Article

Comprehensive Identification of Nuclear andCytoplasmic TNRC6A-Associating Proteins

Masataka Suzawa 1 , Kentaro Noguchi 2 , Kenji Nishi 1 , Hiroko Kozuka-Hata 3 ,Masaaki Oyama 3 and Kumiko Ui-Tei 1, 2
1 - Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
2 - Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo,
Chiba-ken 277-8651, Japan
3 - Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan

Correspondence to Kumiko Ui-Tei: Department of Biological Sciences, Graduate School of Science, The University ofTokyo, Tokyo 113-0033, Japan. ktei@bs.s.u-tokyo.ac.jp
http://dx.doi.org/10.1016/j.jmb.2017.04.017

Abstract
Trinucleotide repeat-containing gene 6A protein (TNRC6A) is an essential protein for microRNA-mediatedgene silencing. TNRC6A functions in the cytoplasm as a platform protein interacting with Argonaute protein,on which microRNA is loaded for RNA silencing, and decapping enzymes or deadenylation protein complexes
to induce mRNA degradation. We previously revealed that TNRC6A shuttles between the cytoplasm andnucleus. However, the function of TNRC6A in the nucleus is unclear. Here, we performed a comprehensive
identification of the nuclear and cytoplasmic interacting proteins of TNRC6A protein by mass spectrometry
and identified multiple proteins involved in the nuclear and cytoplasmic complexes. We found that many RNA
degradation pathway proteins were involved in both nuclear and cytoplasmic TNRC6A complexes,suggesting that RNA silencing may occur via TNRC6A in both nucleus and cytoplasm or that they wereinvolved in other important function in the nucleus. Furthermore, proteins identified in the nuclear TNRC6A
complex were categorized into the spliceosomal pathway. This may mean that TNRC6A regulates splicing in
the nucleus. In contrast, pathogen infection- and RNA transport-associated proteins were identified in thecytoplasmic TNRC6A complex. Thus, TNRC6A may be also involved in these pathways in the cytoplasm.
© 2017 Elsevier Ltd. All rights reserved.

Introduction
Trinucleotide repeat-containing gene 6A protein(TNRC6A) was first identified as an autoantigen
from sera of patient suffering from motor andsensory neuropathies [1]. It was designated as
GW182, since it is a 182-KDa protein containing
multiple glycine-tryptophan repeats, one or moreglutamine-rich (Q-rich) region, and a classical RNA
recognition motif (RRM) at the C terminus. GW182
belongs to an evolutionarily conserved protein family
restricted to metazoans, which includes AIN-1 andAIN-2 from Caenorhabditis elegans [2,3] and Gawky
from Drosophila melanogaster [4]. Vertebrates havethree paralogs, GW182/TNRC6A, TNRC6B, andTNRC6C, which are located in the cytoplasmic
0022-2836/© 2017 Elsevier Ltd. All rights reserved.

processing body [5]. All three members associatewith Argonaute (AGO) family members, AGO1–4,
through their N-terminal GW-motifs in the microRNA(miRNA)-induced silencing complex [6]. TNRC6A/B/
C then promotes translational repression and/or
degradation of miRNA targets mediated by their
C-terminal silencing domains. Extensive in vitro
studies showed that the interaction of GW182 withother components of the processing body, such as
PAN2‐PAN3 and carbon catabolite repression 4(CCR4)-CCR4-associated factor 1 (CAF1) deadenylase complexes, promotes mRNA deadenylation [7].
Recently, we revealed that human TNRC6A has a
nuclear localization signal (NLS) and a nuclear
export signal (NES) and shuttles between the
nucleus and cytoplasm [8]. In several human
J Mol Biol (2017) 429, 3319–3333

3320

Identification of TNRC6A-ssociating Proteins

(a)

NLS
GW-I

GW-II

GW-III

NES

UBA-like

Q rich

PAM2

RRM

(b)

(c)

Fig. 1. Domain structure and subcellular localization of human TNRC6A protein and cell fractionation procedure. (a)Domain structure of TNRC6A protein. Gray boxes indicate GW repeats (GW-I, GW-II, GW-III domains), UBA-like domain,a Q-rich region, a poly(A)-binding protein binding motif 2 (PAM2), and an RRM. Numbers denote the amino acid position
relative to the N terminus. Red box indicates NLS, and sequences of NLS and NLS-mut were shown below the domain
structure. Blue box indicates NES, and sequences of NES and NES-mut were shown below the domain structure. (b)Subcellular localization of TNRC6A-WT, TNRC6A-NES-mut, and TNRC6A-NLS-mut proteins in the cells transfected witheach expression plasmid. Upper panels show the cells stained with Cy3-conjugated anti-HA antibody. Lower panels show
the merged images with DAPI (blue). Bar represents 10 μm. (c) Procedure for cell fractionation. pFHS-TNRC6A-NLS-mut
or pFHS-TNRC6A-NES-mut was transfected into HeLa cells, and the cells were lysed with digitonin and centrifuged. The
supernatant was used as the cytoplasmic fraction. The pellet was further lysed with NP-40, and the supernatant following
centrifugation was used as the nuclear fraction. Both fractions were immunoprecipitated with anti-Flag antibody.

Identification of TNRC6A-ssociating Proteinscancers, such as gastric, colorectal, prostate, andesophageal, TNRC6A protein is mainly localized in
the nucleus [9,10]. Thus, the major subcellular
localization of TNRC6A is not the same in all cell
types, although the functional purpose of subcellular
localization remains unknown.
To unlock the nuclear function of TNRC6A, we
comprehensively identified proteins interactingwith nuclear and cytoplasmic TNRC6A by immunoprecipitation (IP) and mass spectrometry (MS)analyses. Both nuclear and cytoplasmic TNRC6A
commonly interact with multiple proteins associated with RNA degradation. In addition, we also
revealed that nuclear and cytoplasmic TNRC6A
interact with different sets of proteins associatedwith splicing, pathogen infection, and RNA transport pathways.

