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.. We
Trang 1TIA-1-related/ TIA-1-containing ribonucleoproteic
complexes and contributes to post-transcriptional
repression of gene expression
Nathalie Delestienne1, Corinne Wauquier1, Romuald Soin1, Jean-Franc¸ois Dierick2,*,
Cyril Gueydan1,and Ve´ronique Kruys1,
1 Laboratoire de Biologie Mole´culaire du Ge`ne, Faculte´ des Sciences, Universite´ Libre de Bruxelles, Gosselies, Belgium
2 Biovalle´e, 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
Mole´culaire du Ge`ne, Institut de Biologie
et de Me´decine Mole´culaires, Universite´
Libre de Bruxelles, 12 rue des Profs Jeener
et Brachet, 6041 Gosselies, Belgium
Fax: +32 2 6509800
Tel: +32 2 6509804
E-mail: vkruys@ulb.ac.be
*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 impli-cated in several steps of RNA metabolism In the nucleus, TIAR contrib-utes to alternative splicing events, whereas, in the cytoplasm, it acts as a translational repressor on specific transcripts such as adenine and uridine-rich 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 indi-cate 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 addi-tion, 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.
Trang 2In 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-transcrip-tional regulation in the control of gene expression As
each step of the RNA metabolism is tightly regulated,
the regulation of mRNA export, stability and
transla-tion 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-tran-scriptional regulation could play a predominant role in
adapting the eukaryotic cell to minor environment
per-turbations [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
uridine-rich 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]
RNA-binding proteins comprise other key components of
the post-transcriptional regulation of gene expression
These proteins are predominantly composed of
well-conserved RNA-binding domains mediating RNA
con-tact, and auxiliary domains involved in protein–protein
interactions and sub-cellular targeting [3,4] TIA-1-related (TIAR) protein belongs to the RNA recogni-tion 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 pre-mRNAs 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
metallinoproteinase-13 and b2-adrenergic receptor are translationaly repressed by TIAR [9,10] In addition to the transla-tional 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 high-lighted 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 post-transcriptional regulation machinery, acts within large ribonucleoproteic complexes and that its functions could depend on its recruitment in such complexes,
l MINT-7715277 : TIAR (uniprotkb: P70318 ) physically interacts ( MI:0915 ) with p68 ⁄ Ddx5 (uniprotkb: Q61656 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-7715293 : TIAR (uniprotkb: P70318 ) physically interacts ( MI:0915 ) with hnRPN M (uniprotkb: Q3THB3 ) by anti tag coimmunoprecipitation ( MI:0007 )
l 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 )
l MINT-7715427 : TIA-1 (uniprotkb: P52912 ) physically interacts ( MI:0915 ) with ASF ⁄ SF2 (uni-protkb: Q6PDM2 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-7715264 : TIAR (uniprotkb: P70318 ) physically interacts ( MI:0915 ) with ASF ⁄ SF2 (uni-protkb: Q6PDM2 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-7715309 : TIAR (uniprotkb: P70318 ) physically interacts ( MI:0915 ) with Ddx21 (uni-protkb: Q8K2L4 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-7715416 : TIAR (uniprotkb: P70318 ) physically interacts ( MI:0915 ) with ASF ⁄ SF2 (uni-protkb: Q07955 ) by anti tag coimmunoprecipitation ( MI:0007 )
Trang 3as well as on the interactions taking place in these
par-ticles
The present study aimed to identify the proteins
assembling with TIAR and to characterize their role in
TIAR cytoplasmic functions We used a tandem
affin-ity purification (TAP) approach to isolate
TIAR-asso-ciated 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
identi-fied proteins are known to be involved in RNA
metab-olism and belong to three large families of
RNA-binding proteins These proteins contribute to
messenger ribonucleoprotein particule formation
and⁄ or remodelling: the heterogenous nuclear
ribonu-cleoprotein (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
expres-sion of reporter gene bearing an ARE in its 3¢ UTR
We showed that overexpression of ASF⁄ SF2
specifi-cally down-regulated the expression of an ARE
repor-ter 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
compo-nent of stress granules and a novel RNA-binding
protein involved in ARE-mediated post-transcriptional
