Distinguishable In Vitro Binding Mode of Monomeric
TRBP and Dimeric PACT with siRNA
Tomoko Takahashi1, Takuya Miyakawa2, Shuhei Zenno3, Kenji Nishi1, Masaru Tanokura2,
Kumiko Ui-Tei1*
1 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan, 2 Department of Applied Biological Chemistry, Graduate
school of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan, 3 Department of Biotechnology, Faculty of Engineering, Maebashi Institute of Technology,
Gunma, Japan

Abstract
RNA interference (RNAi) is an evolutionally conserved posttranscriptional gene-silencing mechanism whereby smallinterfering RNA (siRNA) triggers sequence-specific cleavage of its cognate mRNA. Dicer, Argonaute (Ago), and either TARRNA binding protein (TRBP) or a protein activator of PKR (PACT) are the primary components of the RNAi pathway, and they
comprise the core of a complex termed the RNA-induced silencing complex (RISC)-loading complex (RLC). TRBP and PACT
share similar structural features including three dsRNA binding domains (dsRBDs), and a complex containing Dicer andeither TRBP or PACT is considered to sense thermodynamic asymmetry of siRNA ends for guide strand selection. Thus, both
TRBP and PACT are thought to participate in the RNAi pathway in an indistinguishable manner, but the differences in siRNAbinding mode and the functional involvement of TRBP and PACT are poorly understood. Here, we show in vitro binding
patterns of human TRBP and PACT to siRNA using electrophoresis mobility shift analysis and gel filtration chromatography.
Our results clearly showed that TRBP and PACT have distinct in vitro siRNA binding patterns from each other. The results
suggest that monomeric TRBP binds to siRNA at the higher affinity compared to the affinity for own homodimerization. In
contrast, the affinity between PACT and siRNA is lower than that of homodimerization or that between TRBP and siRNA.
Thus, siRNA may be more readily incorporated into RLC, interacting with TRBP (instead of PACT) in vivo.
Citation: Takahashi T, Miyakawa T, Zenno S, Nishi K, Tanokura M, et al. (2013) Distinguishable In Vitro Binding Mode of Monomeric TRBP and Dimeric PACT withsiRNA. PLoS ONE 8(5): e63434. doi:10.1371/journal.pone.0063434
Editor: Andreas Hofmann, Griffith University, Australia
Received February 15, 2013; Accepted April 1, 2013; Published May 2, 2013
Copyright: ß 2013 Takahashi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Core Research Project forPrivate University: matching fund subsidy to K.U.-T. and a Research Fellowship for Young Scientists of the Japan Society for the Promotion of Science to T.T. The
funders had no role in study, design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ktei@bi.s.u-tokyo.ac.jp

thermodynamic properties of the helix [10]. Thus, Dicer itself cansense this asymmetry, and functionality is activated upon
association with either TRBP or PACT. Furthermore, TRBP
and PACT are known to interact with each other and associatewith Dicer to facilitate dsRNA cleavage [14], [15].
TRBP and PACT also share structural similarities, eachcontaining three dsRNA binding domains (dsRBDs), namely,
dsRBD1, dsRBD2, and dsRBD3. TRBP was first identified as afactor that binds to human immunodeficiency virus-1 (HIV-1)
TAR RNA [16]. RNA binding analyses of the three TRBP
dsRBDs revealed that the first and second dsRBDs (dsRBD1 anddsRBD2) bind TAR RNA [17] and siRNA [18], [19], respectively,
while the third (dsRBD3) binds neither of these RNAs andinteracts with Dicer [9], [20]. Furthermore, TRBP forms
heterodimers with PKR via dsRBD1 and dsRBD2 without
dsRBD3, and inhibits the function of PKR [21]. However, PACT
was initially thought to be a PKR activator [22]. Either PACT
dsRBD1 or dsRBD2 are required for strong binding to PKR, anddsRBD3 is necessary and sufficient to activate PKR. Thus, PACT
and TRBP have opposite roles in PKR regulation, although their
PKR binding patterns are very similar. Meanwhile, PACT also
binds to Dicer through dsRBD3, and stimulates the cleavage ofdsRNA to siRNA [14] or enhances the function of siRNA strand

Introduction
RNA interference (RNAi) and related pathways are evolutionally conserved mechanisms whereby single-stranded guide RNA
triggers sequence-specific cleavage or translational repression of its
cognate mRNA through the RNA-induced silencing complex(RISC) [1–3]. Silencing is initiated by long double-stranded RNA(dsRNA) or hairpin-structured precursor-microRNA (premiRNA), which are processed by the RNaseIII enzyme Dicer toproduce 21–23 nucleotide (nt) short interfering RNAs (siRNAs) or
microRNAs (miRNAs), respectively [4–6]. These small RNAs arethen loaded onto Argonaute2 (Ago2), the slicer of target mRNA[7], [8]. In human cells, both Dicer and the trans-activation
response (TAR) RNA binding protein (TRBP) or the protein
activator of protein kinase R (PKR) (PACT) are associated with
RISC loading [8–10]. These proteins, along with Ago2, comprisethe core of a complex referred to as the RISC-loading complex(RLC) [11–13]. In human core RLC, a heterodimer containing
Dicer and either TRBP or PACT can sense the thermodynamic
asymmetry of siRNA ends in vitro [10]. Dicer interacts with the lessstable end of the duplex, and TRBP/PACT interacts with themore stable end. Within these heterodimeric complexes, siRNAs
undergo significant repositioning following dicing, thereby biasing
their orientation for guide strand selection according to the
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In Vitro siRNA-Binding Modes of TRBP and PACT

