However, the mechanism underlying thecontrol of endo-siRNA sorting by R2D2 remains
unknown. Here, we show that R2D2 controls endosiRNA sorting by localizing Dcr-2, and presumably
endo-siRNA duplexes, to cytoplasmic foci, D2
bodies. Ago2, but not Ago1, localized to D2 bodies.
dsRNA-binding-deficient mutant, but not wild-type,R2D2 failed to localize D2 bodies and caused endosiRNA misdirection to Ago1 in R2D2-depleted cells.
However, R2D2 was dispensable for sorting miRNAs
and exogenous siRNAs onto Ago1 and Ago2, respectively, in vivo. Endo- and exo-siRNA guide selection
also occurred R2D2 independently. The functionsof R2D2 are required to avoid endo-siRNA misdirection to Ago1, because Ago1 is capable of loading
incompletely complementary miRNA duplexes andendo-siRNA duplexes.

INTRODUCTIONIn RNA silencing, a small noncoding RNA associates with an
Argonaute protein, the effector protein in the RNA silencing
machinery, to form the RNA-induced silencing complex (RISC)
and guides the RISC to their target RNAs to regulate cellulargene networks and physiological processes in living organisms(Siomi and Siomi, 2009). Animals express three types of endogenous small RNAs: microRNAs (miRNAs), PIWI-interacting RNAs
(piRNAs), and endogenous small interfering RNAs (endosiRNAs) (Ghildiyal and Zamore, 2009; Kim et al., 2009; Siomi
et al., 2011). These small RNAs can be classified based on their
origins, processing factors, and Argonaute binding partners(Ghildiyal and Zamore, 2009; Kim et al., 2009; Czech and Hannon, 2011).
miRNAs are processed from their primary precursors by two
RNase III enzymes, Drosha and Dicer, in tandem, in the nucleus
and the cytoplasm, respectively (Denli et al., 2004; Gregory et al.,
2004; Lee et al., 2003). Drosophila possesses two Dicer genes,
Dicer-1 (Dcr-1) and Dicer-2 (Dcr-2), and miRNA processing
requires Dcr-1, but not Dcr-2 (Lee et al., 2004). Upon maturation
following Dcr-1 processing, miRNAs in Drosophila are principally
associated with Argonaute 1 (Ago1) to form the miRISC (Miyoshi
et al., 2009; Okamura et al., 2004).
Like piRNAs, endo-siRNAs originate from intergenic DNA elements. However, in contrast to piRNA biogenesis, which occurs
Dicer independently, endo-siRNA biogenesis in Drosophila
requires Dcr-2 (Vagin et al., 2006; Czech et al., 2008; Ghildiyal
et al., 2008; Kawamura et al., 2008; Okamura et al., 2008).
Dcr-2 was originally identified as an enzyme responsible forprocessing exogenous siRNAs (exo-siRNAs) from long doublestranded RNA (dsRNA) precursors such as viral genomes (Bernstein et al., 2001; Lee et al., 2004; Liu et al., 2003). Therefore, it ismost likely that the endo-siRNA precursors are double-stranded
RNAs. After maturation, endo-siRNAs are loaded onto Ago2 toform the endo-siRISC, which silences transposons and some
protein-coding genes (Czech et al., 2008; Ghildiyal et al., 2008;
Kawamura et al., 2008; Okamura et al., 2008).
RNase III enzymes associate with dsRNA-binding proteins(dsRBPs) during small RNA biogenesis. Drosophila Dcr-1 associates with Loquacious (Loqs; also known as R3D1) (Förstemann et al., 2005; Jiang et al., 2005; Saito et al., 2005). The
Loqs gene gives rise to four isoforms, Loqs-PA, Loqs-PB,
Loqs-PC, and Loqs-PD (Hartig et al., 2009). The longest isoform, Loqs-PB, facilitates miRNA processing by Dcr-1 (Hartig
et al., 2009; Marques et al., 2010; Miyoshi et al., 2010; Zhou
et al., 2009). Mutations in Loqs-PD cause a severe reductionin the accumulation of endo-siRNAs in vivo, but the level ofexpression of Dcr-2 is unchanged by Loqs-PD depletion, suggesting a direct role of Loqs-PD in endo-siRNA processing(Hartig et al., 2009; Marques et al., 2010; Miyoshi et al., 2010;
Zhou et al., 2009).
Another dsRBP, R2D2, associates with Dcr-2 but is dispensable for exo-siRNA excision from precursors in vitro (Liu et al.,

