5256–5269 Nucleic Acids Research, 2014, Vol. 42, No. 8
doi:10.1093/nar/gku153

Published online 20 February 2014

Interactions between the non-seed region of siRNAand RNA-binding RLC/RISC proteins, Ago and
TRBP, in mammalian cells
Tomoko Takahashi1, Shuhei Zenno2, Osamu Ishibashi3, Toshihiro Takizawa3,
Kaoru Saigo1 and Kumiko Ui-Tei1,*1Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan, 2Department of Biotechnology, Faculty of Engineering, Maebashi Instituteof Technology, 460-1 Kamisadori-cho, Maebashi-shi, Gunma 371-0816, Japan and 3Department of Molecular
Medicine and Anatomy, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan

ABSTRACTINTRODUCTION

Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing
in various organisms. We previously showed that
8-nt-long 50 proximal nucleotides, which include
seed sequence (positions 2–8 from the 50 end ofguide strand), and the complementary sequence ofthe passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides
without any substantial loss of gene silencing. In
the present study, examination was made of RNA requirements in the non-seed region of siRNA. The
non-seed region of siRNA was found to be
subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable withcognate deoxyribonucleotides without reducing
RNAi activity. However, RNA sequences at positions
9-12 and 15-18 were essential for effective genesilencing, and these two double-stranded RNA
cores are required for binding of the trans-activation
response RNA-binding protein (TRBP). The terminal
RNA (positions 19–21) provided Argonaute protein
binding sites. Argonaute binding was enhancedby the presence of RNAs at positions 15–18.
Knockdown experiments showed that, unlike
Argonaute and TRBP, Dicer was dispensable forRNAi. Based on these observations, we discusspossible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.

RNA interference (RNAi) is a phenomenon whereby
double-stranded RNA (dsRNA) leads to posttranscriptional gene silencing through base-pairing interaction(1). RNAi and related RNA silencing mechanisms occur
in a variety of evolutionarily diverse organisms (2).
In nonmammalian cells, long dsRNAs, which trigger theinterferon response in mammalian cells (3,4), are digestedby the RNase III endonuclease Dicer (Dcr) to 21–22 nucleotide (nt) small interfering RNA (siRNA) helices with
2-nt 30 overhangs (5,6). Transfected siRNAs are also
capable of triggering RNAi (7). RNAi is directed by thesiRNA guide strand acting in concert with several proteinsin the RNA-induced silencing complex (RISC) (8). RISC
has been puriﬁed from ﬂy and human cells mainly using
immunoafﬁnity puriﬁcation of Argonaute (Ago) protein.
As with Drosophila, mammalian RISC is formed throughdynamic changes in protein–protein and protein–RNA
interactions in RISC-loading complexes (RLCs) (9),but the precise mechanism of in vivo maturation
processes of RISC in mammalian cells remain to beelucidated.
Human RLC may include Ago2, Dcr and dsRNA
binding proteins such as trans-activation-responsive
RNA binding protein (TRBP) as major components(10–12). The RLC for microRNA (miRNA)-based
RNAi may include a precursor miRNA (pre-miRNA)
hairpin, which is presumed to be processed within thecomplex during the RISC formation (10). Mammalian
RLC activity may be functionally similar to thatobserved in Drosophila embryonic extracts (13).

*To whom correspondence should be addressed. Tel: +81 3 5841 3043; Fax: +81 3 5841 3044; Email: ktei@bi.s.u-tokyo.ac.jp
Present addresses:
Osamu Ishibashi, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan.
Kaoru Saigo, Bio-Think Tank Co., Ltd., 4-1-4 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
ß The Author(s) 2014. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on May 13, 2014

Received January 16, 2014; Revised February 3, 2014; Accepted February 4, 2014

Nucleic Acids Research, 2014, Vol. 42, No. 8 5257

the PAZ pocket owing to topological constraints, following propagation of the duplex toward the 30 end of theguide strand (8).
TRBP consists of three domains similar in amino acidsequence, two of which (dsRBD1 and dsRBD2) are responsible for RNA binding. The remaining C-terminal
domain (dsRBD3) is considered to be in charge ofbinding to Merlin, Dcr and PACT, but not dsRNA (49).
In Drosophila, photo-crosslinking studies indicated thatthe Dcr-2/R2D2 heterodimer determines which siRNA
strand associates with Ago and serves as a guide strand(50). Dcr-2 is presumed to bind to the thermodynamicallyless stable siRNA-end, whereas R2D2 binds to morestable end before loading onto Ago2. In a previous experiment, we suggested that Dcr-free TRBP rather binds tothe double-stranded trunk portion of siRNA (51).
In the present study, we systematically introduced
2–3 bp DNA substitutions in the non-seed-duplex regionof siRNA with a dsDNA-substituted seed duplex, andidentiﬁed two subdomains that cannot undergo DNA
substitutions without a profound loss of RNAi activity.
We found the RNA subdomain capable of enhancing thebinding of the Ago-PAZ domain to the terminal non-seedregion of siRNA. We also showed that both of thesedsRNA cores are essential for effective binding of Dcrfree TRBP to siRNA. RNAi based on siRNA with a
dsDNA-substituted seed duplex was found to be completely dispensable for Dcr. These results enable therapeutic approaches for harnessing the untapped potentialof RNAi based on RNA–protein interaction.
MATERIALS AND METHODS
Cell culture
Chinese hamster ovary CHO-K1 (RIKEN Cell Bank) andhuman HeLa cells were cultured in Dulbecco’s Modiﬁed
Eagle’s Medium (DMEM; Gibco BRL) at 37 C. Mediafor both cell lines were supplemented with 10% heatinactivated fetal bovine serum (Mitsubishi Kagaku) andantibiotics (10 units/ml penicillin (Meiji) and 50 mg/ml
streptomycin (Meiji). Mouse embryonic stem (ES) cells
E14TG2a were cultured in DMEM supplemented with
20% heat-inactivated fetal bovine serum (Hyclone),
0.1 mM 2-mercaptoethanol (Wako), 8 mg/ml adenosine,
8.5 mg/ml guanosine, 7.3 mg/ml cytidine, 7.3 mg/ml
uridine, 2.4 mg/ml thymidine, 0.1 mM each nonessential
amino acid and 1000 units/ml leukemia inhibitor factor(Chemicon International). Drosophila S2 cells were
cultured in Schneider’s Drosophila medium (Gibco BRL)at 25 C.
Preparation of siRNA and DNA-substituted siRNA
Passenger- and guide-strand RNA or DNA-substituted
RNA oligonucleotides were chemically synthesized(Sigma), mixed in a 1:1 fashion in 10 mM NaCl and
20 mM Tris–HCl (pH 7.5), and annealed by incubation
at 95 C for 15 min, 37 C for 30 min and 25 C for
30 min. Annealed products were examined using 3%
agarose gel electrophoresis in Tris-borate-EDTA buffer,
which can separate 21-bp long double-stranded siRNA