values of siLuc-36 and siLuc2-153 (5.30 and 4.00 nM, respectively), strongly suggesting that the nucleotides at 39 overhangs does
not affect the binding activity with TRBP, and TRBP mainly bindto a double stranded portion in siRNA. Consistent with theseresults, TRBP-WT protein did not bind the 21 nt double-stranded
DNA (dsDNA) (Figure S1G and H) and 21 nt single-stranded
RNA (Figure S1I and J).
A supershift analysis was performed to confirm that the complex
1 was indeed formed with 32P-labeled siLuc-36 or siLuc2-153, as
well as the TRBP-WT protein. The complex 1 (formed at 1.3 nM
TRBP-WT protein concentration) was supershifted by anti-myc
antibody, which recognizes the C-terminal myc-tag of TRBP-WT
protein, but not by control anti-Flag antibody (Figures 1E andS1B), indicating that the complex 1 is composed of 32P-labeledsiRNA and the TRBP-WT protein. The result that the complex 2
was formed from about 20 nM of TRBP-WT protein suggests thatthe complex 2 consists of the components contained in thecomplex 1 and additionally supplied TRBP-WT protein, because
TRBP-WT protein was abundant but the majority of free siRNAs
were absent when the complex 2 appeared (Figures 1B, S1A andC-F).
To identify components of the complexes 1 and 2 observed in
TRBP-WT EMSA experiments, we performed gel filtration
analysis using a Superdex 200 HR 10/300 column. Initially, weevaluated the elution pattern of siRNA on the gel filtration
column. When free siLuc-36 alone was applied to the column, asingle clear peak was detected (Figure 2A). The elution position
calculated based on the protein molecular weight markers
corresponded to a position of about 23 kDa protein. Because
siLuc-36 forms a single siRNA pair (,14 kDa) by agarose gelelectrophoresis (Figure 2A, inset), a 21 nt RNA duplex was likely
detected at a molecular weight of about 23 kDa (Figure 2A). Whenpurified TRBP-WT (1,300 nM) was pre-incubated with siRNA(300 nM), two peaks at 110 kDa and 23 kDa were observed(Figure 2B). Because the molecular weight of TRBP-WT
containing His- and myc-tags is approximately 44 kDa, the fastest
110 kDa peak of the ribonucleoprotein complex, corresponding tocomplex 2, may be composed of two molecules of TRBP-WT
protein (88 kDa) and a siRNA (23 kDa) pair as predicted by
EMSA experiments. This peak was detected between the positionof molecular weight size markers of 158 and 67 kDa, but TRBPWT protein band was observed alone at about 44 kDa by Western
blot (Figure 2B), suggesting that TRBP-WT proteins are actually
dimerized in the complex. Furthermore, to investigate thecomponent of complex 2 in more detail, non-labeled siRNA at
concentrations from 0 to 600 nM was incubated with the complex
2 (Figure 1F). According to the increase of additional siRNAconcentrations, the second step band of complex 2 was graduallydisappeared and the first step band of complex 1 became
prominent. These results suggest that, one molecule of siRNAfree TRBP-WT protein, which may not directly interact withsiRNA but dimerized with a TRBP-WT protein, in the complex 2
is capable of binding a siRNA. However, upon the siRNA-free
TRBP-WT protein binds to a siRNA, the TRBP-WT homodimers
were dissociated into the monomers consisting of the one moleculeof TRBP-WT and a siRNA, and free 32P-labeled siRNAs weredetected from about 600 nM concentration of non-labeled siRNAs
at which free siRNAs might be saturated. Thus, the affinity of
TRBP with siRNA is stronger than that for own dimerization. The
second peak in Figure 2B was found in the same elution position as
free siLuc-36, indicating that this peak contains free siRNA alone.
Although the complex 1 is substantially observed by EMSA at
TRBP protein concentrations lower than 330 nM (Figure 1B), we
could not detect the peak at about 67 kDa, which corresponding

selection by Dicer [10] in a very similar manner as TRBP.
Furthermore, both PACT and TRBP form homodimer by theinteraction within each of dsRBD1 and dsRBD2 domains, or in aninterchangeable manner [21]. However, the siRNA binding modeof PACT remains unclear.
To distinguish between TRBP and PACT in the RNA silencing
process, we compared the fundamental characteristics of human
TRBP and PACT for siRNA binding using electrophoresis
mobility shift analysis (EMSA) and gel filtration chromatography
in vitro. Here, we propose that TRBP and PACT have uniquesiRNA binding modes in vitro, although they have similar structuraland functional features of RNA silencing.

Results
Monomeric TRBP binds siRNA at low concentrations
To investigate the binding pattern of human TRBP protein tosiRNA, a recombinant N-terminal His- and C-terminal myctagged wild-type TRBP protein (TRBP-WT) was expressed in
Escherichia coli (E. coli) Rosetta (DE3) pLysS and purified on NiNTA agarose. This recombinant TRBP-WT protein was soluble
and the homogeneity was evaluated by SDS-PAGE (Figure 1A).
The binding ability of TRBP-WT protein to siRNA (0.50 nM) was
examined by EMSA with 21 nt siRNA containing a 32P-labeled
guide strand. The migrating ribonucleoprotein complexes composed of TRBP-WT protein and siRNA against firefly luciferase(siLuc-36) were obtained in two steps with TRBP-WT concentrations ranging from 0 to 1,300 nM (Figure 1B). We referred thecomplex in the first binding step as complex 1 and that in thesecond binding step as complex 2.
The same experiments were performed using the other fivesiRNAs against firefly luciferase (siLuc2-153, siLuc-30, siLuc-48,
siLuc-18, and siLuc-1430) with different sequences (Figure S1A,
C–F). In our previous reports, siLuc-36 and siLuc2-153 werecategorized into highly functional siRNAs, siLuc-30 and siLuc-48
were siRNAs with intermediate degrees of silencing efficiencies,
and siLuc-18 and siLuc-1430 were not functional with little or nosilencing activities [23]. Regardless of their silencing efficiencies,
all siRNAs showed similar binding patterns to the highly
functional siRNAs, siLuc-36 (Figure 1B) and siLuc2-153 (Figure S1A). The siRNA-binding curve derived from the EMSA
experiments performed at TRBP-WT concentrations of 0 to
1,300 nM revealed dissociation constants (Kd) for the formation ofcomplex 1 were ranged from 5.30 to 33.0 nM (mean 10.0 nM),
and from 44.0 to 440 nM (mean 203 nM) for the formation ofcomplex 2 (Table 1). These results indicate that TRBP-WT
binding efficiencies to siRNAs are slightly varied in a sequencedependent manner but not necessarily reflecting the silencing
efficiencies. TRBP is shown to sense the thermodynamic
asymmetry of siRNA duplex ends in vitro and interacts with morestable end of the duplex [24]. Then we examined the effects of 2 nt
39 overhang nucleotides in both terminals using 21 nt dsRNAs
with blunt ends (siLuc-36B and siLuc2-153B) and siRNAs
containing DNAs at both 39 overhangs (siLuc-36D and siLuc2153D). The binding patterns of siLuc-36B and siLuc2-153B to
TRBP-WT were similar to those of siLuc-36 and siLuc2-153(Figures 1C and S1K) and the Kd values of siLuc-36B and siLuc2153B calculated from siRNA-binding curves were 4.50 and
5.10 nM, respectively, for the formation of complex 1 (Table 1).
siLuc-36D and siLuc2-153D also showed similar binding patterns
to those of siLuc-36 and siLuc2-153 (Figures 1D and S1L) andtheir Kd values for the complex 1 formation were 11.0 and
5.50 nM, respectively (Table 1). The Kd values of blunt-ended
dsRNAs and siRNAs with DNAs at 39 overhangs were close to the
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Figure 1. Purification and EMSA of TRBP-WT protein. (A) Coomassie brilliant blue stained pattern of purified TRBP-WT protein resolved by SDSPAGE. Arrowhead indicates 44 kDa of TRBP-WT protein. M represents the protein size markers. (B–D) The results of EMSA of TRBP-WT protein with
32
P-labeled siLuc-36 (B), siLuc-36B (C), and siLuc-36D (D). 32P-labeled siRNA (0.50 nM) was incubated with increasing amounts of TRBP-WT protein, as
indicated. Lower cases in siLuc-36D sequence in D indicate DNAs. (E) Supershift analysis of TRBP-WT protein (1.3 nM) with no antibodies, control antiFlag, and anti-myc antibodies. (F) The result of EMSA of TRBP-WT protein (1,300 nM) mixed with 32P-labeled siLuc-36 (0.50 nM) incubated withincreasing amount of non-labeled siLuc-36. In B–F, arrows indicate positions of the first and second step migrating complexes, corresponding to
TRBP-WT complexes 1 and 2, respectively, and the supershifted complex, in addition to siRNA and ATP.
doi:10.1371/journal.pone.0063434.g001

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Table 1. The Kd values (nM) of TRBP-WT and PACT-WT and their mutants.

