Tài liệu Báo cáo khoa học: The splicing factor ASF/SF2 is associated with TIA-1-related/ TIA-1-containing ribonucleoproteic complexes and contributes to post-transcriptional repression of gene expression doc

The splicing factor ASF/SF2 is associated with
TIA-1-related/ TIA-1-containing ribonucleoproteic
complexes and contributes to post-transcriptional
repression of gene expression
Nathalie Delestienne1, Corinne Wauquier1, Romuald Soin1, Jean-Francois Dierick2,*,
¸
´
Cyril Gueydan1,  and Veronique Kruys1, 
´
`
´
´
1 Laboratoire de Biologie Moleculaire du Gene, Faculte des Sciences, Universite Libre de Bruxelles, Gosselies, Belgium
´
2 Biovallee, Proteomics Unit, Charleroi, Belgium

Keywords
AU-rich elements; hnRNP, heterogenous
nuclear ribonucleoprotein; ribonucleoprotein
complexes; RNA metabolism; RNA-binding
proteins; stress granules
Correspondence
V. Kruys, Laboratoire de Biologie
´
`
Moleculaire du Gene, Institut de Biologie
´
´
´
et de Medecine Moleculaires, Universite
Libre de Bruxelles, 12 rue des Profs. Jeener

et Brachet, 6041 Gosselies, Belgium
Fax: +32 2 6509800
Tel: +32 2 6509804
E-mail:
*Present address
GSK Biologicals, Wavre, Belgium
 
These authors contributed equally to this
work

(Received 10 January 2010, revised 10
March 2010, accepted 25 March 2010)
doi:10.1111/j.1742-4658.2010.07664.x

TIA-1-related (TIAR) protein is a shuttling RNA-binding protein implicated in several steps of RNA metabolism. In the nucleus, TIAR contributes to alternative splicing events, whereas, in the cytoplasm, it acts as a
translational repressor on specific transcripts such as adenine and uridinerich element-containing mRNAs. In addition, TIAR is involved in the
general translational arrest observed in cells exposed to environmental
stress. This activity is encountered by the ability of TIAR to assemble
abortive pre-initiation complexes coalescing into cytoplasmic granules
called stress granules. To elucidate these mechanisms of translational
repression, we characterized TIAR-containing complexes by tandem affinity
purification followed by MS. Amongst the identified proteins, we found the
splicing factor ASF ⁄ SF2, which is also present in TIA-1 protein complexes.
We show that, although mostly confined in the nuclei of normal cells,
ASF ⁄ SF2 migrates into stress granules upon environmental stress. The
migration of ASF ⁄ SF2 into stress granules is strictly determined both by
its shuttling properties and its RNA-binding capacity. Our data also indicate that ASF ⁄ SF2 down-regulates the expression of a reporter mRNA
carrying adenine and uridine-rich elements within its 3¢ UTR. Moreover,
tethering of ASF ⁄ SF2 to a reporter transcript strongly reduces mRNA
translation and stability. These results indicate that ASF ⁄ SF2 and TIA

proteins cooperate in the regulation of mRNA metabolism in normal cells
and in cells having to overcome environmental stress conditions. In addition, the present study provides new insights into the cytoplasmic function
of ASF ⁄ SF2 and highlights mechanisms by which RNA-binding proteins
regulate the diverse steps of RNA metabolism by subcellular relocalization
upon extracellular stimuli.
Structured digital abstract
l
MINT-7715509: ASF ⁄ SF2 (uniprotkb:Q6PDM2) and TIAR (uniprotkb:P70318) colocalize (MI:0403)
by fluorescence microscopy (MI:0416)

Abbreviations
ARE, adenine and uridine-rich element; CBB, calmodulin binding buffer; CP, coat protein; FITC, fluorescein isothiocyanate; Fluc, firefly
luciferase; HA, haemagglutinin; IP, immunoprecipitation; NLS, nuclear localization signal; NPc, nucleoplasmin core domain; Rluc, Renilla
luciferase; RRM, RNA recognition motif; RS, arginine-serine; SG, stress granule; SR, serine-arginine; TAP, tandem affinity purification; TIAR,
TIA-1-related.

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ASF ⁄ SF2 in TIAR-mediated regulatory pathways

N. Delestienne et al.

l

l

l

l

l

l

l

MINT-7715277: TIAR (uniprotkb:P70318) physically interacts (MI:0915) with p68 ⁄ Ddx5
(uniprotkb:Q61656) by anti tag coimmunoprecipitation (MI:0007)
MINT-7715293: TIAR (uniprotkb:P70318) physically interacts (MI:0915) with hnRPN M
(uniprotkb:Q3THB3) by anti tag coimmunoprecipitation (MI:0007)
MINT-7715107: TIAR (uniprotkb:P70318) physically interacts (MI:0914) with hnRNP M
(uniprotkb:Q3THB3), Ddx5 (uniprotkb:B1ARC0), Ddx21 (uniprotkb:Q8K2L4), ASF ⁄ SF2
(uniprotkb:Q6PDM2), Ubf1 (uniprotkb:P25976), Rps25 (uniprotkb:P62852), Rps20(uniprotkb:
P60867), Rps8 (uniprotkb:P62242), Rps4 (uniprotkb:P62702), Rps3 (uniprotkb:P62908), Rpl34
(uniprotkb:Q9D1R9), Rpl31 (uniprotkb:P62900), Rpl30 (uniprotkb:P62889), Rpl23 (uniprotkb:
P62830), Rpl22 (uniprotkb:P67984), Rpl21(uniprotkb:O09167), Rpl18 (uniprotkb:P35980), Rpl15
(uniprotkb:Q9CZM2), Rpl14 (uniprotkb:Q9CR57), Rpl13a (uniprotkb:P19253), Rpl13 (uniprotkb:
P47963), Rpl8 (uniprotkb:P62918) and Rpl5 (uniprotkb:P47962) by tandem affinity purification
(MI:0676)
MINT-7715427: TIA-1 (uniprotkb:P52912) physically interacts (MI:0915) with ASF ⁄ SF2 (uniprotkb:Q6PDM2) by anti tag coimmunoprecipitation (MI:0007)
MINT-7715264: TIAR (uniprotkb:P70318) physically interacts (MI:0915) with ASF ⁄ SF2 (uniprotkb:Q6PDM2) by anti tag coimmunoprecipitation (MI:0007)
MINT-7715309: TIAR (uniprotkb:P70318) physically interacts (MI:0915) with Ddx21 (uniprotkb:Q8K2L4) by anti tag coimmunoprecipitation (MI:0007)
MINT-7715416: TIAR (uniprotkb:P70318) physically interacts (MI:0915) with ASF ⁄ SF2 (uniprotkb:Q07955) by anti tag coimmunoprecipitation (MI:0007)

Introduction
In eukaryotes, the regulation of gene expression occurs
at both transcriptional and post-transcriptional levels.

In the past, transcriptional regulations have been
extensively investigated. However, many recent studies
emphasize the crucial role played by post-transcriptional regulation in the control of gene expression. As
each step of the RNA metabolism is tightly regulated,
the regulation of mRNA export, stability and translation rate is essential for the control of the expression
of mRNAs coding for proteins such as cytokines or
proto-oncogenes. Such regulation allows a very fast
modification of the protein pool in response to specific
stimuli. Indeed, a recent study suggests that post-transcriptional regulation could play a predominant role in
adapting the eukaryotic cell to minor environment perturbations [1]. Post-transcriptional control of gene
expression essentially relies on specific interactions
between cis-acting elements mainly localized in the
UTRs of the transcript and the trans-acting factors
(RNA-binding proteins and noncoding regulatory
RNAs) that bind to these sequences. Among the best
studied regulatory sequences, the adenine and uridinerich elements (AREs) located in the 3¢ UTR of
mRNAs are considered to regulate the stability and ⁄ or
traductibility of 8% of all human mRNAs [2]. RNAbinding proteins comprise other key components of
the post-transcriptional regulation of gene expression.
These proteins are predominantly composed of wellconserved RNA-binding domains mediating RNA contact, and auxiliary domains involved in protein–protein

interactions and sub-cellular targeting [3,4]. TIA-1related (TIAR) protein belongs to the RNA recognition motif (RRM) family of RNA-binding-proteins. It
is a shuttling protein [5] involved in multiple aspects of
RNA metabolism. In the nucleus, this protein acts as a
regulator of the alternative splicing of diverse premRNAs such as those encoding Fas, msl-2, FGFR-2
and calcitonin ⁄ CGRP [6–8]. In the cytoplasm, TIAR
has been shown to regulate the translation of various
mRNAs bearing AREs in their 3¢ UTR. For example,
mRNAs encoding human matrix metallinoproteinase13 and b2-adrenergic receptor are translationaly
repressed by TIAR [9,10]. In addition to the translational regulation of specific mRNAs, TIAR is involved

in a broader translational repression mechanism that
takes place in cells having to overcome environmental
stress such as UV irradiation, thermic variations or
oxidative shock [11]. Thus, although predominantly
nuclear at steady state, TIAR exerts both nuclear and
cytoplasmic functions. Previous studies have highlighted the sequence determinants and mechanisms of
the subcellular distribution of TIAR [5], as well as its
capacity to assemble into cytoplasmic stress granules
(SGs) [12,13]. However, the molecular mechanism by
which TIAR promotes the formation of abortive
pre-initiation complexes still remains unclear. We
hypothesized that TIAR, as a component of the posttranscriptional regulation machinery, acts within large
ribonucleoproteic complexes and that its functions
could depend on its recruitment in such complexes,