Results
Isolation of nuclear and cytoplasmic TNRC6A
protein complexes
Human TNRC6A protein shares a common structural organization with vertebrate and insect homologs
due to five characteristic regions: multiple glycinetryptophan repeats, a central ubiquitin-associated(UBA)-like domain, a Q-rich region, a poly(A)-binding protein binding motif 2, and a C-terminal RRM(Fig. 1a). In our previous report [8], we revealed thathuman TNRC6A shuttles between the nucleus andcytoplasm via the NLS and NES in the central region.
When a wild-type Flag-, HA-, and streptavidin-binding
peptide (FHS)-tagged TNRC6A expression construct(pFHS-TNRC6A-WT) was transfected into human
HeLa cells and detected with anti-HA antibody,
TNRC6A-WT was mainly observed in the cytoplasm
compared to control cells stained with 4′-6-diamidino-2-phenylindole (DAPI; Fig. 1b). However, TNRC6A
with a NES mutation (TNRC6A-NES-mut) was mainly
localized to the nucleus (Fig. 1b), and TNRC6A with an
NLS mutation (TNRC6A-NLS-mut) was predominantly
localized to the cytoplasm, similar to TNRC6A-WT.
Thus, TNRC6A is distributed in the cytoplasm in HeLa
cells but usually shuttles between the nucleus and thecytoplasm. It has been confirmed that the subcellular
localizations of overexpressed TNRC6A proteins with
NLS or NES mutation were essentially consistent withthose expressed at the endogenous levels in ourprevious report [8].
To identify proteins interacting with nuclear andcytoplasmic TNRC6A, we used TNRC6A-NES-mut
or TNRC6A-NLS-mut protein in IP. Nuclear andcytoplasmic fractions were separated from HeLa
cells transfected with either pFHS-TNRC6A-NESmut or pFHS-TNRC6A-NLS-mut plasmid (Fig. 1c).
Cell membranes from cells transfected with

3321
pFSH-TNRC6A-NES-mut or pFSHTNRC6A-NLS-mut were dissolved with digitonin,
and the centrifuged supernatant was designated as
the cytoplasmic fraction. The precipitate from cells
transfected with pFSH-TNRC6A-NES-mut was further lysed with 0.5% NP-40, and the centrifugedsupernatant of nucleoplasmic fraction was designated as the nuclear fraction. The nuclear insoluble
fraction was not used in this study, in whichchromatin, the membranes of endoplasmic reticulum (ER), Golgi apparatus, and mitochondria might
be often involved.
HeLa cells transfected with pFHSTNRC6A-NES-mut were fractionated, and the localization of TNRC6A-NES-mut was detected byWestern blotting using anti-TNRC6A antibody. A
successful fractionation of the nucleus (Fig. 2a) andcytoplasm (Fig. 2b) was confirmed by antibodies
against α-tubulin, a cytoplasmic marker, and poly
(ADP-ribose) polymerase (PARP), a nuclear marker
protein. Endogenous TNRC6A signals were detected in the cells transfected with the FHS-tagged
empty control vector (pFHS), but no exogenous
TNRC6A signals were seen. However, consistent
with our previous results [8], TNRC6A-NES-mut wasabundant in the nuclear fraction, although a slightly
small amount of TNRC6A-NES-mut was observed in
the cytoplasmic fraction (Fig. 2a). Contrary to
TNRC6A-NES-mut, TNRC6A-NLS-mut protein was
predominantly detected in the cytoplasmic fraction(Fig. 2b). We then performed IP using anti-Flag
antibody and the nuclear fraction from cells expressing TNRC6A-NES-mut and the cytoplasmic fraction
f r o m c e l l s e x p r e s s i n g TN R C 6 A - N L S - m u t .
TNRC6A-NES-mut protein was successfully immunoprecipitated from the nuclear fraction. In addition,
AGO2, a typical TNRC6A-interacting protein, wasimmunoprecipitated with TNRC6A-NES-mut (Fig.
2c). TNRC6A-NLS-mut was obtained from thecytoplasmic fraction, and the IP of AGO2 was also
confirmed (Fig. 2d). Silver staining was performed todetect proteins contained in these immunoprecipitates (Fig. 2e and f). We clearly show that multiple
proteins other than AGO protein are contained inboth TNRC6A-NES-mut complexes and
TNRC6A-NLS-mut complexes.
Identification and classification of proteins coimmunoprecipitated with nuclear and cytoplasmic TNRC6A
Proteins co-immunoprecipitated with
TNRC6A-NES-mut from the nuclear fraction orwith TNRC6A-NLS-mut from the cytoplasmic
fraction were analyzed using nano-liquid chromatography-electrospray ionization-MS/MS. In total,
628 and 522 proteins were identified in association
with TNRC6A-NES-mut and TNRC6A-NLS-mut,
respectively, and 247 proteins were associated3322

Identification of TNRC6A-ssociating Proteins

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 2 (legend on next page)3323

Identification of TNRC6A-ssociating Proteins

(a)

(b)

45
40

381
(188)

TNRC6ANLS-mut

247
(53)

275
(168)

35
-log10(P-value)

TNRC6ANES-mut

TNRC6A-NES-mut
TNRC6A-NLS-mut

30
25
20
15
10

0

Ribosome
Spliceosome
Proteasome
RNA degradation
Epstein-Barr virus infection
Protein processing in endoplasmic reticulum
RNA transport
Endocrine and other factor-regulated calcium reabsorption
Pathogenic Escherichia coli infection
Huntington's disease
mRNA surveillance pathway
Protein export
Biosynthesis of amino acids
Gap junction
Pentose phosphate pathway
Synaptic vesicle cycle
Aminoacyl-tRNA biosynthesis
Oocyte meiosis
Viral carcinogenesis
Biosynthesis of antibiotics
Legionellosis

5Fig. 3. Results of MS. (a) Venn diagram of immunoprecipitated proteins categorized into the nuclear fraction of
TNRC6A-NES-mut, the cytoplasmic fraction of TNRC6A-NLS-mut, and overlapped proteins in both fractions. Black
numbers indicate the total number of identified proteins, and red numbers indicate the proteins identified for the first time inthis study. Note that the known proteins were identified by the experiments using wild-type TNRC6A-C or AGO1–4
proteins. (b) Classification of identified proteins into KEGG pathways. Blue bars indicate the proteins in theTNRC6A-NES-mut immunoprecipitate, and red bars indicate the proteins in the TNRC6A-NLS-mut immunoprecipitate.
The vertical axis indicates − log10(P-value), then p b 0.01 means more than 2.0 (dotted line).

with both (Fig. 3a and Supplementary Tables S1 andS2). Furthermore, 381 and 275 proteins were exclusively associated with TNRC6A-NES-mut andTNRC6A-NLS-mut, respectively. Compared to previously identified proteins associated with TNRC6A-C
and AGO1–4 proteins [11–21], 188, 168, and 53
proteins were newly detected in TNRC6A-NES-mut
complex, the TNRC6A-NLS-mut complex, and both,respectively.