Purified
protein

TRBP-WT

complex

Highlyfunctional

Intermediate

Not functional

DNA at 39
overhang**

Blunt-ended*siLuc36

siLuc2- siLuc153
30

siLuc48

siLuc18

siLuc1430

Mean

siLuc36B

siLuc2153B

siLucMean 36D

siLuc2153D

Mean

complex 1

5.30

4.00

6.20

33.0

5.80

5.70

10.0

4.50

5.10

4.80

11.0

5.50

8.25

complex 2

44.0

440

81.0

430

93.0

130

203

60.0

74.0

67.0

243

133

188

TRBPdsRBDmt1

complex

250

260

NT

NT

NT

NT

255

NT

NT

NT

NT

NT

NT

TRBPdsRBDmt2

complex

370

520

NT

NT

NT

NT

445

NT

NT

NT

NT

NT

NT

TRBPdsRBDmt1+2

complex

ND

ND

NT

NT

NT

NT

ND

NT

NT

NT

NT

NT

NT

TRBPDdsRBD3

complex 1

5.00

12.0

NT

NT

NT

NT

8.50

NT

NT

NT

NT

NT

NT

complex 2

180

220

NT

NT

NT

NT

200

NT

NT

NT

NT

NT

NT

PACT-WT

complex 2

220

401

NT

NT

NT

NT

311

222

401

312

1030

502

766

PACTdsRBDmt1

complex 2

3900

1900

NT

NT

NT

NT

2900

NT

NT

NT

NT

NT

NT

PACTdsRBDmt2

complex 2

5700

4600

NT

NT

NT

NT

5150

NT

NT

NT

NT

NT

NT

PACTdsRBDmt1+2

complex

ND

ND

NT

NT

NT

NT

ND

NT

NT

NT

NT

NT

NT

PACTDdsRBD3

complex 1

1500

810

NT

NT

NT

NT

1160

NT

NT

NT

NT

NT

NT

complex 2

ND

ND

NT

NT

NT

NT

ND

NT

NT

NT

NT

NT

NT

*and ** indicate 21nt dsRNA with blunt ends and siRNA with 2nt DNAs at both 39 overhangs, respectively.
ND = not determined.
NT = not tested.
doi:10.1371/journal.pone.0063434.t001

to the complex 1, by gel filtration under the concentration of
330 nM TRBP-WT, probably due to the protein concentration is
below the detection limit. However, when purified TRBP-WT at alow concentration of 5.1 nM was pre-incubated with siLuc-36, the
TRBP-WT protein was detected by Western blot in the fraction 14
which includes the proteins with molecular weight of about 48–
75 kDa (Figure 2C). Thus, this complex might be actually complex
1 containing a TRBP monomer bound to the siRNA.

activities observed at 330 and 1,300 nM of TRBP-dsRBDmt1+2
protein concentrations in Figure 3E might be the results of weakbinding activities of mutated dsRBD1/dsRBD2, or dsRBD3.
Although we tried to construct the expression construct with
mutations in dsRBD1 and dsRBD2 in addition to the deletion ofdsRBD3 (TRBP-dsRBDmt1+2D3) to investigate the contributionof each dsRBDs to the weak binding of TRBP-dsRBDmt1+2
protein, we could not purify TRBP-dsRBDmt1+2D3 protein
because of its high instability. TRBP-DdsRBD3 showed binding
patterns similar to TRBP-WT in two steps from 0.32 and 81 nM,
respectively (Figures 3F and S2D). Gel filtration chromatography
revealed a primary peak at 1,300 nM TRBP-DdsRBD3 protein
concentration was detected at 90 kDa (TRBP-DdsRBD3 complex
2, Figure 2D). The fastest peak was observed between the positionof molecular weight size markers of 158 and 67 kDa, but proteinband was observed alone at about 37 kDa by Western blot,
indicating that this peak might contain two molecules of TRBPDdsRBD3 (,37 kDa) and a single siRNA pair, and the ,27 kDapeak was free siRNA (Figures 2D and 3F). As shown in TRBP-WT(Figure 1F), non-labeled siRNAs at concentrations from 0 to
600 nM was also incubated with TRBP-DdsRBD3 complex 1(Figure 3H). The migrating band of complex 2 was graduallydisappeared and that of complex 1 became prominent according
to the increase of siRNA concentrations in an analogous fashion
with TRBP-WT. Furthermore, free 32P-labeled siRNAs wereappeared from about 600 nM concentration of non-labeledsiRNAs. Thus, one molecule of siRNA-free TRBP-DdsRBD3
protein in the complex 2 (Figure 3F) is also capable of binding to asiRNA. However, upon the siRNA-free TRBP-DdsRBD3 protein

siRNA binding by TRBP with mutations in dsRBD1 and/or
dsRBD2, and deletion of dsRBD3
The roles of the three TRBP dsRBDs in siRNA binding were
examined using purified N-terminal His- and C-terminal myctagged TRBP proteins with mutations in dsRBD1 and/or
dsRBD2, and a deletion of dsRBD3, designated as TRBPdsRBDmt1, TRBP-dsRBDmt2, TRBP-dsRBDmt1+2, and TRBPDdsRBD3, respectively (Figure 3A). The homogeneities of theserecombinant proteins were examined using SDS-PAGE(Figure 3B). TRBP-dsRBDmt1, in which lysine residues at 80
and 81 in dsRBD1 were replaced with alanines, could not bind tosiLuc-36 (Figure 3C) and siLuc2-153 (Figure S2A) at low protein
concentration and bound to them in one step at high protein
concentration. TRBP-dsRBDmt2 substituted with alanines at
positions 210 and 211 also showed one-step binding patterns athigh protein concentration (Figures 3D and S2B). TRBPdsRBDmt1+2 with mutations in both dsRBD1 and dsRBD2
virtually could not bind to siLuc-36 (Figure 3E) or siLuc2-153(Figure S2C), indicating that dsRBD3 does not play a role indsRNA binding, as reported previously [25]. The slight binding
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Figure 2. Gel filtration chromatography of purified TRBP-WT and TRBP-DdsRBD3 proteins with siRNA. Gel filtration chromatography
patterns of non-labeled 300 nM of siLuc-36 alone (A), siLuc-36 with 1,300 nM of TRBP-WT (B), siLuc-36 with 5.1 nM of TRBP-WT(C) and siLuc-36 with1,300 nM of TRBP-DdsRBD3 (D). In A, inset, siLuc-36 was electrophoresed on a 3% agarose gel and stained with EtBr. Lower panels in B-D showed theresults of Western blot (WB) by anti-myc antibody for detecting TRBP proteins in the elution fractions. Histograms below WBs showed the quantified
signal densities of TRBP proteins detected by WB. Arrowheads indicate the positions of molecular weight size markers. In B, each fraction of molecularweight size markers was quantified and represented in histogram after staining the gel with Coomassie brilliant blue.
doi:10.1371/journal.pone.0063434.g002

