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Human TNRC6A is an Argonaute-navigator protein for
microRNA-mediated gene silencing in the nucleus
Kenji Nishi, Ai Nishi, Tatsuya Nagasawa, et al.
RNA 2013 19: 17-35 originally published online November 13, 2012
Access the most recent version at doi:10.1261/rna.034769.112

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Human TNRC6A is an Argonaute-navigator protein
for microRNA-mediated gene silencing in the nucleus
KENJI NISHI,1 AI NISHI, TATSUYA NAGASAWA, and KUMIKO UI-TEI1
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan

ABSTRACTGW182 family proteins play important roles in microRNA (miRNA)-mediated gene silencing. They interact with Argonaute(Ago) proteins and localize in processing bodies, which are cytoplasmic foci involved in mRNA degradation and storage. Here,
we demonstrated that human GW182 paralog, TNRC6A, is a nuclear–cytoplasmic shuttling protein, and its subcellular
localization is conducted by a nuclear export signal (NES) and a nuclear localization signal (NLS) identified in this study.
TNRC6A with mutations in its NES region was predominantly localized in the nucleus in an Ago-independent manner. However,it was found that TNRC6A could bring Ago protein into the nucleus via its Ago-interacting motif(s). Furthermore, miRNAs werealso colocalized with nuclear TNRC6A-Ago and exhibited gene silencing activity. These results proposed the possibility that
TNRC6A plays an important role in navigating Ago protein into the nucleus to lead miRNA-mediated gene silencing.
Keywords: Argonaute; microRNA-mediated gene silencing; nuclear export signal; nuclear localization signal; TNRC6A

INTRODUCTION
GW182 family proteins are components of microRNA(miRNA)-induced silencing complexes (miRISCs) in animal cells and play important roles in miRNA-mediatedgene silencing (for review, see Ding and Han 2007; Eulalio
et al. 2009a). They interact with Argonaute (Ago) proteins,core components of miRISC, and mainly localize in processing (P) bodies, which are cytoplasmic foci that containproteins involved in diverse post-transcriptional processes
such as mRNA degradation, storage, and translational repression (Ding et al. 2005; Jakymiw et al. 2005; Liu et al. 2005;
Meister et al. 2005; Behm-Ansmant et al. 2006; Zhang et al.
2007; Landthaler et al. 2008). There are three GW182 paralogs
in vertebrates (TNRC6A/GW182, TNRC6B, and TNRC6C),
one ortholog in insects (GW182), and two orthologs in
Caenorhabditis elegans (AIN-1 and AIN-2) (Eulalio et al.
2009a). Among them, the vertebrate and insect proteins share acommon structural organization characterized by the following five regions: multiple glycine-tryptophan (GW) repeats in the N-terminal region, a central ubiquitin-associated(UBA) domain, a glutamine-rich (Q-rich) region, a poly(A)-1Corresponding authors
E-mail kennishi@bi.s.u-tokyo.ac.jp
E-mail ktei@bi.s.u-tokyo.ac.jp
Article published online ahead of print. Article and publication date areat http://www.rnajournal.org/cgi/doi/10.1261/rna.034769.112.

binding protein (PABP) binding motif 2 (PAM2), anda C-terminal RNA recognition motif (RRM) (Fig. 1A).
Many recent reports have revealed the molecularfunction of GW182 family proteins in miRNA-mediatedgene silencing in the cytoplasm. The human and Drosophila
GW182 family proteins have multiple Ago interaction sites
in an N-terminal GW-repeat-containing region (Till et al.
2007; Eulalio et al. 2009b; Lazzaretti et al. 2009; Lian et al.
2009; Takimoto et al. 2009). The C-terminal region containing the PAM2 motif and RRM possesses silencing
activity (Chekulaeva et al. 2009; Eulalio et al. 2009b; Lazzaretti
et al. 2009; Zipprich et al. 2009). This region of human
TNRC6A, TNRC6B, and TNRC6C and Drosophila GW182(dGW182) interacts with cytoplasmic poly(A)-binding protein
1 (PABPC1) (Fabian et al. 2009; Zekri et al. 2009; Huntzinger
et al. 2010). The most recent reports show that two regionsof GW182 proteins interact with PABPC1: the first contains
the PAM2 motif, which is a direct binding site for PABPC1(Jinek et al. 2010; Kozlov et al. 2010), and the second
contains the M2 and C-terminal sequences, which mediate
indirect binding to PABPC1 (Huntzinger et al. 2010).
Furthermore, the interaction between Ago and GW182 triggers
mRNA decay by inducing accelerated biphasic deadenylation
mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes(Chen et al. 2009).
The components of miRISC containing miRNAs, Agoand GW182 family proteins are localized in the cytoplasm,
but several instances of evidence indicating their nuclear

RNA (2013), 19:17–35. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2013 RNA Society.

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FIGURE 1. Subcellular localization of TNRC6A fragments and identification of NES and NLS. (A) Schematic representation of TNRC6A
deletion mutants, which were expressed as fusion proteins with myc-GFP. Numbers denote the amino acid position relative to the N terminus.
GW-I, -II, and -III (black boxes) indicate GW-repeated Ago-binding motifs. NLS (blue box) and NES (red box) were identified in this study. B–I,
K, and M at the right side indicate the fragments used in B–I, K, and M. (WT) Wild type. (B–N) Subcellular localization of the indicated myc-GFPtagged TNRC6A fragments (B–M) and the control myc-GFP (N). Fluorescent microscopy images of GFP are shown in the left panels, and their merged
images with DAPI are shown in the right panels, in which the GFP signal is shown in white and the DAPI signal in cyan. An arrow in F indicates the cellin which myc-GFP-TNRC6A-(925–1709) signals are exclusively observed in the nucleus. (*) Cells not transfected with each expression construct. Bars,
20 mm. (O,P) Sequences of the TNRC6A NLS motif and its mutant (NLS-mut) (O), and TNRC6A NES and its mutant (NES-mut) (P). The red lettersrepresent basic amino acids or hydrophobic residues. The blue letters indicate substituted amino acids. (Q) The ratio of cells expressing GFP exclusively
in the nucleus (N, blue), cytoplasm (C, red), or both (N+C, yellow). B–N at the upper side indicate the fragments used in B–N. A blue asterisk indicatesthat the expression of GFP is strongly observed in the nucleus, although a negligible signal is also observed in the cytoplasm.

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Nuclear microRNA silencing navigated by TNRC6A

localization were reported recently (Meister et al. 2004;
Hwang et al. 2007; Till et al. 2007; Rüdel et al. 2008; Politz
et al. 2009; Liao et al. 2010; Taft et al. 2010; Yoo et al. 2010;
Jeffries et al. 2011). In addition, several reports demonstrated that miRISC components are functional in the
nucleus as well. The 7SK RNA level in the nuclear fraction
is reduced by siRNAs targeting 7SK RNA in combination
with Ago2 in the nucleus (Robb et al. 2005). In humancells, transcriptional gene silencing (TGS) induced byexogeneous siRNAs and miRNAs targeting to promoteroverlapping RNAs is dependent on Ago proteins (Kim et al.
2006; Han et al. 2007; Younger and Corey 2011). Furthermore, Ago2 high-throughput sequencing of RNA isolated
by cross-linking immunoprecipitation (HITS-CLIP) datashows that 12% of Ago-mRNA HITS-CLIP tags are mapped
to introns (Chi et al. 2009). These reports imply that miRNAs
associated with Ago protein interact with RNAs in the
nucleus. However, the precise mechanisms defining subcellular localization of the nuclear miRISC components
containing Ago protein consolidating their nuclear function remain unresolved.
In this study, we identified a functional nuclear export
signal (NES) and a nuclear localization signal (NLS) in thecentral region of human GW182 family protein(s) anddemonstrate that TNRC6A is a nuclear–cytoplasmic shuttling protein and navigates Ago2 to translocate into the
nucleus. Furthermore, miRNAs were found to be colocalized with Ago-TNRC6A in the nucleus and showed genesilencing activity.
RESULTS
Motif analyses of a member of human GW182
family proteins, TNRC6A
To investigate motifs involved in the subcellular localization of human GW182 family proteins, we generated theexpression constructs encoding N-terminal myc- and GFPtagged TNRC6A (AY035864), named pmyc-GFP-TNRC6A,
and their deletion mutants (Fig. 1A). These constructs weretransfected into human HeLa cells, and the subcellular localization of their GFP signals was observed under a fluorescent microscope (Fig. 1B–N) and quantified by classifying the cells into three groups: those with GFP signals (1)exclusively in the nucleus, (2) exclusively in the cytoplasm,and (3) in both the nucleus and the cytoplasm (Fig. 1Q).
Myc-GFP-tagged wild-type TNRC6A protein (myc-GFPTNRC6A-[1–1709]) was localized in cytoplasmic foci inalmost all of the transfected cells (Fig. 1B), as previously
reported for endogenous TNRC6A and GFP-TNRC6A(Eystathioy et al. 2002). The N-terminal fragment of mycGFP-TNRC6A-(1–924) and the C-terminal fragment of
myc-GFP-TNRC6A-(1077–1709) were distributed in boththe cytoplasm and the nucleus in >90% of the transfected
cells (Fig. 1E,H,Q), although myc-GFP-TNRC6A-(1–924)