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N. Delestienne et al.

as well as on the interactions taking place in these particles.
The present study aimed to identify the proteins
assembling with TIAR and to characterize their role in
TIAR cytoplasmic functions. We used a tandem affinity purification (TAP) approach to isolate TIAR-associated proteins and identified them by MS [14]. The
association of these proteins with TIAR was further
confirmed by co-immunoprecipitation assays in the

presence of RNAse. Interestingly, most of the identified proteins are known to be involved in RNA metabolism and belong to three large families of
RNA-binding proteins. These proteins contribute to
messenger ribonucleoprotein particule formation
and ⁄ or remodelling: the heterogenous nuclear ribonucleoprotein (hnRNP) family, the DEAD ⁄ H box RNA
helicases and the serine-arginine (SR) proteins. We
then investigated the subcellular localization of these
proteins in relation to TIAR in normal cells and in
cells exposed to oxidative stress. We demonstrated
that, although essentially nuclear in normal cells, the
splicing factor ASF ⁄ SF2 relocalized into the cytoplasm
in response to stress and accumulated in bona fide
SGs. Furthermore, the results obtained in the present
study indicate that ASF ⁄ SF2 migration into SGs
strongly depended on previous nuclear export, with
this latter event relying on the RNA-binding activity
of both RRMs. Finally, the association of ASF ⁄ SF2
with TIAR led us to investigate its role on the expression of reporter gene bearing an ARE in its 3¢ UTR.
We showed that overexpression of ASF ⁄ SF2 specifically down-regulated the expression of an ARE reporter gene. Moreover, when tethered to the 3¢ UTR of a
reporter mRNA, ASF ⁄ SF2 strongly affected mRNA
stability and translation. Altogether, our data suggest
that TIAR can assemble with several different proteins
involved in RNA metabolism. The nuclear splicing
factor ASF ⁄ SF2 is identified both as a novel component of stress granules and a novel RNA-binding
protein involved in ARE-mediated post-transcriptional
regulation. Therefore, the results obtained in the present study support the recent findings showing that
members of the SR proteins family, including
ASF ⁄ SF2 and SRp20, have important roles in the
cytoplasmic control of mRNA metabolism [15–17].

Results

Identification of TIAR-associated proteins
The tandem affinity purification procedure originally
developed in yeast by Rigaut et al. [18] was used to
identify proteins interacting with TIAR in mammalian
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cells. Therefore, plasmids encoding the TAP alone or
fused to the carboxy-terminal extremity of TIAR were
generated and the ability of the TIAR-TAP fusion protein to recapitulate TIAR activities was analyzed
before being used to identify interacting partners. We
thus measured the capacity of the TIAR-TAP protein
with respect to activating the inclusion of TIA-1 alternative exon 6A from a transcript derived from a reporter minigene, as previously described for the wild-type
TIAR protein [19]. 293T cells were transiently transfected with the pCI-6-6A-7 minigene in combination
with plasmids encoding TAP alone or TIAR-TAP.
Inclusion of exon 6A in the reporter transcript upon
TIAR-TAP overexpression was subsequently analyzed
by RT-PCR. As shown in Fig. 1A, the expression of
TIAR-TAP but not of TAP alone led to an increased
accumulation of reporter transcript containing exon
6A, thereby indicating that TIAR-TAP recapitulated
the splicing activity of TIAR wild-type protein. TIAR-TAP
protein displayed the same sub-cellular distribution as
the wild-type protein by biochemical fractionation and
migrated into SGs upon arsenite treatment (data not
shown). TAP and TIAR-TAP constructs were then
stably transfected into NIH 3T3 cell lines. Individual
clones were isolated and analyzed for TIAR-TAP
expression, aiming to select a clone in which TIARTAP expression was comparable to the endogenous
TIAR protein (Fig. 1B, left lane). The proteins interacting with TIAR-TAP were purified in the presence
of RNAse A and then separated by gel electrophoresis

before MS analysis (see Materials and methods). The
same procedure was applied to the control cell
line expressing the TAP alone. Beside TIAR-CBP
(Calmodulin-Binding-Peptide) itself, several other
proteins were detected in the TIAR-TAP purified
fraction. MS analysis unambiguously identified several
proteins, many of them corresponding to ribosomal
proteins, as well as five nonribosomal proteins
corresponding to the transcription factor UBF1, the
ribonucleoprotein hnRNP M, the RNA helicases
RHII ⁄ Gu ⁄ DDX21 and p68 ⁄ DDX5, and the SR
protein ASF ⁄ SF2 (Fig. 1C).
The interactions of TIAR with hnRNP M, DDX21
and DDX5 helicases and ASF ⁄ SF2 were further
assessed by co-immunoprecipitation (IP) assays in the
presence or the absence of RNAse A. TIAR protein
fused to the Flag epitope was co-expressed with
haemagglutinin (HA)-tagged candidates in 293T cells
and Flag-IP products were analyzed by western blot
analysis with anti-Flag and anti-HA sera. The specificity of the interactions was evaluated by IP of the unrelated BOIP-Flag protein [20]. As shown in Fig. 2A,
all the candidates identified except DDX21 were

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A

–
Minigene

–

TIARTAP
TAP
+
+

B

6 6A 7

RT-PCR

6

7

kDa

Inputs
+ –

Eluates
+ –
TIAR-TAP
TIAR-CBP

TIAR

48.7

TIAR-TAP
WB anti-TIAR

WB anti-TIAR

TAP

C

TIAR-TAP

TIAR

kDa
200
116.3
97.4

RHII/Gu/DDX21
UBF1
hnRNP M
p68/DDX5

*
*

66.3

*
*
*
*

TIAR-CBP

*

ASF/SF2

55.4

36.5
31
21.5
14.4

Ribosomal
proteins

Fig. 1. Functional characterization of TIAR-TAP protein and purification of TIAR-TAP complexes. (A) Upper panel: RT-PCR analysis of exon
6A inclusion in minigene reporter transcript upon overexpression of TIAR-TAP protein. The alternatively spliced RNA species are indicated.
Lower panel: analysis of TIAR-TAP expression in 293T cells by western blot analysis using anti-TIAR sera. (B) Western blot analysis of TIARTAP in crude extracts and TIAR-cAMP response element-binding protein-binding protein in TAP-purified products obtained from NIH 3T3 cells
stably expressing TIAR-TAP (+) or the TAP alone ()); 0.02% of crude extracts and 10% of purified products were loaded on the gel. (C) TAP
purified products from NIH 3T3 stably expressing TIAR-TAP or the TAP alone. Bands marked by stars corresponded to proteins identified by
MS analysis. Their identity is indicated. The ribosomal proteins present in the TAP purification corresponded to Rpl5, Rpl7A*, Rpl7*, Rpl8,
Rpl13, Rpl13A, Rpl14, Rpl15, Rpl18, Rpl19*, Rpl21, Rpl22, Rpl23, Rpl30, Rpl31 and Rpl34 for the large ribosomal subunit, and Rps3, Rps4,

Rps6*, Rps7*, Rps8, Rps14*, Rps20 and Rps25 for the small ribosomal subunit. Proteins marked with an asterisk are known as common
contaminants of TAP tag purification. The gel is representative of two independent TAP purifications.