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2003). However, the Dcr-2/R2D2 heterodimer constituting the
RISC-loading complex (RLC) enhances exo-siRNA loading onto
Ago2 (Liu et al., 2003, 2006; Pham et al., 2004; Tomari et al.,2004, 2007). In the loading phase, the RLC senses the thermodynamic asymmetry of both ends of exo-siRNA duplexes (Khvorova et al., 2003; Liu et al., 2003; Schwarz et al., 2003; Tomari
et al., 2004, 2007). The exo-siRNA with the less stable 50 end isselected and preferentially incorporated into the RISC. The otherstrand is eventually degraded in the cellular environment.
R2D2 is unnecessary for endo-siRNA accumulation (Czech
et al., 2008; Ghildiyal et al., 2008; Marques et al., 2010; Okamura
et al., 2008; Zhou et al., 2009). However, endo-siRNAs, whichare 20 -O-methylated at the 30 end, became sensitive to b-elimination in r2d2 mutants (Ghildiyal et al., 2008). b-elimination
sensitivity is the hallmark of miRNAs associated with Ago1 in
Drosophila, suggesting that endo-siRNAs would be misloaded
onto Ago1 without R2D2 expression. Indeed, endo-siRNAs
were misdirected to Ago1 in cells lacking R2D2 (Ameres et al.,2011; Marques et al., 2010; Okamura et al., 2011). Loqs andR2D2 act sequentially in exo-/endo-siRNA processing andloading, respectively (Marques et al., 2010). These observations
support the idea that R2D2, which is dispensable for endosiRNA processing, plays a crucial role(s) in endo-siRNA sorting
and loading. However, the underlying mechanism remains
unknown.
In this study, we found that endogenous Dcr-2 and R2D2
localize to cytoplasmic foci in Drosophila cells. Chemicals
affecting cytoplasmic structures, processing bodies (P bodies)and stress granules (SGs), had little or no effects on the Dcr-2positive foci in Schneider 2 (S2) cells; thus, the bodies were
designated D2 bodies. Both Dcr-2 and R2D2 are necessary forD2 body formation, but in distinct ways: Dcr-2 stabilizes R2D2,
whereas R2D2 localizes Dcr-2 to D2 bodies. Overexpression of
R2D2 restores dotty structures similar to D2 bodies in Dcr-2depleted cells; thus, R2D2 is likely critical for D2 body formation.
endo-siRNAs, but not exo-siRNAs, were misdirected to Ago1 in
R2D2-depleted S2 cells, where the guide strands of both exoand endo-siRNA duplexes were selectively loaded onto Ago2;
thus, R2D2 is dispensable for guide strand selection and
Ago2-specific loading of exo-siRNAs. Wild-type (WT) R2D2,
but not dsRNA-binding-defective mutant R2D2, rescued thedefects in D2 body formation and endo-siRNA sorting onto
Ago2 caused by loss of R2D2. These results suggest that
R2D2 ensures endo-siRNA sorting by localizing Dcr-2, andpresumably endo-siRNA duplexes, to D2 bodies, where Ago2,
but not Ago1, is localized. R2D2 functions are required to avoid
endo-siRNA misdirection to Ago1 because Ago1 is capable forloading incompletely complementary miRNA duplexes andendo-siRNA duplexes.
RESULTS
Dcr-2 Localizes to Cytoplasmic D2 Bodies
Immunostaining of S2 cells with anti-Dcr-2 antibodies (Miyoshi
et al., 2010) revealed that endogenous Dcr-2 accumulates intocytoplasmic foci (Figure 1A). To determine if the dotty signals
reflect the Dcr-2 localization per se, we immunostained Dcr-2depleted S2 cells. While a control siRNA against EGFP did notaffect the Dcr-2-positive foci (Figure 1B; EGFP RNAi), depletionof Dcr-2 caused disappearance of these bodies (Figure 1B;
Dcr-2 RNAi), confirming that Dcr-2 accumulates in cytoplasmic
bodies.
The Dcr-2 punctate signals looked similar to those ofwell-characterized cytoplasmic structures, P bodies and SGs(Eulalio et al., 2007a). To examine the relationship between
the Dcr-2-positive foci and P bodies, S2 cells were double
stained with anti-Dcr-2 and anti-Dcp1 antibodies (Miyoshi
et al., 2009). Dcp1 is a component of an mRNA-decapping
complex and a marker of P bodies (Eulalio et al., 2007a). The
Dcr-2-positive foci partially overlapped with P bodies (Figure 1C). Treatment of S2 cells with two chemicals, cycloheximide that stalls ribosomes on mRNAs and puromycin thatdisassembles ribosomes from translating mRNAs, caused fragmentation and enlargement of P bodies, respectively (Figure 1C), as has been reported previously (Eulalio et al.,2007a, 2007b). Two other components of P bodies, Trailer
hitch (Tral) and Me31B (Barbee et al., 2006), behaved similarly
to Dcp1 (see Figure S1A online). In contrast, Dcr-2-positive
structures were not affected by the chemical treatments (Figure 1C and Figure S1A). Depletion of Dcr-2 did not affectDcp1 localization in S2 cells (Figure S1B). Thus, the Dcr-2positive bodies are distinct from P bodies.
Cellular stresses, such as increased temperature, a highly
oxidative environment, and UV irradiation, cause bulk mRNAs
to be accumulated into SGs and inhibit their translation (Farny
et al., 2009; Kedersha et al., 2005). SGs are formed in very close
proximity to P bodies (Kedersha et al., 2005; Farny et al., 2009),
raising the possibility that the Dcr-2-positive foci might correspond to SGs. To examine this possibility, S2 cells were immunostained with anti-Dcr-2 and anti-dFMR1 antibodies. dFMR1
is a KH-domain-containing RNA-binding protein encoded bydFmr1, a fly ortholog of the fragile X mental retardation gene 1(Fmr1) in mammals (Wan et al., 2000). dFMR1 strongly accumulates in SGs under stress conditions and thus can be used asa SG marker (Farny et al., 2009). Under nonstress conditions,weak dFMR1 signals overlapped with Dcr-2 signals (Figure 1D).
Arsenite treatment leading to an increase in the number of
SGs and their enlargement (Farny et al., 2009) drasticallychanged the dFMR1 staining patterns, but not the Dcr-2 signals(Figure 1D), indicating that the Dcr-2-positive foci are not SGs.
The punctuate Dcr-2 signals did not overlap with those fordGM130, a protein that is strongly attached to Golgi membranes(Figure 1E) (Kondylis et al., 2001; Yano et al., 2005). We designate them D2 bodies.
R2D2, a Component of D2 Bodies, Is Necessaryfor D2 Body Formation
R2D2 tightly associates with Dcr-2 (Liu et al., 2003; Miyoshi et al.,2010); thus, we postulated that R2D2 might also localize to D2
bodies. Immunofluorescence using anti-R2D2 (Figure S2A) andanti-Dcr-2 antibodies revealed that R2D2 colocalizes with
Dcr-2 to D2 bodies (Figure 2A and Figure S2B). Foci similar toD2 bodies were observed with both antibodies in ovarianfollicle cells of a fly line overexpressing Dcr-2, dcr-225708 (Figure
S2B). Nurse cells and oocytes showed no dotty structures
corresponding to D2 bodies (Figure S2C). It remains unknown