was distributed uniformly (Fig. 1E) and myc-GFP-TNRC6A(1077–1709) showed dotted foci (Fig. 1H). The other fragmentsof myc-GFP-TNRC6A-(1–1223), -(1–1076), -(925–1709), and-(935–1709) were localized predominantly in the cytoplasm
in >80% of the cells (Fig. 1C,D,F,G,Q). That the detected
GFP signals were mainly derived from the transfected
constructs was verified by Western blot (Supplemental
Fig. S1A). These results indicate that myc-GFP-tagged
TNRC6A fragments lacking the 925–1076 amino acids,
which are situated between GW-III and a Q-rich domain,
were distributed in both the cytoplasm and the nucleus, but
the other fragments containing the 925–1076 amino acids
were preferentially localized in the cytoplasm.
Identification of NES and NLS sequences in human
GW182 family proteins
In contrast to the exclusive cytoplasmic localization of
myc-GFP-TNRC6A-(935–1709) proteins (Fig. 1G,Q), we
noticed that myc-GFP-TNRC6A-(925–1709) proteins werelocalized exclusively in the nucleus in about 10% of thetotal cells counted (Fig. 1F,Q), suggesting that 10 aminoacids in the 925–934 region mediate nuclear transport. The
amino acid alignment of human GW182 family proteins
showed that both TNRC6B (NP_055903) and TNRC6C(NP_061869) have conserved regions with 935–1026 aminoacids in TNRC6A, although the corresponding region with
the 925–934 amino acids in TNRC6A was not found(Supplemental Fig. S2A). Myc-GFP-TNRC6B-(997–1084)
and myc-GFP-TNRC6C-(928–1020) were localized almost
exclusively in the cytoplasm (Supplemental Fig. S2D,F,H),
indicating that the 997–1084 amino acids in TNRC6B and
928–1020 in TNRC6C might be involved in cytoplasmic
localization.
To examine the detailed function of the region between
GW-III and a Q-rich domain (Fig. 1A; Supplemental Fig.
S2A), the expression patterns of the following three mycGFP-tagged TNRC6A fragments were examined: the 925–
934 fragment containing a TNRC6A-specific region, the935–1026 fragment containing a region commonly conserved in three human GW182 family proteins, and the1027–1076 fragment containing a region deleted in TNRC6B(Supplemental Fig. S2A). In most of the transfected cells, themyc-GFP-TNRC6A-(1027–1076) fragment was detected in
the cytoplasm and the nucleus (Fig. 1M,Q). However, strong
nuclear localization was observed in almost all cells transfected with myc-GFP-TNRC6A-(925–934) (Fig. 1I,Q). On
the contrary, in >98% of the cells, myc-GFP-TNRC6A-(935–1026) was detected exclusively in the cytoplasm (Fig. 1K,Q).
These results suggest that the 925–934 and 935–1026 regionsof TNRC6A function as the NLS and NES, respectively. The
925–934 region is rich in basic amino acids (Fig. 1O), which
represents a characteristic feature of the NLS. To confirm the
NLS function of the 925–934 region, myc-GFP-TNRC6A(925–934) was mutated by replacing four arginine residues
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Nishi et al.

with alanines (TNRC6A-(925–934)-NLS-mut) (Fig. 1O).
This mutated product showed diffused distribution in thecytoplasm (Fig. 1J), which is similar to the control myc-GFP
protein (Fig. 1N), indicating that the 925–934 region containing 10 amino acids is a functional NLS. The NES consensus sequences, F-X2-3-F-X2-3-F-X-F (F = L, I, V, F,
M; X is any amino acid) (Kutay and Güttinger 2005), were
found in the 951–960 region of TNRC6A, 1009–1018 of
TNRC6B, and 943–952 of TNRC6C (Fig. 1P; Supplemental
Fig. S2B,C). Their capabilities of nuclear export were
examined by substituting four amino acid residues withalanines in these putative NES fragments (Fig. 1P; Supplemental Fig. S2B,C). All of the NES-mutated proteins(myc-GFP-TNRC6A-[935–1026]-NES-mut, myc-GFPTNRC6B-[997–1084]-NES-mut, and myc-GFP-TNRC6C[928–1020]-NES-mut) accumulated in the nuclei in addition
to the cytoplasm in most of the cells (Fig. 1L,Q; Supplemental Fig. S2E,G,H). These results indicate that theseregions are functional NESs.
Identified NLS and NES are functionalin the full-length TNRC6A
To investigate whether the identified NLS and NES isfunctional in the full-length context of TNRC6A, the sameamino acids shown in Figure 1, O and P, were substituted
in a full-length TNRC6A (myc-GFP-TNRC6A-NLS-mut
and myc-GFP-TNRC6A-NES-mut). The NES mutant
showed punctate localization exclusively in the nucleus(Fig. 2C,E). This was also confirmed under a confocal
microscope. The NES mutant proteins were distributed in
the middle area in the nucleus, and they were notassociated with the nuclear membrane (Fig. 2F,G). However, the subcellular localization of the NLS mutant was notdistinguished from that of wild type (Fig. 2A,B,E). An NLS
mutation was then additionally introduced into the NES
mutant (myc-GFP-TNRC6A-NLS/NES-mut). The productwas preferentially distributed in the cytoplasm (Fig. 2D,E),
demonstrating that the NLS and NES of TNRC6A are
functional in the full-length context.
However, cells transfected with 500 ng/well of TNRC6A
and its mutant expression constructs resulted in protein
product expression levels approximately 5–13 times higher
than the endogenous levels (data not shown). To exclude
the possibility that the NLS- and NES-regulated subcellular
localization of TNRC6A was due to their high expression
levels, the subcellular localization was examined in cells
transfected with 25 ng/well of expression constructs,
representing a similar or lower level of protein expression
compared to endogenous TNRC6A levels. However, GFP
signals were not detected in these experiments due to thesmall amount of protein expressed by the transfected
expression constructs (Supplemental Fig. S3, GFP). Therefore, these cells were stained using an anti-GFP antibody(Supplemental Fig. S3, anti-GFP). The subcellular localizations
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of TNRC6A and its NLS and/or NES mutant proteins at
endogenous levels were essentially consistent with thoseobserved under the overexpression conditions at 500 ng/
well, although clear dotted foci were not detected (Supplemental Figs. S3, S4). These results suggest that subcellular
localization of endogenous TNRC6A is also conducted by
the NLS and NES identified in this study. Additionally, thedotted foci of TNRC6A and its mutant proteins in bothcytoplasm and nucleus might be formed when they are
expressed excessively.
TNRC6A is exported from the nucleus by Exportin 1
Because Exportin 1 is an export receptor for various NESs
(Kutay and Güttinger 2005), we investigated whether TNRC6A
is exported from the nucleus by Exportin 1. Following
transfection of the plasmids encoding myc-GFP-TNRC6A
or its deletion mutants into HeLa cells, the cells weretreated with Leptomycin B (LMB), a specific inhibitor of
Exportin 1-mediated protein export (Kudo et al. 1999).
Four h after LMB treatment, most of the dotted GFP signalsof wild-type myc-GFP-TNRC6A were detected exclusively
in the nucleus (Fig. 3A,E). Nuclear translocation of afragment containing NES, myc-GFP-TNRC6A-(935–984),
was also observed by LMB treatment (Fig. 3B,E). However,the signals in the cytoplasm remained weak, probably because this fragment does not contain the NLS sequence.
To confirm Exportin 1-dependent cytoplasmic transportof TNRC6A, an RNAi experiment targeting Exportin 1 was
conducted. The siRNA against Exportin 1 (siXPO1) efficiently reduced the expression of Exportin 1 protein (Fig.
3G). Following the transfection of siXPO1 with the plasmids encoding myc-GFP-TNRC6A or myc-GFP-TNRC6A(935–984) into HeLa cells, notable nuclear transport ofmyc-GFP-TNRC6A and myc-GFP-TNRC6A-(935–984)
proteins was observed in >40% and z100% of the cells,
respectively (Fig. 3C,D,F). Another export receptor, Exportin 5, has been shown to be involved in the nuclear
export of pre-miRNA (Yi et al. 2003; Bohnsack et al. 2004;
Lund et al. 2004). Transfection of siXPO5 showed no significant change in the subcellular localization of myc-GFPTNRC6A or myc-GFP-TNRC6A-(935–984) (Fig. 3C,D,F),
although Exportin 5 was efficiently knocked down (Fig. 3H).
Thus, Exportin 1 might be a specific mediator of TNRC6A
export from the nucleus to the cytoplasm.
This result was further confirmed for endogenous TNRC6A.
Because the antibodies against endogenous TNRC6A protein tested so far were not available for immunofluorescence experiments (probably due to a low expression levelof endogenous TNRC6A protein or for technical reasons),
we performed cell fractionation experiments followed byWestern blot analysis to determine the subcellular localization of endogenous TNRC6A protein. Following LMB
treatment, the amount of endogenous TNRC6A was clearly
increased in the nuclear soluble and insoluble fractions

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Nuclear microRNA silencing navigated by TNRC6A

FIGURE 2. Subcellular localization of NES and NLS mutants of TNRC6A in the full-length context. (A–D) Subcellular localization of the mycGFP-TNRC6A (WT; A), myc-GFP-TNRC6A-NLS-mut (B), myc-GFP-TNRC6A-NES-mut (C), and myc-GFP-TNRC6A-NLS/NES-mut (D)proteins. Bars, 20 mm. (E) The ratio of the cells transfected with pmyc-GFP-TNRC6A and its mutants by classification according to theirsubcellular localization. Protein expression was verified by Western blot (Supplemental Fig. S1B). (F) The schematic diagram of observation of thecells expressing myc-GFP-TNRC6A-NES-mut by confocal microscopy. (G) Confocal images of the myc-GFP-TNRC6A-NES-mut at each section
shown in F. Bar, 10 mm.