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Dcr was initially determined to catalyze processing oflong dsRNA to siRNA units and pre-miRNA to mature
miRNA 22 bp in length (14–16). However, increasing evidence suggests that Dcr functions not only in the initiation
phase (siRNA/miRNA generation), but also in the downstream step in the effector phase, modulating posttranscriptional gene silencing (11,17–19). The coupling of thetwo phases of RNAi was established by the identiﬁcationof Dcr-interacting, dsRNA-binding proteins, R2D2 in
Drosophila (20), Rde-4 in Caenorhabditis elegans (21,22),
whose human counterparts are thought to be TRBP (11)and a protein activator of PKR (PACT) (23). Dcr alone
can process dsRNA and pre-miRNA (11,24–26), but Dcr
appears to form a heterodimer with TRBP or PACT (27).
TRBP has been reported to signiﬁcantly enhance
Dcr-dependent dsRNA processing (28). Recruitmentof siRNAs/miRNAs to Ago may require these Dcrinteracting, dsRNA-binding proteins (11). However, it
was revealed recently that Dcr is not always necessary
for processing of miRNA and short-hairpin RNAs(shRNAs). Dcr-independent processing of the premiRNA-451 (29–31) or shRNA of minimal size (32) has
been reported.
Ago2 is the endonuclease of human RISC, and cleaves
target mRNA whose sequence is complementary to theguide strand of siRNA. Ago2 may be sufﬁcient for
loading and accurate guide strand selection in theabsence of Dcr (33–35). In fact, minimal RISC that is
capable of cleaving target RNA has been reconstitutedusing single-stranded siRNA and recombinant Ago2(36). Furthermore, a recent experiment indicated thatthe recombinant human Ago2 alone can acquire a guideRNA strand by ﬁrst associating with siRNA or miRNA
duplex, leading to cleavage of target RNA in vitro (37),although recombinant Ago2 was initially thought to beunable to bind to siRNA duplex (36,38). Active RISCs
are shown to be minimally formed by double-stranded
siRNA and Ago in vitro, but Dcr and TRBP or PACT
may be often considered to be essential for siRNA strand
selection (37).
In humans, there are four different Agos (Ago14)highly similar in amino acid sequence to one another(17). Only Ago2 associates with target RNA cleavage
activity (39). Unlike Drosophila containing two Agoproteins, Ago1 and Ago2, onto which siRNA or
miRNA are loaded, mammalian siRNA and miRNA are
not differentially sorted by Agos. Ago is composed of fourdomains: N-terminal (N), Piwi/Argonaute/Zwille (PAZ),
Middle (MID), P-element-induced wimpy testis (PIWI)(40). An RNase H-like active site situated in the PIWI
domain catalyzes the cleavage of target mRNAs (41–43).
N domain is presumed to be involved in RNA unwinding(44). The 50 end of siRNA is anchored within the binding
pocket in the MID domain (45), while the 30 end isanchored in the PAZ domain (46–48). The current understanding of a mechanistic issue supports a ‘two-state’model wherein both ends of the guide strand areanchored in the pockets of MID and PAZ domains, respectively, during the nucleation step of target recognition
as previously described. However, while its 50 end is stably
anchored in the MID domain, its 30 end is released from

5258 Nucleic Acids Research, 2014, Vol. 42, No. 8

from 21-nt long single-stranded RNA. Almost all RNA
was recovered as dsRNA.
Gene silencing activity assay for ﬁreﬂy luciferase genes

Electrophoresis mobility shift analysis
Ampliﬁcation of the coding sequence of TRBP by polymerase chain reaction (PCR) has been described (51). TRBP
protein was produced in Escherichia coli BL21 codon plus
and Rosetta (DE3) pLysS, as amino-terminal hexahistidine fusion proteins, and puriﬁed with NTA agarose(51). Electrophoresis mobility shift analyses (EMSAs) were
performed in binding buffer with 20 mM Tris (pH 7.5),
150 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol,
1 mM dithiothreitol, 50 ng/ml sonicated salmon sperm
DNA and 0.4 U/ml RNasin (Promega). Puriﬁed fusion
proteins were incubated with siRNA or DNA-substituted
siRNA containing 32P-labeled guide strand (0.5 nM) for
30 min, and samples were electrophoresed on a 9% polyacrylamide gel and analyzed quantitatively using a FLA2000 image analyzer (Fujiﬁlm).
For EMSA analysis of the PAZ domain of human
AGO, the control pGST-His vector expressing 6xHis
and pGST-PAZ-His expressing human Ago1 PAZ
domain were constructed. pGST-His was constructed byinsertion of annealed oligonucleotides (50 -GGCCGCAGGCAGCAGCCACCACCACCACCACCACTGA-30 and
50 -GGCCTCAGTGGTGGTGGTGGTGGTGGCTGCT
GCCTGC-30 ) into the Not I site of pGEX-6 P-2(GE Healthcare). For construction of pGST-PAZ-His, a
DNA fragment corresponding to human Ago1 PAZ
domain (amino acid residues 222–376) was ampliﬁed by
PCR with the following primers: 50 -CCGGATCCTTTTATAAGGCACAGCCAGTGATTGAGTTC-30 and 50 -TC
ATCAGGGCGGCCGCAGGCAGCAGCCACCACCA
CCACCACCACTGACTCCTCCTGTCTGTCTGGAGCGGATCTAGC-30 . The ampliﬁed fragment was digested

RESULTS
Difference in RNAi properties of an authentic siRNA anddsDNA-substituted siRNAAt ﬁrst, we would like to brieﬂy describe the results of ourprevious experiment (55), as this study is its natural extension. We constructed siRNAs with various dsDNA substitutions and found that the duplex region ranging from
nucleotide position 1 to 8, which includes the guide-strand
seed region (nucleotide position 2–8), is replaceable withcognate deoxyribonucleotides without any substantial lossof target gene silencing (Figure 1A) (55). Most part of theremaining non-seed-duplex region could not be replacedwith dsDNA (Figure 1A) possibly due to binding ofRNA-recognition proteins in the RLC/RISC.
We hereafter referred to the DNA-modiﬁed siRNA
schematically shown in the lower half of Figure 1A as
siRNA with a dsDNA seed duplex and used as astarting material for systematic 2–3 bp dsDNA substitutions carried out in the present experiment.
siRNA with a dsDNA seed duplex was found to be
associated with little passenger-strand–dependent genesilencing activity or seed-dependent off-target effects(55). Thus, in RNAi triggered by siRNA with a dsDNA
seed duplex, the guide strand appears to be properly recruited to Agos to generate functional RISCs, while thepassenger strand cannot (Figure 1B).
In contrast to siRNA with a dsDNA seed duplex, theauthentic siRNA is rotationally symmetric with respect to
RNA structure. Because of this, Ago binding may occur in
two orientations, resulting in two forms of functional
RISCs, one including the guide strand and the otherthe passenger strand (Figure1C). Balance in formation
between these two RISCs is presumed to be determinedbased on difference in thermodynamic stability of two
siRNA ends (43,56,57). Thus, siRNA with a dsDNA
seed duplex may provide a much simpler system for biochemical substructure analysis of the non-seed region than
the authentic nonmodiﬁed siRNA.
RNA requirements in the non-seed region of siRNA
To clarify RNA requirements in the siRNA non-seedregion, a 2–3 bp DNA substitution was additionallyintroduced into siRNA with a dsDNA seed duplex andsubjected to RNAi assay using cultured cells. Structures of

