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Hsp90 may have a role in RNAi. However, the functional involveA direct role for Hsp90 in pre-RISC Thus,
ment of Hsp90 at the molecular level in RNAi remains unclear.
To determine for which step Hsp90 is required in RNAi, we disformation in Drosophila

t

b

–

– –

+ + Geld- dsRNA

DMSO

Geld

0 30 60 0 30 60 0 30 60 (min)ag

Fl -EG
ag F
-D P
ic
er
Fl
ag
2
Fl -EG
ag F
-D P
ic
er
2

Flag-IPa

Fl

Duplex -100 Single 50 -

c–

pu
t
Fl
ag
Fl EG
ag FP
-A
Fl go
ag 2
-E
Fl G
ag F
-A P
go
2

- siRNA

20 (nt)Flag-IPd–In

In RNA interference (RNAi), small interfering RNA (siRNA) is associated with Argonaute 2 (Ago2) and guides the protein to its target
mRNAs for silencing1,2. In Drosophila melanogaster, siRNAs are processed from long double-stranded RNA precursors by Dicer2 (refs. 1,2).
Upon processing, siRNAs are still in a duplex form and are associatedwith Dicer2 and R2D2, which are major components of the RNAinduced silencing complex (RISC)-loading complex (RLC)1–3. siRNA
duplexes are then transferred to Ago2 from the RLC to form the precursor of the RISC, or pre-RISC3. It has been shown that pre-RISC
formation occurs in an ATP-dependent manner4. However, it remains
unclear why ATP is needed for this process.
In the pre-RISC, siRNA duplexes are ‘unwound’ by Ago2 endonuclease or Slicer activity; the passenger strand of the duplexes is cleaved by
Slicer and displaced from Ago2 (refs. 5,6). This step is known to occur
in an ATP-independent manner5,7. The guide strand of the duplex
remains associated with Ago2. The resultant Ago2–siRNA complex
is termed RISC and is now active and ready to bind and cleave target
RNAs. In this way, genes targeted by the RNAi machinery are effectively silenced1–3. Ago2 frequently co-purifies with heat-shock protein(Hsp) 90 (refs. 8–13), a chaperone whose activity depends on ATP14.

puHeat-shock proteins (Hsps) are molecular chaperones that
control protein folding and function. Argonaute 2 (Ago2), theeffector in RNA interference (RNAi), is associated with Hsp90;
however, its function in RNAi remains elusive. Here we showthat Hsp90 is required for Ago2 to receive the small interfering
RNA (siRNA) duplex from the RNA-induced silencing
complex–loading complex in RNAi, suggesting a model where
Hsp90 modifies Ago2 conformation to accommodate thesiRNA duplex.

sected the RNAi pathway into multiple steps and performed assays forthe individual steps in the presence or absence of geldanamycin, thespecific Hsp90 inhibitor that mimics ATP binding with the protein14.
In siRNA processing assays, we incubated Flag-tagged Dicer2 isolated
from S2 cells with 32P-labeled dsRNA precursors with or without
geldanamycin. The ability of Dicer2 to excise siRNA duplexes fromthe precursors was not affected by geldanamycin (Fig. 1a). Thus,
Hsp90 is not necessary for siRNA excision from the precursor.
We next examined whether the siRNA-unwinding step in RNAi
requires Hsp90 function. We incubated siRNA duplexes, which werelabeled with 32P at the 5′ end of the guide siRNA within the duplexes,in S2 lysates with or without geldanamycin. In the presence of geldana­
mycin, siRNA duplexes remained as duplexes even after 1 h incubation(Fig. 1b). DMSO, an organic solvent used for dissolving geldanamycin

In

DMSO

–

+

+

Geld
- Target RNA

Geld
100 -

0 30 60 0 30 60 0 30 60 (min)128 ntLuc siRNAtarget sequence- RISC- RLC

50 -- Free20 (nt)-Cleavede

27 nt

Flag-EGFP

Flag-Ago2

Flag-IP

Flag-IP

–

+

Input

Figure 1 Hsp90 is required for RISC formation in Drosophila RNAi.
(a) siRNA processing assays showed that Hsp90 is not necessary for
excising siRNA duplexes from the precursor. Geld, geldanamycin;
IP, immunoprecipitation. (b) siRNA duplex unwinding assays showedthat Hsp90 is required for siRNA unwinding in RNAi. (c) RISC formation
assays show that Hsp90 is necessary to convert the RLC into the RISC.
(d) Hsp90 is not necessary for Ago2 Slicer activity once the RISC has
been formed without Hsp90 inhibition. Luc, luciferase. (e) Hsp90 does
not affect the association between Dicer2 and Ago2 in Drosophila.

Input© 2010 Nature America, Inc. All rights reserved.