(Fig. 3I). This result strongly suggests that endogenous
TNRC6A is also exported from the nucleus by Exportin 1,
as in the case with exogenous myc-GFP-TNRC6A protein.
Direct binding of Exportin 1 to the NES of TNRC6A was
examined by a glutathione S-transferase (GST) pulldown
experiment using a recombinant His- and GST-tagged NES
fragment containing 935–984 amino acids (His-GST-TNRC6A[935–984]). Control His-GST, His-GST-TNRC6A-(935–984),
or His-GST-TNRC6A-(935–984)-NES-mut was immobilizedon glutathione-sepharose, and the purified His-tagged Exportin 1 (His-XPO1) was added in the presence or absenceof His-tagged Ran(Q69L), a GTPase-deficient Ran mutant
locked in the GTP-bound state, to stimulate binding of the
NES to Exportin 1 (Fornerod et al. 1997). His-XPO1 boundto His-GST-TNRC6A-(935–984) in the presence of HisRan(Q69L)GTP but not to that with the NES mutation(Fig. 3J). This indicates that Exportin 1 binds the NES of
TNRC6A. Furthermore, binding of His-XPO1 to the NES
fragment immobilized on the glutathione-sepharose waseliminated by the pretreatment of His-XPO1 with LMB(Fig. 3K), which binds directly to Exportin 1 and inhibits
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FIGURE 3. Nuclear export of TNRC6A by Exportin 1. (A–D) Fluorescent images of HeLa cells transfected with pmyc-GFP-TNRC6A (WT; A,C)or pmyc-GFP-TNRC6A-(935–984) (B,D), treated with LMB (A,B), or siXPO1 and siXPO5 (C,D). Bars, 20 mm. (E,F) The ratio of cells expressing
GFP exclusively in the nucleus (N, blue), cytoplasm (C, red), or both (N+C, yellow). A and B (E), and C and D (F) at the upper side indicate theresults shown in A and B, and C and D, respectively. (G,H) Western blot of Exportin 1 (G) or Exportin 5 (H) in HeLa cells transfected withsiControl and siXPO1, or siControl and siXPO5, respectively. Anti-tubulin antibody was used as a loading control. (I) HeLa cells treated with andwithout LMB for 8 h were harvested, and cell fractionation was performed. Ten mg of the cytoplasmic fraction (C), 5 mg of the nuclear soluble
fraction (NS), or 10% of the nuclear insoluble fraction (NI) were analyzed by Western blot using an anti-TNRC6A antibody. Blue and red
arrowheads indicate the positions corresponding to z210-kDa TNRC6A (NP_055309) and z182-kDa TNRC6A (AAK62026) proteins,respectively. Anti-tubulin, anti-Lamin A/C, and anti-Histone H3 antibodies were used as a cytoplasmic marker, a nuclear soluble and insoluble
marker, and a nuclear insoluble marker, respectively. Asterisks (*1, *2, *3) indicate nonspecific bands. (J) GST pulldown assay of Exportin 1 and
TNRC6A fragments. Equal amounts of His-GST, His-GST-TNRC6A-(935–984), or His-GST-TNRC6A (935–984)-NES-mut were immobilized on
glutathione-sepharose beads and incubated with recombinant Exportin 1 with or without Ran(Q69L)GTP. Bound fractions were analyzed byWestern blot using an anti-XPO1 antibody. The lower panel is the pattern of Coomassie brilliant blue (CBB) staining, which indicates that eachprotein is similarly immobilized. (K) GST-TNRC6A-(935–984) was immobilized on glutathione-sepharose beads and incubated with theindicated amount of LMB treated-Exportin 1 in the presence of Ran(Q69L)GTP. Bound fractions were analyzed by Western blot using antiXPO1. The lower panel shows the CBB staining pattern.

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Nuclear microRNA silencing navigated by TNRC6A

TNRC6A is exported from the nucleus by direct binding toExportin 1 in a RanGTP-dependent manner.
TNRC6A has two strong Ago2 binding motifs
and a weak one
Human TNRC6A has been shown to interact with Agoproteins by using its three Ago-interacting motifs (Eulalio
et al. 2009a; Takimoto et al. 2009). We confirmed that
TNRC6A has three GW-repeated Ago-binding motifs,
named as GW-I (amino acid residues 457–532), -II (716–772), and -III (810–924) in this study (Supplemental Fig.
S5A,B). These regions overlapped with the previously
reported regions and showed high homology with those
involved in TNRC6B and TNRC6C (Supplemental Fig. S5C).
To confirm that GW-I, -II, and -III are functional Ago2binding sites in a full-length TNRC6A, pmyc-GFP-TNRC6A
expression constructs lacking each of or combinations of
GW-I, -II, and -III motifs (pmyc-GFP-TNRC6A-del.GW-I,-II, -III, -II+III, -I+III, -I+II, or -I+II+III) were generated.
These constructs were transfected into HeLa cells, andbinding of their products with endogenous Ago2 was examined by immunoprecipitation with an anti-GFP antibody.
The results of Western blot showed that little or no Ago2
binding with myc-GFP-TNRC6A-del.GW-I+II+III was observed, indicating that the three GW motifs are principal
Ago2 binding sites in a full-length context (Supplemental
Fig. S6). Myc-GFP-TNRC6A-del.GW-II+III, -I+III, and-I+II are considered to bind Ago2 by the GW-I, -II, and-III motifs, respectively. Endogenous Ago2 bound to mycGFP-TNRC6A-del.GW-II+III and -I+III in clearly detectablelevels, but myc-GFP-TNRC6A-del.GW-I+II showed a weakand negligible level of Ago2 interaction (Supplemental Fig.
S6). These results indicate that GW-I and -II are strong
Ago2-binding motifs with similar binding activities, but
GW-III is a weak one. Myc-GFP-TNRC6A-del.GW-I, -II,and -III are considered to bind Ago2 using two of three
Ago2 binding motifs, GW-II+III, -I+III, and -I+II motifs,
respectively. However, their binding activities with Ago2
were similar and indistinguishable from those of myc-GFPTNRC6A-del.GW-II+III, -I+III and wild-type myc-GFPTNRC6A (Supplemental Fig. S6), suggesting that endogenous
Ago2 binds to either of GW-I or -II motif in a full-length
TNRC6A with similar binding affinity in HeLa cells.

Fig. S7B,C). In the cells transfected with the wild-type
TNRC6A expression construct, GFP signals of myc-GFPTNRC6A were mainly observed in the cytoplasmic fociwhere endogenous Ago2 was clearly colocalized (Fig. 4B).
Following the transfection with pmyc-GFP-TNRC6A-NESmut, noticeable Ago2 signals emerged in both the nucleus
and the cytoplasm, and myc-GFP-TNRC6A-NES-mut andAgo2 were colocalized well in nucleoplasmic foci (Fig. 4B).
A similar colocalization of TNRC6A and Ago2 in the nucleus
was observed in the cells transfected with wild-type pmycGFP-TNRC6A followed by LMB treatment (Supplemental
Fig. S7D). Then, Ago2-interacting sites in TNRC6A in the
nucleus were examined using NES-mutated TNRC6A expression constructs lacking either GW-I, -II, or -III motifs,
or combinations of them (Fig. 4A). All the NES-mutated
TNRC6As were localized exclusively in the nucleus in about
90% of cells (Fig. 4B,C). However, the subcellular localization of Ago2 was different according to the deleted GW
motif(s) in TNRC6A. When myc-GFP-TNRC6A-del.GW-I,-II, -III, -II+III, or -I+III with NES mutation were expressedin the cells, no apparent change in the nuclear localization ofendogenous Ago2 was observed (Fig. 4B). However, whenpmyc-GFP-TNRC6A-del.GW-I+II+III-NES-mut lacking allfunctional GW motifs was transfected, Ago2 was not colocalized with TNRC6A in the nucleus (Fig. 4B). An almost
identical result was obtained when myc-GFP-TNRC6Adel.GW-I+II-NES-mut was used (Fig. 4B), probably due
to weak Ago2-binding ability of GW-III. These results
demonstrate that TNRC6A can actively translocate into
the nucleus regardless of the interaction of Ago2.
Next, we examined the translocation of Ago2 proteinin the TNRC6A knockdown cells with or without LMB
treatment (Supplemental Fig. S8A,B), as quantitatively represented in Supplemental Figure S8C. In cells transfected withsiControl, the endogenous Ago2 signals were apparently
translocated into the nucleus in the presence of LMB treatment. However, the amount of Ago2 signals translocatedinto the nucleus in the LMB-treated TNRC6A knockdown
cells was significantly small. This result suggests that Ago2translocation into the nucleus is dependent on TNRC6A.
The small increase in Ago2 signals might have been causedby the residual endogenous TNRC6A that had not beenknocked down (Supplemental Fig. S8D). Alternatively, thetranslocation of Ago2 might be regulated by factors otherthan TNRC6A.

