Proc. Natl. Acad. Sci. USAVol. 92, pp. 1629-1633, February 1995Cell Biology

The RNA-binding protein TIAR is translocated from the nucleusto the cytoplasm during Fas-mediated apoptotic cell deathJEAN-LUC TAuPIN*, QINGSHENG TIAN*, NANCY KEDERSHAt, MICHAEL ROBERTSON*, AND PAUL ANDERSON*t*Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA 02115; tDepartment of Cell Biology, ImmunoGen, Inc., Cambridge, MA 02139; andtDepartment of Rheumatology, Brigham and Women's Hospital, Boston, MA 02115

Communicated by Stuart F. Schlossman, Dana-Farber Cancer Institute, Boston, MA, October 28, 1994

ABSTRACT We have determined the structure, intracel-lular localization, and tissue distribution of TIAR, a TIA-1-related RNA-binding protein. Two related isoforms of TIAR,migrating at 42 and 50 kDa, are expressed in primate cells.Unlike TIA-1, which is found in the granules of cytotoxiclymphocytes, TIAR is concentrated in the nucleus of hema-topoietic and nonhematopoietic cells. Because TIAR can trig-ger DNA fragmentation in permeabilized thymocytes, it is acandidate effector of apoptotic cell death. Consistent with thispossibility, we have found that the expression and intracel-lular localization of TIAR change dramatically during Fas-mediated apoptosis. TIAR moves from the nucleus to thecytoplasm within 30 min of Fas ligation. Redistribution ofTIAR precedes the onset of DNA fragmentation and is not anonspecific consequence of nuclear disintegration. Cytoplas-mic redistribution of TIAR is not observed during cellularactivation triggered by mitogens such as concanavalin A orphytohemagglutinin. Our results suggest that cytoplasmicredistribution of TIAR may be a general feature of the apop-totic program.

TIA-1 and TIAR are structurally and functionally relatedRNA-recognition motif (RRM)-type RNA-binding proteinsthat are candidate effectors of apoptotic cell death (1). TIA-1is a 15-kDa cytotoxic granule-associated protein (p15-TIA-1)whose expression is restricted to cytotoxic T lymphocytes(CTLs) and natural killer (NK) cells (2). Molecular cloning ofa cDNA encoding TIA-1 revealed that it is a member of theRRM family of RNA-binding proteins (3). RRM family mem-bers are modular proteins containing one or more 80- to 90-aaRRMs joined to one or more putative protein-interactiondomains (PIDs) (4-7). The full-length, 40-kDa TIA-1 protein(p40-TIA-1) is composed of three N-terminal RRMs and aC-terminal PID (3). The major granule-associated TIA-1 iso-form is a 15-kDa protein that appears to be derived from theC terminus of p40-TIA-1 by proteolysis (3, 8). Thus, thegranules of cytotoxic lymphocytes contain the isolated PID ofan RRM-type RNA-binding protein. The ability of TIA-1 totrigger DNA fragmentation in permeabilized thymocytes hasimplicated this granule-associated protein as an effector ofCTL-mediated apoptosis (3).The molecular mechanism by which the PID of an RNA-

binding protein might trigger apoptotic cell death is unknown.We have proposed that p15-TIA-1 might affect the function ofTIAR, a TIA-1-related RNA-binding protein that was iden-tified by low-stringency hybridization (3, 8, 9). Like p40-TIA-1,TIAR possesses three N-terminal RRMs and a C-terminalPID. The RRMs ofTIA-1 and TIAR are "90% identical at theamino acid level, and the PIDs are 50% identical. Like TIA-1,TIAR triggers DNA fragmentation in permeabilized thymo-cytes, suggesting its possible involvement in apoptosis (9). Wehave used a panel of monoclonal antibodies (mAbs) reactive

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with TIAR to determine its structure and cellular distributionin normal cells and in cells undergoing apoptosis. Our resultsshow that TIAR is a ubiquitously expressed nuclear proteinthat rapidly moves to the cytoplasm in response to exogenoustriggers of apoptosis.

MATERIALS AND METHODSProduction of mAbs Reactive with TIAR. mAbs reactive

with TIAR were prepared by immunizing BALB/c mice withEscherichia coli-derived recombinant TIAR, purified by affin-ity chromatography on a poly(U)-Sepharose column (Phar-macia) (9).

Cells. Cell lines were grown in RPMI 1640 containing 10%fetal bovine serum, 2 mM glutamine, and gentamicin at SO,ug/ml. Peripheral blood lymphocytes (PBLs) were isolatedfrom leukopheresis residues by Ficoll/Hypaque density cen-trifugation, followed by a 2-hr plastic adherence step to removemacrophages. Various human carcinoma cell lines (MCF7,SCaBER, BT-549, HeLa) and normal human diploid fibro-blasts (27SK) were obtained from the American Type CultureCollection.

Affinity Precipitations. The indicated cell types were lysedin Nonidet P-40 (NP-40) lysis buffer [1% (vol/vol) NP-40/150mM NaCl/1 mM EDTA/1 mM phenylmethylsulfonyl fluo-ride/50 mM Tris HCl, pH 8.0] for immunoprecipitation andimmunoblot analysis as described (3), except immunoreactivebands on the blots were revealed by chemiluminescence (ECLdetection kit; Amersham).

Analysis ofTIAR Expression in Anti-Fas-Treated Jurkat 77Cells. Exponentially growing Jurkat 77 cells were cultured witha 1:400 dilution of an anti-Fas mAb ascities (7C11, mouse IgM)or an isotype-matched control ascities (5H10, anti-PEN5; ref.10). Cells were harvested at the indicated times, and cytoplas-mic and nuclear extracts were prepared essentially as described(11). Briefly, cell pellets were lysed in 200 j,l of a hypotoniclysis buffer (0.25% NP-40/10mM MgCl2/20mM Tris HCI, pH7.5) for 15 min on ice. Whereas the 1% NP-40 lysis bufferdescribed above efficiently extracted TIAR from whole cells,this hypotonic lysis buffer allowed the isolation of intact nucleiwhich retained their TIAR. Nuclei were pelleted by micro-centrifugation for 5 min at 16,000 x g. The supematant waskept as the cytoplasmic extract. The nuclear pellet was washedonce with the lysis buffer and then extracted with 200 ,ul of ahypertonic buffer (0.4M NaCl/1 mM EDTA/20mM Tris-HCI,pH 7.5) for 1 hr at 4°C. Nuclear extract was obtained afterseparating the nonextractable chromatin residue by centrifu-gation at 16,000 x g for 10 min. TIAR content was determinedby immunoprecipitation from nuclear and cytoplasmic extractswith the 6E3 mAb as described above. The expression ofnucleolin and La protein was assessed by direct immunoblot-ting of aliquots corresponding to 106 cell equivalents with

Abbreviations: CIL, cytotoxic T lymphocyte; mAb, monoclonal an-tibody; NK, natural killer; NP-40, Nonidet P-40; PBL, peripheral bloodlymphocyte; PID, protein-interaction domain; RRM, RNA-recognition motif.

1629

Proc. Natl. Acad Sci. USA 92 (1995)

1 R mlg 2G9 IHIO 6E3 3E6 9E4 mig 2G9 IHIO 6E3 3E6 9E4.1 .: . .i1 Va

FIG. 1. Specificity of mAbs reactive with TIA-1and TIAR. Purified recombinant TIA-1 and TIAR (1jLg each; lanes 1 and R) were separated in an SDS/10%polyacrylamide gel, transferred to nitrocellulose, anddetected with mAb 2G9 (Left) or 6E3 (Right). In

-14 addition, PBL lysates were immunoprecipitated withthe indicated mAbs or mouse immunoglobulin (mIg)

-i prior to immunoblotting analysis. Relative migrationof molecular size markers is shown at right.

either a mAb reactive with nucleolin (L3C3Glb, mouse IgM,kindly provided by Mark Pasternack, Harvard Medical School)or a polyclonal antiserum reactive with La obtained from apatient with systemic lupus erythematosus (kindly provided byJack Keene, Duke University). Blots were developed with theECL detection kit (Amersham). DNA fragmentation was as-sessed as described (3). Total protein content was determinedwith the BCA protein assay (Pierce).

Immunofluorescence. Cells were fixed for 15 min at roomtemperature in 2% paraformaldehyde in phosphate-bufferedsaline and then permeabilized by a 15-min incubation inPHEM buffer (60 mM Pipes/25 mM Hepes/5 mM EGTA/2mM MgCl2, pH 6.9; ref. 12) containing 0.5% Triton X-100.Cells were then incubated in PHEM containing 2.5% normalgoat serum (NGS/PHEM) for 30 min to block nonspecificbinding sites and for 1 hr with purified anti-TIAR mAb (5,ug/ml) or isotype-matched control IgG2a diluted into NGS/PHEM. Samples were washed three times (5 min each) inPHEM and incubated for 1 hr in NGS/PHEM containing1:100 biotinylated goat anti-mouse IgG (Southern Biotechnol-ogy Associates). After three additional washes in PHEM,bound antibody was visualized with streptavidin-fluoresceinisothiocyanate conjugate (1:100; Fisher Scientific) and nuclearDNA was stained with Hoechst dye 33258 (bisbenzimide, 0.5ptg/ml; Sigma) for a final hour. Cells were washed thoroughlyin PHEM, mounted in a polyvinyl alcohol-based mountingmedium (13), and viewed through a Nikon FXA microscope.Photographs were taken on Kodak Tri-film.

RESULTSProduction and Characterization of mAbs Reactive with

TIAR. mAbs 6E3, 3E6, and 9E4 preferentially recognizedrecombinant TIAR as compared with recombinant TIA-1 in asolid-phase ELISA (data not shown). In contrast, the previ-ously described (3) mAbs raised against natural (2G9) andrecombinant (1H1O) TIA-1 preferentially recognized recom-binant TIA-1 as compared with recombinant TIAR in thisELISA (data not shown). Immunochemical analysis of targetantigens expressed in PBLs confirmed this specificity (Fig. 1).Proteins immunoprecipitated from PBL lysates with the indi-cated mAb were separated in an SDS/10% polyacrylamide gel,transferred to nitrocellulose, and detected with mAb 2G9(specific for TIA-1) or mAb 6E3 (specific for TIAR). Inaddition, equal amounts (1 ,ug) of purified recombinant TIA-1and TIAR (both of which contain the full-length 42-kDaprotein and a 38-kDa degradation product) were analyzed oneach blot to assess the specificity of the antibodies. Consistentwith the ELISA results described above, 2G9 preferentially

Human Mouse Monkey

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FIG. 2. Nucleic acid binding specificity of endogenous TIAR. CEMcell lysates were precipitated with the indicated Sepharose-immobilized homopolymer or with the indicated mAb and analyzed bySDS/10% polyacrylamide gel electrophoresis followed by immunob-lotting with 6E3.

FIG. 3. Expression of TIAR in cell lines. Detergent lysates pre-pared from the indicated human, mouse, and monkey cell lines wereprecipitated with 6E3 and analyzed by 6E3 immunoblotting as in Fig.2. E. coli-derived TIAR was included in the first lane as a size marker.In addition to the full-length p42-TIAR, E. coli-derived TIAR con-tains a 38-kDa proteolytic fragment.

1 R kDa

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1630 Cell Biology: Taupin et al.

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Proc. Natl. Acad Sci USA 92 (1995)

FIG. 4. Immunofluorescence analysis of TIAR. (A and C) MCF7 cells stained with 3E6 (A) or an isotype-matched control mAb (C). (B andD) Corresponding images show the cell nuclei stained with Hoechst dye.

recognized recombinant TIA-1, whereas 6E3 preferentiallyrecognized recombinant TIAR (Fig. 1). As expected, immu-noprecipitates prepared with antibodies raised against TIA-1(2G9 and 1H10) included 15-kDa and 28-kDa TIA-1 isoformsthat were recognized by 2G9 on the blot (Fig. 1 Left; the28-kDa isoform is faint on this blot). Although the mAbsreactive with TIAR (6E3, 3E6, and 9E4) did not precipitatethese TIA-1 isoforms, they each precipitated two proteinsmigrating at 42 kDa and 50 kDa which were recognized by 6E3(Fig. 1 Right). Because each of these mAbs recognizes adistinct epitope on recombinant TIAR (6E3 binds the secondRNA-binding domain, whereas 3E6 binds the PID; unpub-lished results), this result suggests that p42 and p50 are im-munorelated isoforms of the endogenous TIAR protein. Be-cause recombinant TIAR binds to single-stranded nucleicacids (9), detergent lysates prepared from the T-cell line CEMwere precipitated with poly(C), poly(A), and poly(U) ho-mopolymers prior to immunoblotting analysis using 6E3. Bothof the endogenous TIAR isoforms (p42 and p50) bound thehomopolymers with relative affinities poly(U) > poly(A) >>poly(C) (Fig. 2). CEM cells, like PBLs, expressed 42- and50-kDa isoforms of TIAR that were recognized by mAbsreactive with TIAR (6E3 and 3E6) but not by mAbs reactivewith TIA-1 (2G9 and 1H10) (Fig. 2). Although mAb lH1Orecognized recombinant TIAR in the ELISA, it did not effi-ciently precipitate endogenous TIAR. In some experiments,however, immunoprecipitates prepared with lH10 containedsmall amounts of the 42- and 50-kDa TIAR isoforms (data notshown).

In contrast to TIA-1, whose expression (as measured byreactivity with 2G9) is restricted to cytotoxic lymphocytes (2),TIAR appears to be ubiquitously expressed. Both the 42- and50-kDa TIAR isoforms were expressed in hematopoietic (YT,CEM, Daudi, CTLL, P815, NS-1) and nonhematopoietic(HeLa, 3T3, COS) cell lines (Fig. 3). TIAR was also found inNK cells, T cells, and thymocytes (data not shown). mAb 6E3recognized a 42-kDa protein in murine and simian cell lines,suggesting phylogenic conservation of p42-TIAR. The 50-kDaTIAR isoform was largely absent from the murine cell linesexamined, but it was present in the simian cell line COS-1 (Fig.3). Immunofluorescence analysis showed that, unlike TIA-1,TIAR was concentrated in the nucleus of cells. Fig. 4 shows thedistribution of TIAR in MCF7 cells (a breast carcinoma cellline), which are representative of results obtained with severalcarcinoma cell lines (SCaBER, BT-549, and HeLa), as well asnormal diploid fibroblasts (27SK). In each of these cell types,TIAR was predominantly found in the nucleus, where it was

excluded from the nucleoli. This result indicates that thelysosome-targeting motif at the C terminus ofTIAR (9) is notthe primary determinant of its subcellular localization. How-ever, the small amount of cytoplasmic staining observed inthese cells appeared punctate, suggesting a possible vesicularlocalization.

Altered Localization of TLAR in Cells Undergoing Apopto-sis. The ability of TIA-1 (3) and TIAR (9) to trigger DNAfragmentation in permeabilized thymocytes suggests a possiblerole for these molecules in apoptotic cell death. Because ofthis, we monitored the expression and intracellular distribu-tion of TIAR in Jurkat cells triggered to undergo apoptoticdeath by treatment with a mAb reactive with Fas (7C11, IgMisotype). TIAR was largely (80-90%) confined to the nucleusof Jurkat cells cultured for 300 min in the presence of anonbinding control antibody (Fig. SA, lane 0). In contrast,TIAR moved from the nucleus to the cytoplasm of Jurkat cellscultured in the presence of anti-Fas. Cytoplasmic redistribu-tion began within 30 min of Fas ligation, and by 75 min 80%of TIAR was cytoplasmic. At later times, the expression ofboth nuclear and cytoplasmic TIAR progressively decreased,and after 300 min TIAR was undetectable by immunoblotting.Redistribution of TIAR during Fas-mediated apoptosis pre-ceded DNA fragmentation, which was detected at 60 min (Fig.SB). Redistribution ofTIAR during Fas-mediated apoptosis isnot a general consequence of nuclear disintigration, since thequalitative distribution of nuclear (Fig. SC) and cytoplasmic(Fig. SD) proteins stained with Coomassie blue did not sig-nificantly change over the course of these experiments, andsince the total protein contained in nuclear and cytoplasmicextracts also did not significantly change (data not shown). Anexception to this was the appearance of several low molecularweight (histone-sized) proteins in nuclear extracts after 45 minof anti-Fas treatment. Unlike TIAR, these proteins did notconcomitantly appear in cytoplasmic extracts, indicating thattheir appearance reflected increased extractability rather thanredistribution (data not shown). Further evidence for thespecificity of TIAR redistribution was provided by analyzingthe same nuclear extracts analyzed in Fig. SA for their expres-sion of nucleolin, a 100-kDa nuclear RRM-type RNA-bindingprotein. Nucleolin remained in the nucleus during Fas-mediated apoptosis (Fig. SE; the arrow points to full-lengthnucleolin, which was specifically recognized by this mAb; theidentity of a smaller protein also recognized by this mAb(arrowhead) has not been determined; M. Pasternack, per-sonal communication). In a separate experiment, Fas-mediated apoptosis was found to induce the cytoplasmic trans-

Cell Biology: Taupin et al. 1631

Proc. NatL Acad Sci USA 92 (1995)

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FIG. 5. Intracellular localization of TIAR in anti-Fas-treated c-La N.4,, .Jurkat cells. (A) (Upper) Immunoprecipitation of TIAR from

cytoplasmic and nuclear extracts of Jurkat cells treated with anti-Fas for the indicated times. (Lower) Relative expression of nuclear

(0) and cytoplasmic (0TIAR protein at each time. (B) Time

course of DNA fragmentation triggered by anti-Fas. Relative

migration of marker DNAs is indicated at right. (C) Total protein

extractedfromthenucleusofJurkatcellstreatedwithanti-Fasfor 0 15 30 45 60 75 90 105 120 300the indicated times. (D) Total protein extracted fromthe cytoplasmof: Jurkat cells treated with anti-Fas as described above. (E) Im- Time (mm)

lIlunoblot ainalysis of nucleolin in nuclear extracts of Jurkat cellstrcated with anti-Fas for the indicated times. (F) Subcellular lo-

callization of lIAR and La following Fas ligation. Nuclear (n-TIAR and n-La) and cytoplasmic (c-TIAR and c-La) extracts of Jurkat cells treated

with anti-Fits for the indicated times were examined as described in the text. The 52-kDa La protein is indicated by the arrows.

kDa

-97

-69

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1632 Cell Biology: Taupin et aL

Proc. Natl. Acad Sci. USA 92 (1995) 1633

1 2 3 4 5 6 7 8 9 10 kD

||l ^|l C S ~~~~~~-46FIG. 6. Expression of TIAR in nuclear extracts prepared from

Jurkat cells cultured in medium alone (lane 1) or activated withanti-CD3 (lanes 2, 5, and 8), concanavalin A (lanes 3, 6, and 9), or

phytohemagglutinin (lanes 4, 7, and 10) for 1.5 hr (lanes 2-4), 6 hr(lanes 5-7), or 24 hr (lanes 8-10).

location of TIAR, but not La, another nuclear RRM-typeRNA-binding protein (Fig. 5F). Although TIAR may not bethe only nuclear protein translocated to the cytoplasm duringapoptosis, these results suggest that the nuclear-to-cytoplasmictranslocation of TIAR is relatively specific.

Stimuli which trigger cellular activation, but not apoptosis,did not induce the redistribution of TIAR in Jurkat cells (Fig.6). Compared with untreated cells (lane 1), cells activated withanti-CD3 (lanes 2, 5, and 8), concanavalin A (lanes 3, 6, and9), or phytohemagglutinin (lanes 4, 7, and 10) for 1.5 hr (lanes2-4), 6 hr (lanes 5-7), or 24 hr (lanes 8-10) had similaramounts of nuclear TIAR. This result suggests that redistri-bution of TIAR from the nucleus to the cytoplasm is a featureof apoptosis, but not of cellular activation.

DISCUSSIONTIAR is an RRM-type RNA-binding protein that was iden-tified on the basis of its structural similarity to TIA-1, a

cytotoxic granule-associated effector of CTL-mediated apop-tosis (9). Whereas the expression of TIA-1 is restricted tocytotoxic lymphocytes (2), our results indicate that TIAR is a

ubiquitously expressed nuclear RNA-binding protein. UnlikeTIA-1, which appears to be processed to produce the 15-kDagranule-associated isoform (3), TIAR is expressed as a full-length 42-kDa RNA-binding protein. A 50-kDa immunoreac-tive TIAR isoform is also expressed in primate cells. Althoughthe structure of p50-TIAR is not known, it might arise via (i)covalent modification of p42-TIAR (e.g., phosphorylation,ubiquitination, glycosylation), (ii) alternative splicing of theTIAR gene, or (iii) translation from a closely related butdistinct mRNA species.The intracellular localization of certain RRM proteins can

be altered in response to selected stimuli. For example, theautoantigen La is normally confined to the nucleus, where itfacilitates termination of RNA polymerase III transcripts (14,15). Following poliovirus infection, however, La is recruited tothe cytoplasm, where it facilitates the translation of viralmRNAs (16). Similarly, TIAR is normally confined to thenucleus of cells, but during Fas-mediated apoptosis, it is rapidlytranslocated to the cytoplasm. Cytoplasmic redistribution pre-*cedes the onset of DNA fragmentation and the nuclear archi-tectural changes that facilitate histone extraction. Followingthe onset of DNA fragmentation, TIAR progressively disap-pears from cytoplasmic extracts, indicating that it is eitherdegraded or altered so that it is no longer extractable under theconditions employed. Cytoplasmic translocation of TIAR isrelatively specific, since the expression of most nuclear pro-

teins does not change during apoptosis, and neither nucleolinnor La moves to the cytoplasm during apoptosis. Since thecytoplasmic accumulation of TIAR precedes measurablechanges in the nucleus (DNA fragmentation and histone ex-tractability), it is even possible that cytoplasmic TIAR triggerslater events in the apoptotic pathway.Although our results do not prove that TIAR is required for

apoptotic cell death, the ability of TIAR to trigger DNAfragmentation in permeabilized thymocytes suggests that thisis possible (9). How might the recruitment of TIAR from thenucleus to the cytoplasm trigger apoptosis? One possibility isthat cytoplasmic redistribution brings it into contact with RNAor protein substrates not found in the nucleus. Such a cyto-plasmic TIAR substrate might also be the target of p15-TIA-1,the cytotoxic-granule protein that has been implicated as aneffector of apoptosis during CTL killing (3, 9). Indeed, it isstriking that TIAR, which was identified by virtue of its struc-tural similarity to TIA-1, is a candidate effector moleculewhose subcellular distribution is affected by exogenous trig-gers of apoptotic cell death.

We thank Michel Streuli, Stuart Schlossman, Laura Dember, P. J.Utz, and Gene Lee for helpful advice and review of the manuscript.We thank Mark Pasternack and Jack Keene for providing antibodies.This work was supported by grants from the National Institutes ofHealth (AI3360) and from Apoptosis Technology, Inc. P.A. wassupported by a Pew Scholar Award and a Carl J. Herzog FoundationInvestigator Award from the Cancer Research Institute. J.-L.T. wassupported in part by a fellowship from the Association Frangaise pourla Recherche Contre le Cancer. S. F. Schlossman is a member of thescientific advisory board of Apoptosis Technology.

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