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Figure 1. Dcr-2 Localizes to Cytoplasmic Granules, D2 Bodies, in Drosophila S2 Cells(A) S2 cells immunostained with an anti-Dcr-2 antibody show strong foci exclusively located in the cytoplasm. Nuclei are stained with DAPI (blue).
Scale bars, 5 mm.
(B) The punctate signals in (A) disappeared when Dcr-2 was depleted by RNAi (Dcr-2 RNAi). EGFP dsRNA, used as a control, had little or no effect on Dcr-2 signals(EGFP RNAi). Scale bars, 5 mm.
(C) Cycloheximide and puromycin treatments cause changes in the shape and size of P bodies (Dcp1,) but not those of the Dcr-2-positive foci. The inset (topright), an enlarged image of the structure indicated by a white arrowhead, clearly shows that the signals for Dcr-2 and Dcp1 partially overlap. Nuclei are stainedwith DAPI (blue). Scale bars, 5 mm.
(D) Arsenite treatment enlarges SGs (dFMR1) but not Dcr-2-positive foci. Nuclei are stained with DAPI (blue). Scale bars, 5 mm.
(E) The Dcr-2-positive foci do not overlap with Golgi structures (dGM130). Nuclei are stained with DAPI (blue). Scale bars, 5 mm. See also Figure S1.

if nurse cells and oocytes express endo-siRNAs. Thus, R2D2
colocalization with Dcr-2 is not a specific event in cultured
S2 cells.
To examine the mutual dependency of Dcr-2 and R2D2 in D2
body localization, both proteins were respectively depleted from
S2 cells by RNAi. R2D2 depletion caused disappearance of D2
bodies (Figure 2A). The expression level of Dcr-2 was littlechanged by R2D2 depletion (Figure 2A). This is consistent withthe previous finding that the level of Dcr-2 is maintained in
r2d2 null mutant flies (Liu et al., 2006). These results suggestthat Dcr-2 localization to D2 bodies depends on the presenceof R2D2 in Drosophila cells.
Depletion of Dcr-2 also induced loss of D2 bodies (Figures 1Band 2B and Figure S2D). Western blotting revealed that Dcr-2
RNAi greatly reduced the levels of not only Dcr-2 but also

R2D2 (Figure 2B). This also confirmed the previous finding that
Dcr-2 stabilizes R2D2 in vivo (Liu et al., 2006). The R2D2 dotty
signals were hardly detected in Dcr-2-depleted cells, as expected (Figure 2B and Figure S2D). Thus, D2 body formation
requires both Dcr-2 and R2D2, but in distinct ways; Dcr-2 isrequired for stabilizing R2D2, while R2D2 is required for localizing Dcr-2 to D2 bodies.
We then overexpressed Flag-tagged R2D2 in Dcr-2-depleted
S2 cells. Western blotting confirmed that the expression of FlagR2D2 was maintained in Dcr-2-depleted cells, in which Dcr-2
silencing was still effective (Figure 2C). Flag-R2D2 accumulated
in cytoplasmic foci without Dcr-2 expression, demonstrating that
R2D2 has the ability to form the dotty structures even in theabsence of Dcr-2 (Figure 2C and Figure S2E). Thus, R2D2 might
be a core component of D2 bodies.

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Merge

Figure 2. Formation of D2 Bodies Requires
Both R2D2 and Dcr-2

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EGFP RNAiC(A) (Left) RNAi for R2D2, but not EGFP, causes
disappearance of D2 bodies in S2 cells. Nuclei arestained with DAPI (blue). Scale bars, 5 mm. (Right)
Western blotting shows that R2D2 RNAi downregulates R2D2 greatly but does not alter the
expression level of Dcr-2. Tubulin was detected as
an internal control.
(B) (Left) RNAi for Dcr-2, but not EGFP, causes
disappearance of D2 bodies in S2 cells. Scalebars, 5 mm. (Right) Dcr-2 RNAi downregulates not
only Dcr-2 but also R2D2. Dcr-2 is necessary forstabilizing R2D2.
(C) (Left) Flag-R2D2 overexpressed in Dcr-2depleted cells accumulates in cytoplasmic focisimilar to D2 bodies. Scale bars, 5 mm. (Right)
Western blotting shows that Flag-R2D2 is expressed in S2 cells where Dcr-2 is still downregulated.
(D) Western blotting of total S2 lysates using ananti-R2D2 antibody shows that expression of mycR2D2 (red arrowhead), but not that of myc-EGFP,
causes a severe decrease in the level of endogenous R2D2 (blue arrowheads). Tubulin was detected as an internal loading control. See also
Figure S2.