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One milliliter of a CHO-K1 (3  105 cells/ml), HeLa
(1  105 cells/ml) or E14TG2a (2  105 cells/ml), S2(1  106 cells/ml) cell suspension was inoculated in a
1.5-cm well 24 h before transfection. Cells were transfectedwith pGL3-Control DNA (1 or 0.5 mg; Promega) or
pGL2-Control DNA (1 mg; Promega) encoding the ﬁreﬂyluciferase (luc) gene and pRL-SV40 DNA (0.1 mg;
Promega) encoding the Renilla luc gene with or withoutsiRNA or DNA-substituted siRNA against the ﬁreﬂy luc
gene. Knockdown of each protein was performed bytransfection of speciﬁc siRNA for each protein designedby siDirect2.0 (52–54) 24 h before transfection of the lucencoding vectors. Lipofectamine 2000 reagent (Invitrogen)was used for transfection for CHO-K1, HeLa and
E14TG2a cells, and calcium phosphate precipitation
method was used for transfection for S2 cells. Cells were
harvested 24 h after transfection, and luc activity wasmeasured using the dual-luciferase reporter assay system(Promega). In this system, two luc (ﬁreﬂy and Renilla)
genes were expressed simultaneously in cells. The reduction of ﬁreﬂy luc activity by the addition of siRNA against
the ﬁreﬂy luc gene was normalized to the Renilla luc
activity (internal control).

with BamHI and NotI, and inserted into pGST-His. The
PAZ domain was expressed in E. coli BL21 codon plus
(Stratagene) as a glutathione S-transferase (GST) fusion
protein, and puriﬁed with a glutathione sepharose column(Amersham) according to the manufacturers’ instructions.
PAZ protein (1 pmol/ml) was incubated with siRNA or
DNA-substituted siRNA containing 32P-labeled guidestrands (5 fmol/ml each) in 20 mM Tris–HCl (pH 7.5)
with 100 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol,
5% glycerol, 50 ng/ml poly(dA-dT) and 2 U/ml RNasein(Promega) for 30 min at room temperature. The mobility
shift was analyzed by 5% polyacrylamide gel electrophoresis and quantiﬁed using a FLA-2000 image analyzer(Fujiﬁlm).

Nucleic Acids Research, 2014, Vol. 42, No. 8 5259
A

High

Low

Relative luc activity (%) RNA
silencing
activity

RNA

siRNA with a dsDNA seed duplex
Nucleotide position
Passenger strand
Guide strand
non-seed duplex region seed duplex region(9~21)(-2~8)B

C Passenger strand-RISC
RISC

RISC

5’-

-3’

3’-

3’-

5’-

-3’

5’-

-3’

5’-

-5’

3’-

-5’

3’-

-3’
-5’

-5’

RISC

RISC

Guide strand-RISC

Guide strand-RISC

Figure 1. DNA-substitution–dependent changes in target gene silencing due to DNA-modiﬁed siRNA (A) and asymmetric RISC formation in DNAmodiﬁed siRNA-dependent RNAi (B and C). (A) Effects of dsDNA replacement on RNA silencing activity are shown in the upper panel. The redcircle indicates ribonucleotide, whereas blue one indicates deoxyribonucleotide. In each siRNA with or without DNA modiﬁcation, passenger andguide strands are shown on the top and bottom, respectively. The RNA silencing activity of siRNA with <9 bp DNA substitution from the 50 end ofguide strand is essentially identical to that of nonmodiﬁed siRNA (49). Structure of active siRNA with a dsDNA seed duplex, ranging from position
2 to 8, is shown in the lower half. All ribonucleotides in the seed duplex are replaceable with cognate deoxyribonucleotides without any substantial
loss of RNAi activity (49). Most, if not all, nucleotides in the remaining (nucleotide position 9–21) should be ribonucleotides, possibly due to bindingof RNA-recognition proteins in RLC/RISC. Note that, because of the asymmetry in structure, virtually no off-target effect is associated with siRNAwith a dsDNA seed duplex (49). (B) and (C), respectively, show the RISC formation in RNAi due to siRNA with a dsDNA seed duplex and that due
to nonmodiﬁed siRNA. (B) In the former, the guide strand is recruited to Ago2 to generate a functional RISC but the passenger strand may not be
recruited to Ago2 because of asymmetry in structure. (C) In contrast, both guide and passenger strands of the nonmodiﬁed siRNA can be recruited
to Ago2. Note that the nonmodiﬁed siRNA is structural symmetric.

each siRNA derivative were examined, and changes in
RNAi activity observed are shown in Figures 2A and
Supplementary Figure S1A. One hundred percent inrelative luc activity in Figure 2 and Supplementary
Figure S1 corresponds to the absence of RNAi activity.
We used three mammalian cell lines (human cervicalcancer-derived HeLa, mouse ES E14TG2a and Chinese
hamster ovary-derived CHO-K1 cells) and Drosophila S2cells for transfection. siRNA sequences used were ofhighly functional ones against luc (52), siLuc2-153(Figure 2) and siLuc-36 (Supplementary Figure S1).
These siRNAs were same siRNAs used in our previous

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5260 Nucleic Acids Research, 2014, Vol. 42, No. 8

siLuc2-153 with a dsDNA seed duplex
Nucleotide position
Passenger strand
Guide strand

C G U A C G C G G A A T A C T T C G A T T
G U G C A U G C G C C U U A T G A A G C T

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

Relative luc activity (%)

HeLa

E14TG2aCHO-K1

S2

A

B
1
2
3
4
5
6
7
8
9
10
11
12
13

C
1
2
3
4
5
6
7
8
9
10
11
12
DNA

RNA

Figure 2. Effects of 2-bp-long DNA substitutions in the non-seed region of siRNA (siLuc2-153) with a dsDNA seed duplex on RNAi activity as
determined by luc reporter assays. Except for DNA/RNA difference, the nucleotide sequences of DNA-modiﬁed siRNAs used here were identical tothat of siLuc2-153. Red circle, ribonucleotide; blue circle, deoxyribonucleotide. (A) Both guide and passenger strands were simultaneously replacedwith DNA. (B) DNA replacement was carried out only in the guide strand. (C) DNA replacement was carried out only in the passenger strand.
RNAi was assayed using human HeLa, mouse E14TG2a, Chinese hamster CHO-K1 and Drosophila S2 cells using a dual-luciferase reporter assay
system with nonmodiﬁed or DNA-substituted siRNAs at 50 nM. However, in S2 RNAi (B), the siRNA concentration used was 5 nM. The dotted line
in each graph indicates the level of RNAi due to the parental modiﬁed siRNA. Lane 1, siRNA with a dsDNA seed duplex. Lane 2–lane 13, siRNAwith a dsDNA-modiﬁed seed duplex and two additionally DNA-substituted base pairs in the non-seed-duplex region.

