the rna-binding protein tia-1 is a novel mammalian splicing

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/00/$04.0010

Sept. 2000, p. 6287–6299 Vol. 20, No. 17

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

The RNA-Binding Protein TIA-1 Is a Novel MammalianSplicing Regulator Acting through Intron Sequences

Adjacent to a 59 Splice SiteFABIENNE DEL GATTO-KONCZAK,1 CYRIL F. BOURGEOIS,2 CAROLINE LE GUINER,1

LILIANE KISTER,2 MARIE-CLAUDE GESNEL,1 JAMES STEVENIN,2*AND RICHARD BREATHNACH1*

INSERM U463, Institut de Biologie-CHR, 44093 Nantes Cedex 1,1 and Institut de Genetique et de BiologieMoleculaire et Cellulaire, CNRS/INSERM/ULP, 67404 Illkirch Cedex,2 France

Received 8 May 2000/Returned for modification 2 June 2000/Accepted 13 June 2000

Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependenton the U-rich sequence IAS1 lying immediately downstream from its 5* splice site. We show that IAS1 canactivate the use of several heterologous 5* splice sites in vitro. Addition of the RNA-binding protein TIA-1 tosplicing extracts preferentially enhances the use of 5* splice sites linked to IAS1. TIA-1 can provoke a switchto use of such sites on pre-mRNAs with competing 5* splice sites, only one of which is adjacent to IAS1. Usinga combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1in cell extracts. This binding is stronger if IAS1 is adjacent to a 5* splice site and is U1 snRNP dependent.Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated byTIA-1. Splicing can, however, be activated by a TIA-1–MS2 coat protein fusion, provided that the operator isclose to the 5* splice site. Our results identify TIA-1 as a novel splicing regulator, which acts by binding tointron sequences immediately downstream from a 5* splice site in a U1 snRNP-dependent fashion. TIA-1 isdistantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the twoproteins are discussed.

Many eucaryotic genes are made up of exons and introns(43). They are transcribed into pre-mRNAs, from which theintron sequences are removed by splicing. Exons to be includedin mRNA must be identified as such. This involves interactionof short sequences at or close to the exon’s 59 and 39 splice sites(59ss and 39ss, respectively) with spliceosome components suchas snRNPs and associated proteins (for reviews, see references4, 29, and 43). Exon splicing can be controlled, and severalsequences which participate in the control of tissue-specific ordevelopmentally controlled alternative splicing events havebeen described (for a review, see reference 32). These se-quences are particularly interesting to study, as they may yieldinformation on both splicing activation mechanisms and tissue-specific control mechanisms of gene expression. We have beenstudying fibroblast growth factor receptor 2 (FGFR-2) pre-mRNA splicing for this reason.

FGFR-2 alternative exons K-SAM and BEK are spliced in atissue-specific, mutually exclusive manner, and the two types ofFGFR-2 obtained bind different subsets of FGF family mem-bers (38). The K-SAM exon is under complex control. It hasweak splice sites, and it contains an exon splicing silencer(ESS) which functions by recruiting hnRNP A1 (13). To over-come the activity of this silencer, at least three activating se-quences in the downstream intron are required (6, 10, 12). One

of these, IAS1, lies immediately downstream of the 59ss and isa U-rich sequence (10). In the absence of IAS1 (10), or if IAS1is moved further downstream from the 59ss (F. Del Gatto-Konczak, unpublished data), the K-SAM exon is very poorlyspliced, unless the ESS is inactivated also. IAS1 and the ESSare thus major determinants of K-SAM exon splicing. How-ever, neither element may be directly responsible for the tis-sue-specific splicing of the K-SAM exon. Both elements cancontrol splicing of heterologous exons in cells, and we have notdetected any difference in their activities between cells whichsplice the K-SAM exon and cells which do not (reference 11and our unpublished data).

The necessary proximity of IAS1 to the 59ss suggests a modelfor activation in which a protein bound to IAS1 interacts withU1 snRNP bound itself to the 59ss. Searching for the activatorbased on its ability to bind IAS1 has not proved easy, as manynuclear proteins bind U-rich sequences, including U2AF (52),polypyrimidine tract-binding protein (PTB) (18), or hnRNP C(44). Recent work on splicing in Saccharomyces cerevisiae hassuggested a different approach, however. Yeast U1 snRNP isconsiderably more complex than mammalian U1 snRNP (21),and several yeast U1 snRNP proteins have no known metazoancounterpart. One such protein is Nam8p. Nam8p activity isnecessary for efficient 59ss recognition when U1 snRNP bind-ing to the 59ss is poor (39). In commitment complexes, Nam8pcontacts nonconserved nucleotides in yeast pre-mRNA down-stream of the 59ss. Its activity is optimal when these sequencesare U rich (39, 53). This led to the suggestion that a mamma-lian counterpart of Nam8p could be involved in activation ofweak 59ss followed by U-rich sequences, such as the 59ss of theK-SAM exon (39).