binds to a siRNA, the TRBP-DdsRBD3 homodimers weredissociated into the monomers consisting the one molecule of
TRBP-DdsRBD3 and a siRNA. Their Kd values calculated fromsiRNA-binding curves were about 8.50 and 200 nM, respectively(Table 1), similar to the wild type. However, the fastest migrating
bands of complex 1 were rather broad, probably because the
TRBP-DdsRBD3 protein was slightly unstable (see Figure 3B).

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Mobilities of the ribonucleoprotein complexes composed ofsiRNA and TRBP-WT or its dsRBD mutant proteins werecompared on a native gel at 1,300 nM concentrations (Figure 3G).
Although the molecular weight of TRBP-WT is similar to TRBPdsRBDmt1 and TRBP-dsRBDmt2, neither complex showed thesame mobility, which were rather slow compared to TRBP-WT.
This suggests that the complexes containing TRBP-dsRBDmt1 or
TRBP-dsRBDmt2 might be multimerized. The EMSA experi-

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step from protein concentrations of 160 nM (complexes 2 inFigures 5B and S4D). PACT-dsRBDmt2 replaced with alanines at
177 and 178 also showed a single band from protein concentrationof 630 nM (complexes 2 in Figures 5C and S4E) with rather weakaffinity compared to PACT-dsRBDmt1. PACT-dsRBDmt1+2
with mutations in both dsRBD1 and dsRBD2 had very low
siRNA binding affinity if at all (Figures 5D and S4F), whichsuggests that dsRBD3 is not responsible for siRNA binding in
PACT protein. However, the EMSA experiment using PACTDdsRBD3 protein represented that two different migrating bands
appeared simultaneously at 81 nM, and the fastest band (complex
1) was predominant, and the slowest band (complex 2) was
subsidiary (Fig. 5E and S4G). Their average Kd values were1,160 nM (Table 1). The mobility of the ribonucleoprotein
complexes composed of siRNA and PACT-WT or its dsRBD
mutant proteins were compared on a native gel at protein
concentrations of 1,300 nM (Figure S4H). The mobilities of thecomplexes 2 composed of PACT-WT, PACT-dsRBDmt1, and
PACT-dsRBDmt2 with siRNA were similar. However, themobility of complex 1 containing PACT-DdsRBD3 protein withsiRNA was different to that of complex 2 of PACT-WT and theother mutant proteins, and the mobility of PACT-DdsRBD3
complex 2 was similar to them.
Gel filtration chromatography revealed that PACT-WT,
PACT-dsRBDmt1 and TRBP-dsRBDmt2 proteins formed complexes with similar molecular weights of 130 kDa (Figure 6A–C).
Although these fastest peaks, probably corresponding to complexes
2, were observed between the positions of molecular weight size
markers of 158 and 67 kDa, but protein bands detected by SDSPAGE were shown at about 40 kDa (Figures 4A and 6A–D),
indicating that more than one molecules of PACT-WT proteinsare contained in the complex. The molecular weight of PACTWT, PACT-dsRBDmt1 and PACT-dsRBDmt2 proteins areapproximately 40 kDa, so it is possible that two molecules ofeach of PACT-WT, PACT-dsRBDmt1 and PACT-dsRBDmt2
proteins (,80 kDa) and two molecules of siRNAs (46 kDa) arecontained in the complex 2. However, calculation of the numberof PACT-WT or PACT-DdsRBD3 proteins interacted with one
molecule of siRNA using their molar absorptivities suggested thatthe two molecules of PACT-WT proteins interact with one
molecule of siRNA in complex 2. Thus, we could not determine
the accurate number of siRNA molecules contained in thecomplex 2. The PACT-WT dimer in the complex 2 may interact
with one or two molecules of siRNA(s). The PACT-DdsRBD3
mutant protein and siRNA showed two major peaks, and themolecular weight of the fastest major peak was 64 kDa, indicatingthat the complex might be composed of one molecule of 33 kDa of
PACT-DdsRBD3 and a 23 kDa siRNA, while the second peak
represented free siRNA (Figure 6D). In addition, a negligible small
peak was also observed at about 130 kDa. This band mightprobably correspond to the complex 2 in Figure 5E. Thus, thecomplex 1 is ascertained to be a major complex of the PACTDdsRBD3 mutant protein and siRNA.
As shown in TRBP-WT and TRBP-DdsRBD3 proteins, PACTWT and PACT-DdsRBD3 proteins at 1,300 nM were incubatedwith non-labeled siRNA at concentrations from 0 to 600 nM after
incubation with 32P-labeled siRNA (0.50 nM) (Figure 5F and G).
Unlike TRBP, the amounts of complex 2 in Figure 5F, andcomplexes 1 and 2 in Figure 5G gradually decreased, whereas theamount of free siRNA increased, according to the increase of nonlabeled siRNA concentrations. The results suggest that thecomplex 2 of PACT-WT or PACT-DdsRBD3 is already filledwith siRNA: the PACT-WT complex 2 includes two molecules of
PACT proteins and each of them might contain a siRNA, whilements also suggested that TRBP-dsRBDmt1 and TRBPdsRBDmt2 complexes formed aggregates of large molecularweight (Figures 3C and D, and S2A and B). Gel filtration
chromatography revealed that TRBP-dsRBDmt1 and TRBPdsRBDmt2 readily form aggregates at increasing protein concentrations (Figure S3). Next, the TRBP-dsRBDmt1 and TRBPdsRBDmt2 proteins at concentrations from 330 to 1,300 nM wereincubated with siRNA. The molecular weights of gel filtration
peaks increased from 180 to 250 kDa for TRBP-dsRBDmt1(Figure S3A), and from 120 to 170 kDa for TRBP-dsRBDmt2(Figure S3B). However, the gel filtration peaks of TRBP-WT and
TRBP-DdsRBD3 proteins with siRNA did not increase, evenwhen their protein concentration was increased (Figure S3C and
D). These results suggest that TRBP-dsRBDmt1 and TRBPdsRBDmt2 form aggregates more easily than TRBP-WT and
TRBP-DdsRBD3, possibly because the mutated dsRBD1 anddsRBD2 domains are easy to associate when their interaction withsiRNA is abolished.