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1
2
3
4
5
6
7
8
9
10
11
12
13

Nucleic Acids Research, 2014, Vol. 42, No. 8 5261
siLuc2-153 with a dsDNA seed duplex
and subdomain B (TCA-RNA)
21 1918

Position
Passenger strand
Guide strand

15 14 13 12

98

1

5 - C G U A C G C G G A A T A C T T C G A T T -3
3 - G U G C A U G C G C C U U A T G A A G C T -5

RegionT

C

B

A

seed duplex

Relative luc activity (%) Relative luc activity (%) Relative luc activity (%) Relative luc activity (%)

DNA

RNA

HeLa

E14TG2aCHO-K1

S2

Figure 3. Effects of DNA substitutions in the non-seed-duplex subdomains A, B, C and T on RNAi activity. Except for DNA/RNA difference, thenucleotide sequences of DNA-modiﬁed siRNAs were identical to that of siLuc2-153. Red circle, ribonucleotide; blue circle, deoxyribonucleotide. Seed
duplex, nucleotide position 2–8; subdomain A, position 9–12; subdomain B, position 13–14; subdomain C, position 15–18; subdomain T, position
19–21. RNAi activity was assayed using HeLa, E14TG2a, CHO-K1 and S2 cells, using a dual-luciferase reporter assay system. The concentration ofsiRNA was 50 nM. Lane 1, nonmodiﬁed siRNA; lane 2, TCBA-RNA (the entire non-seed duplex is RNA); lane 3, TCA-RNA; lane 4, CA-RNA;lane 5, A-RNA; lane 6, TC-RNA; lane 7, C-RNA; lane 8, T-RNA; lane 9, RNA in the guide strand non-seed region; lane 10, RNA in the passenger
non-seed region; lane 11, siDNA.

study (55). siRNA concentrations used were 50 or 5 nM.
In all cases, position-dependent changes in RNAi activity
were similar, if not identical to each other.
DNA substitutions at positions 13 and 14 and of the 30
overhang of the guide strand changed little, if any, RNAi
activity (lanes 6, 12, 13). DNA replacement at nucleotide
position 19 gave some RNAi activity reduction in a celltype–dependent manner (lanes 13): gene silencing activity
was considerably reduced in HeLa and E14TG2a cells butthat in CHO-K1 and S2 cells was not signiﬁcant. If nototherwise mentioned, the 30 overhang of the guide strand
and the nucleotide pair at position 19 are collectively
called the subdomain T. Based on these observations, weconsidered that the non-seed region of siRNA are divided
into four subdomains and that two of them, A (from nucleotide position 9 to 12) and C (from position 15 to18)
subdomains, should be RNA for active RNAi (seethe upper margin of Figure 3 and Supplementary FigureS2). Subdomain B corresponds to two nucleotide pairs at
position 13 and position 14. Subdomain B is replaceablewith DNA.
Figure 2B and Supplementary Figure S1B show that luc
gene silencing due to a 2–3 bp dsDNA substitution
is strongly correlated with those induced by a similar
DNA substitution only in the guide strand. In contrast,
transfection with siRNAs with DNA substitution only inthe passenger strand gave a small, if any, effect on luc
target gene silencing in mammalian cell (Figure 2C andSupplementary Figure S1C). Note that all nucleotides ofthe passenger and guide strands, respectively, in Figure 2B

and C (or Supplementary Figure 1B and C) areribonucleotides. Although these results may indicate thatRNA requirement in the non-seed region is due primarily
to interactions with RLC/RISC protein moieties, it should
be noted that 2–3-bp DNA substitutions have nevercompletely abolished relative luc activity (Figure 2 andSupplementary Figure S1), possibly suggesting thateither 2–3-bp-long DNA segments are too short to completely suppress RNAi or there is some ambiguity in positioning of putative RNA-binding proteins in RLC/RISC.
The contribution of the passenger strand to genesilencing is much greater in Drosophila than mammaliancells (Figure 2C and Supplementary Figure S1C).
However, this is not due to the effect of introduction ofan additional 2–3-bp DNA substitution in the non-seedregion, as virtually no difference in gene silencing activity
between parental and additionally DNA-modiﬁed siRNAs(compare lane 1 and other lanes in Figure 2C or lane 1 andother lanes in Supplementary Figure S1C).
DNA-substitution–induced inactivation of three RNArequiring subdomains found in the non-seed-duplex regionof siRNATo make certain that the siRNA non-seed-duplex region
consists of four subdomains (see the upper margins
of Figure 3 and Supplementary Figure S2), various
DNA substitution variants were constructed and theirRNAi-inducing activity was examined (Figure 3 andSupplementary Figure S2). Lane 1 and lane 2 are positive
controls, indicating RNA in the entire non-seed-duplex

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1
2
3
4
5
6
7
8
9
10
11

5262 Nucleic Acids Research, 2014, Vol. 42, No. 8

Relative mRNA levels (%)

A 120
100

siControl
siAgo2siDcr
siTRBP
siPACT

80
60
40
20
0

DNA-modified siLuc2-153(TCBA-RNA)

DNA-modified siLuc2-153(TCA-RNA)

100

10

10

80

8

8

6

6

B

Relative IC50

siLuc2-153

60

*
*

4

40

*

4

*
*

*

2

2

2

0

0

0

DNA-modified siLuc-36(TCBA-RNA)

DNA-modified siLuc-36(TCA-RNA)C

siLuc-36

108Relative IC50

*
*

20
*
*

16

6

30
24

*
*

12

4

4
*
*

*
*
*

6
*
*

4

2

2

2

0

0

0

Involvement of Ago and TRBP but not Dcr in humanRNAi due to siRNA with a dsDNA seed duplex

Figure 4. Effects of Ago2, Dcr, TRBP and PACT knockdown on luc
RNAi due to DNA-modiﬁed or nonmodiﬁed siRNA. Except for DNA/RNA difference, the nucleotide sequences of DNA-modiﬁed siRNAs
were identical to that of siLuc2-153 or siLuc-36 as indicated. Red
circle, ribonucleotide; blue circle, deoxyribonucleotide. (A)Knockdown efﬁciency of siAgo2, siDcr, siTRBP or siPACT was
examined by real-time reverse transcriptase-PCR in HeLa cells.
Changes in expression level of each mRNA normalized with that in
the cells treated with siControl were measured following the addition of
25 nM siAgo2, siDcr, siTRBP or siPACT. (B and C) Relative change inIC50s of Ago2, Dcr, TRBP and PACT knockdown of siLuc2-153 (B)and siLuc-36 (C). Relative IC50 was normalized using that for RNAi
due to control siRNA, siGY441. IC50 values were calculated using thedual-luciferase reporter assay data in Supplementary Figures S3 and S4.
P-values were determined by Student’s t-test (**P < 0.01, *P < 0.05).