Dcr-2

Dcr-2 RNAi

R2D2
Flag-R2D2

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myc-EGFP

Tubulinmyc-R2D2

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In the course of our experiments, we noticed that overexpression of myc-tagged R2D2 in S2 cells resulted in a severe
reduction in the level of endogenous R2D2 (Figure 2D). The
number of D2 bodies looked the same before and after mycR2D2 and Flag-R2D2 overexpression (Figure 2C and Figure S2F). The simplest scenario to explain these observations
is that myc-R2D2 substituted for endogenous R2D2 in D2
bodies and that endogenous R2D2 that had been displaced
from D2 bodies was degraded in the cytosol via an unknown
mechanism. These results suggest that D2 bodies are likely
composed of Dcr-2 and R2D2 in a stoichiometric manner.
Quantification of the Dcr-2 and R2D2 signals in S2 cells revealed that Dcr-2 and R2D2 in D2 bodies make up 8.6%
and 5.5%, respectively, of the total amounts of Dcr-2 andR2D2 per cell, respectively (Figure S2G). Loss of Dcr-2 destabilizes R2D2 (Figure 2B). Thus, R2D2 may interact with Dcr-2
even outside of D2 bodies.
Depletion of Dcr-1, Ago2, and all Loqs isoforms did not affectappearance of D2 bodies (Figure S2H), indicating that thesefactors, including Loqs-PD, are dispensable for D2 bodyformation. myc-Loqs-PD expressed via a transgene did not
specifically localize to D2 bodies (Miyoshi et al., 2010). Thus,
Loqs-PD may not be a constituent of D2 bodies, unlike R2D2.
Association with R2D2 Is Necessary for Dcr-2 toLocalize to D2 Bodies
To determine which region of Dcr-2 is required for its localization
to D2 bodies, Dcr-2 was divided in half to yield two deletionmutants, N950 (residues 1–950) and C951 (residues 951–
1,722) (Figure 3A). Expression of the deletion mutants in S2 cells
revealed that N950, but not C951, localized to D2 bodies, as did
WT Dcr-2 (Figure 3B and Figure S3A). N950 coimmunoprecipitated with R2D2, but C951 failed to bind R2D2 (Figure 3C).
Thus, the D2 body localization of Dcr-2 may coincide with
Dcr-2’s interaction with R2D2.
The N950 mutant was further delineated to produce three
additional mutants, N694 (residues 1–694), N572 (residues 1–572), and N207 (residues 1–207) (Figure 3A). Both N694 andN572 weakly localized to D2 bodies, whereas N207 did not (Figure 3D and Figure S3B). N694 and N572 interacted with R2D2 toa lesser extent than did WT Dcr-2 (Figure 3E). N207 failed to bind
R2D2 under the experimental conditions (Figure 3E). These findings are consistent with the recent finding that Dcr-2 lacking the
N-terminal region (residues 1–551) does not associate with R2D2
(Hartig and Förstemann, 2011). The findings also suggest that
the N-terminal region of Dcr-2 containing the HELN and HELC(RNA helicase) domains maintains its association with R2D2
and localizes to D2 bodies. Dcr-2 localization to D2 bodies
indeed coincides with Dcr-2’s interaction with R2D2. D2 body
localization and R2D2 binding aspects of Dcr-2 WT and mutants
are summarized in Figure 3A.
The dsRNA-Binding Activity and the Auxiliary Domainof R2D2 Are Required for D2 Body Formation
We next examined which part of R2D2 is required for D2 bodyformation. R2D2 WT and two deletion mutants, R1R2 (residues
1–166 consisting of two dsRBD domains) and Aux (residues
167–311 containing the auxiliary domain) (Figure 4A), were ex-pressed in S2 cells, and immunofluorescence was performed.
myc-tagged full-length R2D2 WT localized to D2 bodies, butneither myc-R1R2 nor myc-Aux did (Figure 4B and Figure S4).
Aux was then extended toward the N-terminal end to produce
another mutant, R2Aux, to cover the second dsRNA-binding
domain of R2D2 (dsRBD2) (residues 82–311) (Figure 4A). Thismutant R2Aux accumulated to D2 bodies, as did R2D2 WT (Figure 4B and Figure S4), suggesting that both dsRBD2 and theauxiliary domains are necessary for R2D2 to form D2 bodies.
We then constructed another mutant, R2Aux-M, in which two
residues highly conserved in dsRBDs, A153 and A154, were
substituted with Glu and Asp, respectively (Figure 4A) (Liu et al.,2006; also see the Experimental Procedures). This mutation abolishes the dsRNA-binding activity of R2D2 (Liu et al., 2006).
R2Aux-M did not accumulate to D2 bodies (Figure 4B and Figure S4). Immunoprecipitation experiments showed that WT
R2D2, R2Aux, and R2Aux-M bound Dcr-2 to a similar extent (Figure 4C), while the level of Dcr-2 associated with R1R2 was near
the background level. Aux associated with Dcr-2, but not asstrongly as did WT R2D2 (Figure 4C). Thus, dsRBD2 stabilizes
the association between Dcr-2 and R2D2, but the dsRNA-binding
activity of dsRBD2 is not necessary for maintaining their association. The dsRNA-binding activity of dsRBD2 was required for
R2D2 to localize to D2 bodies (Figure 4B and Figure S4). Previous
studies have shown that the dsRNA-binding activity of R2D2 isrequired for Dcr-2 to efficiently bind siRNA duplexes (Liu et al.,2003, 2006). Thus, D2 body localization of the Dcr-2-R2D2
heterodimer might depend on the dsRNA-binding status of theheterodimer. D2 body localization and Dcr-2 binding aspects of
R2D2 WT and mutants are summarized in Figure 4A.
R2D2 Dependency of Endo-siRNA- andExo-siRNA-Specific Sorting onto Ago2 In Vivo
In r2d2 mutants, very few endo-siRNAs are loaded onto Ago2,
with most being misdirected to Ago1 (Okamura et al., 2011).
We sought to examine if this phenomenon would be recapitulated in S2 cells. R2D2 was depleted from S2 cells by RNAi,and then Flag-Ago1 and Flag-Ago2 were expressed individually.
Northern blotting was performed on RNAs isolated from the
Flag-Ago1 and Flag-Ago2 complexes immunoprecipitatedusing an anti-Flag antibody (Figure S5A). We found that esiRsl-1, an endo-siRNA expressed in S2 cells (Kawamura et al.,2008), was loaded onto Ago2 to a similar extent to that seen in
luciferase (luc) RNAi-treated cells, suggesting that R2D2 isdispensable for endo-siRNA loading onto Ago2 in S2 cells (Figure 5A). D2 bodies are not formed without R2D2 expression (Figure 2A), indicating that endo-siRNA loading onto Ago2 occurs D2body independently in the cytosol.
Endo-siRNAs were aberrantly loaded onto Ago1 when R2D2
was lacking in S2 cells (Figure 5A). Thus, R2D2 is required forpreventing endo-siRNA misdirection onto Ago1 in S2 cells. The
distribution of miR-bantam, an miRNA expressed in S2 cells,
between Ago1 and Ago2 was hardly affected in R2D2-lacking
cells (Figure 5A); thus, Ago1 favors miRNAs even under aberrant
conditions in which endo-siRNAs are also available for Ago1 tobind. The total level of esiR-sl-1 was higher (3-fold) in R2D2depleted S2 cells than in control cells (Figure 5A and Figure S5B).
R2D2 depletion led to D2 body disruption, and Dcr-2 alone was