The identified proteins were classified into
possible functional pathways using the Kyoto
Encyclopedia of Genes and Genomes (KEGG)
database † [22] (Fig. 3b and Supplementary Tables
S1 and S2). Many nuclear and cytoplasmic proteins
immunoprecipitated with TNRC6A were identified
in RNA degradation pathway (Fig. 4). The components of CCR4‐negative on TATA-less (NOT),
poly(A)-binding protein 1, a DEAD-box helicase

Fig. 2. Fractionation and IP of nuclear and cytoplasmic fractions. (a and b) Verification of fractionated samples. pFHS
and (a) pFHS-TNRC6A-NES-mut or (b) pFHS-TNRC6A-NLS-mut was transfected into HeLa cells, and cytoplasmic or
nuclear fraction was separated. Anti-TNRC6A was used for the detection of TNRC6A, and anti-PARP and anti-α-tubulin
were used for nuclear and cytoplasmic markers, respectively. Red asterisks indicate the endogenous TNRC6A protein. (cand d) Western blots of immunoprecipitated fraction with anti-Flag antibody of (c) nuclear fraction of the cells transfectedwith pFHS-TNRC6A-NES-mut, and (d) cytoplasmic fraction of the cells transfected with pFHS-TNRC6A-NLS-mut. Leftpanels indicate the results using anti-Flag antibody, and right panels are results using anti-AGO2 antibody. In the left panelof (c), heavy chain (HC) and light chain (LC) of immunoglobulin (IgG) 1 derived from beads were contaminated. (e and f)
Silver staining of immunoprecipitated fractions. Immunoprecipitated fractions of the cells transfected with pFHS and (e)pFHS-TNRC6A-NES-mut or (f) pFHS-TNRC6A-NLS-mut. Black arrow indicates TNRC6A-NES-mut or TNRC6A-NLS-mut
protein, and white arrows indicate the possible interacting proteins.

3324

Identification of TNRC6A-ssociating ProteinsFig. 4. Identified proteins involved in the KEGG eukaryotic RNA degradation pathway. RNA degradation pathways areclassified into two pathways: the 5′ to 3′ decay pathway, which is followed by decapping that permits the 5′ to 3′
exonucleolytic degradation, and the 3′ to 5′ pathway, in which the exosome plays a key role to induce deadenylation.
Green indicates the proteins immunoprecipitated with anti-Flag antibody in both nuclear and cytoplasmic fractions. Blue,
specific proteins identified in the nuclear fraction. Orange, specific proteins identified in the cytoplasmic fraction.

protein, DDX6 (Rck/p54) [23,24] were commonly
found in both nuclear and cytoplasmic fractions(Fig. 4 and Table 1). Furthermore, TOB, whosefunctions are associated with deadenylase subunits of the CCR4–NOT complex [25], an RNA
helicase DHX36 (RNA helicase associated with
AU-rich element ARE) [26], EDC4, responsible formRNA decapping [27], and 5′ to 3′ exonuclease
XRN2 [28] were identified in the nuclear fraction,and Sm-like protein 4 involved in mRNA degradation [29] was recovered in the cytoplasmic fraction.
Proteins associated with the ribosome pathway
were abundant in both the nuclear and cytoplasmic
TNRC6A complexes (Fig. 3b). The ribosome
pathway proteins usually interact with mRNAs,
which directly associate with miRNAs but indirectly
associate with AGO or TNRC6A proteins. Thus,
many proteins in the ribosomal pathway arethought to be immunoprecipitated by such indirectinteractions, and this may be true for other proteins.
Proteins from the spliceosomal pathway wereabundantly identified in the nuclear TNRC6A complex (Fig. 3b). Most proteins involved in the small
nuclear RNA (snRNA) complexes, containing U1,
U2, U2-related, U4, U5, and U6, were identified (Fig.
5 and Table 2). Proteins categorized into protein
processing in the ER were also significantly detected(Fig. 3b). Since the nuclear fraction obtained using
our fractionation procedure contains ER (Fig. 1c),these proteins were immunoprecipitated with
TNRC6A-NES-mut from the nuclear fraction but
are not considered to function in the nucleus. In
contrast, proteins involved in the Epstein–Barr virus
infection, pathogenic Escherichia coli infection, andRNA transport pathways were abundant in thecytoplasmic TNRC6A complexes (Fig. 3b). Thus,
cytoplasmic TNRC6A may have unidentified functions associated with pathogen infection or RNA
transport in the cytoplasm. Proteins associated with
Huntington's disease, mRNA surveillance, andprotein export pathways were specifically detectedin TNRC6A-NES-mut immunoprecipitates, andthose associated with the biosynthesis of aminoacids, gap junction, pentose phosphate pathway,
synaptic vesicle cycle, aminoacyl-tRNA biosynthesis, oocyte meiosis, viral carcinogenesis, biosynthesis of antibiotics, and legionellosis pathways weredetected in TNRC6A-NLS-mut immunoprecipitates(Fig. 3b). We cannot yet determine the significanceof these results, since only a small population ofproteins involved in each pathway were identified.
Verification of the interaction of TNRC6A withimmunoprecipitated proteins
To verify the interaction between TNRC6A protein
and the immunoprecipitated proteins, we performed
the IP experiment for CCR4-NOT subunit proteins,which were classified into RNA degradation pathway
and were identified as top-ranked proteins in the
immunoprecipitates using both TNRC6A-NES-mut
and TNRC6A-NLS-mut (Table 1). The CCR4-NOT
complex is a multisubunit complex conserved insequence and function throughout the eukaryotes
and is involved in many functions [30,31], including
regulation of gene expression in various processes,
from transcription in the nucleus [32,33] to mRNA
decay, quality control, translational repression, andprotein ubiquitination in the cytoplasm [34,35]. RNA
silencing is thought to occur mainly in the cytoplasm

3325

Identification of TNRC6A-ssociating Proteins

Table 1. Proteins classified into RNA degradation pathway using KEGG database
Bait protein
TNRC6A-NES-mut

TNRC6A-NLS-mut

Protein GI Accession

Description

M.W.