TNRC6A translocates into the nucleus
in an Ago2-independent manner

Nuclear TNRC6A is colocalized with miRNAs

We examined whether TNRC6A localized in the nucleus
can interact with Ago2 by detecting endogenous Ago2
using anti-Ago2 antibody. The endogenous Ago2 signals
were detected slightly, if at all, by anti-Ago2 antibody in the
nucleus of normal HeLa cells (Supplemental Fig. S7A). Thesesignals were confirmed as Ago2 signals, since these signals
were greatly reduced by Ago2 knockdown (Supplemental

The result that TNRC6A can interact with Ago2 in the
nucleus as well as in the cytoplasm raises the question ofwhether Ago2 associates with miRNAs in the nuclear foci.
To clarify this point, pmyc-GFP-TNRC6A and its NES
mutants with or without Ago2-binding motifs were transfected into HeLa cells, and immunoprecipitation with ananti-GFP antibody was carried out. Then, miRNAs in the
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FIGURE 4. Nuclear transport of TNRC6A is Ago2-independent. (A) Schematic representation of myc-GFP-TNRC6A-NES-mut with GW motif
deletions. Note that NES regions were mutated. (B) Fluorescent images of HeLa cells transfected with the expression constructs of myc-GFPTNRC6A (WT) or myc-GFP-TNRC6A-NES-mut with GW motif deletions and stained by an anti-Ago2 antibody, followed by Cy5-conjugatedanti-mouse IgG. Fluorescent images are shown, from the left: GFP signals of TNRC6A fragments; Cy5 signals of the second antibody against antiAgo2 antibody; the merged images of GFP and Cy5, in which GFP is shown in green, the Cy5 in magenta, and the overlapped GFP/Cy5 in white;and DAPI signals. Bars, 5 mm. Protein expression was verified by Western blot (Supplemental Fig. S1C). (C) The ratio of cells expressing GFP
exclusively in the nucleus (N, blue), cytoplasm (C, red), or both (N+C, yellow).

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Nuclear microRNA silencing navigated by TNRC6A

purified RNA from the immunoprecipitates were detectedby RT-PCR. As expected, both of myc-GFP-TNRC6A andmyc-GFP-TNRC6A-NES-mut were immunoprecipitated with
Ago2, but TNRC6A without Ago2 binding motifs, myc-GFPTNRC6A-del.GW-I+II+III and myc-GFP-TNRC6A-del.GWI+II+III-NES-mut were not (Fig. 5A). All of the four miRNAs
examined in this study (let-7g, miR-16, miR-21, and miR-30a) were clearly detected in RNAs purified from the immunoprecipitates from the cells expressing myc-GFP-TNRC6A
and myc-GFP-TNRC6A-NES-mut (Fig. 5B). However, little ornone of miRNAs were detected in the immunoprecipitatesfrom the cells expressing TNRC6A without GW motifs.
To confirm the nuclear localization of miRNAs, we
performed an in situ hybridization analysis using a locked

FIGURE 5. Nuclear TNRC6A is colocalized with miRNAs. (A) Cell lysates from HeLa cells expressing myc-GFP-TNRC6A and myc-GFPTNRC6A-del.GW-I+II+III with or without NES mutation were immunoprecipitated with an anti-GFP antibody or rabbit normal IgG. Then, theimmunoprecipitates (IP) and cell lysates were analyzed by Western blot using a monoclonal anti-Ago2 antibody (upper panel) and an anti-GFP
antibody (lower panel). Asterisks indicate products of myc-GFP-TNRC6A and myc-GFP-TNRC6A-del.GW-I+II+III with or without NES
mutation. (B) The amount of miRNAs contained in the immunoprecipitates was quantified by real-time PCR. Data are the mean and standard
deviation of three independent experiments. (C–H) HeLa cells expressing myc-GFP-TNRC6A (WT; C,D), myc-GFP-TNRC6A-NES-mut (NESmut; E,F), or myc-GFP-TNRC6A-del.GW-I+II+III-NES-mut (del.GW-I+II+III-NES-mut; G,H) were stained by digoxigenin-labeled LNA probetargeting miR-21 (C,E,G) or control probe (D,F,H). Fluorescent images are shown, from the left: GFP signals of TNRC6A; Rhodamine signals of
the antibody against miRNA probes; the merged images of GFP and Rhodamine, in which GFP is shown in green and the Rhodamine in magenta;
and DAPI signals. In the merged images, the dashed lines represent nuclear outlines. Bars, 10 mm.

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nucleic acid (LNA) probe specific to miR-21 (Fig. 5C–H).
In HeLa cells expressing wild-type myc-GFP-TNRC6A,
miR-21 signals were detected as dotted foci overlappedwith GFP signals in the cytoplasm (Fig. 5C), although suchapparent dotted structures of miR-21 were not detected in
the cells without overexpression of myc-GFP-TNRC6A(data not shown). In contrast, no definite signals weredetected by a scrambled LNA miRNA probe (Fig. 5D). In
the cells expressing TNRC6A with NES mutation, miR-21
but not control LNA probe signals were observed in the
nuclear foci containing GFP signals (Fig. 5E,F). Suchnuclear localization of miR-21 was not observed in thecells transfected with TNRC6A without any GW motif (Fig.
5G,H). Altogether, our results indicate that miRNAs might
be associated with Ago2 protein, which is tethered by
TNRC6A in the nuclear foci as well as in the cytoplasmic
P bodies.
miRNA-mediated gene silencing is inducedby TNRC6A with NES and/or NLS mutations
TNRC6A has been shown to be necessary for miRNAmediated gene silencing (Liu et al. 2005). To investigate
whether NES and/or NLS mutations in TNRC6A affectmiRNA-mediated gene silencing, we used a CXCR4 miRNAmediated gene silencing assay system (Doench et al. 2003).
However, since TNRC6A, TNRC6B, and TNRC6C are known
to be functionally redundant (Eulalio et al. 2009a), absolutemRNA quantification of TNRC6s was carried out. Endogenous TNRC6A and TNRC6B were found to be expressed
abundantly in HeLa cells (160 and 112 copies per ng total
RNA, respectively), but the amount of TNRC6C was verylow (12 copies per ng total RNA). Then, to eliminate theeffects of endogenous TNRC6A and TNRC6B, they were
knocked-down by the simultaneous transfection of siRNAs
against TNRC6A (siTNRC6A) and TNRC6B (siTNRC6B#1
or siTNRC6B#2) targeting their 39 UTRs. Both combinationsof siTNRC6A/siTNRC6B#1 and siTNRC6A/siTNRC6B#2 effectively reduced the expression levels of endogenous TNRC6A
and TNRC6B transcripts at z35%–50% (Fig. 6A,B). The
reduction of endogenous TNRC6A and TNRC6B remarkably repressed miRNA-mediated gene silencing activity (Fig.
6C), indicating that TNRC6s are essential for miRNAmediated gene silencing, as shown in the previous reports(Liu et al. 2005; Eulalio et al. 2009a; Zipprich et al. 2009).
Under these conditions, the introduction of myc-GFPTNRC6A and its NES and/or NLS mutants showed substantial silencing activities (Fig. 6C). However, control mycGFP exhibited almost no silencing activity. The protein
expression of myc-GFP, myc-GFP-TNRC6A and its mutants
was verified by Western blot (Supplemental Fig. S9A).
Moreover, under the same conditions shown in Figure 6C,
the unambiguous changes in subcellular localization of
TNRC6A or TNRC6A-NES-mut were not observed (Fig.
6D,E). In addition, whereas NES-mutated TNRC6A having
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at least the GW-I or GW-II motif exhibited miRNA-mediatedgene silencing activities, little or no silencing activities wereobserved by NES-mutated TNRC6A without any GW motifs
or with the weakest GW-III motif (Fig. 6F; Supplemental Fig.
S9B), indicating that interaction of TNRC6A with Ago2 might
be essential for silencing activity in the nucleus.

TNRC6A with NES mutation enhances RNAi activity
against a nuclear-specific noncoding RNA, MALAT-1
To confirm that RNA silencing in the nucleus is associatedwith the nuclear Ago2, navigated by TNRC6A with NES
mutation, we generated TNRC6A- and TNRC6A NES
mutant-inducible cells using the Flp-In T-REx system in
HEK293 cells and examined the effects of their overexpression. Under control of the tetracycline-inducible promoter,
expression of FLAG-HA-SBP-tagged TNRC6A (FLAG-HASBP-TNRC6A) or its NES mutant was induced by doxycycline (dox) treatment in HEK293 cells. Then, we examined
the RNAi activity against metastasis-associated in lungadenocarcinoma transcript 1 (MALAT-1), a nuclear-specificnoncoding RNA that is known to be stably retained in the
nucleus (Miyagawa et al. 2012). FLAG-HA-SBP-TNRC6A
and its mutants were induced by dox in a dose-dependent
manner (Fig. 7A). In cells transfected with siRNA against
MALAT-1 (siMALAT-1), a nearly identical level of MALAT-1
repression was observed as that found in cells transfectedwith either TNRC6A or its mutant protein expression constructs in the absence of dox (Fig. 7B–E). Induction of FLAGHA-SBP-TNRC6A-NES-mut increased RNAi activity for
MALAT-1 (Fig. 7D). In contrast, FLAG-HA-SBP-TNRC6ANLS-mut decreased this activity (Fig. 7E). However, theempty vector or TNRC6A-WT did not show any remarkable effects (Fig. 7B,C). The results suggest that the overexpression of TNRC6A with NES mutation navigates many
Ago2 proteins into the nucleus, resulting in the increase in
RNAi activity for nuclear RNA, whereas TNRC6A with NLS
mutation retains Ago2 in the cytoplasm, thereby decreasing
RNAi activity in the nucleus.