specifically immunoprecipitated with TIAR-Flag, both
in the absence and the presence of RNAse A, thereby
indicating that TIAR association with these proteins is
specific and reliant on protein–protein interactions. By
contrast, DDX21 became undetectable in the TIARFlag IP pellet upon RNAse A treatment, suggesting
that its association with TIAR occurs via RNA intermediates.
Subcellular localization of TIAR-associated
proteins
As noted above, TIAR exerts both nuclear and cytoplasmic functions. In the cytoplasm, it is an invariant
component of SGs appearing in response to diverse
environmental stresses that induce a general translation

arrest [12]. We investigated the capacity of TIAR interacting candidates to migrate into SGs upon oxidative
stress. COS cells were transiently transfected to express
the HA-tagged partners and were subsequently treated
with arsenite (1 mm for 30 min) to induce SGs. Indirect fluorescence microscopy revealed that TIAR-associated proteins predominantly accumulated in the
nucleus in normal conditions. However, upon oxidative stress, only ASF ⁄ SF2 protein migrated in TIARpositive cytoplasmic foci (Fig. 2B).
ASF/SF2 is associated with TIAR and TIA-1 protein
complexes and is a bona fide SG component
Because ASF ⁄ SF2 and TIAR shared similar localization patterns both in normal and stressed cells, we

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A

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ASF/SF2
Inputs

p68

IP

Inputs

IP

TIAR-flag +

–

+

–

+

–

TIAR-flag +

–

+

–

BOIP-flag –

+

–

+

–

+

HA-ASF/SF2 +
+
w/o
RNaseA

+

+
w/o

BOIP-flag –
+

+
HA-p68 +
w/o
RNaseA

–
+

+
+
w/o

+
+
with

WB anti-flag

–

–
+
+
+
with

WB anti-flag

WB anti-HA

+

WB anti-HA

hnRNP M
Inputs
TIAR-flag +
BOIP-flag –

DDX21

IP

–
+

+
–

–
+

HA-hnRNP M +
+
RNaseA
w/o

+

+

w/o

Inputs
+
–

–
+

+
+
with

IP

TIAR-flag +

–

+

–

+

–

BOIP-flag –

+

–

+

–

+

+
HA-DDX21 +
w/o
RNaseA

+

+
w/o

WB anti-flag

WB anti-flag

WB anti-HA

+
+
with

WB anti-HA

B

HA

TIAR

Merged

Fig. 2. (A) Analysis of the identified interactions by co-immunoprecipitation. 293T cells
were transiently transfected with DNA
constructs encoding TIAR or BOIP (control)
proteins fused to the Flag epitope in combination with HA-tagged interacting
candidates. Cells were lysed and flag-tagged
proteins were immunoprecipitated with
sepharose beads coupled with M2 anti-flag
serum. Inputs and immunoprecipitates (IP)
were analyzed by SDS-PAGE and western
blot analysis (WB) with anti-flag or anti-HA
sera. Transfected DNAs are indicated. The
experiments were performed in the absence
or presence of RNAse A in the cell lysate.
(B) Sub-cellular distribution of TIAR partners.
COS cells were transfected with the DNA
constructs encoding the HA-tagged interacting candidates and were treated with
arsenite (1 mM for 30 min). Cells were fixed
and stained with mouse anti-HA and goat
anti-TIAR sera. Secondary Alexa
594-coupled donkey anti-mouse (red) and
FITC-coupled anti-goat sera (green) were

used to reveal HA-tagged proteins and TIAR,
respectively. Merged figures correspond to
superpositions of signals corresponding to
HA-tagged proteins and TIAR. Nuclei were
stained with 4¢,6-diamidino-2-phenylindole
(blue).

HA-ASF/SF2

HA-p68

HA-hnRNP M

HA-DDX21

focused the present study on this TIAR-interacting candidate. Figure 3A shows that endogenous ASF ⁄ SF2
protein co-immunoprecipitates with TIAR-Flag. We
2500

DAPI

then determined whether ASF ⁄ SF2 could be associated
with TIA-1 protein, the closest homologue of TIAR.
Co-immunoprecipitation assays revealed that ASF ⁄ SF2

FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS


ASF ⁄ SF2 in TIAR-mediated regulatory pathways

N. Delestienne et al.

Inputs

A
TIAR-flag

–

–

RNaseA

+

+

+

BOIP-flag

IP

–

w/o

–

+

+

–

w/o

–
+
with

WB anti-flag
WB anti-ASF/SF2

Inputs

B

IP

TIA1-flag

+

–

+

–

+

–

BOIP-flag

–

+

–

+

–

+

HA-ASF/SF2

+

+

+

+

+

RNaseA

w/o

w/o

+
with

WB anti-flag
WB anti-HA

Fig. 3. (A) Co-immunoprecipitation of endogenous ASF ⁄ SF2 with
TIAR. The experiment was performed as described in Fig. 2A
except that 293T cells were transfected with BOIP-flag or TIARFlag constructs only. ASF ⁄ SF2 detection was performed by western blot analysis using anti-ASF ⁄ SF2 serum. (B) Co-immunoprecipitation of ASF ⁄ SF2 with TIA-1 protein. The experimental procedure
was identical to that described in Fig. 2A.

is also associated with TIA-1 (Fig. 3B), thereby indicating that ASF ⁄ SF2 can interact with both TIA proteins.
Western blot analysis on whole cell extracts and
purified nuclear and cytoplasmic fractions revealed
that ASF ⁄ SF2 cytoplasmic accumulation upon oxidative stress was a result of the relocalization of the protein and not an increase of ASF ⁄ SF2 gene expression
(Fig. 4A). These data are further supported by the
capacity of ASF ⁄ SF2 to migrate into SGs in cells treated with arsenite in combination with puromycin (data
not shown). Furthermore, ASF ⁄ SF2 co-localized exclusively with TIAR and not with Dcp1-positive Pbs [21],
thereby confirming that ASF ⁄ SF2 is a genuine SG
component (Fig. 4B). To determine whether other
cellular stresses led to the migration of ASF ⁄ SF2 into
SGs, COS cells expressing HA-ASF ⁄ SF2 were exposed
to cytoplasmic stresses such as heat or osmotic shock.
Both conditions led to the migration of ASF ⁄ SF2 into

cytoplamic aggregates corresponding to SGs based on
their content in eIF3b, another SG marker (Fig. 4C).
Several SG components can assemble into SGs upon
overexpression. This is the case for G3BP [22], as well
as RNA-binding proteins such as TIA-1, TIAR [23],
FMRP [24], CPEB1 [25] and CIRP [26]. Therefore, we

evaluated the capacity of ASF ⁄ SF2 to assemble SGs
upon overexpression by transiently transfecting COS
cells with high amounts of ASF ⁄ SF2-expressing plasmid. We observed that the overexpression of ASF ⁄ SF2
induced the spontaneous formation of eiF3b-positive
cytoplasmic aggregates, indicating that, when overexpressed, ASF ⁄ SF2 shares the capacity to promote
SG assembly with other RNA-binding proteins
(Fig. 4D).
Characterization of ASF/SF2 domains controlling
subcellular localization and migration to SGs
Truncated and point-mutated forms of ASF ⁄ SF2 fused
to the HA epitope were generated to determine the
motifs mediating ASF ⁄ SF2 sub-cellular distribution
and recruitment into SGs (Fig. 5A). These constructs
were transfected into COS cells and the intracellular
distribution of the expressed proteins was analyzed by
fluorescence microscopy (Fig. 5B). Previous studies
[27] reported that the deletion of the carboxy-terminal
arginine-serine (RS)-rich domain markedly increased
the proportion of ASF ⁄ SF2 accumulated in the cytoplasm. We observed that the RS1 sub-domain appears
to be the main nuclear import determinant within the
RS domain because the mutant lacking this motif
(DRS1) accumulated in the cytoplasm. By contrast,
ASF ⁄ SF2 nucleo-cytoplasmic distribution is modified

neither by the removal of the RS2 sub-domain, nor by
point mutations disrupting RRM1 (FF-DD mutant)
[28] or RRM2 RNA-binding activity (W134A mutant)
[29]. However, combined inactivation of RRM1 and 2
RNA-binding activities led to a major accumulation of
ASF ⁄ SF2 in the cytoplasmic compartment.
ASF ⁄ SF2 nuclear export determinants were investigated by analyzing the nucleo-cytoplasmic distribution
of wild-type and mutated forms of ASF ⁄ SF2 after
exposure of the transfected cells to actinomycin D and
cycloheximide. This treatment inhibits the transcription-dependent nuclear import of ASF ⁄ SF2, allowing
the observation of ASF ⁄ SF2 nuclear export [30]. As
previously observed, a massive relocalization of wildtype ASF ⁄ SF2 to the cytoplasmic compartment was
detected upon actinomycin D exposure. By contrast,
the W134A mutant remained mostly nuclear under the
same conditions (Fig. 5C), similar to the FF-DD
mutant [30]. Altogether, these data suggest that
ASF ⁄ SF2 nucleo-cytoplasmic shuttling requires intact
RNA-binding activity. Because the nuclear fraction of
ASF ⁄ SF2 is phosphorylated [31,32] and ASF ⁄ SF2
nuclear export is conditioned by a dephosphorylation
process [33,34], we analyzed the sub-cellular distribution
of a phosphomimetic mutant of ASF ⁄ SF2 in which the