Tomohiro Miyoshi1, Akiko Takeuchi1, Haruhiko Siomi1 &Mikiko C Siomi1,2–+ Geld- Dicer2- Flag-Ago2- R2D2

1Keio University School of Medicine, Tokyo, Japan. 2Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama, Japan.

Correspondence should be addressed to M.C.S. (siomim@sc.itc.keio.ac.jp).
Received 22 April; accepted 21 June; published online 18 July 2010; doi:10.1038/nsmb.1875

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BRIE F COMMUNICATIONSasiRNA1I
P-UCGAAGUAUUCCGCGUACGUG
AUAGCUUCAUAAGGCGCAUGC-P–

Geld

0 10 30 60 90

0 10 30 60 90 (min)siRNA2
P-UCGAAGUAUUCCGCGUACGUG
AUAGCUUCAUAAGGCGCAUGC-P
I
–
Geld

siRNA3I
P-UCGAAGUAUUCCGCGUACGUG
AUAGCUUCAUAAGGCGCAUGC-P–

Geld

0 10 30 60 90

0 10 30 60 90

0 10 30 60 90

0 10 30 60 90 (min)(min)- Dicer2- Ago2

21
17

7
12
19

(%)

31
65
92
10
3
10
0

34

21
10
0
82
59
40

57
32

0
83

(%)

10

0
10
0
81
61
50

10

41
28

0
10

81
58

- R2D2(%)

cb
Flag-IP

Dicer2

Dicer2

R2D2

Flag-Ago2
RLC
Dicer2R2D2© 2010 Nature America, Inc. All rights reserved.

si

R
N
A
G
el
d
+
si
si
R
R
N
N
A
A
+
si
G
R
e
N
ld
A
G
el
d
si + si
R
N RN
A
A
+
G
el
d

Flag-EGFP

Figure 2 Hsp90 is required for pre-RISC
formation. (a) Three siRNA duplexes, siRNA1,
siRNA2 and siRNA3, were individually incubatedwith S2 cell lysates in the presence or absence of
geldanamycin (geld). Ago2 association with thesiRNA duplexes was severely affected by Hsp90
inhibition, although R2D2 and Dicer2 association
with the duplexes was barely affected. Dicer2
association with the duplexes was slightly affected.
I, iodine-uridine. Red, siRNAs end-labeled with
32P for visualization. Densitometric analysis of
the cross-link is shown by percentages. The
numbers indicated on the gel image represent
siRNA incubation time in lysates before 10 min
UV cross-link (Supplementary Methods). IP,
immunoprecipitation. (b) Ago2 in S2 lysates
preincubated with geldanamycin was not loadedwith guide siRNAs. (c) A model for Hsp90
function in the RNAi pathway. Hsp90 promotes
a conformational change in Ago2, enabling it toreceive siRNA duplex from the RLC.