Dimerized PACT binds to siRNA at high concentrations
Both PACT and TRBP bind to Dicer, and are thought to be
involved in the RNA silencing pathway. Furthermore, the aminoacid sequences of dsRBD1, dsRBD2, and dsRBD3 in TRBP share
56%, 59%, and 58% identities and 76%, 83%, and 75%
similarities, respectively, with those of the corresponding PACT
domains.
To compare the dsRNA binding mode of PACT protein withthat of TRBP protein, a recombinant N-terminal His- and Cterminal myc-tagged PACT protein (PACT-WT) was also
expressed in E. coli and purified by Ni-NTA agarose. Recombinant
PACT-WT protein was soluble and the homogeneity was
ascertained by SDS-PAGE (Figure 4A). The binding affinity of
PACT-WT protein to siRNA was examined by EMSA experiments with 32P-labeled siRNA. The single migrating band
(referred to as complex 2) was observed starting at 20 nM
concentration of PACT-WT protein with siLuc-36 (Figure 4B) or
siLuc2-153 (Figure S4A). The siRNA-binding curve derived fromthe EMSA experiments performed with PACT-WT at concentrations of 0–1,300 nM indicated that the average Kd value was
331 nM, similar to the second binding step band of TRBP-WT
protein (Table 1). As shown in the experiment using TRBP protein(Figure 1C and D), we performed EMSA with 21 nt dsRNA withblunt ends (siLuc-36B and siLuc2-153B) and siRNA containing
2 nt DNAs at both 39 overhangs (siLuc-36D and siLuc2-153D) toexamine the effects of 2 nt single-stranded RNA at 39 overhangson the binding activity with PACT protein. Single migrating bands
were observed by siLuc-36B and siLuc2-153B and their Kd values
were 222 and 401 nM, respectively, for the formation of complex
2 (Table 1), closely similar to the results of siLuc-36 and siLuc2153 (Figures 4C and S4B). siLuc-36D and siLuc2-153D also
showed similar binding patterns to those of siLuc-36 and siLuc2153 (Figures 4D and S4C) and their Kd values were almost similar
or slightly high at 502 nM (siLuc2-153D) and 1,030 nM (siLuc36D), respectively, for the formation of complex 2 (Table 1),
suggesting that 2 nt DNAs at 39 overhangs of siRNAs may notaffect the binding activity with PACT, if any.
The role of three dsRBDs in PACT for siRNA binding were
examined using purified N-terminal His- and C-terminal myctagged PACT protein with mutations in dsRBD1 and/or dsRBD2,or a deletion of dsRBD3, which were termed as PACTdsRBDmt1, PACT-dsRBDmt2, PACT-dsRBDmt1+2, and
PACT-DdsRBD3, respectively, as shown in Figure 5A. PACTdsRBDmt1, in which lysine residues at 84 and 85 in dsRBD1 were
replaced with alanines, bound to siLuc-36 and siLuc2-153 in one
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In Vitro siRNA-Binding Modes of TRBP and PACT

Figure 3. Structure, purification and EMSA of TRBP mutant proteins. (A) Schematic representation of TRBP-WT and its mutant proteins.
Amino acids at positions 80 and 81 were substituted from lysines (KK) to alanines (AA) in TRBP-dsRBDmt1, and those at 210 and 211 were also
substituted from KK to AA in TRBP-dsRBDmt2. TBRP-dsRBDmt1+2 contains both substitutions. The dsRBD3 was deleted in TRBP-DdsRBD3. All
mutants were tagged with N-terminal His and C-terminal myc, in the same way as TRBP-WT. (B) Coomassie brilliant blue stained patterns of purified
TRBP mutant proteins resolved by SDS-PAGE. Arrows indicate the migration position of TRBP-dsRBDmt1, TRBP-dsRBDmt2, and TRBP-dsRBDmt1+2.
Arrowhead indicates TRBP-DdsRBD3 protein. M represents the protein size markers. (C–F) EMSA patterns of TRBP-dsRBDmt1 (C), TRBP-dsRBDmt2 (D),TRBP-dsRBDmt1+2 (E) and TRBP-DdsRBD3 (F) with 32P-labeled siLuc-36. 32P-labeled siRNA (0.50 nM) was incubated with increasing amounts of TRBP
protein, as indicated. (G) The comparison of mobilities of siRNA complexes with TRBP-WT, TRBP-dsRBDmt1, TRBP-dsRBDmt2, TRBP-dsRBDmt1+2 and
TRBP-DdsRBD3 proteins (1,300 nM), respectively. (H) EMSA pattern of TRBP-DdsRBD3 protein (1,300 nM) mixed with 32P-labeled siLuc-36 (0.50 nM)
incubated with increasing amount of non-labeled siLuc-36.
doi:10.1371/journal.pone.0063434.g003

ating into monomer, no additional siRNA could not be
incorporated into the complex 2. The dsRBD3 domain, which isone of the essential regions for dimerization, was deleted in the
PACT-DdsRBD3 protein, so their dimerized form might be very
unstable, and equilibrium state of complexes 1 and 2 observed in
Figure 5E showed that complex 1 is more stable compared tocomplex 2. Thus, these results suggest that the intermolecular

PACT-DdsRBD3 complex 1 composed of one molecule of PACTDdsRBD3 and a siRNA. Then, only the replacements of 32Plabeled siRNA to non-labeled siRNA occurred in the complexes 1
and 2 according to the increase of additional non-labeled siRNAconcentrations. Alternatively, the complex 2 might contain two
molecules of PACT-WT proteins and one molecule of siRNA.
However, because PACT-WT dimer constructed very stable
dimer formation and cannot incorporate siRNA without dissoci-

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In Vitro siRNA-Binding Modes of TRBP and PACT

interactions due to dsRBD3 in PACT proteins are stronger
compared to the interaction between PACT and siRNA.