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A

B

Arsenite (min)

0

30

Untreated

60 120

ASF/SF2
ASF/SF2

TIAR

Merged

TIAR

Merged

RFP-Dcp1

Merged

Actin
Arsenite

0

Arsenite (min)
N

120
C

N

ASF/SF2

C
ASF/SF2
Arsenite

P105
Myc

ASF/SF2

C

Heat shock

elF3b

Merged

DAPI

HA-ASF/SF2

elF3b

Merged

DAPI

HA-ASF/SF2

elF3b

Merged

DAPI

HA-ASF/SF2

Osmotic shock

D

Fig. 4. Expression and sub-cellular localization of ASF ⁄ SF2 in normal and arsenite-treated cells. (A) NIH3T3 cells were treated for increasing
time periods with arsenite (1 mM) and ASF ⁄ SF2 accumulation was determined by western blot analysis using anti-ASF ⁄ SF2 serum on 15 lg
of total protein extracts. Actin was detected for loading control (upper panel). ASF ⁄ SF2 nucleo-cytoplasmic distribution was determined by

western blot analysis on cytoplasmic and nuclear fractions (15 lg of protein extract). The fractionation was verified using anti-myc serum,
which allows the detection of an exclusively cytoplasmic p105 protein in addition to Myc nuclear protein (lower panel). (B) Endogenous
ASF ⁄ SF2 migrates into SGs and not into processing bodies. COS cells were treated (or not) with arsenite (1 mM for 1 h) before immunostaining to detect endogenous ASF ⁄ SF2 in combination with TIAR (upper and middle panels). Dcp1-RFP-transfected COS cells were stained
with anti-ASF ⁄ SF2 serum after exposure to arsenite (bottom panels). Merged figures correspond to superpositions of signals detected in the
left and middle panels. (C) ASF ⁄ SF2 migration into SGs is induced by several cellular stresses. COS cells were transfected with a DNA construct encoding HA-fused ASF ⁄ SF2 protein and were exposed to heat shock (43 °C for 50 min) or osmotic shock with sorbitol (600 mM for
2 h 30 min). HA-ASF ⁄ SF2 sub-cellular localization was revealed by indirect immunofluorescence using mouse anti-HA serum and alexa 594coupled donkey secondary anti-mouse serum (red). Endogenous eIF3b was detected with goat anti-eiF3b and FITC-coupled donkey anti-goat
serum (green). The merged image corresponds to the superposition of red and green signals. (D) Overexpression of ASF ⁄ SF2 leads to SG
assembly. COS cells were transiently transfected with high amounts of DNA (3 lg instead of 1 lg) encoding HA-ASF ⁄ SF2. Cells were analyzed as described in (C). Arrowheads indicate eIF3b-positive foci detectable in HA-ASF ⁄ SF2 overexpressing cells.

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serine (S) residues in the RS domain were replaced by
negatively charged aspartic acid (D) residues. Interestingly, this phosphomimetic mutant was relocalized to
the cytoplasm to the same extent as the wild-type protein upon cycloheximide ⁄ actinomycin D treatment,
demonstrating the ability of this mutant to efficiently
exit the nucleus (ASF ⁄ SF2 RD; Fig. 5C). We then
analyzed the capacity of ASF ⁄ SF2 mutants to migrate
into SGs upon arsenite exposure and observed that all
of them migrated into SGs, except the double FF-DD
W134A mutant (Fig. 5D). These observations reveal
the importance of ASF ⁄ SF2 RNA-binding ability for
its sub-cellular distribution and the independent capacity of both RRMs to address ASF ⁄ SF2 to SGs. Moreover, the fact that FF-DD and W134A mutants
cannot exit the nucleus (Fig. 5C) but are able to

migrate to SG (Fig. 5D) suggests that ASF ⁄ SF2
migrating in SGs originates from the cytoplasmic fraction or that arsenite induces an alternative nuclear
export pathway relying on other export determinants.
To test this hypothesis, we generated DNA constructs
expressing wild-type or mutated ASF ⁄ SF2 in fusion
with a protein normally confined to the nucleus. This
protein corresponds to the nucleoplasmin core domain
(NPc) fused to the classical nuclear localization signal
(NLS) of hnRNP K. Several studies have shown that,
once carried into the nucleus, this protein does not
passively cross the nuclear envelope into the cytoplasm
[35–37]. We observed that this reporter protein was
predominantly nuclear as previously described [5,38]
and was not recruited into SGs following arsenite
treatment (Fig. 5E). By contrast, the fusion of NPcNLS with ASF ⁄ SF2 induced a detectable redistribution of the protein in the cytoplasmic compartment in
normal cells, as well as its aggregation into SGs upon
oxidative stress. We then analyzed the sub-cellular distribution of mutants defective for RNA binding. Point
mutations disrupting the RNA-binding capacity of any
of the two RRMs led to the nuclear sequestration of
the reporter protein, confirming the inability of such
mutants to exit the nucleus. Moreover, these mutants
were unable to migrate into SGs upon stress (Fig. 5E).
Altogether, these observations indicate that the cytoplasmic accumulation of ASF ⁄ SF2 strongly depends
on the RNA-binding ability of both RRMs and suggest that cytoplasmic accumulation rather than activation of an alternative export pathway is a pre-requisite
for ASF ⁄ SF2 migration into SGs upon stress.
Because both RRMs contributed to ASF ⁄ SF2 cytoplasmic redistribution, we investigated their intrinsic
capacity to do so by fusing them independently to
NPc-NLS reporter protein. Wild-type but not FF-DDmutated RRM1 induced a significant relocalization of

the protein in the cytoplasm and subsequent migration

into SGs upon stress (Fig. 5E). By contrast, the
RRM2 by itself could not recapitulate these properties.
Interestingly, both RRMs (NPc-RRM1RRM2-NLS)
synergized to induce a massive accumulation of the
reporter protein in the cytoplasm, exceeding that of
the reporter protein fused with the full-length
ASF ⁄ SF2 protein, or with the RRM1 alone. Most
likely, this difference is partly a result of the absence
of the RS domain that contributes to nuclear import.
Altogether, our results indicate that ASF ⁄ SF2 nuclear
export relies on the RNA-binding capacity of both
RRMs, whereas the RS domain is dispensable. Moreover, RRM1 but not RRM2 is necessary and sufficient
to promote this cytoplasmic redistribution. However,
in the context of the wild-type ASF ⁄ SF2 protein, both
RRMs play equally important roles.
Overexpression of ASF/SF2 down-regulates the
expression of an ARE-containing reporter mRNA
The association of ASF ⁄ SF2 with TIAR and TIA-1
led us to investigate whether ASF ⁄ SF2 might modulate
the expression of ARE-containing genes. Accordingly,
we tested the effect of overexpressing ASF ⁄ SF2 on the
expression of Renilla luciferase (Rluc) reporter genes
carrying (or not) eight AUUU direct repeats in the 3¢
UTR. These reporter genes were placed under the control of a bidirectional cytomegalovirus promoter mediating the transcription of another reporter gene
encoding firefly luciferase (Fluc) (Fig. 6A). This strategy ensured that the ratio between the control (Fluc)
and the reporter (Rluc) genes was strictly conserved in
all experiments. These reporter constructs were transfected in 293T cells in combination with plasmids
encoding ASF ⁄ SF2, TTP (a mRNA destabilizing
ARE-BP) [39] or the unrelated protein BOIP. The
activity of the Rluc reporter genes containing

