Supplementary Information for

A direct role for HSP90 in pre-RISC formation in Drosophila
Tomohiro Miyoshi1, Akiko Takeuchi1, Haruhiko Siomi1 and Mikiko C. Siomi1,2*1

Keio University School of Medicine, Tokyo 160-8582, Japan, 2JST, CREST, Saitama

332-0012, Japan* Corresponding author
Phone +81-3-5363-3498; Fax +81-3-5363-3266; E-mail siomim@sc.itc.keio.ac.jp
This PDF file includes:
Methods
References for Supplementary Information
Supplementary Figure 1
Methods
Plasmid constructs. To obtain Argonaute 2 (Ago2) cDNA, poly(A)+ RNAs werepurified from S2 cells and reverse transcription-polymerase chain reaction (RT-PCR)
was carried out using a ProSTAR Ultra-HF RT-PCR system (Stratagene). To produce
3x flag-tagged Ago2, the full-length Ago2 cDNA was subcloned into the pRH3xflag
vector1.
siRNA processing assay. In vitro siRNA processing assays were performed aspreviously described2. Geldanamycin (Tokyo Chemical Industry co., LTD) was added
to the assay reaction at 100 µM.
siRNA unwinding assay. siRNA unwinding assays were performed essentially as
previously described3. S2 lysates were pre-incubated with DMSO or geldanamycin (100µM) for 20 min at 26ºC. After the luciferase siRNA duplex was incubated in S2 celllysates, 10 µL of the reaction mixture was mixed with 2 µL of stop solution (30 mM
HEPES-KOH (pH 7.4), 30 mM EDTA, 30% glycerol, 3% SDS, 300 nM siRNA guidestrand DNA, 3 mg per mL proteinase K), incubated for 10 min at 37ºC, electrophoresed
onto 15 % native polyacrylamide gels at 4ºC.

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Nature Structural & Molecular Biology: doi:10.1038/nsmb.1875

RISC formation assay. RISC formation assays were performed essentially aspreviously described3. Briefly,

32

P-radiolabeled luciferase siRNA (guide strand;

5’-UCG AAG UAU UCC GCG UAC GUG-3’) and non-labeled passenger strand(5’-CGU ACG CGG AAU ACU UCG AAA-3’) were annealed and the resultant siRNA
duplex was incubated in S2 lysates at 26 ºC with DMSO (control) or geldanamycin (100µM). After incubation, samples were adjusted to 6% (w/v) glycerol and resolved bysubmarine native agarose gel electrophoresis at 4ºC. Gels were dried under a vacuum
onto Hybond-N+ nylon membrane (GE Healthcare) and exposed to an imaging plate ofa BAS 2500 (Fuji Film).
Immunoprecipitation. To examine the interaction between Ago2 and Dicer2, lysatesof S2 cells expressing Flag-Ago2 were prepared in a NP-40 buffer containing 30 mM
HEPES (pH7.4), 150 mM potassium acetate, 5 mM magnesium acetate, 5 mM DTT,
0.1% NP40, 2 µg per mL pepstatin, 2 µg per mL leupeptin, and 0.5% aprotinin. Celllysates were then incubated with DMSO or geldanamycin (100 µM) for 20 min at 26ºC.
The luciferase siRNA duplex (0.1 µM) was added to the cell lysates andmmunoprecipitation performed using anti-Flag M2 agarose beads (Sigma-Aldrich).
Beads were washed five times with NP-40 buffer. Western blotting was performed asdescribed4. Monoclonal antibodies against R2D2 were produced against full-length
R2D2 tagged with GST. Anti-Ago1, anti-Ago2 and anti-Dicer2 antibodies weredescribed previously4,5. Anti-Hsp90 (rabbit polyclonal) was purchased from Stressgen.
Target RNA cleavage assay. Target RNA cleavage assays were performed essentiallyas

previously

described4.

ThesequenceoftheluctargetRNA

wasgggcgaauuggguaccCAAGUACGCGGAAUACUUCGAgaauuccugcagcccgggggauccacu
aguucuagagcggccgccaccgcgguggagctccagcuuuuguucccuuuagugaggguuaauu, in which
the sequence complementary to luc siRNA is indicated by capital letters. Geldanamycin
was added to the assay reaction at 100 µM.
Northern blotting. RNAs were isolated from immunoprecipitated fractions using
ISOGENE-LS (Nippon Gene). Northern blotting was performed as described
previously1, except that the RNAs were run on 15% acrylamide-denaturing gels,transferred onto Hybond-N membrane (GE Healthcare) and cross-linked with
2Nature Structural & Molecular Biology: doi:10.1038/nsmb.1875

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide6. The DNA Oligo used as a siRNA
guide strand probe was: 5’-CAC GTA CGC GGA ATA CTT CGA-3’.
Photo-crosslinking assay. 5-iodide-uracil7 siRNA photo-crosslinking assays wereperformed using a modified version of a previously reported method8. S2 cell lysates
were prepared as in the RISC formation assays and then incubated with DMSO,geldanamycin or Radicicol (Sigma-Aldrich) for 20 min at 26ºC. luciferase siRNA
duplexes were added to the lysates and then irradiated by 312 nm UV for 10 min andresolved on 7.5% SDS-polyacrylamide gels. The band signal in each lane (10, 30, 60 or90 min) in the siRNA1 and siRNA2 panels was compared with that of ‘0 (min)’ lane. Inthe siRNA3 geldanamycin - panel (the second from the right hand side), the band signal
in each lane (0, 10, 30 or 60 min) was compared with that in ‘90 (min)’ lane. The bandsignal in the siRNA3 with geldanamycin panel (far right) was compared with that in the‘90 (min)’ lane in the siRNA3 without geldanamycin panel.