regulation Therefore, the results obtained in the
pres-ent study support the recpres-ent 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
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 pro-tein 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 alter-native exon 6A from a transcript derived from a repor-ter minigene, as previously described for the wild-type TIAR protein [19] 293T cells were transiently trans-fected 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 TIAR-TAP expression was comparable to the endogenous TIAR protein (Fig 1B, left lane) The proteins inter-acting 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 specific-ity of the interactions was evaluated by IP of the unre-lated BOIP-Flag protein [20] As shown in Fig 2A, all the candidates identified except DDX21 were
Trang 4specifically 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
TIAR-Flag IP pellet upon RNAse A treatment, suggesting
that its association with TIAR occurs via RNA
inter-mediates
Subcellular localization of TIAR-associated
proteins
As noted above, TIAR exerts both nuclear and
cyto-plasmic 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 inter-acting 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 Indi-rect fluorescence microscopy revealed that TIAR-asso-ciated proteins predominantly accumulated in the nucleus in normal conditions However, upon oxida-tive stress, only ASF⁄ SF2 protein migrated in TIAR-positive 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 localiza-tion patterns both in normal and stressed cells, we
A
Minigene
kDa
RHII/Gu/DDX21
*
*
*
**
*
UBF1 hnRNP M p68/DDX5
TIAR-CBP
ASF/SF2
Ribosomal proteins
200
116.3 97.4
66.3 55.4
36.5
21.5 14.4 31
48.7
– TIAR-TAP
Inputs Eluates
WB anti-TIAR
TIAR-TAP TIAR-CBP TIAR TAP
6 6A 7
RT-PCR
WB anti-TIAR
TIAR-TAP
TIAR
C
B
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 TAP expression in 293T cells by western blot analysis using anti-TIAR sera (B) Western blot analysis of TIAR-TAP in crude extracts and TIAR-cAMP response element-binding protein-binding protein in TIAR-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.
Trang 5focused the present study on this TIAR-interacting
can-didate Figure 3A shows that endogenous ASF⁄ SF2
protein co-immunoprecipitates with TIAR-Flag We
then determined whether ASF⁄ SF2 could be associated with TIA-1 protein, the closest homologue of TIAR Co-immunoprecipitation assays revealed that ASF⁄ SF2
A
TIAR-flag
+
+ –
+ + + + +
ASF/SF2
p68
BOIP-flag
TIAR-flag BOIP-flag HA-p68
WB anti-flag
WB anti-HA
HA-ASF/SF2
+
+ –
+ + + + + +
–
+ + –
+ + + + + +
–
WB anti-flag
WB anti-HA
TIAR-flag
BOIP-flag
HA-hnRNP M
RNaseA
WB anti-flag
WB anti-HA
TIAR-flag BOIP-flag HA-DDX21 RNaseA
WB anti-flag
WB anti-HA
B
HA-ASF/SF2
HA-p68
HA-hnRNP M
HA-DDX21
Fig 2 (A) Analysis of the identified interac-tions by co-immunoprecipitation 293T cells were transiently transfected with DNA constructs encoding TIAR or BOIP (control) proteins fused to the Flag epitope in combi-nation 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 interact-ing candidates and were treated with arsenite (1 m M 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).
Trang 6is also associated with TIA-1 (Fig 3B), thereby
indicat-ing 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
oxida-tive stress was a result of the relocalization of the
pro-tein 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
trea-ted with arsenite in combination with puromycin (data
not shown) Furthermore, ASF⁄ SF2 co-localized
exclu-sively 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 plas-mid We observed that the overexpression of ASF⁄ SF2 induced the spontaneous formation of eiF3b-positive cytoplasmic aggregates, indicating that, when over-expressed, 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 cyto-plasm 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 investi-gated 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 transcrip-tion-dependent nuclear import of ASF⁄ SF2, allowing the observation of ASF⁄ SF2 nuclear export [30] As previously observed, a massive relocalization of wild-type 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
TIAR-flag
A
B
+
+
–
+
BOIP-flag
RNaseA
TIA1-flag
BOIP-flag
RNaseA
WB anti-flag
WB anti-flag
HA-ASF/SF2
WB anti-HA
WB anti-ASF/SF2
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
TIAR-Flag constructs only ASF ⁄ SF2 detection was performed by
wes-tern blot analysis using anti-ASF ⁄ SF2 serum (B)
Co-immunoprecipi-tation of ASF ⁄ SF2 with TIA-1 protein The experimental procedure
was identical to that described in Fig 2A.