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A
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R2D2 binding

WT
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694
572

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Figure 3. Dcr-2 N Terminus Interacts with R2D2 and Localizes to D2 Bodies(A) The domain structure of Dcr-2 WT and deletion mutants. D2 body localization and R2D2 binding of Dcr-2 WT and mutants are summarized on the right.
(B) A Dcr-2 deletion mutant, N950, consisting of the N-terminal half of Dcr-2, localizes to D2 bodies, as does Dcr-2 WT. By contrast, the C-terminal half of Dcr-2,
C951, fails to localize to D2 bodies. Dcr-2 WT, N950, and C951 were tagged with a 3xFlag peptide (green). Endogenous R2D2 is detected by anti-R2D2 antibody(magenta). Nuclei are stained with DAPI (blue). Scale bars, 5 mm.
(legend continued on next page)

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released into the cytosol (Figure 2A). Dcr-2 floating in the cytosolmight have enhanced endo-siRNA processing, resulting ina higher level of endo-siRNAs in the cells. Northern blottingwith a DNA probe detecting esiR-sl-1* (the antisense strandof esiR-sl-1) failed to detect esiR-sl-1* in Ago1 and Ago2
complexes (Figure S5C), suggesting that strand selection ofendo-siRNAs can still be maintained without R2D2. The factors
controlling strand selection in the absence of R2D2 remain to
be determined.
We set out to determine if exo-siRNAs are loaded onto Ago2
R2D2 independently. R2D2-depleted S2 cells were transfectedwith exo-siRNA along with a vector for expressing Flag-Ago1 or
Flag-Ago2. Northern blotting was performed on total RNAs in
the immunoprecipitated Flag-Ago1 and Flag-Ago2 complexes
using two probes recognizing the guide and passenger strands,
individually. Both strands were detected in immunoprecipitatesobtained from R2D2-depleted S2 cells expressing Flag-Ago2,
but not in those obtained from cells expressing Flag-Ago1, in
R2D2-depleted cells, suggesting that exo-siRNAs are loaded
specifically onto Ago2 R2D2 independently (Figure 5B and Figure S5D). Depletion of Dcr-2, which destabilizes both Dcr-2 andR2D2 (Figure 2B), did not affect exo-RNA loading onto Ago2
(Figure 5B and Figure S5D). Thus, both exo-siRNA sorting
onto Ago2 and miRNA sorting onto Ago1 occur R2D2/Dcr-2
independently. The guide strand, but not the passenger strand,of exo-siRNA duplex was loaded onto Ago2 (Figure S5E). Thus,
guide strand selection of siRNAs occurs R2D2 independently.
exo-siRNA duplexes are fully complementary, while endosiRNA duplexes show mostly incomplete complementarity(Czech et al., 2008; Ghildiyal et al., 2008; Kawamura et al.,2008; Okamura et al., 2008). This may be the cause of thedifference between exo-siRNAs and endo-siRNAs in terms of
their R2D2 dependency of Argonaute sorting. It has beenshown that small RNA duplexes with full complementarity
were loaded onto Ago2, while those with incomplete complementarity were loaded onto Ago1 in in vitro assays (Tomari
et al., 2007).
The Ability of R2D2 to Localize Dcr-2 to D2 Bodies
Is Required for Endo-siRNA Sorting onto Ago2
We then examined if expression of RNAi-resistant R2D2 is able
to rescue the R2D2 defective phenotype in S2 cells. Endogenous
R2D2 was knocked down by a specific R2D2-siRNA, and thenRNAi-resistant R2D2 WT (R2D2 WT-R) was expressed (Figure S5F). As expected, R2D2 depletion caused endogenous
Dcr-2 to become dispersed in the cytosol (Figure 5C and Figure S6A). Expression of R2D2 WT-R in R2D2-depleted cells
restored D2 bodies (Figure 5C and Figure S6A). Northern blottingof total RNAs isolated from Flag-Ago1 and Flag-Ago2 complexes
showed that R2D2 WT-R was able to restore specific endosiRNA loading onto Ago2 (Figure 5D).