References
1. Saito, K., Ishizuka, A., Siomi, H. & Siomi, M.C. Processing of pre-microRNAs by
the Dicer-1-Loquacious complex in Drosophila cells. PLoS Biol. 3, e235 (2005).
2. Miyoshi, K., Miyoshi, T., Hartig, J.V., Siomi, H. & Siomi, M.C. Molecularmechanisms that funnel RNA precursors into endogenous small-interfering RNA andmicroRNA biogenesis pathways in Drosophila. RNA 16, 506-515 (2010).
3. Okamura, K., Ishizuka, A., Siomi, H. & Siomi, M.C. Distinct roles for Argonaute
proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655-1666(2004).
4. Miyoshi, K., Tsukumo, H., Nagami, T., Siomi, H. & Siomi, M.C. Slicer function ofDrosophila Argonautes and its involvement in RISC formation. Genes Dev. 19,2837-2848 (2005).
5. Miyoshi, K., Okada, T.N., Siomi, H. & Siomi, M.C. Characterization of themiRNA-RISC loading complex and miRNA-RISC formed in the Drosophila miRNA
pathway. RNA 15, 1282-1291 (2009).
6. Pall, G.S. & Hamilton, A.J. Improved northern blot method for enhanced detection ofsmall RNA. Nat Protoc. 3, 1077-1084 (2008).
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Nature Structural & Molecular Biology: doi:10.1038/nsmb.1875

7. Meisenheimer, K.M. & Koch, T.H. Photocross-linking of nucleic acids to associatedproteins. Crit. Rev. Biochem. Mol. Biol. 32, 101-140 (1997).
8. Tomari, Y., Matranga, C., Haley, B., Martinez, N. & Zamore, P.D. A protein sensor
for siRNA asymmetry. Science 306, 1377-1380 (2004).

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Miyoshi et al.
Supplementary Figure 1daGeld
0459004590min

siRNA3I
P-UCGAAGUAUUCCGCGUACGUG
AUAGCUUCAUAAGGCGCAUGC-P
0 2 10 50 250 Radicicol (µM)

- Ago2- Tubulin- Ago2

Ago2

Ago1IP

Dicer2IP
R2D2besiRNA3 mutant

I
P-UCGAAGUAUUCCGCGUACGUG
AUAGCUUCAUAAGGCGCAUGC-POMec

0 10 30 60 90

siRNA3siRNA3siRNA2siRNA10 10 30 60 90

Geldmin-Ago2siRNA3I
P-UCGAAGUAUUCCGCGUACGUG
AUAGCUUCAUAAGGCGCAUGC-P
0 2 10 50 250 Geld (µM)

f- Ago2

Input

Flag-IP++Flag-Ago2- Flag-Ago2- Hsp90

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1875

Supplementary Figure 1 Geldanamycin inhibitory effect on RNAi
Supplementary Figure 1. (a) Western blot analysis revealed that addition ofgeldanamycin to S2 cell lysates causes little or no instability of Ago2 in the lysates.
Tubulin was used as a loading control. (b) Immunoprecipitation of Ago14 and Ago24
was performed from S2 lysates where siRNA3 (see Fig. 2a) was pre-incubated andphotocrosslinked. siRNA3 was photocrosslinked to Ago2; however, Ago1 was hardly
photocrosslinked with siRNA3. Immunoprecipitation with anti-R2D2 and anti-Dicer25
antibodies confirmed the interaction of R2D2 and Dicer2 with siRNA1 and siRNA2,
respectively. (c) The geldanamycin inhibitory effect was observed when geldanamycin
was added at the concentration of 10µM or higher. (d) Radicicol, another Hsp90
inhibitor, interferes with siRNA duplex association with Ago2. (e) In photocrosslinking
assays, siRNA3 mutant, in which the passenger strand was modified with a 2’-O-methylgroup at the 10th nucleotide from the 5’ end and thus interferes with Ago2 Slicer activity,
behaved very similarly to siRNA3 (see Fig. 2a). (f) The protein-protein interaction
assay shows that Ago2 is able to interact with Hsp90 in S2 cells. Flag-tagged Ago2 wasexpressed in S2 cells by transfection prior to the lysate preparation.

Nature Structural & Molecular Biology: doi:10.1038/nsmb.1875