Human RLC/RISC may include Ago, TRBP, PACT and
Dcr as major components (10–12). Thus, examination wasmade of whether these gene products are functionallyrequired for RNAi due to three different types of antiluc siRNA, which consist of authentic siRNA, siRNAwith a dsDNA seed duplex (TCBA-RNA) and siRNAwith simultaneously dsDNA-replaced seed-duplex andsubdomain B (TCA-RNA) (see the upper margin of
Figure 4B and C). siGY441, an siRNA speciﬁc to GFP,
was used as an siRNA control. As shown in Figure 4A,

80% of the expression of any one of RLC/RISC genes
examined was found to be abolished on transfection ofcorresponding siRNA, indicating a successful inactivationof relevant genes to occur.
RNAi activity against a background of RLC/RISCrelated gene knockdown was quantitatively analyzed(Supplementary Figures S3 and S4). In each case, the concentration of 50% inhibition (IC50) was normalized by

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region to give a highly active RNAi activity. Lane 11 is anegative control and the relative luc activity induced was
virtually 100%, indicating no or little functional RNAi to
be induced by siRNA in which all ribonucleotides are
replaced with cognate deoxyribonucleotides (siDNA). If necessary, DNA substitution variants are hereafter simply
referred to as ‘an RNA domain combination’. Thus, forexample, TC-RNA means a DNA-substitution variant ofsiRNA in which only subdomains T and C are RNA.
As expected, the introduction of DNA into subdomain
B (TCA-RNA) resulted in only a slight, if any, reductionof RNAi activity (lane 3), indicating that RNA in
subdomain B is not essential for functional RNAi inall cell types examined. DNA substitutions in bothsubdomains B and T (CA-RNA) (lane 4) induced a considerable reduction of RNAi in human and Drosophila
cells but not in mouse and Chinese hamster cells, suggesting that RNAi activity due to DNA substitution in
subdomain T to occur in a cell-type–dependent manner.
We interpret these ﬁndings as suggesting the presence ofsome difference in siRNA binding mode among threemammalian and Drosophila cells during RLC/RISC
development.
RNA only in subdomain A (A-RNA; lane 5) or only insubdomain C (C-RNA; lane 7) could not trigger effective
RNAi, whereas the introduction of RNA into subdomain
T to the C-RNA derivative slightly improve RNAi activity
in particular in the cases of siLuc-36 derivatives (lane 6 in
Supplementary Figure S2). But RNA in subdomain T
could not solely recover the abolished RNAi activity atall (compare lane 8 and lane 11). Thus, we consider thatsynergistic interactions among three RNA subdomains A,
C and T may be important for maintaining siRNA-based
RNAi activity.
In the above section, we showed that the guide strand ismuch more important to the DNA-dependent suppressionof RNAi activity than the passenger strand. To further
extend this notion, two types of DNA-modiﬁed siRNAcontaining a dsDNA seed duplex were constructed. In
one of them, DNA substitution was introduced only inthe four subdomains of the passenger strand (lane 9),
while in the other, the guide strand domains (lane10).
Again, we noted the guide strand preference. In fact, apartial and cell-type–dependent RNAi activity was
found to be associated with the presence of RNA in the
non-seed-duplex guide strand but not the non-seed-duplex
passenger strand.

Nucleic Acids Research, 2014, Vol. 42, No. 8 5263

Ago protein binding to the non-seed duplex is enhanced bysubdomain C, one of the central dsRNA subdomains
X-ray crystallographic analysis indicated the PAZ domainof Ago protein to contain a pocket capable of binding to
the 30 end of single-stranded RNA (46–48). However, weare not yet sure about how Ago-PAZ protein domain canrecognize all or part of four subdomains in the non-seed
duplex of siRNA for binding. In human, there are
four Ago paralogs (Ago14) whose PAZ domains are
similar in amino acid sequence to one another [(17);Supplementary Figure S5]. Thus, to clarify possible interactions between non-seed-duplex subdomains and the
PAZ domain of Ago protein, examination was made ofinteractions between the PAZ domain of Ago1, taggedwith GST, and subdomains of the siRNA non-seed
duplex. RNA–protein interaction was examined using
EMSA assay.
As shown in lane 1 and lane 12 in Figure 5, GST-PAZ
was found to be capable of binding to siRNA but not
siDNA, suggesting that PAZ can interact with doublestranded siRNA but not siDNA. Subdomain B is an
siRNA domain replaceable with DNA without any substantial loss of RNAi activity and hence, is presumed to beunrelated to PAZ binding. In contrast, the three remaining
subdomains, A, C and T should be RNA for effective
RNAi. In fact, a strong interaction was detected
between GST-PAZ and DNA-modiﬁed siRNA with
RNA in all three subdomains (TCA-RNA; lane 2).

T C B Aseed
duplexFraction ofbound siRNA
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ
Buffer
GST
GST-PAZ

1
2
3
4
5
6
7
8
9
10
11
12
DNA

0.89
0.24
0.00
0.00
0.24
0.00
0.02
0.22
0.00
0.32
0.04
0.00

RNA

Figure 5. EMSA proﬁles of DNA-substituted siRNAs associated withrecombinant PAZ domain of human AGO1. EMSA experiments werecarried out using recombinant human AGO1 PAZ domain with 32Plabeled authentic siLuc-36 or its DNA-substituted derivatives. Relative
fractions of bound siRNA are shown by numerals on the left. Lane 1,
nonmodiﬁed siRNA; lane 2, TCA-RNA; lane 3, CA-RNA; lane 4,
A-RNA; lane 5, TC-RNA; lane 6, C-RNA; lane 7, T-RNA; lane 8,
RNA in the guide strand non-seed region; lane 9, RNA in the passenger non-seed region; lane 10, 10-nt 30 protrusion of passenger strand;lane 11, 5-nt 50 protrusion of passenger strand; lane 12, siDNA.