Immunofluorescence analyses showed that expression ofR2Aux in R2D2-depleted cells was able to restore D2 bodyformation, but that expression of R2Aux-M was not (Figure 5C).
We then asked if these mutants are able to rescue misloading ofendo-siRNA onto Ago1 in R2D2-depleted cells. R2Aux, but not
R2Aux-M, rescued the defective phenotype (Figure 5E and Figure S6B). These findings suggest that Dcr-2 localization to D2
bodies is directly linked to prevention of endo-siRNA misloading
onto Ago1.
Ago1, which forms a complex with miRNAs and their target
mRNAs, accumulates in P bodies, where the miRNA target is
eventually degraded (Behm-Ansmant et al., 2006). By immunofluorescence, Flag-Ago1 showed a similar distribution to Dcp1,
Tral, and Me31B in S2 cells with and without cycloheximide
and puromycin treatments (Figure 1B, Figures S1A and S6C).
Thus, Ago1 is not a component of D2 bodies. Flag-Ago2 in S2
cells was mostly localized in the cytosol (Figure S6D). However,Flag-Ago2 was also found in D2 bodies, and these signals wereresistant to cycloheximide treatment (Figure S6E). Ago2 might
localize to D2 bodies in S2 cells, albeit transiently.
DISCUSSION
In this study, we show that R2D2 localizes Dcr-2, presumably
together with endo-siRNA duplexes, to D2 bodies to ensure
endo-siRNA-specific loading onto Ago2 (and to prevent endosiRNA misloading onto Ago1) in S2 cells (Figure 6A). Depletionof R2D2 causes D2 bodies to collapse. Under this circumstance,
endo-siRNAs can still be loaded onto Ago2; however, concomitantly, Ago1 starts misloading endo-siRNAs in the cytosol (Figure 6B). This is because Ago1 has the ability to accommodate
siRNA duplexes showing incomplete complementarity (Tomari
et al., 2007; Czech and Hannon, 2011). In regular S2 cells, exosiRNAs might be loaded onto Ago2 in a fashion equivalent tothat for endo-siRNAs (Figure 6A). In cells lacking D2 bodies,
exo-siRNAs are still loaded onto Ago2 (Figure 6B), because
Ago1 avoids binding fully complementary small RNA duplexes(Tomari et al., 2007; Czech and Hannon, 2011). miRNA loading
onto Ago1 does not seem to require such a regulatory system
for loading, because Ago1 prefers to bind small RNA duplexes
with incomplete complementarity. We propose that D2 bodies
are the cellular location where endo-siRNA loading onto Ago2
occurs (Figure 6A), although we cannot exclude the possibility
that some endo-siRNAs may be loaded onto Ago2 in the cytosol,
outside of D2 bodies, Dcr-2-R2D2 dependently or independently. The ‘‘inside D2 bodies’’ theory is favorable, because
localized concentration of the necessary factors might enhance
the rate of the loading reaction. Upon loading of an siRNA duplex
onto Ago2, the Dcr-2/R2D2 heterodimer might leave D2 bodies
to associate with siRNA duplexes in the cytosol, and then go
back to D2 bodies (Figure 6A). This model for intracellular,(C) Immunoprecipitation followed by western blotting shows that Flag-Dcr-2 WT and Flag-N950, but not Flag-C951, bind R2D2. Flag-EGFP was used asa negative control.
(D) N950 was further delineated to produce the mutants N694, N572, and N207 (see A). All Dcr-2 mutants and WT Dcr-2 are Flag tagged (3xFlag) and weredetected using anti-Flag antibody (green). N694 and N572 weakly localize to D2 bodies, but N207 did not. Endogenous R2D2 is shown in magenta. Nuclei arestained with DAPI (blue). Scale bars, 5 mm.
(E) Flag-N694 and Flag-N572 weakly associate with R2D2, while Flag-N207 failed to bind R2D2. Flag-EGFP was used as a negative control. See also Figure S3.

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Roles of R2D2 in the Endo-siRNA Pathway

A

R2D2

D2 body localization
dsRBD1

Dcr-2 binding

dsRBD2

WT
R1R2

166

Aux
R2Aux
R2Aux-M

167
82

xx
endo
Dcr-2

Merge

C

Input

IP
EGFP
WT
R1R2
R2Aux
Aux
R2Aux-M

R2D2

WT

B

EGFP
WT
R1R2
R2Aux
Aux
R2Aux-M

A153E A154D

R1R2

Dcr-2

R2Aux-M

R2Aux

Aux

EGFP
WT
R1R2
R2Aux
Aux
R2Aux-M

Figure 4. The dsRNA-Binding Activity and the Auxiliary Domain of R2D2 Are Required for Association with Dcr-2 and D2 Body Localization(A) The domain structure of R2D2 WT and its deletion mutants. D2 body localization and Dcr-2 binding of R2D2 WT and mutants are summarized on the right.
(B) R2D2 WT localizes to D2 bodies, but two deletion mutants, R1R2 and Aux, failed to do so. By contrast, R2Aux containing dsRBD2 and Aux localizes to D2
bodies. To abolish the dsRNA-binding activity of R2Aux, A153 and A154 were substituted with Glu and Asp, respectively, in the context of R2Aux. This mutant
R2Aux-M no longer accumulates in D2 bodies, suggesting that the dsRNA-binding activity is necessary for R2D2 to localize to D2 bodies. R2D2 WT and all
mutants are myc tagged. Scale bars, 5 mm.
(C) Immunoprecipitation followed by western blotting shows that myc-R2D2 WT, myc-R2Aux, and myc-R2Aux-M, but not myc-R1R2, bind Dcr-2 to a similar
extent. myc-Aux binds Dcr-2, but weakly compared with myc-R2D2 WT. myc-EGFP was used as a negative control. See also Figure S4.

dynamic translocation of the Dcr-2/R2D2 heterodimer is consistent with the observations that heterodimers lacking dsRNAbinding activity do not localize to D2 bodies (Figure 4B) andthat only a minor portion of Dcr-2 and R2D2 is localized to D2
bodies (Figure S2G). It remains unclear how D2 body formationis triggered in the cells. Identification of D2 body components

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Roles of R2D2 in the Endo-siRNA Pathway