concentrations starting from 20 nM (Figures 5E and S4G).
Because the molecular weight of the PACT-DdsRBD3 complex
1 was about 64 kDa (Figure 6D), this complex representmonomeric PACT-DdsRBD3 protein (33 kDa) binding to one
molecule of siRNA, while the slowest PACT-DdsRBD3 complex 2
represent the dimeric form binding one or two siRNA molecules(Figure 5E). Although dsRBD3 was not essential for siRNAbinding, dsRBD3 is important for PACT homodimerization asshown previously [27]. Because the monomeric form (PACTDdsRBD3 complex 1) was predominant (see Figures 5E and S4G),
siRNA binding of monomeric PACT-DdsRBD3 might be morestable than dimeric PACT-DdsRBD3 binding to siRNA.
The fastest migrating complex 1, shown in EMSA of TRBPWT and siRNA (Figure 1B), was not observed when TRBPdsRBDmt1 and TRBP-dsRBDmt2 were used (Figure 3C and D).
This suggests that both dsRBD1 and dsRBD2 in TRBP bindsiRNA simultaneously in the TRBP-WT complex 1, while themutated dsRBD1 and dsRBD2 of TRBP could not bind siRNA.
As shown in Figure S3, TRBP-dsRBDmt1 and TRBP-dsRBDmt2
readily form aggregates at increasing protein concentrations. The
molecular weights of aggregated protein sizes were larger for
TRBP-dsRBDmt1 compared to TRBP-dsRBDmt2 at the same
concentration, indicating that the interaction in dsRBDmt1
domains might be more stable than that in the TRBP-dsRBDmt2
domains.
PACT-dsRBDmt1 and PACT-dsRBDmt2 formed a complex
with siRNA in EMSA (PACT-dsRBDmt1 complex 2 and PACTdsRBDmt2 complex 2) at high protein concentrations startingfrom 160 nM (Figure 5B and C). However, PACT-WT formed
PACT-WT complex 2 at concentrations as low as 5.1 nM(Figure 4B), suggesting that both dsRBD1 and dsRBD2 in PACT
protein bind siRNA simultaneously in the PACT-WT complex 1,
while the mutated dsRBD1 and dsRBD2 of PACT could not bindsiRNA, similar to TRBP. However, PACT-dsRBDmt1 and
PACT-dsRBDmt2 did not form large aggregates (Figure 6B andC) even when the protein concentration increased, unlike the
TRBP-dsRBDmt1 and TRBP-dsRBDmt2 proteins. This suggeststhat the PACT homodimer is very stable probably due to the rigid
interaction via dsRBD3 domains.
We found that the binding modes of human TBRP and PACT
to siRNA are unique, although they have similar structuralfeatures and contain three dsRBDs (Figure 7). Monomeric TRBP
protein can bind siRNA with dsRBD1 and dsRBD2 (Figure 7A).
However, at higher concentrations of TRBP-WT proteins, TRBPWT formed dimers in which one molecule of proteins probably
did not interact with siRNA. However, when excess amounts ofsiRNAs were added, TRBP-WT dimer was dissociated intomonomeric form containing a single molecule of siRNA. Thus,
TRBP-WT have the higher affinity with siRNA compared to theaffinity of own dimerization. A similar binding pattern was also
observed in the TRBP protein lacking dsRBD3, namely, TRBPDdsRBD3 (Figure 7B). In contrast, because PACT-WT protein
did not bind siRNA as a monomer, PACT is likely to have thehigher dimerization affinity compared to that for siRNA-binding
via a stable interaction between own dsRBD3s (Figure 7C).
However, PACT protein lacking dsRBD3 can bind siRNA as
either a monomer or dimer (Figure 7D), similar to TRBPDdsRBD3 protein. In addition, the affinity of siRNA binding of
TRBP-DdsRBD3 (mean 8.50 nM, Table 1) was considerably
higher compared to that of PACT-DdsRBD3 (mean 1,160 nM),
indicating that the siRNA binding affinities of dsRBD1 anddsRBD2 of TRBP protein are significantly strong. Thus, TRBP
may be readily incorporated into the gene-silencing pathway
triggered by small RNAs.

Discussion
Our results clearly show that TRBP-WT and PACT-WT
proteins bind siRNA, although their binding patterns based on
EMSA differed (Figures 1B, 4B, S1A, C-F and S4A). The dsRBD1
or dsRBD2 mutant, and the dsRBD deletion mutant (TRBPdsRBDmt1, TRBP-dsRBDmt2, and TRBP-DdsRBD3, PACTdsRBDmt1, PACT-dsRBDmt2, and PACT-DdsRBD3) also
bound siRNA (Figures 3C, D, F, 5B, C, E, S2A, B, D, S4D, E
and G). However, TRBP-dsRBDmt1+2 with mutations in bothdsRBD1 and dsRBD2 showed little binding activities to siRNA if
at all (Figures 3E and S2C), as shown in the previous report [17],[19]. We revealed that PACT-dsRBDmt1+2 also showed little
binding activities to siRNA (Figures 5D and S4F), indicating that
PACT proteins also bind siRNA with dsRBD1 and dsRBD2, while
dsRBD3 is not involved in siRNA binding. The mean Kd values of
TRBP-dsRBDmt1 and TRBP-dsRBDmt2 binding to siRNA were
255 and 445 nM, respectively, and those of PACT-dsRBDmt1
and PACT-dsRBDmt2 were 2,900 and 5,150 nM, respectively(Table 1), indicating that dsRBD2 has a higher affinity to siRNAthan dsRBD1.
TRBP-WT bound siRNA in two sequential steps (Figures 1B
and S1A). During the first binding step, TRBP-WT formed
complex 1 with siRNA at high affinities at concentrations as low as
0.32 nM. The TRBP-WT complex 2 formed at lower affinities at
20 nM protein concentrations. According to the increase in
TRBP-WT concentration, the amount of complex 1 gradually
decreased while complex 2 increased. Gel filtration chromatography and Western blot analysis suggested that complex 1 consistedof monomeric TRBP-WT, which binds a siRNA (Figures 1B,
2C and S1A), while complex 2 composed of two molecules of
TRBP-WT, either one of TRBP-WT proteins is considered tobind a single molecule of siRNA, although we cannot omit thepossibility that TRBP binds to two separate sites on the siRNA.
Interestingly, when increasing amounts of siRNAs were added tothe complex 2, the migrating band of TRBP-WT protein was
shifted to the position of complex 1, suggested that the siRNA-free
TRBP-WT protein contained in the homodimer of complex 2
dissociated to the monomers of complex 1 by incorporating siRNA(Figure 1F). TRBP is known to form homodimers via dsRBD1 anddsRBD2 without dsRBD3 [14]. Our results suggested that theaffinity of TRBP binding to siRNA is higher than its affinity forhomodimerization. Their Kd values were 5.30,33.0 nM forcomplex 1 and 44.0,440 nM for complex 2 (Table 1). Althoughthese values were slightly higher than previously reported values of0.24 [19] or 0.77 [26] nM for complex 1 equivalent and 13.3 nM[19] for complex 2 equivalent, this may be derived from thedifferent experimental conditions such as protein preparation,buffer composition, experimental method (EMSA or isothermal
titration calorimetry), protein tags (His/Myc-tag, His-tag, or MBPtag), and/or siRNA sequence.
PACT-WT protein bound to siRNA in one step (Figures 4B and
S4A). PACT-WT could not form a complex with siRNA at low
protein concentrations, but formed complexes at high concentrations starting from 20 nM (PACT-WT complex 2). This complex
was likely to be formed with dimeric PACT-WT protein binding
to one or two siRNA molecule(s) by gel filtration chromatography
and Western blot analysis (Figure 6A), suggesting that affinity of
PACT-WT dimerization is higher than that of siRNA binding
affinity. However, when dsRBD3 was deleted, the PACTDdsRBD3 protein formed two complexes at high protein
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In Vitro siRNA-Binding Modes of TRBP and PACT