We introduced an additional DNA replacement in
subdomain T (lane 3) and found that resultant DNAmodiﬁed siRNA with RNA in subdomains A, C
(CA-RNA) was found to be incapable of binding to
GST-PAZ at all. Similarly, all other siRNAs with DNA
in subdomain T failed to show any GST-PAZ binding(lane 4, lane 6). Contrary to these, DNA-modiﬁed
siRNAs with RNA in subdomain T were associated with
GST-PAZ binding (lane 2, lane 5, lane7). Thus, weconclude that it is the terminal subdomain T thatprovides PAZ binding sites.
Lane 5 and lane 7 in Figure 5 showed that GST-PAZ
binding activity to increase >10 times when DNAmodiﬁed siRNA possesses RNA sequences not only insubdomain T but also in subdomain C (TC-RNA),
indicating that subdomain C serves as an enhancer for
GST-PAZ binding to subdomain T. As no improvementin GST-PAZ binding activity was detected whensubdomain A was of RNA (lane 2), suggesting that,
unlike subdomain C, subdomain A possesses little
enhancer activity, if any. In a previous section, we
showed that, in RNAi due to siRNA with a dsRNA
seed duplex, there is a strong preference for the guidestrand. Thus, strand preference in interactions between
the non-seed region of siRNA and PAZ domain of Ago

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on May 13, 2014

that for siGY441 treatment and the resultant relative IC50s were shown for comparison. Although relative IC50
values considerably varied depending on target luc sequences, it is virtually evident that Ago2 and TRBP areprofoundly involved in RNAi due to all three types ofsiRNAs used and that Ago2 knockdown results inchanges much larger than TRBP knockdown (Figure 4B
and C). In contrast, siDcr transfection exhibited no or
little effect on RNAi activity not only due to DNAmodiﬁed siRNA but due to authentic siRNA as well.
This ﬁnding was further conﬁrmed by transfection ofmouse mutant cells lacking Dcr activity (T.T., unpublished observation). Thus, Dcr may not be involved in
mammalian RNAi based on 21-bp-long authentic
siRNA or those due to DNA-modiﬁed siRNAs exhibitedhere (TCBA-RNA and TCA-RNA). Alternatively, Dcr isinvolved in mammalian RNAi but totally redundant infunction. In fact, there is a body of literature on Dcrindependent processing of miRNAs or shRNAs (29–32).
In this connection, it may be noteworthy to point out that
Dcr has been shown to be necessary for shRNA-based
RNAi, which includes maturation processes of a small
RNA component (33).
The PACT-containing complex is shown to enhance thestrand selection for some miRNAs and inhibits the processing of pre-siRNA substrates (37). At present we are
not yet sure about whether PACT is involved in RNAi
due to siRNA with or without a dsDNA seed-duplex
substitution, as relative IC50 values except for one
case (Figure 4C, TCA-RNA) appeared small, if any(Figure 4B and C).

5264 Nucleic Acids Research, 2014, Vol. 42, No. 8

Requirements of two central, non-seed-duplex siRNA
subdomains, A and C, for effective TRBP binding
TRBP knockdown experiments may suggest that TRBP
contribution to RNAi due to siRNA with a dsDNA
seed duplex is similar, if not identical, to that due tononmodiﬁed siRNA (see Figure 4B and C). Our previous
experiments showed interactions between recombinant
TRBP and authentic siRNA to be carried out in a twostep fashion (51). The ﬁrst-step complex consists of
TRBP monomer and one molecule of authentic siRNA,
whereas the second-step complex, TRBP homodimer andsiRNA monomer. Here we analyzed putative complex formation between TRBP protein and siRNA with a dsDNA
seed duplex using a similar strategy. In both cases, complex
formation was analyzed by EMSA assays (Figure 6A and B
and Supplementary Figures S6A and B and S7).
At a ﬁrst glance, TRBP binding to siRNA with a dsDNA
seed duplex strikingly differed from that to the authentic
siRNA (Figure 6C and Supplementary Figure S6C). As
shown previously (51), in the case of nonmodiﬁed siRNAbinding, the TRBP concentration giving 50% binding ofsiRNA was estimated at 4.05.3  103 mM, whereas thatfor siRNA with a dsDNA seed duplex (TCBA-RNA),
1526 mM (Figure 6C and Supplementary Figure S6C).
Note that the latter is 500 times as high as the former
concentration. As described in ‘Discussion’ section, we interpret these results as suggesting that TRBP binding tosiRNA with a dsDNA seed duplex within cells is not
weak, and that only TRBP associated with an unidentiﬁed
RNA-binding protein ‘X’, which is capable of strong
binding to siRNA with a dsDNA seed duplex, can effectively bind to siRNA with a DNA-modiﬁed guide sequence.
Next, we examined TRBP capability for binding to four
subdomains consisting of the non-seed region of siRNA.
As shown in Figure 6C and Supplementary Figure S6C,
introduction of DNA substitutions into subdomain B gave
somewhat increased TRBP concentration giving 50%
binding of siRNA or exhibited a slightly lower afﬁnity

to siRNA (compare lane 2 and lane 3), and it appearedslightly increased by an additional DNA replacement in
subdomain T (lane 4), suggesting low levels of contribution of RNA in subdomains B and T for TRBP binding.
In contrast, as expected, siDNA exhibited virtually no
siRNA binding activity (lane 5).
DNA-modiﬁed siRNA containing RNA only in one of
the non-seed-duplex subdomains other than subdomain B
gave much reduced values of the TRBP concentration
giving 50% binding of siRNA (lane 1, lane 3 and lane 4
in Figure 6D and Supplementary Figure S6D). siRNAs
with RNA in (T+C) subdomains (TC-RNA) also gave
low values (lane 2 in Figure 6D and Supplementary
Figure S6D). Taken together, these results may indicatethat the physical association between TRBP protein andthe non-seed duplex of siRNA requires a simultaneouspresence of nonterminal subdomains, A and C.
Finally, we examined the effect of guide- and passengerstrand-speciﬁc DNA-replacement in the non-seed region
on the activity of TRBP binding to siRNA. Consistent
with a previous experiment (51), we could ﬁnd no appreciable difference between them, although TRBP binding
was low (lane 5 and lane 6 in Figure 6D and
Supplementary Figure S6D).
DISCUSSION
A highly functional siRNA may consist of two regions, theseed duplex region, which is capable of being totallyreplaced with DNA without substantial loss of genesilencing activity, and the non-seed-duplex region, mostof which should be RNA and possibly provide bindingsites for RNA-binding RLC/RISC proteins (55). Genesilencing due to siRNA with a dsDNA seed duplex has
been shown to be similar, if not identical, to RNAi due toauthentic or nonmodiﬁed siRNA (55). Taking advantageof these ﬁndings, the present study was carried out anddemonstrated the non-seed region of siRNA to consist offour subdomains with respect to RNA requirements not
only in mammalian cells such as human HeLa, mouse
E14TG2a and Chinese hamster CHO-K1 cells but inDrosophila S2 cells as well (see the upper margin of
Figure 3). These ﬁndings may indicate that subdomain
structure found in siRNA is one of the skeletal mechanisms of RNAi.
Our results (see Figure 5) showed that the terminal
subdomain (subdomain T) and its neighbor (subdomainC), respectively, provide a human Ago (PAZ domain)binding site and human Ago binding enhancer function
and that RNA in two of the three central subdomains
is essential for human TRBP binding (Figure 7A). In
contrast to Ago and TRBP, Dcr activity was not
required for any step of RNAi, which is triggered by
21-bp-long, highly functional, nonmodiﬁed siRNA or
siRNA with a dsDNA seed duplex within human cells,
indicating that Dcr is redundant in function or is uninvolved in human RNAi examined in the present study. We
used two nonoverlapping target sequences of luc (targets
for siLuc2-153 and siLuc-36) for general value andobtained an identical conclusion.