RNAi

Flag-Ago2

Flag-Ago1

Flag-Ago2

Dcr-2
Flag-EGFP

Flag-Ago1

Flag-Ago1

R2D2
Flag-EGFP

EGFP

RNAi
Flag-Ago2

Flag-Ago1

Flag-EGFP

R2D2
Flag-Ago2

Flag-Ago1

Flag-EGFP

IP

B

IP
luc

Flag-Ago2

A- esiR-sl-1- guidestrand- miR-bantam

C

R2D2

Dcr-2

- passeger
strand

D

Merge

IP

-

WT-R
R2D2
Flag-Ago2

Flag-Ago1

Flag-EGFP

Flag-Ago2

Flag-Ago1

Flag-EGFP

Flag-Ago2

Flag-Ago1-

RNAi- esiR-sl-1

WT-R

Anti-R2D2 detectionlucrescueWT-R- miR-bantam

rescueFlag-Ago2

Flag-Ago1

Flag-Ago2

R2Aux-M

Flag-Ago1

R2Aux

Flag-EGFP

IP

Flag-EGFP

R2Aux

Anti-myc detection

E

R2Aux-M- esiR-sl-1- miR-bantam

Figure 5. R2D2 Ensures Endo-siRNA Sorting onto Ago2 by Sequestering Dcr-2 to D2 Bodies(A) Flag-Ago1 and Flag-Ago2 were expressed after R2D2 was deleted by RNAi. The endo-siRNA esiR-sl-1 is still loaded onto Ago2 in the absence of R2D2,
demonstrating that R2D2 is not necessary for endo-siRNA loading onto Ago2. Under the same conditions, Ago1 also associates with esiR-sl-1. endo-siRNA
misdirection onto Ago1 (Okamura et al., 2011) was also induced in S2 cells. miR-bantam was not misloaded onto Ago2.
(B) exo-siRNA was specifically loaded onto Ago2 in the absence of R2D2 and the Dcr-2/R2D2 heterodimer. The amount of proteins immunoprecipitated is shownin Figure S5D.
(C) The expression of an R2D2-siRNA-resistant form of R2D2 WT, WT-R, and R2Aux restored D2 body formation in R2D2-depleted cells, but R2Aux-M failed to do
the same. R2D2 WT-R, R2Aux, and R2Aux-M were myc tagged and detected by anti-R2D2 and/or anti-myc antibodies, as indicated (green). Endogenous Dcr-2
was detected using anti-Dcr-2 antibody (magenta). Nuclei are shown in blue by DAPI staining. Scale bars, 5 mm.
(D) Expression of R2D2 WT-R rescues the R2D2-defective phenotype, endo-siRNA misloading onto Ago1, in R2D2-depleted cells.
(E) Expression of R2Aux, but not of R1Aux-M, rescues the R2D2-defective phenotype, endo-siRNA misloading onto Ago1, in R2D2-depleted cells. See also
Figures S5 and S6.

other than Dcr-2 and R2D2 may help to understand the underlying mechanism, which is currently under investigation in ourlaboratory.

Depletion of R2D2 from S2 cells caused disappearance of D2
bodies, but in this circumstance, endo-siRNAs still associatedwith both Ago1 and Ago2, suggesting that endo-siRNA loading

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Roles of R2D2 in the Endo-siRNA Pathway

endo-siRNA
precursor

A Wild-type

Dcr-2/Loqs-PD

exo-siRNA
duplexendo-siRNA
duplexD2 body
Dcr-2

Dcr-2

Dcr-2
R2D2

B R2D2depleted

Dcr-2

R2D2

R2D2

miRNA
duplex

R2D2

Ago2

Ago2

Ago1

exo-siRISC

endo-siRISC

miRISC

endo-siRNA
precursor
Dcr-2/Loqs-PD

exo-siRNA
duplexendo-siRNA
duplex

Ago2

Ago2

exo-siRISC

miRNA
duplexAgo1

endo-siRISC

Ago1

miRISC

Figure 6. A Model for R2D2’s Function in Controlling Endo-siRNA
Sorting in Drosophila S2 Cells(A) In Drosophila S2 cells (wild-type), R2D2 localizes Dcr-2, and presumably
endo-siRNA duplexes, into D2 bodies to ensure endo-siRNA loading onto
Ago2. This reaction yields the endo-siRISC. Ago1 does not localize to D2
bodies; thus, endo-siRNA misdirection onto Ago1 is avoided. exo-siRNAs
might also be loaded onto Ago2 in a fashion equivalent to that for endosiRNAs. This reaction yields the exo-siRISC. miRNAs are loaded onto Ago1 ina manner independent of D2 bodies, which yields the miRISC. Upon loading ofsiRNA duplex onto Ago2, the Dcr-2/R2D2 heterodimer leaves D2 bodies toassociate with siRNA duplexes in the cytosol and then goes back to D2 bodies.
(B) In R2D2-depleted Drosophila cells (R2D2-depleted), D2 bodies collapse
and Dcr2 is orphaned in the cytosol. In this circumstance, endo-siRNAs areloaded onto not only Ago2 but also Ago1. This loading might occur without thehelp of Dcr-2, because R2D2, or its dsRNA-binding activity, is required for Dcr2 to efficiently bind siRNA duplexes and load them onto Ago2 (Liu et al., 2003,
2006). Strand selection is still maintained, although the underlying mechanism
remains unknown. exo-siRNAs are specifically loaded onto Ago2, while
miRNAs are still specifically loaded onto Ago1.

onto Argonaute proteins does not absolutely require R2D2. It has
been reported that R2D2 plays a crucial role in the asymmetricalselection of guide exo-siRNAs from exo-siRNA duplexes (Tomari
et al., 2007). However, in our study, esiR-sl-1* was not detectedin Ago1 and Ago2 complexes, unlike esiR-sl-1, which was detected in R2D2-depleted cells. We also found that guide exosiRNA was also selectively loaded onto Ago2 in Dcr-2- andR2D2-depleted cells. Thus, R2D2 is not absolutely required forsiRNA strand selection in vivo. One possibility is that Argonaute