Figure 4. Purification of PACT-WT and its mutant proteins, and EMSA of PACT-WT protein. (A) Coomassie brilliant blue stained pattern ofpurified PACT-WT (left panel), and its mutant proteins (right panel) resolved by SDS-PAGE. Arrows indicate the migration position of PACT-WT, PACTdsRBDmt1, PACT-dsRBDmt2, and PACT-dsRBDmt1+2 proteins. Arrowhead indicates PACT-DdsRBD3 protein. M represents the protein size markers.
(B–D) EMSA patterns of PACT-WT with 32P-labeled siLuc-36 (B), siLuc-36B (C), and siLuc-36D (D). 32P-labeled siRNA (0.50 nM) was incubated withincreasing amounts of PACT-WT as indicated. Lower cases in D indicate DNAs.
doi:10.1371/journal.pone.0063434.g004

differences in the binding modes of TRBP and PACT proteins incomparison. Although we did not carry out the experiments forinvestigating TRBP and PACT binding to miRNAs and hairpin
precursor miRNAs, the results might have important biologicalsignificance. Our results could offer a suggestion that the binding
modes of TRBP and PACT to miRNAs, which often containbulges or internal loops, might be different from those to siRNA,since TRBP and PACT proteins could not bind to single-stranded
RNAs. TRBP is shown to affect the kinetics or sites of precursor
miRNA processing by Dicer in RNA-structure-specific manner[29]. It is likely that these effects might be the results of different
binding modes of TRBP depending on the structures of miRNAs.

In vivo, the TRBP-PACT-Dicer interactions (or other factors)
may regulate the affinity of siRNA binding of TRBP and PACT
under various circumstances. Although TRBP and PACT are
known to directly interact with Dicer [14], monomeric TRBP
binding and dimeric PACT binding to siRNA may result in the
differences in the RISC-loading step of small RNA, the formation
and/or stability of RLC, or the cleavage reaction of small RNA by
Dicer [28], [29]. Furthermore, phosphorylation of serine 287 in
PACT increases its affinity for PKR by weakening PACT’sinteraction with TRBP. PKR activation by PACT leads tophosphorylation of translation initiation factor eIF-2a, which
reduces protein synthesis [30–32]. Phosphorylation of TRBP
mediated by the mitogen-activated protein kinase (MAPK) Erk is
known to enhance miRNA production by increasing the stabilityof the Dicer-TRBP complex [33]. These factors may regulate thesiRNA binding affinity of TRBP and PACT in vivo.
In this study, we performed the most simple experiments usingsiRNAs with completely-matched base-pairings to examine the
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Materials and Methods
Oligonucleotides
Guide strand and passenger strand RNA oligonucleotides for
EMSA and gel filtration experiments were chemically synthesized

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In Vitro siRNA-Binding Modes of TRBP and PACT

Figure 5. Structure and EMSA of PACT-WT and PACT mutant proteins. (A) Schematic representation of PACT-WT and its mutant proteins.
Amino acids at positions 84 and 85 were substituted from lysines (KK) to alanines (AA) in PACT-dsRBDmt1, and those at 177 and 178 were also
substituted from KK to AA in PACT-dsRBDmt2. PACT-dsRBDmt1+2 contained both substitutions. The dsRBD3 was deleted in PACT-DdsRBD3. All
mutants were tagged with N-terminal His and C-terminal myc, in the same way as PACT-WT. (B–E) EMSA patterns of PACT-dsRBDmt1 (B), PACTdsRBDmt2 (C), PACT-dsRBDmt1+2 (D), and PACT-DdsRBD3 (E) with 32P-labeled siLuc-36. (F, G) EMSA patterns of PACT-WT (F) and PACT-DdsRBD3 (G)(1,300 nM) mixed with 32P-labeled siLuc-36 (0.50 nM) incubated with increasing amount of non-labeled siLuc-36.
doi:10.1371/journal.pone.0063434.g005(Proligo), as shown in Table S1, and purified by polyacrylamide
gel electrophoresis. The guide strand was labeled with [c32P] ATP(6,000 Ci/mmol; Perkin Elmer). Hybridizations were performed
by incubating at 95uC for 5 min, and annealed products were
examined using 15% polyacrylamide gel electrophoresis in TBE
buffer, which can separate 21 bp double-stranded siRNA from
21 nt single-stranded RNA. Almost all RNA was recovered as
dsRNA.

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Plasmid construction
Fragment encoding a full length human TRBP was amplified
using a cDNA mixture of total RNA extracted from human HeLa
cells by PCR with primers containing restriction enzyme sites of
HindIII and NotI. The amplified fragment was digested with thesame enzyme sites and cloned into pcDNA3.1-myc/His A vector(Invitrogen). C-terminal myc-tagged TRBP fragment was amplified from pcDNA3.1-TRBP using primer with restriction enzyme
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In Vitro siRNA-Binding Modes of TRBP and PACT

Figure 6. Gel filtration chromatography of purified PACT-WT and PACT mutant proteins with siRNA. Gel filtration chromatography
patterns of non-labeled siLuc-36 (300 nM) with PACT-WT (A), PACT- dsRBDmt1 (B), PACT-dsRBDmt2 (C), and PACT-DdsRBD3 (D). Lower panels
showed the results of Western blot (WB) by anti-myc antibody for detecting PACT proteins in the elution fractions. Histograms below WBs showed
the quantified signal densities of PACT proteins detected by WBs. Arrowheads indicate the positions of molecular weight size markers.
doi:10.1371/journal.pone.0063434.g006

PACT-dsRBD1 and 177 and 178 in PACT-dsRBD2, corresponding positions mutated in TRBP-dsRBD1 and TRBP-dsRBD2,
were replaced with alanines.
The used primers were shown in Table S2.

sites NheI and HindIII. The amplified fragments were digested
with same enzyme sites and cloned into pET-28a vector(Novagen).
The PACT CDS was also amplified from a cDNA mixture of
total RNA extracted from human HeLa cells by PCR using
primers with restriction enzyme sites of BamHI and SalI. The
amplified fragment was digested with same enzyme sites andcloned into pET-28a vector amplified using primers with same
enzyme sites.
For the mutational analysis of each dsRBDs of TRBP and
PACT, the site-directed mutagenesis was carried out. The lysine
residues at 80 and 81 in TRBP-dsRBD1 and 210 and 211 in
TRBP-dsRBD2 were replaced with alanines in accord with theprevious reports [17–19]. The lysine residues at 84 and 85 in

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Protein purification
The TRBP and PACT expression plasmids were transformed
into E. coli Rosetta (DE3) pLysS (Novagen), and cultured to an
OD600 of 0.6. After 6 h of induction in 0.3 mM isopropyl b-dthiogalactoside, cells were lysed by sonication and the hexahistidine-fusion proteins were purified with Ni-NTA agarose(QIAGEN) according to the manufacturer’s instruction. The
samples were exchanged to the buffer containing 20 mM Tris
(pH8.0) and 300 mM NaCl using PD-10 Desalting columns (GE11