(Rluc AU8) (or not) the AU repeats (Rluc AU0) was
normalized by the corresponding Fluc activity and the
normalized Rluc AU8 ⁄ Rluc AU0 ratios were calculated and expressed relative to the value obtained upon
overexpression of BOIP protein. As shown in Fig. 6B,
TTP strongly reduced the AU8 ⁄ AU0 expression ratio
compared to BOIP, thereby confirming the capacity of
TTP to down-regulate ARE-containing mRNAs. Likewise, ASF ⁄ SF2 significantly down-regulated Rluc AU8
mRNA, although to a lesser extent than TTP. Interestingly, although mutations precluding ASF ⁄ SF2
RNA-binding capacity did not significantly alter
ASF ⁄ SF2 down-regulating activity, the deletion of the
C-terminal RS domain almost completely alleviated
this suppressive effect. Western blot analysis revealed

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Untreated
HA

RRM1

RS1
RRM2

8

ActD/CHX
HA

24

0
22

7

3

19

GSWQDL

RG

ASF/SF2

18

85
10

PFAFV

6

C

11

A

RS2
RS

HA-ASF/SF2 WT

HA-ASF/SF2 WT

HA-ASF/SF2 FF-DD

HA-ASF/SF2 FF-DD

HA-ASF/SF2 W134A

HA-ASF/SF2 W134A

HA-ASF/SF2 RD

HA-ASF/SF2 RD

ASF/SF2 ΔRS1
ASF/SF2 ΔRS2
PDADV

ASF/SF2 FFDD
GSAQDL

ASF/SF2 W134A
PDADV

GSAQDL

ASF/SF2 FFDD-W134A
ASF/SF2 RD
RD
RDPSYG(RD)8NDRDRDYSPRRDRGSPRYSPRHDRDRDRT

Untreated

B
HA

HA-ASF WT

HA-ASF ΔRS1

HA-ASF ΔRS2

HA-ASF FF-DD

TIAR

Arsenite

D
Merged

HA

TIAR

Merged

HA-ASF/SF2

HA-ASF ΔRS1

HA-ASF ΔRS2

HA-ASF FF-DD

HA-ASF W134A

HA-ASF W134A

HA-ASF FF-DD W134A

HA-ASF FF-DD W134A

Fig. 5.

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E

Untreated
Flag

TIAR

Arsenite
Merged

Flag

NPc-NLS

NPc-ASF-NLS

NPc-ASF FFDD-NLS

NPc-ASF FFDD-NLS

NPc-ASF W134A-NLS

NPc-ASF W134A-NLS

NPc-RRM1-NLS

NPc-RRM1-NLS

NPc-RRM1 FFDD-NLS

NPc-RRM1 FFDD-NLS

NPc-RRM2-NLS

NPc-RRM2-NLS

NPc-RRM1 RRM2-NLS

Merged

NPc-NLS

NPc-ASF-NLS

TIAR

NPc-RRM1 RRM2-NLS

Fig. 5. Subcellular distribution of ASF-SF2 mutants in actinomycin D- or arsenite-treated COS cells. (A) Schematic representation of
ASF ⁄ SF2 mutants. The amino acids bordering the different domains composing ASF ⁄ SF2 as well as the mutated residues are indicated. The
dotted lines indicate the deleted region in the different mutants. (B, D) Subcellular distribution of ASF ⁄ SF2 mutants in untreated COS cells
(B) and in COS cells treated with arsenite (D). Cells were fixed and the localization of the proteins was performed as described in Fig. 2B.
(C) Subcellular distribution of ASF ⁄ SF2 wild-type and mutated forms upon inhibition of transcription. Transfected COS cells were treated for
3 h with cycloheximide (20 lgỈmL)1) and actinomycin D (5 lgỈmL)1). HA-fused ASF ⁄ SF2 wild-type and mutant proteins were detected with

mouse anti-HA serum and alexa 594-coupled donkey secondary anti-mouse serum. (E) Subcellular distribution of Npc-NLS-Flag alone or in
fusion with ASF ⁄ SF2 domains. The experiment was performed as described in (C), except that Npc-NLS-Flag proteins were detected with
anti-Flag serum.

that all the tested proteins were expressed at similar
levels in Rluc AU8 and Rluc AU0 transfected cells
(Fig. 6C). Altogether, these results indicate that

ASF ⁄ SF2 acts as a negative regulator on ARE-containing mRNAs and that this activity relies on its RS
domain rather than on its RRMs.

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A

Globin 3′UTR

Renilla luciferase

Firefly luciferase
Bidirectional CMV

0

(AUUU)8

B 1.40
Normalized ratio AU8/AU0

1.20
1.00
0.80

***

***
0.60
0.40

1.00

***

0.95
0.54

0.60

HA-ASF/SF2

HA-ASF/SF2
FF-DD W134A

0.20

0.29

0.00
BOIP flag

C

Rluc

TTP flag

AU0

AU8

AU0

AU8

WB anti-flag
BOIP-Flag
Rluc

AU0

AU8

TTP-Flag
AU0

AU8

AU0

AU8

WB anti-HA

HA-ASF/SF2

HA-ASF/SF2
FF-DD W134A

HA-ASF/SF2
SF2ΔRS

ASF/SF2 down-regulates the expression of a
reporter luciferase mRNA when tethered to the
3¢ UTR
The function of ASF ⁄ SF2 in mRNA metabolism was
further analyzed by a tethering approach. This proce-

HA-ASF/
SF2ΔRS

Fig. 6. Overexpression of ASF ⁄ SF2
down-regulates the expression of a reporter
containing AU-rich elements. (A) Schematic
representation of the bidirectional FLuc ⁄
RLuc reporter constructs bearing (or not)

eight repeats of the AUUU sequence with
its globin 3¢ UTR. (B) The reporter constructs (500 ng) were transiently transfected
in 293T cells in combination with plasmids
(500 ng) encoding wild-type or mutated
forms of ASF ⁄ SF2 fused to the HA epitope.
Flag-tagged TTP and BOIP proteins were
included in the experiment as positive and
negative controls, respectively. The Rluc
activity was normalized by Fluc values and
a normalized Rluc AU8 ⁄ Rluc AU0 was calculated for each overexpressed protein, attributing the value of 1 for BOIP protein
(mean ± SD of at least three independent
transfections). Mean values of TTP-Flag,
HA-ASF ⁄ SF2 and HA-ASF ⁄ SF2FF-DDW134A samples were tested for statistical
significance compared to a BOIP-Flag sample using a two-tailed Student’s test
(*P < 0.05, **P < 0.01, ***P < 0.001).
(C) Expression of the tagged proteins in the
co-transfection experiment was monitored
by western blot using anti-Flag or anti-HA
sera.

dure is based on the high affinity of the bacteriophage
MS2-coat protein (CP) for a defined hairpin MS2
RNA sequence. When a protein is expressed in fusion
with MS2-CP, it is directly addressed onto the RNA
harbouring the MS2 sequence, allowing the function of
this protein to be analyzed without competition from

Fig. 7. Tethering of ASF ⁄ SF2 to the 3¢ UTR of a reporter gene strongly down-regulates its stability and translation. (A) 293T cells were transfected with Rluc 0MS2 or 8MS2 plasmids in combination with constructs encoding MS2-CP alone or in fusion with TTP, ASF ⁄ SF2 or
ASF ⁄ SF2 lacking the RS domain. The luciferase activities measured in the cell extracts are reported as the ratio of the Rluc to Fluc activities
(mean ± SD of at least four independent transfections). Ratios obtained upon TTP-CP and ASF ⁄ SF2-CP expression were tested for statistical

significance compared to the ratio obtained upon CP expression using a two-tailed Student’s test. Dark grey bars, 0MS2; light grey bars,
8MS2. (B) Expression of the tagged proteins in the co-transfection experiment was monitored by western blot using anti-Flag serum. (C)
Sub-cellular localization of ASF ⁄ SF2-MS2-CP in normal and arsenite-treated cells. COS cells were transiently transfected with ASF ⁄ SF2-MS2CP encoding plasmid and were subsequently treated (or not) with arsenite. Cells were fixed and ASF ⁄ SF2-MS2-CP protein was detected
with anti-Flag serum as described in Fig. 4. (D) Quantification of Rluc mRNA accumulation normalized to Fluc mRNA values. After transfection, cytoplasmic RNA was isolated and analyzed by northern blot using Fluc and Rluc riboprobes. The radioactive signals were quantified by
the STORM 820 equipment and IMAGEQUANT software (Molecular Dynamics). The Rluc ⁄ Fluc ratios are indicated as a percentage of the normalized value obtained for Rluc 0MS2. (E) The effect of tethering ASF ⁄ SF2 or TTP to Rluc reporter on the protein expression per cytoplasmic mRNA was calculated as the ratios of the normalized Rluc activities compared to the normalized ratio of Rluc cytoplasmic mRNA of one
representative experiment.