proteins alone can implement endo-siRNA and exo-siRNA
strand selection. We are currently exploring this possibility in
our laboratory.
The level of miRNAs associated with Ago1 was hardly
changed by R2D2 depletion in S2 cells (Figure 5A). A recent
study showed that miRNAs are loaded onto Ago1 as efficiently
in r2d2 mutant flies as they are in WT flies (Okamura et al.,2011). One explanation for these observations is that the expression level of Ago1 is so high that endo-siRNA misloading onto
Ago1 does not disturb Ago1-miRNA binding and Ago1 functions.
Another explanation is that there is an inherent mechanism incells that forces Ago1 to favor miRNAs even in circumstancesin which endo-siRNAs and miRNAs are equally available for
Ago1 to bind.
If the latter possibility is correct, what would be the biologicalrelevance for Drosophila? There are potentially two scenarios:the ‘‘endo-siRNA function protection’’ scenario and the ‘‘miRNA
function protection’’ scenario. It has been shown that Ago1’s
Slicer activity (endonuclease activity to cleave its target transcripts) is much weaker than that of Ago2 (Förstemann et al.,2007). This means that endo-siRNAs misdirected to Ago1 would
not be able to exhibit sufficiently the intrinsic activity of endosiRNAs to silence transposons by cleaving the transcripts.
Many species, including flies, nematodes, and plants, are equipped with an siRNA-mediated gene silencing pathway (or RNAi)
as an innate immune system to protect themselves from lifethreatening, invasive viruses and/or transposons randomly inserting copies of themselves into the host genome (Ding andVoinnet, 2007). To control this system, animals and plants havedeveloped systems to fully maintain siRNA functions (the‘‘endo-siRNA function protection’’ scenario).
Endo-siRNAs act as an innate immune system, while miRNAs
function to maintain cellular gene networks by regulating theexpression of many of the genes involved in them. In mammals,
the two pathways share some proteins as common factors: for
instance, Argonaute proteins. However, Drosophila basicallymaintains these pathways independently. This notion is strongly
supported by the observations that Drosophila distinguishably
uses Dcr-1 and Ago1, and Dcr-2 and Ago2, which are dedicatedto the miRNA and siRNA pathways, respectively, although it
should be noted that some crosstalk between these pathways
has been previously reported (Ghildiyal and Zamore, 2009;
Ghildiyal et al., 2010). Keeping the independency of these pathways is crucial for Drosophila, because, unlike mammals, flies
do not have other immune systems for protecting themselvesfrom infectious viruses. Thus, Drosophila has had to develop
mechanisms to maintain the independency of the miRNA andsiRNA pathways. One such mechanism is the one preventing
endo-siRNA misloading onto Ago1 (the ‘‘miRNA function protection’’ scenario).
EXPERIMENTAL PROCEDURES
Immunofluorescence
Immunofluorescence was performed after fixing S2 cells with 2% formaldehyde for 15 min at room temperature. Cells were permeabilized with 0.1%
Triton X-100 and then, after washing, stained with anti-Dcr-2 (Miyoshi et al.,2009), anti-R2D2 (this study), anti-Dcp1 (Miyoshi et al., 2009), anti-dFMR1(Okamura et al., 2004), and anti-dGM130 (Yano et al., 2005) antibodies. To

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detect Flag-Dcr-2 WT and mutants, cells were stained with anti-Flag (M2) antibody (Sigma-Aldrich). To detect myc-tagged R2D2 WT and mutants, anti-myc(9E10) antibody (Sigma-Aldrich) was used. Alexa Fluor 488/555 goat antimouse IgG1/IgG2a/IgG2b (Invitrogen) and Alexa Flour 488 goat anti-rabbit
IgG (H+L) were used as secondary antibodies. Monoclonal antibodies against
R2D2 were produced using full-length R2D2 tagged with GST as an antigen, as
described previously (Miyoshi et al., 2009). S2 cells were treated with cycloheximide (10 mg/ml) and puromycin (100 mg/ml) for 1 hr (Eulalio et al., 2007b)
before immunofluorescence. Arsenite treatment was performed at final
concentration of 500 mM for 2 hr (Farny et al., 2009).

ACKNOWLEDGMENTS

Plasmid Construction
Expression vectors of Flag-Dcr-2 WT and deletion mutants were generated byinserting the corresponding coding regions into the pRH3xflag vector(Saito et al., 2005). DNA oligos used for Dcr-2 mutant construction are shownin Table S1. Expression vectors of myc-R2D2 WT and deletion mutants weregenerated by inserting the corresponding coding region into the pAcM vector(Saito et al., 2009). DNA oligos used for R2D2 mutant construction are shownin Table S1. Liu et al. (2006) described that A153 and A154 were mutated to
two lysines. However, we found by sequencing the original mutant constructfrom that group that they were actually mutated to Glu and Asp, respectively,
which was confirmed by Q. Liu (Liu et al., 2006). To yield RNAi-resistant R2D2
WT, two primers, R2D2-R-F and R2D2-R-R, were used (Table S1) for PCR.
PCR was carried out using KOD plus DNA polymerase (Toyobo).

Received: July 16, 2012
Revised: November 16, 2012
Accepted: December 26, 2012
Published: January 31, 2013

RNAi
RNAi was performed in S2 cells as described previously (Saito et al., 2005).
dsRNA used for R2D2 depletion corresponded to nucleotides 1–820 of theR2D2 coding sequence. dsRNAs for Dcr-2 and EGFP were described previously (Saito et al., 2005). S2 cells (3 3 106 cells) were suspended in 100 ml of
Solution V of the Cell Line Nucleofector Kit V (Amaxa Biosystems) with 200
pmol of siRNA duplex. Transfection was conducted in electroporation
cuvettes using Nucleofector (Amaxa Biosystems). After transfection, cells
were transferred to fresh medium and incubated at 26 C for 3 days for further
experiments. The siRNAs used for RNAi are summarized in Table S1. Luc
siRNA (Saito et al., 2009) was used as a control. The sequences of siRNAs
used in the experiments are shown in Table S1.
Northern Blot Analysis
Northern blot analysis was carried out essentially as described previously
using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Saito et al.,2010). The sequences of the DNA oligos for esiR-sl-1 and miR-bantam areindicated in Kawamura et al. (2008). The DNA oligos used to detect siRNA
guide and passenger strands are 50 -AAATAGATAATAGTCATTGGC-30 (guide)and 50 -AAGCCAATGACTATTATCTAT-30 (passenger) (Figure 5B).

We thank Q. Liu for the R2D2 mutant plasmid; S. Goto for the dGM130 antibody; and T.N. Okada, A. Takeuchi, and T. Sunohara for technical assistanceand reagents. We are thankful to M. Isogai and M. Shimura (PerkinElmer
Japan) for their assistance with fluorescence quantification. We also thankthe other members of the Siomi laboratories for discussions and commentson the manuscript. This work was supported by MEXT grants (to H.S., K.M.,
and M.C.S.) and by grants from the Uehara Memorial Foundation (to K.M.).
M.C.S. is supported by CREST from JST.

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