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In Vitro siRNA-Binding Modes of TRBP and PACT

healthcare), and determined the concentration using Protein Assay(BIO-RAD).

increasing amounts of each protein as indicated. (B) Supershift
analysis of TRBP-WT (1.3 nM) with no antibodies, control antiFlag, and anti-myc antibodies. Lowercases in siLuc2-153 sequence
indicate DNAs. Arrows in A–F, K, and L indicate positions ofcomplexes 1, complexes 2, and supershifted complex, in addition
to siRNA and ATP.
(TIFF)

Electrophoresis mobility shift assay
EMSAs were performed in binding buffer with 20 mM Tris
(pH7.5), 150 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, 1 mM dithiothreitol (DTT), 50 ng/ml sonicated salmon sperm
DNA, and 0.4 U/ml RNasin (Promega). The purified fusionproteins were incubated with siRNA containing 32P-labeled guidestrand (0.5 nM) for 30 min on ice, and the samples were
electrophoresed on a 9% polyacrylamide gel and analyzed
quantitatively using FLA-2000 image analyzer (Fujifilm). For thesupershift analysis, anti-myc antibody or anti-Flag antibody (Cell
Signaling) were pre-incubated with purified proteins for 1 h on ice
and incubated with 32P-labeled siRNAs for 30 min on ice, thenanalyzed by polyacrylamide gel electrophoresis.

Figure S2 EMSAs of TRBP mutant proteins. EMSA
patterns of TRBP-dsRBDmt1 (A), TRBP-dsRBDmt2 (B), TRBPdsRBDmt1+2 (C), and TRBP-DdsRBD3 (D) with 32P-labeled
siLuc2-153 (0.50 nM), incubated with increasing amounts of
TRBP mutants, as indicated. Arrows indicate positions of the firstand second step migrating complexes 1 and 2 and supershifted
complex, in addition to siRNA and ATP.
(TIFF)
Figure S3 Gel filtration chromatography of purified
TRBP-dsRBDmt1, TRBP-dsRBDmt2, TRBP-WT, and
TRBP-DdsRBD3 proteins with siRNA. Gel filtration chromatography patterns of non-labeled siLuc-36 (300 nM) withincreasing amounts of TRBP-dsRBDmt1 (A) and TRBPdsRBDmt2 (B) (330, 650, 1,300 nM), TRBP-WT (C) and
TRBP-DdsRBD3 (D) (650, 1,300 nM). Red lines indicate thepositions of siRNA peaks. Blue dotted lines, the peaks of TRBPdsRBDmt1 (A), TRBP-dsRBDmt2 (B), TRBP-WT (C), and
TRBP-DdsRBD3 (D) with 330 nM siRNA. The shifted areas ofthese peaks in A and B were represented as blue dashed lines. Notethat the peaks in C and D were not shifted even when these
protein concentrations increased.
(TIFF)

Gel filtration chromatography
Gel filtration chromatography was performed in gel filtrationbuffer with 20 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA,
1 mM 2-mercaptoethanol, and 1 mM DTT. The purified fusionproteins were incubated with chemically synthesized siRNA(300 nM) for 30 min on ice, and samples were applied to a
Superdex 200 HR 10/30 column (GE healthcare) equilibrated ingel filtration buffer. The amount of siRNA was quantified bymeasuring the radioactive intensity of the migrated band separated
in PAGE. The protein molecular weight markers used were as
follows: 232 kDa, catalase; 158 kDa, aldolase; 67 kDa, albumin;
43 kDa, ovalbumin; 13.7 kDa, ribonulease A.

Western blot

Figure S4 EMSA patterns of PACT-WT and its mutant
proteins. (A–G) EMSA patterns of PACT-WT with 32P-labeled
siLuc2-153 (A), siLuc2-153B (B), and siLuc2-153D (C), and thoseof PACT-dsRBDmt1 (D), PACT-dsRBDmt2 (E), PACTdsRBDmt1+2 (F), and PACT-DdsRBD3 (G) with 32P-labeled
siLuc2-153. 32P-labeled siRNA (0.50 nM) was incubated withincreasing amounts of PACT-WT and its mutant proteins, as
indicated. Lower cases in C in siLuc2-153D sequence indicate
DNAs. Arrows indicate positions of the migrating complexes 1 and
2, in addition to siRNA and ATP. (H) Comparison of siRNAcomplex mobilities of PACT-WT, PACT-dsRBDmt1, PACTdsRBDmt2, PACT-dsRBDmt1+2 and PACT-DdsRBD3 protein(1,300 nM).
(TIFF)The elution fractions of gel filtration chromatography was
mixed with the equal volume of 2 6 SDS PAGE Sample Buffer(4% SDS, 0.1 M Tris-hydrochloric acid (HCl), [pH6.8], 12% 2mercaptoethanol, 20% glycerol, 0.01% bromophenol blue). Afterboiling for five minutes, the sample was separated by SDS-PAGE
and transferred to PVDF membrane using Trans-Blot Turbo
Transfer System (BIO-RAD). The membrane was blocked for
1 hour in TBS-T (20 mM Tris-HCl [pH7.5], 150 mM NaCl,
0.2% Triton X-100) supplemented with 5% DifcoTM Skim Milk
(Becton, Dickinson and Company), and incubated with 62,000diluted anti-myc antibody (Cell Signaling) at 4uC overnight. The
membrane was washed three times with TBS-T, and reacted with
620,000-diluted HRP-linked anti-rabbit antibody (GE healthcare)at room temperature for 1 h. After being washed three times with
TBS-T, the membrane was reacted with ECL Prime Western
Blotting Detection Reagent (GE healthcare), and visualized with
LAS3000 (FUJIFILM). Each band was quantified with Image
Gauge 4.0 (FUJIFILM).

Table S1 siRNA sequences used in this study.

(TIFF)
Table S2 PCR primers used in this study.

(TIFF)

Supporting Information

Acknowledgments

Figure S1 EMSAs of TRBP-WT protein with various

We thank Dr. Kaoru Saigo for helpful comments and discussion, andreading the manuscript.

siRNAs, dsDNA, and ssRNA. (A, C–L) The results of EMSAs
of TRBP-WT protein, with 32P-labeled siRNA siLuc2-153 (A),siLuc-30 (C), siLuc-48 (D), siLuc-18 (E), or siLuc-1430 (F),dsDNAs, dsDNA_Luc-36 (G), dsDNA_Luc2-153 (H), ssRNA_Luc-36 (I), ssRNA_Luc2-153 (J), siLuc2-153B (K), and siLuc2153D (L). 32P-labeled siRNA (0.50 nM) was incubated with

Author Contributions
Conceived and designed the experiments: TT TM SZ KN MT KUT.
Performed the experiments: TT TM. Wrote the paper: TT KUT.

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Figure 7. Model of TRBP and PACT binding to siRNA. (A, B) TRBP-WT (A) or TRBP-DdsRBD3 (B) binds one molecule of siRNA as a monomer at
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