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other cellular proteins for the RNA-binding site and
independent of any intermediate docking factor
[26,40]. DNA constructs encoding CP alone or in
fusion with ASF ⁄ SF2 or TTP were co-transfected in

A

293T cells with plasmids encoding Fluc and Rluc
proteins under the control of the bidirectional cytomegalovirus promoter. In these experiments, the b-globin 3¢ UTR flanking the Rluc coding sequence

0MS2

Protein

8MS2

Rluc/Fluc (% of control)

120
100

100

100

100

100
65.4

80

**

60

39.1

40

**

**

6.3

20

4.6

0
CP

B

TTP-CP

ASF/SF2-CP ASF/SF2ΔRS-CP

D Cytoplasmic RNA

0MS2 8MS2 0MS2 8MS2 0MS2 8MS2 0MS2 8MS2

TTP-CP

ASF/
ASF/
SF2-CP SF2ΔRS-CP

Rluc/Fluc (% of control)

WB anti-flag

CP

Untreated

Arsenite 1 mM

100

100

100
65.52

80
60

*

40

20.01

*

9.55

20

CP

TTP-CP

ASF/SF2-CP

E
DAPI

Protein/Cytoplasmic RNA (% of control)

C Localization

8MS2
100

0

ASF/SF2-CP

0MS2

120

Protein/Cytoplasmic RNA
120

0MS2
8MS2

100 100.81

100

100

100

82.94
80
60
40
16.03

20
0

CP

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ASF/SF2-CP

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contained eight or zero (control) repeats of the MS2
sequence [41]. The effect of ASF ⁄ SF2-CP tethering to

the reporter mRNA was evaluated by comparing Rluc
activities obtained with constructs containing (or not)
MS2 repeats. The values were divided by Fluc activity
values to normalize the transfection and recovery efficiencies. Tethering of TTP was tested in parallel. It
was found that the expression of ASF ⁄ SF2-CP led to
a marked decrease ( 95%) of the Rluc ⁄ Fluc ratio for
the 8MS2-containing reporter gene compared to the
0MS2 control (Fig. 7A). This effect was comparable to
TTP-tethering and was mostly specific to ASF ⁄ SF2
because CP alone only marginally altered the Rluc ⁄ Fluc ratio of the 0MS2 reporter gene compared to the
0MS2 control. Western blot analysis revealed that all
the tested proteins were expressed at similar levels in
Rluc 8MS2 and Rluc 0MS2 transfected cells (Fig. 7B).
In addition, ASF ⁄ SF2-CP spatial distribution was similar to endogenous ASF ⁄ SF2 because it is mainly
nuclear at equilibrium and migrates into SGs upon
stress (Fig. 7C). The effect of ASF ⁄ SF2-CP on cytoplasmic Rluc mRNA accumulation was measured and,
to normalize the transfection and recovery efficiencies,
the accumulation of Fluc mRNA was measured in the
same RNA samples (Fig. 7D). CP and TTP-CP were
included as controls. The steady-state level of Rluc
mRNA containing eight MS2 binding sites was slightly
reduced upon expression of CP alone. The tethering of
TTP led to a decrease of 8MS2 Rluc reporter mRNA
to the same extent as that at the protein level
(Fig. 7E), therefore confirming the mRNA-destabilizing activity of TTP as previously described [39,42].
ASF ⁄ SF2 significantly decreased 8MS2 mRNA accumulation, although not to the same extent as that
observed at the protein level (four- to five-fold less),
thereby suggesting that ASF ⁄ SF2 down-regulates both
mRNA stability and translation (Fig. 7D, E). It is
worth noting that the deletion of the RS domain significantly alleviated ASF ⁄ SF2 repressive activity as

observed for ARE reporter mRNA (Fig. 7A).

Discussion
In the present study, we identified new candidate proteins associated with TIAR. They include ribosomal
proteins, the transcription factor UBF1 and proteins
involved in RNA metabolism, such as hnRNP M and
ASF ⁄ SF2, as well as DDX21 and DDX5 RNA helicases. The physiological relevance of the presence of
ribosomal proteins in the TIAR-TAP pellet is difficult
to address because ribosomal proteins are common
contaminants of TAP assays [43,44]. On the other
hand, the presence of ribosomal subunits in the TIAR2508

TAP pellet might result from uncompleted RNA degradation by RNAse A in the purification process and
reflect the capacity of TIAR to be associated with
polysomes. Although the association of DDX21 with
TIAR relies on RNA intermediate, DDX5, hnRNP M
and ASF ⁄ SF2 are associated with TIAR by protein–
protein interaction. It is worth noting that TIAR,
hnRNP M and p68 ⁄ DDX5 are components of a
ribonucleoproteic complex including tumour necrosis
factor-a mRNA, a prototype of ARE-containing
mRNAs, in macrophage cell extracts [45]. Similar to
TIAR, all three proteins display a major nuclear localization, suggesting that the association of TIAR with
these proteins most likely occurs in the nuclear compartment. Upon stress, only the splicing factor
ASF ⁄ SF2 migrates with TIAR into cytoplasmic stress
granules. Moreover, we observed that the expression
of a TIAR mislocalized mutant strongly disturbed
ASF ⁄ SF2 nuclear accumulation, further demonstrating
the existence of an interacting event between TIAR
and ASF ⁄ SF2 proteins (Fig. S1). ASF ⁄ SF2 is also

associated with TIA-1, the protein sharing the highest
degree of structural conservation and overlapping
functions with TIAR [12,19,46,47]. This suggests that
TIAR and TIA-1 protein complexes including
ASF ⁄ SF2 might be involved in redundant molecular
processes. However, the physiological existence of
ASF ⁄ SF2-TIA complexes is conditioned by the tissue
distribution of TIA protein [46], which is clearly more
restricted compared to ASF ⁄ SF2 [48].
ASF ⁄ SF2 migration into SGs strongly depends on its
transit in the cytoplasm, as well as on its ability to bind
RNA. Therefore, this process most likely results from
the sequestration of ASF ⁄ SF2-bound transcripts in
such cytoplasmic structures. Because both RRMs can
independently mediate ASF ⁄ SF2 migration into SGs, it
can be speculated that, although acting synergistically
for optimal interaction with RNA, both RRMs mediate
interactions with distinct motifs present in mRNA molecules addressed to SGs upon stress. ASF ⁄ SF2 is a
bona fide SG component and does not get associated
with other cytoplasmic structures such as processing
bodies. Similar to other SG components, it displays the
capacity of spontaneous SG assembly upon overexpression. However, down-regulation of ASF ⁄ SF2 expression does not alter SG assembly upon stress (Fig. S2).
Altogether, these results suggest that ASF ⁄ SF2 and
TIA proteins participate in common mechanisms of
translational repression in response to stress.
The data obtained in the present study further
describe the molecular determinants for ASF ⁄ SF2 subcellular distribution. We demonstrated that the
removal of the RS1 domain, but not of the RS2

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domain, recapitulated the cytoplasmic redistribution
observed for the mutant lacking the whole RS region
in accordance with RS1 phosphorylation-dependent
ASF ⁄ SF2 nuclear import [49,50]. ASF ⁄ SF2 RRMs
were reported to cooperate with respect to RNA-binding affinity and selectivity. Moreover, point mutations
disrupting RRM1 RNA-binding activity (FF-DD)
were shown to effectively inhibit ASF ⁄ SF2 nuclear
export [28,51]. Structural and functional analysis
of RRM2 highlighted the importance of W134 for
RRM2 RNA-binding activity [29]. In the present
study, we demonstrated that, although RRM1 and
RRM2 display remarkable structural differences [29],
the RNA-binding activities of each RRM equally contribute to ASF ⁄ SF2 nuclear export. It can thus be
speculated that ASF ⁄ SF2 nuclear export is conditioned
by a strong association with RNA requiring both
RRMs. By contrast to an earlier study [52], we
observed that the phosphomimetic RD mutant of
ASF ⁄ SF2 exits the nucleus at a rate comparable to the
wild-type protein under conditions blocking ASF ⁄ SF2
nuclear import. The results obtained suggest that this
phosphomimetic mutant bypasses the dephosphorylation step prior to nuclear export. Alternatively,
ASF ⁄ SF2 may exit the nucleus by two distinct mechanisms, one of which is dephosphorylation-independent.
Overexpression of ASF ⁄ SF2 specifically down-regulates the expression of a reporter gene bearing AREs
in its 3¢ UTR, suggesting the participation of