Trang 7Arsenite (min)
A
C
D
B
Arsenite (min)
0
30 60 120
Untreated
ASF/SF2 TIAR Merged
ASF/SF2 TIAR Merged
ASF/SF2 RFP-Dcp1 Merged
Arsenite
Arsenite
Heat shock
HA-ASF/SF2 elF3b Merged DAPI
HA-ASF/SF2 elF3b Merged DAPI
HA-ASF/SF2 elF3b Merged DAPI Osmotic shock
ASF/SF2
ASF/SF2
P105 Myc Actin
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 m M ) 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 m M for 1 h) before immuno-staining 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 con-struct encoding HA-fused ASF ⁄ SF2 protein and were exposed to heat shock (43 C for 50 min) or osmotic shock with sorbitol (600 m M for
2 h 30 min) HA-ASF ⁄ SF2 sub-cellular localization was revealed by indirect immunofluorescence using mouse anti-HA serum and alexa 594-coupled donkey secondary anti-mouse serum (red) Endogenous eIF3b was detected with goat anti-eiF3b and FITC-594-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 ana-lyzed as described in (C) Arrowheads indicate eIF3b-positive foci detectable in HA-ASF ⁄ SF2 overexpressing cells.
Trang 8serine (S) residues in the RS domain were replaced by
negatively charged aspartic acid (D) residues
Interest-ingly, this phosphomimetic mutant was relocalized to
the cytoplasm to the same extent as the wild-type
pro-tein 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
capac-ity of both RRMs to address ASF⁄ SF2 to SGs
More-over, 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
frac-tion 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
NPc-NLS with ASF⁄ SF2 induced a detectable
redistribu-tion 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
dis-tribution 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
cyto-plasmic accumulation of ASF⁄ SF2 strongly depends
on the RNA-binding ability of both RRMs and
sug-gest that cytoplasmic accumulation rather than
activa-tion of an alternative export pathway is a pre-requisite
for ASF⁄ SF2 migration into SGs upon stress
Because both RRMs contributed to ASF⁄ SF2
cyto-plasmic redistribution, we investigated their intrinsic
capacity to do so by fusing them independently to
NPc-NLS reporter protein Wild-type but not
FF-DD-mutated 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 More-over, 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 con-trol of a bidirectional cytomegalovirus promoter medi-ating the transcription of another reporter gene encoding firefly luciferase (Fluc) (Fig 6A) This strat-egy ensured that the ratio between the control (Fluc) and the reporter (Rluc) genes was strictly conserved in all experiments These reporter constructs were trans-fected 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 calcu-lated 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 Like-wise, ASF⁄ SF2 significantly down-regulated Rluc AU8 mRNA, although to a lesser extent than TTP Inter-estingly, 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
Trang 911 85 106 183 197 220 248
ASF/SF2 ΔRS1
ASF/SF2
RRM1
PDADV
GSAQDL
GSAQDL
RDPSYG(RD)8NDRDRDYSPRRDRGSPRYSPRHDRDRDRT
PDADV
RD
Untreated
ActD/CHX
ASF/SF2 ΔRS2
ASF/SF2 FFDD
ASF/SF2 W134A
ASF/SF2 FFDD-W134A
ASF/SF2 RD
HA
HA-ASF ΔRS1
HA-ASF ΔRS2
HA-ASF FF-DD
HA-ASF W134A
HA-ASF FF-DD W134A
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
HA-ASF ΔRS1
HA-ASF ΔRS2
HA-ASF FF-DD
HA-ASF W134A
HA-ASF FF-DD W134A
TIAR
Untreated
Arsenite
Fig 5.
Trang 10that 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-con-taining mRNAs and that this activity relies on its RS domain rather than on its RRMs
Untreated
Flag
NPc-ASF W134A-NLS NPc-ASF W134A-NLS
NPc-RRM1 FFDD-NLS NPc-RRM1 FFDD-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 lgÆmL)1) and actinomycin D (5 lgÆmL)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.