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on May 13, 2014

protein was examined (lane 8 and lane 9). Again, we foundthat RNA in the guide strand is much more preferable forAgo-siRNA binding than that in the passenger strand. As
described above, DNA substitutions in 2-nt 30 overhang of
the guide strand and subdomain A did not signiﬁcantly
change PAZ-siRNA binding. Thus, we conclude that theposition-19 nucleotide of the guide strand and RNA
sequence in subdomain C of the guide strand, respectively,
are primarily important for PAZ binding and enhancer
function for PAZ binding to siRNA in human HeLa cells.
Finally, we examined some structural features of siRNAends for PAZ binding. Lane 10 and lane 11, respectively,
showed the results of PAZ binding when 30 or 50
protruding RNA were used as the substrates forbinding. We found that the end with 50 protrusion ismuch less efﬁcient in PAZ protein binding than the end
with 30 protrusion, suggesting that PAZ prefers to a 30
protruding siRNA end for binding and that 50 protrusionin the siRNA end may prevent PAZ from interacting withsiRNA. Note that DNA substitution in the lane 10 siRNA
is opposite in orientation to all other siRNAs shown here.

Nucleic Acids Research, 2014, Vol. 42, No. 8 5265

A DNA-modified siLuc2-153

B(TCBA-RNA)

DNA-modified siLuc2-153(TCA-RNA)

5䇻- C G U AC G C G G A A T A C T T C G A T T -3’
3䇻- G U G C A UG C G C C U U A T G A A G C T -5䇻

5䇻- CG U AC G C G G A A T A C T T C G A T T -3䇻
3䇻- G U GC A UG C G C C U U A T G A AG C T -5䇻

TRBP (µM)

TRBP (µM)

siRNA/TRBP
complexsiRNA/TRBP
complex

siRNA

siRNA

ATP

ATP

C
seed
T C B A duplex

100
80

TRBP concentration (µM)
giving 50% binding of
siRNA

5'3'-

-3'
-5'

4.0㽢10-3

2

5'3'-

-3'
-5'

26

3

5'3'-

-3'
-5'

70

4

5'3'-

-3'
-5'

59

5

5'3'-

-3'
-5'

KD

60
40
20

DNA

0
0

50
100
TRBP (µM)

RNA

150
seed
T C B A duplex

D 100
1

5'3'-

-3'
-5'

2

5'3'-

-3'
-5'

60

3

5'3'-

-3'
-5'

40

4

5'3'-

-3'
-5'

5

5'3'-

-3'
-5'

6

5'3'-

-3'
-5'

80

20
0
0

50
100
TRBP (µM)

150

DNA

RNA

Figure 6. EMSA of DNA-substituted siRNAs associated with recombinant human TRBP protein. Except for DNA/RNA difference, the nucleotide
sequence of DNA-modiﬁed siRNA was identical to that of siLuc2-153. Red circle, ribonucleotide; blue circle, deoxyribonucleotide. (A and B) EMSA
experiments. Puriﬁed recombinant TRBP and 32P-labeled siRNA with DNA substitutions (0.5 nM) were incubated with increasing amounts of wildtype TRBP protein, as indicated. In both (A) and (B), seed duplex region was replaced with dsDNA. In (B), dsDNA substitution was also carried out
in subdomain B. (C and D) A bound fraction (%) of siRNA was plotted against the input concentration of TRBP. Note that the binding activities of
DNA-modiﬁed siRNAs were signiﬁcantly lower than that of the nonmodiﬁed siRNA. Structures of DNA modiﬁcations are shown in the rightmargin (C and D). In (C), observed TRBP concentrations giving 50% binding of nonmodiﬁed siRNA and siRNA with a dsDNA seed duplex arealso shown in the right margin.

It is known that the PAZ domain speciﬁcally recognizes
an RNA with a 2-nt 30 overhang. Then, if the subdomain
T is RNA, this substrate would not be expected toperform a binding defect with Ago-PAZ protein domain.
Thus, the mechanistic implications of a weak Ago binding
to the DNA-substituted siRNA variant with RNA only insubdomain T (T-RNA) (Figure 5, lane 7) and theenhancer function of subdomain C (Figure 5, lane5)
remain unclear. To our knowledge, we are not surewhether PAZ domain is sensitive to differing geometries,

one of the possible explanations may be that the regional
geometry of subdomain T is perturbed away from atypical A-form RNA duplex toward B-form by the
DNA base pairs in the neighboring subdomain C. In the‘two-state’ model, the MID/PIWI domains have important function in substrate recognition, while the 30 end isreleased from PAZ pocket during nucleation steps. Thus,
although the PAZ domain is less important to canonicalRNAi function, it does not rule out possibility that thePAZ domain playing an important role in substrate

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on May 13, 20141

5266 Nucleic Acids Research, 2014, Vol. 42, No. 8
A
TRegionC

B

A

seed duplex

Position
Passenger strand
Guide strand

minimum enhancer
Ago binding region
TRBP binding regions

?

B

Dicer
TRBP

pre-RISC

“X”
TRBP

5’3’-

-3’
-5’

PAZ
3’-

5’-

-3’
-5’

Ago

mature RISC

PAZ
3’-

seed region-5’

Ago
Figure 7. Presumed subdomain structure of siRNA (A) and possiblemodels for functional RISC formation (B) A deduced subdomain structure of siRNA is shown in (A). Subdomain A is required for TRBP
binding. Subdomain B is a region replaceable with DNA without asubstantial loss of RNAi activity. Subdomain C is not only required
for TRBP binding but also serves as an enhancer for Ago2/siRNAbinding. Subdomain T provides Ago2 binding. (B) Possible case of
RISC formation is depicted. A model consists of three steps. Prebinding step includes an unidentiﬁed factor, ‘X’, which is presumed
to be capable of binding to TRBP and recruiting it to siRNA. Notethat one of the possible candidates of ‘X’ might be Ago. Dcr is totallydispensable or redundant in function to ‘X’. Although in some cases,
RNAi due to siRNA with a dsDNA seed duplex may include neither‘X’ nor Dcr. In pre-RISC, DNA-modiﬁed siRNA duplex is recognizedwith the PAZ domain of Ago and, in the mature RISC, consisting of
Ago2 and the guide strand, is formed.

recognition and/or discrimination when Ago is interactingwith a noncanonical siRNA-like substrate such as the
DNA-substituted duplexes used in our study.
Drosophila RLC consisting of Dcr-2, Ago2 and R2D2
promotes siRNA loading (58). In the case of siRNA-based
RNAi, guide strand selection appears carried out according to RNA duplex thermodynamics (52,56,57). Before
loading onto Ago2, R2D2 binds to the more stable end,
whereas Dcr-2 binds to the less stable end (50). Although
Ago2/Dcr complexes associated with TRBP or PACT
might be the primary complexes involved in RISC