ASF ⁄ SF2 in ARE-mediated post-transcriptional regulation. The significant but less pronounced effect of
ASF ⁄ SF2 compared to TTP might be a result of the
differential capacity of the two proteins to be
recruited to AREs. It is already well established that
AREs can recruit several different ARE-BPs, depending on their type of AU-rich motifs, as well as the
abundance of each ARE-BP [53]. Interestingly, the
down-regulating effect of ASF ⁄ SF2 is preserved upon
inactivation of its RNA-binding ability by point mutations in both RRM RNA-binding motifs (FF-DD
W134A mutant). These results suggest that ASF ⁄ SF2
is recruited to the ARE reporter mRNA by intermediate protein–protein interactions, in contrast to its
migration into SGs, which directly relies on RRM
RNA-binding activities. The removal of the RS
domain completely reversed ASF ⁄ SF2 down-regulating activity on the ARE reporter mRNA. The mutant
lacking the RS domain might become associated with
the ARE reporter mRNA via interactions with AREBPs but is inactive in down-regulating mRNA translation and stability. Of note, the mutant lacking the
RS domain still becomes associated with TIAR in
co-immunoprecipitation assays (Fig. S3). However,

down-regulation of the ARE reporter mRNA by
ASF ⁄ SF2 could not be abrogated by the expression of
a RNA-binding defective TIAR mutant (data not
shown), therefore suggesting that the targeting of
ARE-containing mRNAs by ASF ⁄ SF2 can occur via
several different interactions.
The capacity of ASF ⁄ SF2 to down-regulate mRNA
expression was further confirmed by artificial tethering
of a reporter mRNA to the 3¢ UTR. In this assay, the
down-regulating activity of ASF ⁄ SF2 was almost complete ( 95% inhibition) and was comparable to that
obtained upon tethering of TTP to the same reporter
mRNA. These results indicate that ASF ⁄ SF2 is a

potent repressing factor in the absence of competing
factors. It is worth noting that the deletion of the RS
domain strongly alleviated ASF ⁄ SF2 repressing activity, confirming the importance of this domain in the
down-regulating activity of ASF ⁄ SF2. Although TTP
induces mRNA destabilization as previously described
[39], ASF ⁄ SF2 appears to act by a mechanism combining mRNA degradation and translational repression.
Previous studies revealed the ability of ASF ⁄ SF2 to
induce the destabilization of the mRNA encoding
PKCI-related protein by direct binding to a purine-rich
sequence present in PKCI-r mRNA 3¢ UTR [15]. On
the other hand, other studies indicated that ASF ⁄ SF2
activates the translation of mRNAs bearing ASF ⁄ SF2
binding sites within the coding region in intact cells
[16,54]. The capacity of ASF ⁄ SF2 to promote mRNA
translation when tethered to mRNA 3¢ UTR in cellfree systems or upon microinjection into Xenopus
oocytes was also reported [16]. The apparent contradiction between these previous observations and those
obtained in the present study might reflect the importance of the nuclear origin of the target mRNA for the
functional outcome of ASF ⁄ SF2 recruitment to
mRNA 3¢ UTR.
In conclusion, the present study highlights the
involvement of ASF ⁄ SF2 in post-transcriptional downregulating mechanisms in both normal and stressed
cells. It appears that, similar to other RNA-binding
proteins, such as AUF1 [55] and HuR [56], ASF ⁄ SF2
differentially modulates the fate of transcripts with
which it becomes associated.

Materials and methods
Materials
Enzymes were purchased from Invitrogen (Carlsbad, CA,
USA) and Roche (Basel, Switzerland); oligonucleotides

were obtained from Sigma (St Louis, MO, USA); and cell
culture media were obtained from Invitrogen.

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DNA constructs
A pcDNA3.1-TAP construct was obtained by inserting the
sequence coding for TAP into the SfuI ⁄ PmeI sites of
the pcDNA3.1(+) plasmid. The sequence coding for the
C-terminal TAP tag was amplified by PCR using pBS1479
as template DNA. pBS1479 was kindly provided by D.
´
Lafontaine (Universite Libre de Bruxelles, Belgium) and
has been described previously [57]. pcDNA3.1-TIAR-TAP
construct was generated by inserting murine TIARb coding
region (accession number: AAC52870) between the EcoRI
and BamHI sites of the pcDNA3.1-TAP plasmid. pcDNABOIP-Flag construct was generated by inserting PCR product coding for BOIP (accession number: AAI69777) [20] in
fusion with flag epitope into pcDNA3.1()) plasmid. DNA
constructs used to express HA-tagged TIAR-interacting
proteins were generated by introducing PCR-amplified coding regions into the EcoRI ⁄ BamHI sites of pRK5-NHA
plasmid (BD Pharmingen, San Diego, CA, USA)
(ASF ⁄ SF2 accession number: NP_775550; Ddx21:
AAH30895; Ddx5: CAM18571; hnRNP M: CAX15839).

DNA constructs used to express ASF ⁄ SF2 in fusion with
the nucleoplasmin-core domain fused to the classical NLS
of hnRNP K were generated by introducing PCR-amplified
wild-type, truncated or point mutated ASF ⁄ SF2 into EcoRI
site of a pcDNA-NPc-NLS-Flag construct [5].
The bidirectional Renilla ⁄ firefly luciferase constructs containing none, eight MS2-CP-binding sites, or an ARE, were
kindly provided by L. Paillard (Rennes, France) and are
described elsewhere [41]. The MS2-CP (pCMS2) plasmid
was described previously [58]. The plasmids encoding
TTP-MS2-CP (accession number: NP_035886), ASF ⁄ SF2MS2-CP and ASF ⁄ SF2DRS-MS2-CP were obtained by
inserting full-length or truncated coding sequences flanked
by the Flag epitope into the BamH1 site of pCMS2. All the
constructs were subsequently sequenced.

Stable cell line generation
NIH 3T3 cells were transfected with the pcDNA3.1-TIARTAP or the pcDNA3.1-TAP construct carrying the NeoR
gene. After 48 h of transfection, drug selection was started
by adding G418 (1 mgỈmL)1) to the cell culture medium.
After approximately 3 weeks of selection, when isolated colonies appeared, drug resistant clones were isolated by limit
dilutions of transfection pools.

Purification of TIAR-TAP complexes
NIH 3T3 cells (6 · 108) stably transfected with the
pcDNA3.1-TAP or with the pcDNA3.1-TIAR-TAP construct were lysed in 20 mL of lysis buffer containing 50 mm
Tris (pH 8.0), 150 mm NaCl, 0.1% NP40 and a cocktail of
protease inhibitors (Roche). The lysate was cleared by centrifugation for 30 min at 8000 g. The supernatant (200 mg

2510

of protein extract) was incubated overnight with 150 lL

rabbit IgG Sepharose 6 Fast Flow (GE Healthcare UK Ltd
Amersham, Little Chalfont, UK) agarose beads equilibrated in IgG binding buffer (IgG BB: 0.15 m NaCl, 0.1%
NP40, 50 mm Tris, pH 8.0) in the presence of RNAse A
(10 lgỈmg)1 extract). After 15 h of binding at 4 °C, beads
were washed twice with 1 mL of IgG BB and once with
1 mL of Tev cleavage buffer (Tev CB: 25 mm Tris, pH 8.0,
0.15 m NaCl, 0.1% NP40, 0.5 mm EDTA and 1 mm dithithreitol). Three hundred units of Tev protease (Invitrogen)
were added and cleavage of the TAP tag was performed in
1 mL of Tev CB for 3 h at room temperature. Proteins
released from the beads were collected in two fractions of
1 mL, and the eluate was adjusted to 2 mm CaCl2 before
the addition of three volumes of calmodulin binding buffer
(CBB: 10 mm Tris, pH 8.0, 0.15 m NaCl, 10 mm 2-mercaptoethanol, 1 mm Mg acetate, 1 mm imidazole, 2 mm CaCl2,
0.1% NP40) and loaded onto calmodulin Sepharose 4B
affinity resin (Amersham) equilibrated in the same buffer.
After 2 h of binding at 4 °C, beads were washed once with
1 mL of CBB and once with modified CBB (0.02% NP40).
Elution of the bound proteins was performed by addition
of 5 · 600 lL of calmodulin elution buffer (10 mm Tris,
pH 8.0, 0.15 m NaCl, 0.02% NP40, 10 mm 2-mercaptoethanol, 1 mm Mg acetate, 1 mm imidazole, 2 mm EGTA). Proteins were concentrated by trichloracetic acid precipitation
and separated on 4–12% gradient polyacrylamide gel (Invitrogen). The gels were stained with Sypro Ruby reagent
(Molecular Probes, Carlsbad, CA, USA). The output of
three TIAR-TAP purifications was loaded on the gel.