that, at least in Drosophila cells Hsp90
has little or no effect on the Dicer2-Ago2
Northern
association. This is in contrast to mammalian
Ago2
Ago2
ADP Hsp90- Flag-Ago2
cells, where Hsp90 activity was required for the
RISC
pre-RISC
Conformational change- Flag-EGFP
Argonaute-Dicer interaction16.
in Ago2Western
Two models regarding Hsp90 function in
RNAi can now be proposed: the first is thatin the lysates, did not affect the activity (Fig. 1b). Thus, Hsp90 is Hsp90 functions in displacing the siRNA duplex from the RLC, whereas
required for unwinding siRNA duplexes into single-stranded siRNAs. the second is that Hsp90 is required for Ago2 to receive the siRNAWe then speculated that the RISC would not be assembled in S2 duplex from the RLC, after the siRNA duplex has been pro­perly dislysates when Hsp90 was inhibited. To test this, we performed RISC for- placed from the RLC. To examine which model is correct, we performed
mation assays. Whereas DMSO alone did not show any obvious effect, siRNA-protein interaction experiments using three siRNA duplexes,­geldanamycin severely inhibited RISC formation (Fig. 1c). RLC for- siRNA1, siRNA2 and siRNA3, each of which was composed of themation was not affected by geldanamycin treatment (Fig. 1c). These same sequence but contained an iodine-uridine at different positions
results indicate that Hsp90 has an important role in the particular step(s) within the duplex (Fig. 2a). We individually incubated siRNA duplexes
necessary for converting RLC into RISC. A recent study has shown that in S2 lysates for the time periods indicated in Figure 2a, and then thegeldanamycin significantly reduces the levels of Argonaute proteins in mixtures were exposed to UV light to cross-link the siRNA duplexes
mammalian cells and thus reduces the programming of the RISC12. with the ­proteins physically associated with them. Previous studies have
However, the stability of Ago2 was barely changed in S2 lysates even after indicated that Dicer2 and R2D2, the second known component of thewe added geldana­mycin (Supplementary Fig. 1a). Thus, the reduced RLC, are mainly cross-linked at the 3′ end of the guide and passenger
RISC-forming activity was likely not due to reduced Ago2 stability.
strands of the duplex, respectively17,18. Ago2 was shown to be crosssiRNA duplexes remained as duplexes in the presence of geldana­mycin linked predominantly at the 5′ end of the guide strand of the duplex17.
(Fig. 1b), implying that geldanamycin inhibits Ago2 Slicer activity. To We confirmed that the main bands observed in the left, center andtest this, we performed target RNA cleavage assays. After expressing right panels of Figure 2a corresponded to R2D2, Dicer2 and Ago2,
Flag-tagged Ago2 or Flag-tagged enhanced green fluorescent protein respectively (Supplementary Fig. 1b). We found that the association(EGFP) in S2 cells, we lysed the cells and assembled the RISC in the of R2D2 and Dicer2 with siRNA1 and siRNA2, respectively, was not
lysates by adding siRNA duplexes. We isolated Flag-Ago2 and Flag- significantly altered by Hsp90 inhibition (Fig. 2a). However, Hsp90
EGFP from the lysates and added target RNAs to the complexes in the inhibition drastically affected siRNA duplex association with Ago2;
presence or absence of geldanamycin. The Flag-tagged Ago2 complex in the presence of geldanamycin, Ago2 barely interacted with siRNA3
cleaved the target RNA in both cases (Fig. 1d). Thus, Hsp90 was not (Fig. 2a, right). We observed the inhibitory effect when we added
required for Ago2 to cleave the target RNAs once a functional RISC was geldanamycin at a concentration of 10 µM or higher (Supplementary
properly formed. An earlier report showed that Hsp90 inhibition impairs Fig. 1c). Radicicol, another Hsp90 inhibitor, also interfered with siRNA
RNAi in Drosophila ovary extracts15. However, in these experiments, duplex association with Ago2 (Supplementary Fig. 1d). A siRNA
siRNAs and geldanamycin were added simultaneously to the lysates to duplex mutant (siRNA3 mutant), in which the passenger strand was
examine the inhibitory effect of the functional loss of Hsp90 (ref. 15).
modified with a 2′-O-methyl group at the 10th nucleotide from theConversion of the RLC into the pre-RISC requires siRNA duplexes 5′ end and thus interferes with Ago2 Slicer activity, behaved similarly toto be transferred from the RLC to Ago23. At this step, Dicer2, the siRNA3 (Supplementary Fig. 1e). In addition, we showed that Hsp90
main component of the RLC, should interact with Ago2, although was present in immuno­precipitated Ago2 complex (Supplementary
this interaction may be transient. Thus, we examined whether inhi- Fig. 1f). These results suggest that Hsp90 is required for Ago2 to receive
bition of Hsp90 activity interferes with the association between siRNA duplexes from the RLC in the RNAi pathway. In siRNA-Ago2
Dicer2 and Ago2. Flag-tagged Ago2 interacted with Dicer2 simi- binding assays, the guide siRNAs were barely associated with Ago2
larly either with or without geldanamycin (Fig. 1e). This indicated when we added geldanamycin first to the lysates prior to the addition
Ago2- Guide RNA

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of the siRNA duplexes (Fig. 2b). These results ­further ­support the ideathat Hsp90 is needed for Ago2 to bind the siRNA duplex. Structural
analysis of the eubacterial Ago protein has revealed that the cavity in
the Ago protein that accommodates the siRNA duplex is too small to
be bound by an RNA molecule19, and a conformational change in the
Ago protein would be required for binding. In the current study, ourresults suggest that Hsp90 acts as the driving force for changing theconformation of Ago2, likely by hydolyzing ATP as an energy source,
as geldanamycin is known to inhibit the ATPase activity of Hsp90
by occupying the N-terminal ATP binding pocket of the protein20,
enabling it to accommodate siRNA duplex from the RLC (Fig. 2c).
This step is crucial in RNAi, and so, without Hsp90, the pre-RISC is
not formed and RNAi is impaired.

© 2010 Nature America, Inc. All rights reserved.

Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
Acknowledgments
We thank K. Miyoshi and T. Yoshihisa for providing technical advice and othermembers of the Siomi laboratory for discussions. This work was supported by
Ministry of Education, Culture, Sports, Science and Technology of Japan grants to
H.S. and M.C.S. M.C.S. is supported by Core Research for Evolutional Science andTechnology from the Japan Science and Technology Agency.
AUTHOR CONTRIBUTIONS
T.M. and A.T. conducted experiments; T.M., A.T., H.S. and M.C.S. designed
experiments, interpreted data and prepared the manuscript.

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COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/nsmb/.
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/.
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