Downloaded from http://nar.oxfordjournals.org/ at University of Tokyo on May 13, 2014pre-binding

and
/or

loading in humans (12), we are not yet sure about
whether similar complexes play an essential role instrand selection in humans (59). Furthermore, at least
for some siRNAs and miRNAs, Ago2 has been shown
to be sufﬁcient for loading and guide strand selection in
the absence of Dcr (33–35). Note that the RNAi systemdepending on siRNA with a dsDNA seed duplex isrequired little, if any, in intracellular strand-selection
mechanism described above, since the fate to become
guide or passenger strands has been determined almost
immediately after cell transfection (Figure 1B). Thus,
our RNAi system due to DNA-modiﬁed siRNA mightprovide some important insight into Dcr-independent
RNAi mechanism.
One of the most surprising ﬁndings in the present study
may be that, although effective RNAi triggered by siRNAwith a dsDNA seed duplex appeared to require TRBP
activity similar, if not identical, to that due to nonmodiﬁed
authentic siRNA, the former exhibited a much lower
afﬁnity to puriﬁed recombinant TRBP than the latter(see Figure 6C and Supplementary Figure S6C). The
TRBP concentration giving 50% binding of siRNA witha dsDNA seed duplex was estimated at 50 mM(see Figure 6C and Supplementary Figure S6C), whichroughly corresponds to one molecule in a sphere of
400 Å diameter, leading us to the notion that a functional
intracellular TRBP should be associated with an unidentiﬁed RNA-binding RLC/RISC protein. One of thepossible candidates may be Ago protein. However, asinteraction between TRBP and Ago protein is conﬂicting,the other unidentiﬁed protein(s) ‘X’, which is a possible
substitute for Dcr and stimulates the access of TRBP
protein to siRNA as in the case of Drosophila R2D2/
Dcr-2 complexes, might not be excluded [Figure 7B prebinding step; (50)].
Human TRBP contains two dsRNA-binding domains,
dsRBD1 and dsRBD2, both of which include a-b-b-ba–folds and three amino acids presumably involvedin dsRNA binding in any 16-nt region (60,61).
Mutagenesis along with structural analysis indicated thateach TRBP domain is capable of interacting with dsRNA
via at least two sites in minor and major grooves, whichare <11 nt apart from each other and close to the distance
between the distal ends of siRNA subdomains A and C(see the upper margin in Figure 3). Furthermore, we
showed both dsRBD1 and dsRBD2 to be necessary for
effective TRBP binding to siRNA (51). In the dsRNA
helix, a minor groove is located on the opposite-side
surface of a major groove, and a major groove is locatedon the opposite-side surface of a minor groove. Thus, it
may be suggested that two sets of major and minor
grooves aligned with the non-seed-duplex region face-toface so that two dsRBDs of TRBP can hold siRNA stably
from both sides.
Drosophila R2D2 has been shown to be localized in thevicinity of the nucleotide at position 20 in the 30 overhangof the guide strand. However, we are not yet sure about
whether human TRBP is similarly capable of recognizing
the 30 overhang of the guide strand, although RNA in
the 30 terminal domain (subdomain T) of the guidestrand may slightly increase TRBP binding (see Figure 6

Nucleic Acids Research, 2014, Vol. 42, No. 8 5267

siRNA, unwinding siRNA and incorporating the guidestrand to form an active mature RISC for silencing theintended target gene [see Figure 7B; (37)].
In conclusion, we used DNA replacement for the
analysis of subdomain structures of siRNA not only in aprevious study (55) but in the present study as well, anddemonstrated that siRNA consists of a seed duplex andfour non-seed-duplex subdomains, three of which areessential for Ago2 and TRBP binding.

SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS
The authors thank K. Nishi for helpful suggestions, and
A. Juni, A. Tanaka and N. Fukunishi for technicalsupport.

FUNDING
This work was supported by the Ministry of Education,
Culture, Sports, Science and Technology of Japan, Cell
Innovation Program (MEXT) [21310123 and 21115004]
and Core Research Project for Private 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.). Funding for open
access charge: Research grant form Ministry of
Education,Culture, Sports, Science and Technology ofJapan [21115004 to K.U.-T.] and JSPS (to T.T.).
Conﬂict of interest statement. None declared.

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and Supplementary Figure S6). The nucleotide duplex
geometry may be important for TRBP binding tosiRNA. TRBP can bind to A-form duplex but not to
B-form duplex. So, it seems likely that the differences in
afﬁnity of TRBP for different types of DNA-substituted
siRNAs may somewhat correlate with a gradual shift
away from A-form duplex geometry toward B-form
geometry according to the increase of DNA content.
Our data shown in Figure 2 indicate RNArequirement–dependent subdomain structure in the
non-seed-duplex region to be primarily due to that in
the guide-strand sequence but not in the passengerstrand sequence in all cells examined, indicating differential involvement of siRNA guide and passenger strands in
RNAi pathway. One might imagine that strand selection
occurs at an early stage of RLC/RISC development sothat the contribution of the passenger-strand to RNAi
may be virtually completely excluded. However, thismay not be the case. Except for the seed duplex region,
nucleotides of the passenger strand as a duplex partner ofthe guide strand with a 2–3-bp DNA modiﬁcation are allribonucleotides and hence, all potential protein-bindingsites on the passenger strand remain intact.
The passenger strand is eventually eliminated from theRISC (37) so that any information from the passenger
strand should become dispensable from the time of unwinding of siRNA duplex or passenger elimination(mature RISC step in Figure 7B). Our data in Figure 5
indicated Ago strongly binds to the 30 terminal subdomain
T through the enhancer function due to the subdomain-C
RNA sequence in the guide strand, thus indicating thatguide and passenger strands have been correctly
recognized at this stage of RLC/RISC development. We
presume interactions between Ago-PAZ domain andsiRNA subdomains (T+C) to occur at pre-RISC step in
Figure 7B. Lane 11 in Figure 5 showed the presence of 50
protruded RNA strand to prevent the PAZ domain of
Ago protein from binding to the siRNA terminal
subdomain and hence, PAZ of Ago may recognize thesiRNA subdomain T, which is associated with 30 RNA
protrusion of the guide strand, as a binding site.
Knockdown of RLC/RISC-related gene activity (seeFigure 4) clearly showed the involvement of both Ago2
and TRBP in RNAi under our experimental conditions.
However, increment in relative IC50 due to knockdownof Ago2 was, in general, much higher than that due
to knockdown of TRBP. Thus, in some cases, TRBP
function may be replaced by other dsRNA binding
protein such as PACT (see Figure 4C). However, PACT
and TRBP do not always work in redundant manner;
PACT in complex with Dcr is shown to inhibit the processing of pre-siRNA substrates when compared with Dcr
and a Dcr-TRBP complex (37). Knockdown of TRBP
may generate a state where Dcr preferentially binds to
PACT, and inhibits the siRNA-promoting capability
during initial substrate recognition, Dicer processing andpossibly RISC loading. Alternatively, neither dsRNA
binding protein nor presumed unidentiﬁed RLC/RISC
protein, ‘X’, may be involved in our RNAi system. In
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