Protein identification by MS
Protein spots were excised from the gel before in-gel tryptic
digestion [43] and the resulting peptides were analyzed by
MALDI-TOF MS. MALDI-TOF peptide mapping was carried out on a M@LDI LR instrument (Waters ⁄ Micromass
UK Ltd, Manchester, UK) after purification ⁄ concentration
of the tryptic peptides on home-made nanoscale reversedphase columns [59]. MS data were searched against the

mouse sequence databank ( />mouse/) using proteinlynx Global server 2 software
(Waters ⁄ Micromass UK Ltd).

Co-immunoprecipitation and western blotting
HEK 293T cells were transiently transfected using
FUGENE-6 reagent (Roche) in accordance with the manufacturer’s instructions. pcDNA3.1-BOIP-Flag, pcDNA3.1TIAR-Flag or pcDNA3.1-TIA-1-Flag (accession number:
AAC52871) [5] plasmids were co-transfected with pRK5NHA constructs encoding the candidate partners in
1 · 106 cells. Forty eight hours after transfection, cells were
washed twice in ice-cold NaCl ⁄ Pi and lysed in IP buffer
(50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.2% NP40,

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ASF ⁄ SF2 in TIAR-mediated regulatory pathways

N. Delestienne et al.

protease inhibitors) for 30 min at 4 °C. Flagged-proteins
were immunoprecipitated by incubating protein extract
(2 mg) with BSA (1%) blocked-anti-Flag M2 (20 lL) affinity gel (Sigma) for 2 h at 4 °C. The beads were washed
three times in IP buffer and were then incubated for 30 min
at room temperature in IP buffer containing RNAse A
(10 lgỈmg)1 extract) and washed once in IP buffer. Bound
proteins were eluted in Laemmli gel sample buffer, separated on 12.5% SDS-PAGE and transferred to nitrocellulose for western blot analysis. Western blot analysis of HAand Flag-tagged proteins was performed as described previously [60] using anti-HA serum (dilution 1 : 5000) and M2
monoclonal anti-Flag serum (dilution 1 : 2000), respectively.

Cell culture and treatments
COS, NIH 3T3, 293T and MEF cells (kindly provided
by P. Anderson, Harvard Medical School, USA) cells were

grown in DMEM Glutamax (Invitrogen, Carlsbad, CA,
USA) supplemented with 10% fetal bovine serum, penicillin
(50 mL)1) and streptomycin (50 mgỈmL)1). All cell lines
were transiently transfected using Fugene-6 (Roche) in accordance with the manufacturer’s instructions. The arsenite
treatment was performed by removing culture medium and
immediately adding fresh culture medium containing 1 mm
or 500 lm arsenite and cells were incubated for 30 min or
1 h, respectively.

Cell fractionation
Cytoplasmic and nuclear extracts from NIH3T3 cells were
prepared as described previously [61].

Cell fixation and immunofluorescence
Cells were seeded on glass coverslips (18 · 18 mm). Twenty
four or 48 h post transfection, cells were rapidly washed
twice with ice-cold NaCl ⁄ Pi and fixed with NaCl ⁄ Pi
containing 2% paraformaldehyde at room temperature for
10 min. The fixed cells were washed three times for 5 min
with NaCl ⁄ Pi, permeabilized for 5 min with NaCl ⁄ Pi, 0.5%
Triton-X 100 at 4 °C and washed again as described above.
Blocking was performed with NaCl ⁄ Pi containing 10%
BSA for 30 min. After blocking, the coverslips were incubated for 1 h with antibodies diluted in NaCl ⁄ Pi, 0.1%
Tween 20. Antibodies were used at a dilution of 1 : 50 for
anti-ASF ⁄ SF2 (Zymed Laboratories; Invitrogen), goat antiTIAR C18 and anti-eiF3b (Santa Cruz Biotechnology,
Santa Cruz, CA, USA) sera; at a dilution of 1 : 5000 for
mouse anti-FLAG M2 serum; and at a dilution of 1 : 30
000 for anti-HA serum (Sigma). The coverslips were subsequently washed three times for 10 min with NaCl ⁄ Pi, 0.1%
Tween 20 and incubated with the secondary antibody in

NaCl ⁄ Pi, 0.1% Tween 20. Alexa594-conjugated donkey
anti-goat serum was used at a dilution of 1 : 25 000. In
double staining experiments, a donkey anti-goat serum
conjugated with fluorescein isothiocyanate (FITC) was used
(dilution 1 : 1000). After 1 h, coverslips were washed three
times as above, rapidly rinsed with desionized water
and mounted on glass slides with the Gel MountÔ Aqueous
Mounting Medium (Sigma-Aldrich, St Louis, MO, USA)
containing 4Â,6-diamidino-2-phenylindole (100 pgặmL)1) and
examined by uorescence microscopy with a Leica
DM4000B microscope with a · 63 HCXPL APO objective
(numerical aperture 1.40–0.60) (Leica Microsystems, Wetzlar, Germany). Digital pictures were acquired with a Leica
DFC320 camera, using leica software. The images were processed and assembled with photoshop 7.0 (Adobe Systems
Inc., San Jose, CA, USA). Controls without primary antibodies were always included for comparison.

Luciferase assay
Cell lysis and luciferase assays were performed using the
Promega Dual-luciferase system (Promega, Madison, WI,
USA) and a TD-20 ⁄ 20 luminometer (Turner Designs,
Sunnyvale, CA, USA). Each assay was performed in
triplicate.

Northern blot analysis
To isolate cytoplasmic RNA, cells were trypsinized, washed
in NaCl ⁄ Pi, resuspended in ice-cold isotonic buffer (0.14 m
NaCl, 1.5 mm MgCl2, 10 mm Tris, pH 8.6, and 100 mL)1
heparin), and lysed by the addition of an equal volume of
this buffer, containing 0.5% NP-40 detergent. The nuclei
were pelleted, two-thirds of the cytoplasmic extract was
recovered and mixed with an equal volume of SDS buffer

(0.2 m Tris, pH 7.5, 0.3 m NaCl, 25 mm EDTA, 2% SDS)
and twice extracted with phenol ⁄ chloroform, once extracted
with chloroform, and precipitated with ethanol and sodium
acetate [62]. The quality of the RNA samples was verified
by agarose gel electrophoresis before loading on a 1.5%
agarose gel and conducting northern blot analysis. Renilla
and firefly luciferase antisense RNA probes were generated
by in vitro transcription using linearized DNA templates in
the presence of 80 lCi [a-32P]UTP (800 CiỈmmol)1) and
20 lm UTP. Quantitative analysis of northern blots was
performed using a phosphorimager (STORM 820; Molecular Dynamics, Sunnyvale, CA, USA) and imagequant
software (Molecular Dynamics).

Acknowledgements
We thank Sylvain Lestrade for providing excellent
technical assistance in the proteomic analysis, Dominique Weil for the Dcp1-RFP construct, Luc Paillard

FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS

2511


ASF ⁄ SF2 in TIAR-mediated regulatory pathways

N. Delestienne et al.

for the bidirectional reporter genes and Fabienne
Konczak for helping us test the splicing activity of
TIAR-TAP protein. This work was funded by the
´

DGTRE (Region Wallonne), the Fund for Medical Scientific Research (Belgium, grant 2.4.511.00.F), and the
´
‘Actions de Recherches Concertees’ (grants 00-05 ⁄ 250
and AV.06 ⁄ 11-345). N. Delestienne was supported by a
PhD FRIA fellowship from the FNRS (Belgium).

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Supporting information
The following supplementary material is available:
Fig. S1. Expression of a C-terminal truncated mutant
of TIAR leads to ASF ⁄ SF2 mislocalization.
Fig. S2. Down-regulation of ASF ⁄ SF2 expression does
not alter stress-induced SG formation.
Fig. S3. Co-immunoprecipitation of TIAR with ASF ⁄
SF2 mutant lacking the RS domain.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.

FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS