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,  ´ ` ´ ´ Laboratoire de Biologie Moleculaire du Gene, Faculte des Sciences, Universite Libre de Bruxelles, Gosselies, Belgium ´ 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 6509800 Tel: +32 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 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 2496 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 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, FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS 2497 ASF ⁄ SF2 in TIAR-mediated regulatory pathways 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 2498 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 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 A – Minigene – TIARTAP TAP + + B 6A RT-PCR 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 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 FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS 2499 ASF ⁄ SF2 in TIAR-mediated regulatory pathways A N Delestienne et al 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 (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 (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 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 FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS 2501 ASF ⁄ SF2 in TIAR-mediated regulatory pathways N Delestienne et al A B Arsenite (min) 30 Untreated 60 120 ASF/SF2 ASF/SF2 TIAR Merged TIAR Merged RFP-Dcp1 Merged Actin Arsenite 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 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 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 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 lg) encoding HA-ASF ⁄ SF2 Cells were analyzed as described in (C) Arrowheads indicate eIF3b-positive foci detectable in HA-ASF ⁄ SF2 overexpressing cells 2502 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 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 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 FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS 2503 ASF ⁄ SF2 in TIAR-mediated regulatory pathways N Delestienne et al Untreated HA RRM1 RS1 RRM2 ActD/CHX HA 24 22 19 GSWQDL RG ASF/SF2 18 85 10 PFAFV 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 2504 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 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 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 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 FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS 2505 ASF ⁄ SF2 in TIAR-mediated regulatory pathways N Delestienne et al A Globin 3′UTR Renilla luciferase Firefly luciferase Bidirectional CMV (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 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 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 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 (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 2506 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 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 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 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 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 CP FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS TTP-CP ASF/SF2-CP 2507 ASF ⁄ SF2 in TIAR-mediated regulatory pathways N Delestienne et al 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 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 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 FEBS Journal 277 (2010) 2496–2514 ª 2010 The Authors Journal compilation ª 2010 FEBS 2509 ASF ⁄ SF2 in TIAR-mediated regulatory pathways N Delestienne et al 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 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 at 8000 g The supernatant (200 mg 2510 of protein extract) was incubated overnight with 150 lL rabbit IgG Sepharose 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 °C, beads were washed twice with mL of IgG BB and once with 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 mm dithithreitol) Three hundred units of Tev protease (Invitrogen) were added and cleavage of the TAP tag was performed in mL of Tev CB for h at room temperature Proteins released from the beads were collected in two fractions of mL, and the eluate was adjusted to 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, mm Mg acetate, mm imidazole, mm CaCl2, 0.1% NP40) and loaded onto calmodulin Sepharose 4B affinity resin (Amersham) equilibrated in the same buffer After h of binding at °C, beads were washed once with mL of CBB and once with modified CBB (0.02% NP40) Elution of the bound proteins was performed by addition of · 600 lL of calmodulin elution buffer (10 mm Tris, pH 8.0, 0.15 m NaCl, 0.02% NP40, 10 mm 2-mercaptoethanol, mm Mg acetate, mm imidazole, 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 (http://www.genedb.org/genedb/ mouse/) using proteinlynx Global server 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 · 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, 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 protease inhibitors) for 30 at °C Flagged-proteins were immunoprecipitated by incubating protein extract (2 mg) with BSA (1%) blocked-anti-Flag M2 (20 lL) affinity gel (Sigma) for h at °C The beads were washed three times in IP buffer and were then incubated for 30 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 : 5000) and M2 monoclonal anti-Flag serum (dilution : 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 mm or 500 lm arsenite and cells were incubated for 30 or 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 The fixed cells were washed three times for with NaCl ⁄ Pi, permeabilized for with NaCl ⁄ Pi, 0.5% Triton-X 100 at °C and washed again as described above Blocking was performed with NaCl ⁄ Pi containing 10% BSA for 30 After blocking, the coverslips were incubated for h with antibodies diluted in NaCl ⁄ Pi, 0.1% Tween 20 Antibodies were used at a dilution of : 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 : 5000 for mouse anti-FLAG M2 serum; and at a dilution of : 30 000 for anti-HA serum (Sigma) The coverslips were subsequently washed three times for 10 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 : 25 000 In double staining experiments, a donkey anti-goat serum conjugated with fluorescein isothiocyanate (FITC) was used (dilution : 1000) After 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) References Halbeisen RE & Gerber AP (2009) Stress-dependent coordination of transcriptome and translatome in yeast PLoS Biol 7, e105 Bakheet T, Williams BR & Khabar KS (2003) ARED 2.0: an update of AU-rich element mRNA database Nucleic Acids Res 31, 421–423 Biamonti G & Riva S (1994) New insights into the auxiliary domains of eukaryotic RNA binding proteins FEBS Lett 340, 1–8 ´ Clery A, Blatter M & Allain FH (2008) RNA recognition motifs: boring? 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