Colocalization of Dcp1 and p54 with cytoplasmic
TNRC6A but not with nuclear TNRC6A
We investigated the subcellular localization of the marker
proteins of the cytoplasmic P bodies Dcp1 and p54, which
are known to be colocalized with TNRC6A or Ago2 in thecytoplasm (Eystathioy et al. 2003; Chu and Rana 2006).
Dcp1 and p54 proteins were clearly colocalized with wildtype myc-GFP-TNRC6A in the cytoplasmic foci, but suchcolocalization was not observed in the nuclear foci formedby the expression of myc-GFP-TNRC6A-NES-mut or wildtype myc-GFP-TNRC6A after LMB treatment (Fig. 8).
Thus, the components of cytoplasmic and nuclear focidiffered from one another.

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Nuclear microRNA silencing navigated by TNRC6A

FIGURE 6. miRNA-mediated gene silencing activity of TNRC6A with NES and/or NLS mutations. (A,B) TNRC6A (A) and TNRC6B (B) mRNAlevels in HeLa cells transfected with siTNRC6A/siTNRC6B#1 or siTNRC6A/siTNRC6B#2. (C) One d after the transfection with siControl,
siTNRC6A/siTNRC6B#1 or siTNRC6A/siTNRC6B#2, cells were further transfected with a mixture of the following four plasmids: psiCHECKCXCR4 target, pGL3-Control, pSUPER-CXCR4 or control pSUPER-DsRed, and myc-GFP or the indicated TNRC6A expression constructs.
Two d after second transfection, luciferase activities were measured. The activities were shown as the mean and standard deviation of three or four
independent experiments. P-values were determined by Student’s t-test. (*) P < 0.05. (D,E) Fluorescent microscopy images of the cells expressing
WT (D) or NES-mut (E) under the same conditions shown in C. WT was exclusively localized in the cytoplasm, but NES-mut was in the nucleus.
Bars, 20 mm. (F) Relative luciferase activities by the transfection of NES-mutated TNRC6As lacking various combinations of GW motifs.

DISCUSSION
In this study, we found that human TNRC6A protein isa nuclear–cytoplasmic shuttling protein, and its subcellular
localization is conducted by its NES and NLS sequences.

There are functional NESs in the central regions between
GW-III and Q-rich domains in TNRC6A as well as in
TNRC6B and TNRC6C (Supplemental Fig. S2A). TheseNES sequences in human TNRC6A–C are completely conserved in their mouse counterparts, MmTNRC6A–C, and theirwww.rnajournal.org

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Nishi et al.

the function of the UBA domain. Thus,
it is not negligible that the miRNA-mediated gene silencing activity via EDD isdisturbed.
TNRC6A also has a NLS in the region
between GW-III and a Q-rich region(Supplemental Fig. S2A), but neither
TNRC6B nor TNRC6C has NLS sequencein this corresponding region. No NLSlike region has been found in the GW182
family proteins of C. elegans or Drosophila melanogaster. Thus, the NLS might be
not essential for all of the GW182 family
proteins. However, Till et al. (2007) reported
that GFP-TNRC6B iso3 (NP_001020014;
z110 kDa) substantially accumulates
in the nucleus by LMB treatment, indicating that TNRC6B iso3 is a nuclear–cytoplasmic shuttling protein. Since any
proteins larger than z40 kDa require
NLS sequences for their nuclear import(Davis 1995), TNRC6B or its interacting protein(s) might have a NLS. The
best understood pathway of nuclear
transport is conducted by a heterodimeric import receptor consisting of the
FIGURE 7. Effects of overexpression of TNRC6A and its NES or NLS mutants on RNAi b-karyopherin importin b, which meactivity against MALAT-1. (A) The induction of FLAG-HA-SBP-TNRC6A or its mutant diates interactions with the nuclear poreproteins by the treatment of the indicated concentration of dox for 24 h in HEK293 cells stably complex, and the adaptor protein imtransfected with either empty vector, FLAG-HA-SBP-TNRC6A-WT, FLAG-HA-SBP-TNRC6ANES-mut, or FLAG-HA-SBP-TNRC6A-NLS-mut expression constructs was verified by Western portin a, which directly binds to theblot using anti-TNRC6A antibody. Anti-tubulin antibody was used as a loading control. (B–E) NLS motif (Lange et al. 2007). Weinmann
Stable HEK293 cells with pcDNA5/FRT/TO/FLAG-HA-SBP (empty vector; B), and expressing et al. (2009) reported that Importin 8
FLAG-HA-SBP-TNRC6A (C), FLAG-HA-SBP-TNRC6A-NES-mut (D), and FLAG-HA-SBP(Imp8) binds to Ago in P bodies and
TNRC6A-NLS-mut (E) were transfected with siControl2 or siMALAT-1 and cultured in thepresence of the indicated concentration of dox for 24 h. MALAT-1 RNA levels were shown as the shuttles Ago protein to the nucleus.
mean and standard deviation of three independent experiments. P-values were determined by Therefore, it is probable that Ago transStudent’s t-test. (*) P < 0.05.
locates into the nuclei independent of
TNRC6A. However, they described that,even when Imp8 was depleted, a significant portion of Ago remained in the nucleus, suggesting
consensus sequences are also found in C. elegans AIN-1 andthat alternative nuclear import pathways for Ago proteinsAIN-2, and Drosophila dGW182 (Supplemental Fig. S10),
may exist. Although we could not identify an import receptor
suggesting that the NES is functionally essential in diverseof TNRC6A in this study, TNRC6A might be a strong canorganisms. In human GW182 family proteins, the NES
didate for an Ago-navigator protein into the nucleus by somemotif is included in the previously reported UBA domain
other type of importin receptor that is able to bind to the NLS(Supplemental Fig. S2A). However, because four Drosophilasequence.
putative NESs in dGW182 did not overlap its UBA domain,
Human GW182 family proteins have three GW motifs.
the UBA domains of human and mouse GW182 family
Takimoto et al. (2009) demonstrated that GW182 could
proteins might have an alternative function other than theassociate with multiple Ago proteins, but each of them can
NES. A recent report revealed that each of UBA domains of
independently bind to Ago2. Our results indicate thathuman TNRC6A and TNRC6C can interact with a UBA
binding activities of GW-I, -II, and -III with Ago2 weredomain of mammalian hyperplastic discs protein EDD,
different in the full-length context (Supplemental Fig. S6).
which is an E3 ubiquitin ligase and participates in miRNAThe results of nuclear translocation with Ago2 indicatedmediated gene silencing by recruiting downstream effectorsthat the GW-I and GW-II motifs in TNRC6A showed(Su et al. 2011). As shown in Supplemental Figure S2A, thestrong binding activities, but the GW-III motif has little orUBA domain in TNRC6A is overlapped with the identified
negligible activity (Fig. 4B). Furthermore, NES-mutated
NES. The NES mutant of TNRC6A could potentially destroy
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FIGURE 8. Dcp1 and p54 were colocalized with cytoplasmic but notwith nuclear TNRC6A. (A,C) HeLa cells transfected with pmyc-GFPTNRC6A were treated with or without LMB, and stained with antiDcp1 (A) or anti-p54 (C) antibody. Fluorescent images are shown,from the left: GFP signals of TNRC6A; Cy5 signals of the second
antibody against anti-Dcp1 (A) or anti-p54 (C) antibody; the merged
images of GFP and Cy5, in which GFP is shown in green and the Cy5
in magenta; and DAPI signals. Bars, 10 mm. (B,D) HeLa cells
transfected with pmyc-GFP-TNRC6A-NES-mut were stained with
anti-Dcp1 (B) or anti-p54 (D) antibody. Fluorescent images areshown, from the left: GFP signals of TNRC6A; Cy5 signals of thesecond antibody against anti-Dcp1 (B) or anti-p54 (D) antibody; themerged images of GFP and Cy5, in which GFP is shown in green and
the Cy5 in magenta; and DAPI signals. Bars, 10 mm.

TNRC6A having at least GW-I and/or GW-II motif(s)showed substantial silencing activities, but the mutants
lacking both the GW-I and GW-II motifs exhibited no or
little silencing activities (Fig. 6F). Thus, at least one of the
GW-I or GW-II motifs in TNRC6A might be functionallysufficient for nuclear–cytoplasmic shuttling of endogenous
Ago2 and gene silencing activity in the nucleus.

We investigated the effects of NES and/or NLS mutationsin TNRC6A using the CXCR4 miRNA-mediated gene silencing assay system (Fig. 6). Under knockdown conditionsof the endogenous GW182 family proteins, the expressionof NES-mutated TNRC6A showed adequate silencing activity, and this activity was eliminated when Ago2-binding
motifs were deleted. The result strongly suggested that
TNRC6A exerts miRNA-mediated gene silencing activity
with Ago2 in the nucleus. However, because a small
amount of NES-mutated TNRC6A protein was observedin the cytoplasm (Supplemental Fig. S4C), the possibility
that the NES-mutated TNRC6A exhibits silencing activity
in the cytoplasm could not be excluded. Therefore, we
carried out an RNAi assay targeting nuclear-specific MALAT-1
RNA (Fig. 7). RNAi activity against MALAT-1 was increasedby the induction of NES-mutated TNRC6A expression,
although little or no effect was observed by the induction of
wild-type TNRC6A proteins. The result suggests that theAgo2 protein is navigated into the nucleus by binding with
TNRC6A to enhance the RNAi activity in the nucleus.
Under the overexpression condition of wild-type TNRC6A,
clear dotted foci were observed in the nucleus by Exportin 1
knockdown or LMB treatment (Fig. 3). Furthermore, not only
TNRC6A but also TNRC6C was proved to produce nuclear
foci, and both proteins were shown to be colocalized in thesame nuclear foci (L Badertscher and W Filipowicz, pers.
comm.). As shown previously (Eystathioy et al. 2003; Chu
and Rana 2006), we confirmed that the subcellular localization of the P-body marker proteins Dcp1 and p54 werecolocalized with the wild-type myc-GFP-TNRC6A in thecytoplasmic foci, but such colocalization was not observedin the nuclear foci formed by the expression of myc-GFPTNRC6A-NES-mut or wild-type myc-GFP-TNRC6A after
LMB treatment (Fig. 8). Thus, we revealed that the components of cytoplasmic and nuclear foci were different fromone another, suggesting that the miRNA-mediated silencing
mechanism in the nucleus might not be parallel to that in
the cytoplasm, although the possibility that the nuclear fociare formed due to the overexpression of TNRC6A could not
be excluded. So far, conceivable silencing mechanisms in the
nucleus are as follows: (1) Target mRNAs are tethered bymiRNA-Ago-TNRC6 complex in the nucleus to avoid translocation into cytoplasm for translation; (2) TNRC6A destabilizes poly(A) RNAs in the nucleus, since the components
involved in miRNA-mediated deadenylation, PABPC1,
Ccr4-Caf1, and Pan2-Pan3 (Chen et al. 2009; Fabian et al.
2009), were shown to be nuclear–cytoplasmic shuttlingproteins (Afonina et al. 1998; Yamashita et al. 2005); and (3)miRNA associates with promoter RNAs to induce miRNAmediated TGS or heterochromatin formation (Kim et al. 2008;
Younger and Corey 2011).
In summary, we found that: (1) nuclear–cytoplasmictransport of human TNRC6A is conducted by its NLS andNES (Fig. 9); (2) its nuclear export is mediated by Exportin
1; (3) TNRC6A can translocate into the nucleus with or
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FIGURE 9. TNRC6A regulates miRNA-mediated gene silencing in
the nucleus and cytoplasm by navigating Ago protein. TNRC6A
translocates from the cytoplasm to the nucleus via its own NLS
sequence. Its nuclear export is mediated by Exportin 1 (XPO1), which
directly binds to its NES sequence. In the cytoplasm, interaction
between Ago and TNRC6A triggers translational repression and/or
mRNA degradation by a known mechanism (see Introduction). In the
nucleus, the miRNA-Ago-TNRC6A complex might induce miRNAmediated gene silencing targeting nuclear RNA by an unknown
mechanism. See the text for details.

without Ago2 interaction; (4) in the nucleus, miRNAs arefound to be colocalized with TNRC6A which interacts with
Ago2; and (5) small RNA-containing Ago2 protein navigated into the nucleus by TNRC6A might cleave MALAT-1
RNA or hold target RNAs in the nucleus. Our results
proposed the possibility that GW182 family proteins could
regulate miRNA-mediated gene silencing in the nucleus
as well as in the cytoplasm by navigating Ago proteins,
although the silencing mechanism in the nucleus remains
unknown.
MATERIALS AND METHODS
Plasmid construction
The expression plasmids of TNRC6A, B, and C and their derivatives, Exportin 1 and Ran(Q69L) were constructed as follows:
pIRESneo-FLAG/HA-Ago2 (Meister et al. 2004), kindly provided
by Dr. Thomas Tuschl through Addgene, was used as pFLAG/HAAgo2 in this study. All PCR primers used for plasmid construction
are shown in Supplemental Table S1.
Myc-GFP-TNRC6A expression construct, pmyc-GFP-TNRC6A:
A fragment encoding EGFP was amplified using pEGFP-N3(Clontech) as a template by PCR with a forward primer containing
the myc-tag and a reverse primer. An amplified fragment wasdigested with NheI and HindIII and inserted into the NheI/
HindIII-digested pcDNA3.1-myc-Dicer (Provost et al. 2002). A
5130-bp fragment encoding TNRC6A, which was amplified using

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pCMV-SPORT/FLAG-GW182 (Wakiyama et al. 2007) by PCR, wasdigested with HindIII and XbaI and inserted into pcDNA3.1-mycGFP-Dicer by two-step ligation: (1) A 1994-bp fragment was inserted into pcDNA3.1-myc-GFP-Dicer digested with HindIII and
XbaI; and (2) a 3136-bp fragment was inserted into the HindIII site.
The orientation of the insert was determined by nucleotide sequence analysis.
Myc-GFP-TNRC6B expression construct, pmyc-GFP-TNRC6B:
Fragment encoding TNRC6B was amplified using cDNA derived
from HEK293 cells. A 5172-bp fragment of TNRC6B was digested
with HindIII and XbaI and inserted into the HindIII/XbaIdigested pcDNA3.1-myc-GFP-Dicer.
Myc-GFP-TNRC6C expression construct, pmyc-GFP-TNRC6C:
A 5073-bp fragment of TNRC6C amplified using cDNA from
HEK293 cells was digested with HindIII and XbaI, and inserted into
pcDNA3.1-myc-GFP-Dicer by two-step ligation: (1) A 3202-bp
fragment was inserted into pcDNA3.1-myc-GFP-Dicer digested
with HindIII and XbaI; and (2) a 1871-bp fragment was inserted
into the XbaI site. Deleted constructs of TNRC6A, B, and C were
amplified using pmyc-GFP-TNRC6A, B, and C, respectively, as
templates. They were digested with HindIII and XbaI, and inserted
into the HindIII/XbaI-digested pcDNA3.1-myc-GFP-Dicer. Aminoacid-substituted constructs and several deletion constructs of
TNRC6A, B, and C were generated using the corresponding
pmyc-GFP-TNRC6A, B, and C by site-directed mutagenesis using
PCR.
pET-Exportin 1 and pET-Ran(Q69L): A fragment encoding
Exportin 1 or Ran was amplified by PCR using cDNA derived
from total RNA of HEK293 cells. They were then digested with
NheI and NotI, and inserted into NheI/NotI sites of pET28a(Novagen). pET-Ran(Q69L) was generated from pET-Ran by sitedirected mutagenesis using PCR.
pET-GST, pET-GST-TNRC6A-(935–984) and pET-GSTTNRC6A-(935–984)-NES-mut: A fragment encoding GST was
amplified by PCR using pGEX-6P2 (GE Healthcare) as a template.
It was digested with NheI and HindIII, and inserted into the NheI/
HindIII sites of pET28a. A fragment encoding GFP-TNRC6A(935–984) or TNRC6A-(935–984)-NES-mut was amplified frompmyc-GFP-TNRC6A derivatives. They were digested with NheI
and NotI, and inserted into the NheI/NotI sites of pET28a. A
fragment encoding GST was amplified by PCR using pGEX-6P2.
It was then digested with NheI and HindIII, and inserted into
pET-GFP-TNRC6A-(935–984) or pET-GFP-TNRC6A-(935–984)NES-mut digested with NheI/HindIII.
pcDNA5/FRT/TO/FLAG-HA-SBP-TNRC6A and its mutant
expression constructs: A fragment encoding TNRC6A, which
was amplified from pCMV-SPORT/FLAG-GW182 by PCR, wascloned into pcDNA5/FRT/TO/FLAG-HA-SBP (Okada-Katsuhata
et al. 2012) using an In-Fusion HD Cloning Kit (Clontech). Aminoacid-substituted constructs were generated using pcDNA5/FRT/
TO/FLAG-HA-SBP-TNRC6A by site-directed mutagenesis using
PCR.
The siRNA expression constructs: The expression constructs ofchemokine receptor CXCR4 siRNA (Doench et al. 2003) orcontrol DsRed siRNA, named pSUPER-CXCR4 or pSUPERDsRed, were constructed by inserting annealed synthetic oligonucleotides consisting of two strands of single-stranded 67-meroligonucleotides (Supplemental Table S1) into BamHI/HindIIIsites in the pSUPER.retro.Puro vector (OligoEngine). From theDownloaded from rnajournal.cshlp.org on December 17, 2012 - Published by Cold Spring Harbor Laboratory Press

Nuclear microRNA silencing navigated by TNRC6A

inserted oligonucleotides, the transcript was encoded in thefollowing order: (1) a 21-nt siRNA guide strand; (2) a human
miR-23a loop; and (3) a 19-nt passenger strand of the identicalsiRNA lacking a 39 overhang.
The siRNA target-containing construct: The plasmid expressing
the CXCR4 siRNA target site (Doench et al. 2003) was constructedby inserting oligonucleotides with a central bulge (Supplemental
Table S1) into the XhoI/EcoRI sites in the 39 UTR of psiCHECK-1(Promega), which encodes the Renilla luciferase gene (psiCHECKCXCR4 target).

Cell culture and LMB treatment
HeLa and HEK293 cells were cultured in Dulbecco’s Modified
Eagle Medium (DMEM; Invitrogen) with 10% fetal bovine serum(FBS; Sigma) at 37°C with 5% CO2. Transfection was conductedusing Lipofectamine 2000 (Invitrogen). For LMB (Calbiochem)
treatment, 2 d after transfection, HeLa cells were washed twicewith DMEM with 10% FBS and incubated in DMEM with 10%
FBS containing 10 ng/mL LMB or the same volume of solvent for
4 h, except for Figure 3I.

Fluorescence microscopy
HeLa cells were inoculated on a glass coverslip in a well of a 12well culture plate at 1 3 105 cells/mL/well at 1 d before transfection. Various GFP-tagged TNRC6A expression constructs (500ng/well) were transfected into the cells. At 2 d after transfection,
cells were washed with phosphate-buffered saline (PBS) and fixedwith 4% paraformaldehyde at room temperature for 15 min. The
cells were mounted in ProLong Gold antifade reagent with DAPI
(Invitrogen). GFP and DAPI signals were observed under a Zeiss
Axiovert 200 fluorescence microscope, using a 633 objective lens,or Olympus Fluoview FV1000 confocal microscopy system, using
a 603 objective lens. In confocal imaging, optical sections weretaken at 1-mm intervals. Quantification of fluorescence microscopy images was performed using Image J software (NIH). The
antibody staining was performed as previously reported (Nishi
and Saigo 2007). Briefly, the fixed cells permeabilized with PBS
containing 0.2% Triton X-100 were incubated with the firstantibody (1:200–400 dilution) in PBS, and this was followed bytreatment with the second antibody (1:400 dilution) in PBS. They
were mounted with DAPI and observed under fluorescencemicroscope. In subcellular localization assays, the numbers ofcounted cells were listed in Supplemental Table S2. The firstantibodies used for staining the cells were as follows: Living
Colors Full-Length Aequorea victoria polyclonal GFP antibody(Clontech), anti-human AGO2 monoclonal antibody, 4G8 (Wako),mouse monoclonal antibody to Dcp1a (Abcam; ab57654), and antiRck/p54 polyclonal antibody (MBL). The second antibodies were as
follows: Cy5-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories), and goat anti-mouse IgG conjugated with
Cy5 (Amersham Pharmacia Biotech).

Western blot
Cells were lysed with ice-cold lysis buffer (10 mM Hepes-NaOH[pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 140 mM NaCl, 0.5 mM
DTT, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 0.5% NP40, and13 complete protease inhibitor cocktail [Roche]) on ice for 30
min and centrifuged. The supernatants were mixed with same

volume of 23 SDS-PAGE sample buffer and boiled for 5 min.
Except for Western blot of TNRC6A, samples were separated by
SDS-PAGE, and the gels were transferred to BioTrace PVDF
membrane (Pall Corp.) with an iBlot Dry Blotting System (Invitrogen). The membranes were blocked for 1 h in TBST (20 mM
Tris-HCl [pH 7.5], 150 mM NaCl, and 0.2% Triton X-100 or 0.1%
Tween-20) supplemented with 5% skim milk (Difco) and washedonce with TBST. Following incubation with the first antibody(1:200–2000 dilution) in TBST containing 1% liquid block (GEHealthcare) at 4°C overnight, the membranes were washed threetimes with TBST and treated with the second antibody (1:10,000
dilution) in TBST containing 1% liquid block. After being washedthree times with TBST, the membranes were incubated with CDPStar detection reagent (GE Healthcare), and signals were detectedby an LAS3000 imaging system (FUJIFILM). For Western blot of
TNRC6A, samples were separated by SDS-PAGE, and the gels weretransferred to PVDF membranes using a Trans-Blot Turbo Transfer
System (Bio-Rad). The membranes were blocked for 1 h in TBST
(20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween-20)
supplemented with 2% ECL advance blocking agent (GE Healthcare). Following incubation with the first antibody (1:2000 dilution) in TBST containing 2% ECL advance blocking agent at 4°C
overnight, the membranes were washed three times with TBST andtreated with the second antibody (1:10,000 dilution) in TBST
containing 2% ECL advance blocking agent. After being washedthree times with TBST, the membranes were reacted with an ECL
advance Western blotting detection kit (GE Healthcare), and signals
were detected by an LAS3000 imaging system.
The first antibodies used for Western blot were as follows:polyclonal anti-Ago2 antibody (Upstate), myc-tag rabbit antibody(Cell Signaling Technology), anti-human AGO2 monoclonal antibody, 4G8, Living Colors Full-Length Aequorea victoria polyclonal GFP antibody, anti-CRM1 (Exportin 1) rabbit polyclonal
IgG, H-300 (Santa Cruz Biotechnology), anti-Exportin 5 rabbit
polyclonal IgG, H-300 (Santa Cruz Biotechnology), Monoclonal
Anti-a Tubulin antibody (ICN/CAPPEL Biomedicals), mouseanti-Lamin A/C monoclonal antibody (BD Transduction Laboratories), Anti-Histone H3 antibody (Abcam; ab1791), and Rabbit
anti-GW182 (TNRC6A) Antibody (Bethyl; A302-329A). The
second antibodies were as follows: alkaline phosphatase-conjugated goat affinity purified antibody to rabbit IgG (ICN/CAPPEL
Biomedicals), alkaline phosphatase-conjugated goat affinity purified antibody to mouse IgG (ICN/CAPPEL Biomedicals), and ECL
Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody(GE Healthcare).

Knockdown of Exportin 1 and Exportin 5
The siRNAs against human Exportin 1 (siXPO1) and Exportin 5(siXPO5) were designed by our functional siRNA selection
algorithm (Ui-Tei et al. 2004, 2008) using siDirect2.0 (Naito et al.
2009) and purchased from Sigma. Their sequences are describedin Supplemental Table S3. HeLa cells inoculated on a glass
coverslip at 1 3 105 cells/mL/well in wells of 12-well culture plate
were transfected twice at 24-h intervals with 50 nM siRNA. At thesecond transfection, 500 ng/well pmyc-GFP-TNRC6A or its
mutant constructs were simultaneously transfected with eachsiRNA. One day after the second transfection, cells were fixedand observed under the fluorescent microscope or harvested forWestern blot.
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Nishi et al.

Cell fractionation
Nuclear soluble and cytoplasmic fractions were isolated using
NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo
scientific) according to the manufacturer’s recommendation. The
remaining pellets were sonicated in 23 SDS sample buffer and usedas nuclear insoluble fractions.

Purification of recombinant proteins
His-GST, His-GST-TNRC6A-(935–984), His-GST-TNRC6A-(935–984)-NES-mut, His-Exportin 1, and His-Ran(Q69L) were expressedfrom the pET constructs (see ‘‘Plasmid construction’’ section) in
Escherichia coli Rosetta (DE3) pLysS (Novagen). Cells were firstgrown in L Broth with 50 mg/mL kanamycin and 34 mg/mL
chloramphenicol at 37°C overnight. The culture was diluted 1:20
in the same medium and grown to A600 = 0.6–1.0 at room temperature, and isopropyl b-D-thiogalactoside was added to a final
concentration of 0.5 mM. After an additional 12 h of culture, thecells were collected by centrifugation at 4°C. The pellets werefrozen and stored at 20°C. Cells were resuspended in lysis buffer(50 mM sodium phosphate [pH 8.0], 10 mM imidazole, and 300
mM NaCl) and disrupted by sonication. The lysate was centrifuged at 12,000g for 1 h at 4°C. The supernatant was loaded ontoa Ni-NTA column (Qiagen) equilibrated with lysis buffer. It waswashed with lysis buffer and then wash buffer (50 mM sodium
phosphate [pH 8.0], 20 mM imidazole, and 300 mM NaCl), andfinally eluted with the elution buffer (50 mM sodium phosphate[pH 8.0], 250 mM imidazole, and 300 mM NaCl). Eluted proteins
were dialyzed against PBS containing 10% glycerol on a PD10
column (GE Healthcare). Purified protein was stored at 80°C.

In vitro binding assay
His-GST, His-GST-TNRC6A-(935–984), His-GST-TNRC6A(935–984)-NES-mut, His-Exportin 1, and His-Ran-GTP(Q69L)
were prepared as described in the ‘‘Purification of recombinant
proteins’’ section. A total of 600 pmol GST fusion proteins werebound to 20 mL of glutathione-Sepharose 4B (GE Healthcare) in
PBS containing 10% glycerol for 2 h at 4°C. They were washedthree times with PBS containing 10% glycerol, and 500 mL PBS
containing 20 pmol Exportin 1, 190 pmol Ran(Q69L), 10 nmol
GTP, and 10% glycerol was added. After incubation for 2 h at 4°C,
they were washed three times with PBS containing 500 mM NaCl,
2 mM GTP, 0.2% Triton X-100, and 10% glycerol and resuspended in 23 SDS sample buffer. Bound proteins were eluted byboiling the beads for 5 min and separated by SDS-PAGE. To
detect the inhibitory effect of LMB on Exportin 1 binding toTNRC6A NES, 20 pmol Exportin 1 protein was treated with 20 or
100 pmol LMB or the solvent in PBS for 1 h on ice before thebinding assay.

Immunoprecipitation
HEK293 cells were plated in a 9-cm dish at 2 3 106 cells/10 mL
1 d before transfection. Three mg of pFLAG/HA-Ago2 weretransfected into the cells with 3 mg of pmyc-GFP-TNRC6A or
its mutant expression constructs. Two days after transfection, cells
were washed once with PBS and lysed with ice-cold lysis buffer (10mM Hepes-NaOH [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 140
mM NaCl, 0.5 mM DTT, 1 mM EDTA, 1 mM Na3VO4, 10 mM

32RNA, Vol. 19, No. 1

NaF, 0.5% NP40, and 13 complete protease inhibitor cocktail[Roche]). Lysates were centrifuged at 20,000g for 10 min at 4°C,
and the supernatants were centrifuged at 48,000g for 30 min at
4°C. The supernatants were incubated with 30 mL anti-c-Myc
agarose conjugate beads (Sigma) with constant rotation for 2 h at
4°C. The beads were washed six times with the lysis buffer
supplemented with 300 mM NaCl, and resuspended in 30 mL
23 SDS sample buffer. Bound proteins were eluted by boiling thebeads for 5 min and separated by SDS-PAGE (SuperSep 5%–20%[Wako]).
HeLa cells were plated in a 9-cm dish at 2 3 106 cells/10 mL 1 d
before transfection. The cell lysates from the cells transfected with
5 mg of pmyc-GFP-TNRC6A or its mutants were prepared as described above. Thirty mL of protein A agarose (MILLPORE) was
equilibrated with 500 mL of lysis buffer and incubated with 2.5 mgof anti-GFP antibody in lysis buffer for 1 h at 4°C. Then, the celllysates were added to the antibody-bound protein A agarose andincubated with constant rotation for 2 h at 4°C. The beads were
washed twice with the lysis buffer containing 300 mM NaCl,
once with lysis buffer, and resuspended in 30 mL 23 SDS sample
buffer. Bound proteins were eluted by boiling the beads for 5 min
and separated by SDS-PAGE.

Knockdown of Ago2 or TNRC6A
The sequences of siRNAs against Ago2 (siAgo2) and TNRC6A(siTNRC6A) are described in Supplemental Table S3. For verification of immunofluorescence signals of Ago2, HeLa cells inoculated
on a glass coverslip at 1 3 105 cells/mL/well in wells of 12-wellculture plate were transfected twice at 24-h intervals with 50 nM
siAgo2. One day after the second transfection, cells were fixed,
stained with an anti-Ago2 antibody, and observed under the
fluorescence microscope or harvested for Western blot. For
TNRC6A knockdown, HeLa cells were transfected once with 50
nM siTNRC6A. Two days after the transfection, cells were treatedwith LMB, stained with an anti-Ago2 antibody, and observed.

miRNA quantification
Quantification of miRNAs contained in immunoprecipitates prepared from HeLa cells transfected with pmyc-GFP-TNRC6A or its
mutant expression constructs was carried out. Sixty mL of beads
carrying the immunoprecipitates were resuspended in 60 mL lysis
buffer. Eighty mL of suspension was used for RNA extraction, andthe remaining 40 mL suspension was used for protein analysis.
RNA was extracted by using the mirVana miRNA Isolation Kit(Ambion). MiRNAs were quantified using a modified procedureof the stem–loop RT-PCR reported previously (Chen et al. 2005).
The reverse transcriptase reactions were performed in a 10-mL
reaction mixture containing 2.5 mL of eluted RNA, 1.5 pmoles of
RT primers specific for each of the miRNAs, 13 RT buffer(RETROscript Kit; Ambion), 0.25 mM dNTPs, 35 U of MultiScribe
reverse transcriptase (Applied Biosystems), and 20 U of RNase OUT(Invitrogen). The mixture was incubated at 16°C for 30 min, 42°C
for 30 min, 85°C for 5 min, and then held at 4°C. A mixture of thesynthesized cDNA, FastStart Universal SYBR Green Master (Roche),and respective primers for each miRNA was incubated at 95°C for
10 min. The 40-cycle PCR reaction was then carried out at 95°C for
15 sec and 60°C for 1 min in triplicate. The levels of PCR products
were monitored with an ABI PRISM 7000 sequence detection

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Nuclear microRNA silencing navigated by TNRC6A

system (Applied Biosystems). The stem–loop RT primers and PCRprimers are listed in Supplemental Table S4.

Fluorescence in situ hybridization
Fluorescence in situ hybridization was performed essentiallyaccording to the procedure reported by Nathans et al. (2009)
with slight modifications. LNA hybridization probes with 59
digoxigenin modification complementary to miR-21 (59-TCAA
CATCAGTCTGATAAGCTA-39) and control scramble-miR (59GTGTAACACGTCTATACGCCCA-39) were purchased from
Exiqon. Briefly, HeLa cells fixed with 4% paraformaldehyde wererehydrated in PBS for 10 min and prehybridized for 15 min with
40% formamide in 23 SSC. Each probe (20 ng) was mixed with
E. coli tRNA (5 mg) and salmon sperm DNA (5 mg) in 40 mL of
80% formamide, heated for 5 min at 95°C, and immediately mixed
with an equal volume of hybridization buffer (2 mg/mL BSA, 20%
dextran sulfate in 43 SSC). Cells were incubated with 60 mL ofprobe in hybridization buffer for 3 h at 37°C, and washed. Cells werethen incubated with 1:100 diluted Anti-Digoxigenin-Rhodamine Fabfragments (Roche) in PBS at 4°C overnight, and washed three times
with PBS. They were visualized under a fluorescence microscope.

ences). One d after transfection, cells were selected with hygromycin B (50 mg/mL) for 7–8 wk. Obtained stable HEK293 cells
were maintained in DMEM supplemented with 10% Tet System
Approved FBS (Clontech).
Stable HEK293 cells were plated at 1.3 3 105 cells/well in a wellof a 24-well plate. Cells were transfected with 50 nM control
siRNA (siControl2) or 50 nM siMALAT-1. Their sequences aredescribed in Supplemental Table S3. After transfection, cells were
cultured in the presence or absence of dox for 24 h. Knockdown
efficiency of MALAT-1 was quantified by real-time RT-PCR. The
expression of MALAT-1 was normalized to that of GAPDH. The
primers’ sequences are listed in Supplemental Table S4.

Statistical analysis
All statistical analyses were performed using the two-tailed unpaired Student’s t-test.

SUPPLEMENTAL MATERIAL
Supplemental material is available for this article (10 figures andfour tables).

Gene silencing activity assay
HeLa cells were plated at 0.6 3 105 cells/well in a well of a 24-wellplate. Cells were transfected with 50 nM control siRNA (siControl),25 nM siTNRC6A and 25 nM siRNA#1 against TNRC6B(siTNRC6B#1), or 25 nM siTNRC6A and siTNRC6B#2. Theirsequences are described in Supplemental Table S3. Twenty-four hafter siRNA transfection, the cells were simultaneously transfectedwith psiCHECK-CXCR4 target (10 ng), pGL3-Control (100 ng;
Promega) encoding firefly luciferase gene, pSUPER-CXCR4 or
pSUPER-DsRed (100ng), and pmyc-GFP-TNRC6A or its mutant
expression constructs (500 ng). The cells were harvested 48 h after
the second transfection, and the relative Renilla/firefly luciferase
activity was determined using a Dual-Luciferase Reporter Assay
System (Promega).
Two d after siRNA transfection, knockdown efficiency of
TNRC6A and TNRC6B by each siRNA was quantified by realtime RT-PCR using total RNA isolated using an RNeasy Mini kit(Qiagen). The expression of TNRC6A and TNRC6B was normalized to that of GAPDH. The primers’ sequences are listed in
Supplemental Table S4.

Absolute quantification of TNRC6s’ mRNAs
cDNA was synthesized from 1 mg of the total RNA of HeLa cellsby SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). The PCR primers used are listed in Supplemental Table
S4. The copy numbers of mRNA was calculated from serial dilutionsof known copy numbers of pCMV-SPORT/FLAG-GW182, pmycGFP-TNRC6B, or pmyc-GFP-TNRC6C.

Generation of stable HEK293 cells and knockdown
experiment for MALAT-1
Flp-In T-REx 293 cells (Invitrogen) were transfected with
pcDNA5/FRT/TO/FLAG-HA-SBP, pcDNA5/FRT/TO/FLAG-HASBP-TNRC6A, or its mutant expression constructs along with
pOG44 (Invitrogen) using Polyethylenimine ‘‘Max’’ (Polysci-

ACKNOWLEDGMENTS
We thank Drs. Patrick Provost, Shigeyuki Yokoyama, Thomas
Tuschl, and Akio Yamashita for kindly providing plasmids, andDr. Tetsuya Kojima for conducting the confocal microscopy
imaging. This work was partially supported by grants from the
Ministry of Education, Culture, Sports, Science and Technology ofJapan (MEXT) and the Cell Innovation Project (MEXT) to K.U.-T.
Received June 7, 2012; accepted October 18, 2012.

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