Score

18860916
28872722
42716275
388454220
13123772
76573857657387115583679
85067507
31542315
4885579
164664518
167830433
45827771
24234688
31542947
334191701
46367787
208431836
770673942716275
376319247
76573857657387115583679
85067505
31542315
4885579
164664518
355477275
24234688
48762920
4505749
11321601
46367787
208431836

5′–3′ exoribonuclease 2
protein BTG3 isoform b
CCR4-NOT transcription complex subunit 1 isoform a
CCR4-NOT transcription complex subunit 1 isoform c
CCR4-NOT transcription complex subunit 10 isoform 1
CCR4-NOT transcription complex subunit 2
CCR4-NOT transcription complex subunit 3
CCR4-NOT transcription complex subunit 6-like
CCR4-NOT transcription complex subunit 7 isoform 1
CCR4-NOT transcription complex subunit 8
Cell differentiation protein RCD1 homolog isoform 2
Probable ATP-dependent RNA helicase DDX6
Probable ATP-dependent RNA helicase DHX36 isoform 1
Enhancer of mRNA-decapping protein 4
Stress-70 protein, mitochondrial precursor
60-kDa heat shock protein, mitochondrial
6-phosphofructokinase type C isoform 2
Polyadenylate-binding protein 1
Polyadenylate-binding protein 4 isoform 3
Protein Tob2
CCR4-NOT transcription complex subunit 1 isoform a
CCR4-NOT transcription complex subunit 10 isoform 2
CCR4-NOT transcription complex subunit 2
CCR4-NOT transcription complex subunit 3
CCR4-NOT transcription complex subunit 6-like
CCR4-NOT transcription complex subunit 7 isoform 2
CCR4-NOT transcription complex subunit 8
Cell differentiation protein RCD1 homolog isoform 2
Probable ATP-dependent RNA helicase DDX6
U6 snRNA-associated Sm-like protein LSm4 isoform 2
Stress-70 protein, mitochondrial precursor
6-phosphofructokinase, liver type6-phosphofructokinase, muscle type isoform 26-phosphofructokinase type C isoform 1
Polyadenylate-binding protein 1
Polyadenylate-binding protein 4 isoform 3

108.5129.10
266.77266.21
82.26
59.70
81.82
62.96
32.72
33.52
33.61
54.38
114.69151.57
73.63
61.02
85.26
70.63
69.53
36.61
266.77
79.33
59.70
81.82
62.96
28.21
33.52
33.61
54.38
13.80
73.63
84.96
85.13
85.54
70.63
69.53

929.64143.58
10,175.84
10,078.17
956.212353.47
1770.92
660.131340.29692.131218.70
564.43720.9038.68
4575.105278.91278.743278.55
2188.69201.272930.45
156.43633.15196.17234.82219.58102.64533.02
45.47
53.10
35.49
1006.30209.28894.49960.43308.64

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●
●

The list of proteins co-immunoprecipitated with TNRC6A-NES-mut from nuclear fraction (upper rows) or TNRC6A-NLS-mut fromcytoplasmic fraction (lower rows). “Score” indicates the sum of the scores of the unique peptides for that protein based on the calculated
probability, P, that the observed match between the experimental data and the database sequence is a random event. The numericalvalue in the score column is −10Log(P). Black circles indicate proteins detected in both immunoprecipitates of TNRC6A-NES-mut andTNRC6A-NLS-mut.

by interaction of TNRC6A protein with the
CCR4-NOT complex [14,18,36,37]. However, since
many components essential for RNA silencing
involving CCR4-NOT complex proteins were also
detected in the immunoprecipitates in the nuclear
fraction, our results suggest that the similar phenomenon involving RNA silencing components isable to occur in the nucleus when TNRC6A istranslocated into the nucleus. Among the subunit
proteins of the CCR4-NOT complex, seven (CCR4,
CNOT1, CNOT2, CNOT3, CNOT7/8, CNOT9, and
CNOT10) were commonly immunoprecipitated inboth cytoplasmic and nuclear fractions with TNRC6A(Fig. 4 and Table 1). Among them, since the CNOT1
is the largest subunit of the complex and functions asa scaffold protein in the assembly of other components, we used CNOT1 for validation experiment. To
clarify the interaction between TNRC6A and CNOT1,
we transfected each of pFHS-TNRC6A-WT or

pFHS-TNRC6A-NES-mut into HeLa cells. Then, thesubcellular localization of CNOT1 and TNRC6A wasinvestigated by western blotting. Since there was a
possibility that the nuclear fraction obtained using ourcell fractionation procedure contained ER, we modified
the procedure to segregate ER fraction from thenuclear fraction using 0.3% NP-40 before lysis of
nuclear membrane by 0.5% NP-40 (see Fig. 1c) for
validation experiment. Successful fractionation was
confirmed by the nuclear marker (anti-PARP), the ER
marker (anti-calreticulin), and the cytoplasmic marker(anti-α-tubulin; Fig. 6a). Wild-type TNRC6A protein
was preferentially detected in the cytoplasmic fraction
by anti-TNRC6A antibody, and the small amount wasobserved in the nuclear fraction and also in the ER
fraction. Although a large amount of TNRC6A with
NES mutation was also detected in the cytoplasm, thelarger amount of NES mutant protein was apparently
observed in the nuclear and ER fractions compared to3326

Identification of TNRC6A-ssociating ProteinsFig. 5. Identified proteins involved in the KEGG spliceosome pathway. In splicing, introns are excised and exons arejoined by the spliceosome components. The spliceosome is usually composed of five snRNPs, U1, U2, U4, U5, and U6
snRNPs, and several spliceosome-associated proteins. Green indicates the spliceosomal proteins immunoprecipitatedwith anti-Flag antibody in both nuclear and cytoplasmic fractions. Blue, specific proteins identified in the nuclear fraction.
Orange, specific proteins identified in the cytoplasmic fraction.

wild-type TNRC6A, suggesting that NES mutant
protein preferably translocates from the cytoplasm to
the nucleus through ER (Fig. 6a). CNOT1 protein was
mainly detected in the cytoplasmic fractions ofwild-type TNRC6A and its NES mutant (Fig. 6a), andtheir amounts were slightly higher in both ER andnuclear fractions from the cells expressing NES mutant
of TNRC6A compared to those from the cells
expressing wild-type TNRC6A. The essentially similar
results were also observed for AGO2 proteins,
although the differences in amounts of AGO2 proteinsin both nuclear and ER fractions were not so definitive
compared to CNOT1. IP using anti-Flag andanti-TNRC6A antibodies revealed that the large
amounts of TNRC6A and TNRC6A-NES-mut proteins
were located in the cytoplasm, and the amounts of
NES mutant proteins were clearly higher in the
nucleus and ER fraction compared to those ofwild-type TNRC6A (Fig. 6b). CNOT1 wasco-immunoprecipitated with wild-type TNRC6A
and its NES mutant in the cytoplasm, and the amountof co-immunoprecipitated CNOT1 with
TNRC6A-NES-mut protein was higher than that withwild-type TNRC6A protein in both nuclear and ER

fractions. Thus, it was suggested that the cytoplasmic
CNOT1 was translocated into the nucleus through ER,
associating with TNRC6A. The clear differences in theamounts of AGO2 proteins co-immunoprecipitatedwith wild-type TNRC6A or TNRC6A-NES-mut protein
were undetermined.

Discussion
We have previously revealed that TNRC6A is a
protein that shuttles between the cytoplasm andnucleus [8]. The components involved in the nuclear
and cytoplasmic TNRC6A complexes were identified
for the first time in this study using
TNRC6A-NES-mut and TNRC6A-NLS-mut. Our
results revealed that many proteins involved in the
RNA degradation pathway were immunoprecipitatedwith TNRC6A in both the cytoplasmic and nuclear
fractions (Fig. 3b and Table 1). TNRC6A alsointeracts with many distinct proteins between the
nucleus and cytoplasm.
To verify the MS results, we examined theinteraction between TNRC6A and CNOT1. Multiple

3327

Identification of TNRC6A-ssociating Proteins

Table 2. Proteins classified into spliceosome pathway using KEGG database
Bait protein

Protein GI
Accession

Description

TNRC6A-NES-mut 4758138
68509926
256000749
14249678
56699409
122937227
119226260
217272894
7661920
194248072
57298774504445117190174
14165435
157412270
14141161
45050874505343
222352151
221136939
91208426
4506901
59021024759158
4,759,160
29568103
40217847
50593002
475915638149981
450712745071314507125
5803167
5411211755749531
54112121
50320697706326
345197228
TNRC6A-NLS-mut 355477275
194248072
13676857
5729877
117190174
14165435
157412270
14141161
4505343
222352151
221136939
91208426
59021024759158475916029568103
40217847
38149981
45071294507125
54112121

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●
●

Probable ATP-dependent RNA helicase DDX5
Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15
Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX16 isoform
2
Splicing factor 45
RNA-binding motif protein, X chromosome isoform 1U2 snRNP-associated SURP motif-containing protein
Calcium homeostasis ER protein
116-kDa U5 small nuclear ribonucleoprotein component isoform b
Eukaryotic initiation factor 4A–III
Heat shock 70-kDa protein 1A/1B
Heat shock cognate 71-kDa protein isoform 1
Heterogeneous nuclear ribonucleoprotein A1 isoform a
Heterogeneous nuclear ribonucleoproteins C1/C2 isoform b
Heterogeneous nuclear ribonucleoprotein K isoform b
Heterogeneous nuclear ribonucleoprotein M isoform b
Heterogeneous nuclear ribonucleoprotein U isoform b
Protein mago nashi homolog
Nuclear cap-binding protein subunit 1
Poly(rC)-binding protein 1
U4/U6 small nuclear ribonucleoprotein Prp31
Pre-mRNA-processing-splicing factor 8
Serine/arginine-rich splicing factor 3
Small nuclear ribonucleoprotein Sm D1
Small nuclear ribonucleoprotein Sm D2 isoform 1
Small nuclear ribonucleoprotein Sm D3
U1 small nuclear ribonucleoprotein 70 kDa
U5 small nuclear ribonucleoprotein 200-kDa helicase
U2 small nuclear ribonucleoprotein A′
U1 small nuclear ribonucleoprotein A
U2 small nuclear ribonucleoprotein B″
U1 small nuclear ribonucleoprotein C
Small nuclear ribonucleoprotein F
Small nuclear ribonucleoprotein-associated proteins B and B′ isoform B
Splicing factor 3A subunit 3
Splicing factor 3B subunit 1 isoform 1
Splicing factor 3B subunit 2
Splicing factor 3B subunit 3
Splicing factor 3B subunit 4
Pre-mRNA branch site protein p14
Transformer-2 protein homolog beta isoform 2
U6 snRNA-associated Sm-like protein LSm4 isoform 2
Heat shock 70-kDa protein 1A/1B
Heat shock-related 70-kDa protein 2
Heat shock cognate 71-kDa protein isoform 1
Heterogeneous nuclear ribonucleoproteins C1/C2 isoform b
Heterogeneous nuclear ribonucleoprotein K isoform b
Heterogeneous nuclear ribonucleoprotein M isoform b
Heterogeneous nuclear ribonucleoprotein U isoform b
Nuclear cap-binding protein subunit 1
Poly(rC)-binding protein 1
U4/U6 small nuclear ribonucleoprotein Prp31
Pre-mRNA-processing-splicing factor 8
Small nuclear ribonucleoprotein Sm D1
Small nuclear ribonucleoprotein Sm D2 isoform 1
Small nuclear ribonucleoprotein Sm D3
U1 small nuclear ribonucleoprotein 70 kDa
U5 small nuclear ribonucleoprotein 200-kDa helicase
U2 small nuclear ribonucleoprotein B″
Small nuclear ribonucleoprotein E
Small nuclear ribonucleoprotein-associated proteins B and B′ isoform B
Splicing factor 3B subunit 3

M.W.

Score

69.1
278.8190.88 526.73
112.38 101.21
44.93
42.31
118.22103.64105.32
46.84
70.01
70.85
34.18
32.32
50.94
73.57
88.92
17.15
91.78
37.47
55.42
273.43
19.32
13.27
13.52
13.91
51.53
244.35
28.40
31.26
25.47
17.38
9.72
23.64
58.81
145.74100.16135.49
44.36
14.58
21.92
13.80
70.01
69.98
70.85
32.32
50.94
73.57
88.92
91.78
37.47
55.42
273.43
13.27
13.52
13.91
51.53
244.35
25.47
10.80
23.64
135.4990.63
361.7485.48
36.42
122.29474.392170.98
3465.38
359.30652.682037.892191.56
1041.5378.71
516.72205.972260.5927.54
27.21
436.5367.91
136.1262.65
224.1276.19
206.91
192.53
46.04
58.88
74.94
73.60
584.47451.78629.54114.02320.92169.0253.10
2170.98
1111.413465.38652.682037.892191.56
1041.53516.72205.972260.5927.54
436.5367.91
136.1262.65
224.12
192.5328.70
74.94
629.54The list of proteins co-immunoprecipitated with TNRC6A-NES-mut from nuclear fraction (upper rows) or TNRC6A-NLS-mut from cytoplasmic
fraction (lower rows). “Score” indicates the sum of the scores of the unique peptides for that protein based on the calculated probability, P, thatthe observed match between the experimental data and the database sequence is a random event. The numerical value in the score column is −10Log(P). Black circles indicate proteins detected in both immunoprecipitates of TNRC6A-NES-mut and TNRC6A-NLS-mut.

3328

Identification of TNRC6A-ssociating Proteins

(a)

(b)

Fig. 6. Verification of the result of MS using CNOT1. (a) Subcellular localization examined by the modified cell
fractionation procedure, which separates cells into nuclear, ER, and cytoplasmic fractions. The cells transfected with
pFHS, pFHS-TNRC6A-WT, or pFHS-TNRC6A-NES-mut were used. TNRC6A protein was detected by anti-TNRC6A
antibody. CNOT1, anti-CNOT1 antibody. AGO2, anti-AGO2 antibody. PARP was used as a nuclear marker and detectedby anti-PARP antibody. Calreticulin, an ER marker, anti-calreticulin antibody. α-tubulin, a cytoplasmic marker,
anti-α-tubulin antibody. Red asterisks indicate nonspecific bands. (b) Immunoprecipitation using the nuclear, ER, andcytoplasmic fractions of the cells transfected with pFHS, pFHS-TNRC6A-WT, or pFHS-TNRC6A-NES-mut. Immunoprecipitation was carried out using anti-Flag antibody, and TNRC6A, CNOT1, and AGO2 proteins were detected. The
experiments were performed two times, and the typical results were shown.

subunit proteins contained in the CCR4-NOT complex were significantly immunoprecipitated with
TNRC6A-NES-mut and TNRC6A-NLS-mut protein(Table 1). When pFHS-TNRC6A-WT was transcfected into HeLa cells, both TNRC6A and CNOT1

were mainly observed in the cytoplasm, and small
amounts were in the nucleus (Fig. 6a). Thus, these
proteins are able to shuttle between cytoplasm andnucleus but are preferably localized in the cytoplasm
in the normal condition of HeLa cells. However,Identification of TNRC6A-ssociating ProteinspFHS-TNRC6A-NES-mut was transfected, and theamounts of CNOT1 protein co-immunoprecipitatedwith NES-mut in the nucleus and ER fractions
evidently increased (Fig. 6b). Thus, it was strongly
suggested that CNOT1 is recruited from cytoplasm
to nucleus through ER by interacting with TNRC6A.
In addition to the CCR4-NOT complex components,
PABP, which plays an important role in RNA
silencing by interacting with CNOT1 [38,39], andDDX6 (Rck/p54) that functions in mRNA decapping[23,24], were also found in both nuclear andcytoplasmic immunoprecipitates. Furthermore,
TOB, RHAU, EDC4, and XRN2 were identified
among the TNRC6A-associating proteins in the
nucleus. TOB interacts with deadenylase subunitsof the CCR4-NOT complex [25], RHAU is an RNA
helicase DHX36 that is necessary for deadenylation[26], and EDC4 (also known as Hedls, Ge-1, or
RCD-8) enhances mRNA decapping [27]. XRN2 is a
5′ to 3′ exonuclease [28], and Sm-like protein 4 isassociated with mRNA degradation [29]. Thus, theseresults strongly suggest that the some unknown
mechanism using RNA silencing components is
induced via TNRC6A in the nucleus as RNA
silencing in the cytoplasm. The isoforms of immunoprecipitated proteins in the nucleus and thecytoplasm were not necessarily the same. The
isoforms of CNOT1, CNOT7, and CNOT10 differed
between the nucleus and cytoplasm (Table 1). The
isoforms of CNOT1, CNOT7, and CNOT10 immunoprecipitated with the nuclear TNRC6A-NES-mut
were CNOT1 isoform c, CNOT7 isoform 1, and
CNOT10 isoform 1. Isoforms immunoprecipitatedwith cytoplasmic TNRC6A-NLS-mut included
CNOT7 isoform 2 and CNOT10 isoform 2. The
common isoform a of CNOT1 was found in both
fractions. CNOT1 is the largest subunit of the
CCR4-NOT complex and functions as a scaffold
protein in the assembly of other components. The
N-terminal region of CNOT1 interacts with CNOT11,
which interacts with CNOT10 [40]; the middle region
directly interacts with CNOT7 or CNOT8 [40–42];
and the C-terminal region interacts with CNOT2/
CNOT3 [40,43]. Thus, the isoforms of CNOT1,
CNOT7, and CNOT10 are important subunits.
CNOT7 and CNOT8 are paralogous genes, known
as hCAF1a and hCAF1b, respectively. The hCAF1z
isoform complex differs from the conventional
CCR4-NOT deadenylase complex [44] and isconcentrated in nuclear Cajal bodies shuttling
between the nucleus and cytoplasm. It subjects
substrate RNAs to rapid deadenylation and slow
exonucleolytic degradation from the 3′ end in vitro. It
is possible that such functional changes also occur
via the nuclear and the cytoplasmic isoforms of
CNOT1, CNOT7, and CNOT10.
Proteins involved in the spliceosome pathway
were identified in the nuclear fraction of cells
expressing TNRC6A-NES-mut protein, and those3329
involved in the Epstein–Barr virus infection, RNAtransport, and pathogenic E. coli infection pathways
were detected in the cytoplasmic fraction of the cells
expressing TNRC6A-NLS-mut. Thus, it is possiblethat TNRC6A has various other roles in the nucleus
and the cytoplasm in addition to RNA silencing: thesplicing in the nucleus and pathogenic infection or
RNA transport in the cytoplasm. Many proteins
involved in the splicing machinery were detected in
the nuclear fraction. Most components of snRNAcomplexes containing U1, U2, U2-related, U4/U6,
U5, exon junction complex/transcription export
complex, and common components of the spliceosome were identified (Fig. 5 and Table 2). Some ofthese components were detected in both thecytoplasmic and nuclear fractions, because snRNAs
shuttle between the nucleus and cytoplasm [45].
It has been reported that vertebrate (TNRC6A/B/
C) and insect GW182 proteins share a common
domain organization characterized by two annotatedstructural domains: a central UBA-like domain and a
C-terminal RRM [5], and NES is positioned in the
UBA-like domain. Although these domains areembedded in regions predicted to be unstructured,
it might be possible that the mutations in NESchange the structure of TNRC6A and the interacting
proteins. However, in the validation experiment, werevealed that CNOT1 protein was able to be
co-immunoprecipitated with wild-type TNRC6A, although the co-immunoprecipitated amount ofwild-type TNRC6A protein was smaller comparedto TNRC6A with NES mutation (Fig. 6b). Thus, atleast CNOT1 can interact with TNRC6A even when
its NES was mutated.
It is clear that AGO proteins also regulate nuclear
processes in association with small RNAs [46]. In
Schizosaccharomyces pombe, AGOs participate in theassembly of heterochromatin at centromeric regions
via histone methylation [47]. Also, in mammalian cells,
AGO1 and/or AGO2, in association with small RNAs
complementary to genomic regions, repress [48,49] oractivate gene expression [50,51] and control alternative splicing [11,52] and DNA double-strand breakrepair [53,54]. Although it is unclear how AGO2translocates into the nucleus, importin 8, a memberof the karyopherin β family, mediates thecytoplasm-to-nuclear transport of mature miRNAs
with help from the AGO2 complex [16,55]. Our MS
and Western blot did not discriminate whether the
splicing is regulated by AGO or TNRC6A directly.
In most cells, TNRC6A is mainly observed in thecytoplasm, even though it can shuttle between thecytoplasm and the nucleus. However, TNRC6A is
mainly localized in the nucleus in several humancancers, such as gastric, colorectal, prostate, andesophageal cancers [9,10]. In this study, we revealed
that TNRC6A protein interacts with CNOT1 in the
nucleus. Human CNOT1 protein is known to recruitother CCR4-NOT subunits into the nucleus and3330
function as a transcriptional repressor of nuclear
estrogen receptor α in hormone-dependent fashion[33]. Furthermore, the depletion of endogenous
CNOT1 in breast cancer cells results in the deregulation of endogenous estrogen receptor α target genes.
Thus, TNRC6A may be important for the understandingof molecular mechanism involved in various cancers
containing breast cancer.

Materials and Methods
Cell culture and transfection
HeLa cells derived from human cervical cancer
were cultured in Dulbecco's modified Eagle's medium(Wako) supplemented with 10% fetal bovine serum(GIBCO) and antibiotics [Penicillin–Streptomycin
Solution (× 100; Wako)] at 37 °C in 5% CO2.
Cells were seeded at 2.0 × 10 6 cells/ml into each of
90-mm dishes 1 day before transfection. Transfection
was performed using Polyethylenimine “MAX” (Polyscience Inc.) with 10 μg of total plasmid DNA per dishaccording to the manufacturer's protocol.

Identification of TNRC6A-ssociating Proteins

IP for MS
For MS analysis, IP of nuclear TNRC6A complexes was performed using HeLa nuclear fraction
transfected with pFHS-TNRC6A-NES-mut or pFHS
as a control. IP of cytoplasmic TNRC6A complexes
was performed using HeLa cytoplasmic fraction
transfected with pFHS-TNRC6A-NLS-mut or pFHS.
Each extract was centrifuged at 48,000g for 30 min
at 4 °C, and the supernatant was precleared using
Sepharose 4B (Sigma, 4B200), rotating for 1 h at
4 °C. Extracts were transferred to 60 μl of anti-FLAG
M2 affinity gel (Sigma, A2220) and incubated withrotation for 2 h at 4 °C. The affinity gel was washedtwice with wash buffer (same as ice-cold lysis buffer
but we increased 50 mM NaCl to 300 mM NaCl),
once with nuclear lysis buffer wash, and twice withTBS buffer [10 mM Tris–HCl (pH 7.5) and 150 mM
NaCl]. The bound proteins to the affinity gel were
eluted with 50 μl TBS containing 1 mg/ml Flag
peptide (Sigma, F-3290), with shaking for 1 h at
4 °C. The eluates were separated from the gel by
centrifugation at 800g for 2 min at 4 °C. An aliquot of
the eluate was subjected to SDS-PAGE, andco-immunoprecipitated proteins were stained usinga Silver Stain II kit (Wako).

Cell fractionation
MS analysis
Cells were lysed in ice-cold lysis buffer [10 mM
Hepes-NaOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl,
50 mM NaCl, 0.5 mM DTT, 1 mM EDTA, 1 mM
Na3Vo4, 10 mM NaF, and 1 x complete proteaseinhibitor cocktail (Roche)] supplemented with digitonin(130 μg/1.0 × 10 7 cells). After incubation for 10 min on
ice, cells were centrifuged and supernatant was usedas cytoplasmic fraction. Precipitated nuclei were
washed two times with ice-cold lysis buffer. To makenuclear extract, we resuspended nuclei in nuclear lysis
buffer (same as ice-cold lysis buffer but we increased
50 mM NaCl to 140 mM NaCl and added 0.5% NP40).
After incubation for 10 min on ice, cells were centrifuged and supernatant was used as nuclear fraction.
For the experiment in Fig. 6a and b, cells were lysedin ice-cold lysis buffer supplemented with digitonin(130 μg/1.0 × 10 7 cells). After incubation for 10 min
on ice, cells were centrifuged and supernatant wasused as cytoplasmic fraction. Precipitates were
washed with ice-cold lysis buffer. To isolate ER
fraction, we washed the precipitate three times in
ice-cold lysis buffer with 50 mM NaCl with 0.3%
NP-40, and the first supernatant was recovered as
ER fraction. Then, to make nuclear extract, we
resuspended nuclei in high salt nuclear lysis buffer(same as ice-cold lysis buffer but we increased 50 mM
NaCl to 420 mM NaCl and added 0.5% NP40). Afterincubation for 10 min on ice, cells were centrifuged
and supernatant was diluted by the addition of twovolumes of diluted buffer (same as ice-cold lysis buffer
without 50 mM NaCl) and used as nuclear fraction.

The remained eluate was digested with trypsin
desalted using ZipTipC18 (Millipore) and centrifugedin a vacuum concentrator. Shotgun proteomic
analyses by a linear ion trap-orbitrap MS (LTQ-Orbitrap Velos; Thermo Fisher Scientific) coupled with ananoflow liquid chromatography system (Dina-2A;
KYA Technologies) were performed following theprevious report [56]. Briefly, trypsinized peptides
were injected into a 75-μm reversed-phase C18
column at a flow rate of 10 μl/min. Elution was
performed with a linear gradient of solvent A (2%acetonitrile and 0.1% formic acid in H2O) to solvent B(40% acetonitrile and 0.1% formic acid in H2O) at
300 nl/min. Peptides were sequentially sprayed froma nanoelectrospray ion source (KYA Technologies)
and analyzed by collision-induced dissociation using
full-scan MS spectra (from m/z 380 to 2000). The
analyses were carried out in a data-dependent
mode. The 20 most intense ions at a thresholdlevel above 2000 were fragmented in the linear ion
trap. The orbitrap analyzer was operated to perform
shotgun detection with high accuracy. Protein
identification was conducted using MS and MS/MS
data against a comprehensive, integrated,
non-redundant, and well-annotated set of reference
sequence including genomic, transcript, and proteinof the National Center for Biotechnology Information
using Mascot version 2.4.1 (Matrix Science). A
maximum of two missed cleavages was allowed,
while the mass tolerance was set to 3 ppm for3331

Identification of TNRC6A-ssociating Proteins

peptide masses and 0.8 Da for MS/MS peaks, andwe applied a filter to satisfy a false discovery rate
lower than 1%.
Western blot
The samples were mixed with same volume of 2 x
SDS-PAGE sample buffer [4% (wt/vol) SDS, 0.1 M
Tris–HCl (pH 6.8), 12% (vol/vol) β-mercaptoethanol,
20% (wt/vol) glycerol, and 0.01% (wt/vol) bromophenol
blue], boiled for 5 min, and separated by SDS-PAGE.
The gels were transferred to PVDF membrane using a
Trans-Blot Turbo Transfer System (Bio-Rad). The
membranes were blocked for 1 h at room temperature
with 5% skim milk in TBST [20 mM Tris–HCl (pH 7.5),
150 mM NaCl, and 0.1% Tween-20].
Blocked membranes were incubated with thefollowing primary antibodies at 4 °C overnight in
TBST + 5% skim milk: anti-Ago2 at 1:500 dilution(Wako, 015–22,031), anti-GW182 (TNRC6A) at
1:2000 (Bethyl Laboratoried, A302-329A), anti-alphatubulin at 1:1000 (Sigma, T9026), anti-PARP at 1:2000(Abcam, ab32071), anti-Flag at 1:500 (Sigma, F1804),
anti-GFP at 1:2000 (Clontech, 632,592), andanti-CNOT1 at 1:1000 (Proteintech, 14,276–1-AP).
After primary antibody incubation, the membranes
were washed three times for 10 min at room temperature with TBST and then incubated for 1 h at room
temperature with horseradish peroxidase-conjugatedanti-mouse IgG (GE Healthcare, NA931V) at 1:6000 or
anti-rabbit IgG (GE Healthcare, NA934V) at 1:6000 in
TBST. After being washed three times for 10 min in
TBST at room temperature, the membrane wasincubated with ECL Prime Western Blotting Detection
Reagent (GE Healthcare, RPN2232), and signals weredetected by an ImageQuant LAS 4000 (GEHealthcare).
Data analysis of MS
For the classification of identified proteins by MS
analysis, the database for annotation, visualization,and integrated discovery was used [57,58]. Accession numbers allocated to each protein were pasted
into functional annotation tool (step 1), selected in
PROTEIN_GI_ACCESSION (step 2) and Gene List
(step 3), and then submitted (step 4). Significantly
enriched pathways in KEGG database tested by themodified Fisher's exact test were listed in Fig. 3b,
and pathway maps were shown in Figs. 4 and 5.
IP for validation
Cells were seeded at 2.0 × 10 6 cells/ml into eachof 90-mm dishes 1 day before transfection. Then, 10 μg
of pFHS, pFHS-TNRC6A-WT, or
pFHS-TNRC6A-NES-mut was transfected into HeLa
cells. We harvested the cells 2 days after transfection,and cell fractionation into cytoplasm, ER, and nuclear

fractions was carried out. Each fraction was centrifuged
at 48,000g for 30 min at 4 °C, and the supernatant was
precleared using Sepharose 4B, rotating for 1 h at
4 °C. Extracts were transferred to 20 μl of anti-Flag M2
affinity gel and incubated with rotation for 2 h at 4 °C.
The affinity gel was washed three times with nuclear
lysis buffer and resuspended in 30 μl of 2 x SDS-PAGE
sample buffer. We boiled the beads for 5 min andseparated it by SDS-PAGE.
Fluorescence microscopy
Immunofluorescence was essentially performed
as previously reported [8]. Briefly, HeLa cells
transfected with various FHS-tagged TNRC6A
expression constructs were cultured on 12-wellculture plate with a 15-mm glass at the bottom. Cells
were fixed with 4% paraformaldehyde and thenpermeabilized with 0.2% Triton X-100. Cells wereincubated with the anti-HA tag monoclonal antibody(MBL, M180–3, 1:800 dilution) in PBS and washed.
Then, they were incubated with the Cy3-labeledgoat anti-mouse IgG (Amersham biosciences,
PA43002, 1:800 dilution) and washed again. They
were mounted with DAPI (Invitrogen, P36931) andobserved under Zeiss Axiovert 200 fluorescencemicroscope.

Acknowledgments
We thank Dr. Akira Yasui for kindly providing
antibodies. This work was supported by the grants
from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (21310123,
21115004, 15H04319, and 16H14640) and the
grants provided from Suzuken Memorial Foundation to K.U.-T.
Author contributions: K.U.-T. designed thestudy, and M.S., K.N., and K.U.-T. discussed theprocedures and results. M.S. and K.N. prepared themass spectrometry samples, and H.K.-H. and M.O.
performed mass spectrometry. M.S. performed data
analysis, and M.S. and K.N. carried out validation
experiments. The manuscript was drafted by
K.U.-T., and M.S. and K.U.-T. completed themanuscript. All authors read and approved thefinal manuscript.
Conflict of interest: The authors declare nocompeting financial interests.

Appendix A. Supplementary Data
Supplementary data to this article can be foundonline at http://dx.doi.org/10.1016/j.jmb.2017.04.
017.

3332

Identification of TNRC6A-ssociating Proteins

Received 28 December 2016;
Received in revised form 18 April 2017;
Accepted 24 April 2017
Available online 3 May 2017
Keywords:
GW182;
TNRC6A;
mass spectrometry;RNA degradation;
spliceosome† http://www.genome.jp/kegg/pathway.html
Abbreviations used:
TNRC6A, trinucleotide repeat-containing gene 6A protein;
Q-rich, glutamine-rich; RRM, RNA recognition motif; AGO,
Argonaute; miRNA, microRNA; CCR4, carbon catabolite
repression 4; CAF1, CCR4-associated factor 1; NLS,
nuclear localization signal; NES, nuclear export signal; IP,
immunoprecipitation; MS, mass spectrometry; UBA,
ubiquitin-associated; FHS, Flag-, HA-, and streptavidinbinding peptide; DAPI, 4′-6-diamidino-2-phenylindole;
pFHS-TNRC6A-WT, wild-type FHS-tagged TNRC6A expression construct; TNRC6A-NES-mut, TNRC6A with a
NES mutation; TNRC6A-NLS-mut, TNRC6A with an NLS
mutation; ER, endoplasmic reticulum; PARP, poly(ADPribose) polymerase; KEGG, Kyoto Encyclopedia of Genes
and Genomes; NOT, negative on TATA-less; snRNA,
small nuclear RNA; RHAU, RNA helicase associated with
AU-rich element ARE.

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