The known mammalian proteins most closely related toNam8p are a pair of very similar proteins called TIA-1 (47) and

* Corresponding author. Mailing address for Richard Breathnach:INSERM U463, Institut de Biologie-CHR, 9 Quai Moncousu, 44093Nantes Cedex 1, France. Phone: 33 (0)2 40 08 47 50. Fax: 33 (0)2 40 3566 97. E-mail: [email protected]. Mailing address for JamesStevenin: Institut de Genetique et de Biologie Moleculaire et Cellu-laire, CNRS/INSERM/ULP, 67404 Illkirch Cedex, France. Phone:33(0)3 88 65 33 61. Fax: 33(0)3 88 65 32 01. E-mail: [email protected].

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the related protein TIAR (27). TIA-1 was originally believedto be a precursor to a cytotoxic T-lymphocyte granule protein.However, it is now known that this is not the case (34). TIA-1and TIAR are widely expressed RNA-binding proteins (3, 33).Both TIA-1 and TIAR are involved in stress-induced transla-tional arrest, colocalizing after stress with poly(A)1 RNA inthe cytoplasmic foci known as stress granules (28), and it hasbeen reported previously (22) that TIAR binds to the transla-tional regulatory AU-rich element of tumor necrosis factoralpha mRNA in macrophages and may be involved in transla-tional repression. However, under normal conditions, the pro-teins are in general located mainly in the nucleus (28; C. LeGuiner, unpublished data). Predominantly nuclear localizationhas also been described previously for UBP1, a recently char-acterized TIA-1–TIAR relative in plants, which enhances splic-ing of suboptimal introns and also protects mRNAs from exo-nucleolytic degradation (30).

Like Nam8p, TIA-1 and TIAR are composed of an N-ter-minal domain containing three RNA recognition motifs, linkedto a C-terminal domain (27, 47). The similarity betweenNam8p and TIA-1–TIAR (approximately 26% sequence iden-tity) is limited to their RNA recognition motif-containing do-mains. Both TIA-1 and TIAR bind to RNA, with the preferredbinding sequence being U rich (14). These observations en-couraged us to test if TIA-1 is able to activate splicing of exonslinked to a U-rich sequence like IAS1. In this article, we showthat TIA-1 activates 59ss use and that activation depends onthe intron sequence downstream from the 59ss, with IAS1being a preferred sequence. We show that TIA-1 can bind toIAS1 in cell extracts; this binding is optimal if a 59ss is adjacentto IAS1, and the binding is U1 snRNP dependent. We discussthe functional similarities between TIA-1 and Nam8p.

MATERIALS AND METHODS

Plasmids. (i) Splicing constructs. b-Tropomyosin constructs for in vitro syn-thesis of pre-mRNA substrates were derived from the previously describedTropo 6A-7 clone (1) using standard techniques. Constructs with two competing59ss were derived from a truncated adenovirus E1A gene (Sp1), in which the 13S59ss (D1 site) is replaced with a polylinker allowing insertion of a region con-taining two 59ss (5).

(ii) Expression vectors. A mouse TIA-1 cDNA clone was obtained as anI.M.A.G.E. consortium clone (identification no. 1261161) containing the TIA-1coding sequence lacking alternative exon 5. It was used to make pTIA-1, anexpression vector for an N-terminal FLAG-tagged TIA-1, and pTIA-coat, anexpression vector for an N-terminal FLAG-tagged TIA-1–coat fusion. The coatexpression vector pcoat (or pCI-MS2) and the hnRNP A1-coat fusion vectorhave been described elsewhere (13). The entire coding sequence of hnRNP C1was introduced into the StuI site of pCI-MS2-NLS-FLAG (13) to make anexpression vector for an N-terminal FLAG-tagged hnRNP C1-coat fusion (inwhich coat sequences are C terminal). The hnRNP C1 expression vectorphnRNP C1 was obtained from it by eliminating coat sequences by BamHIdigestion and religation.

(iii) Minigenes. Rat preprotachykinin minigene pBPSVpA12-7 (37) was a giftfrom P. J. Grabowski. The CD44 minigene was obtained by cloning a 6.9-kbClaI-SmaI fragment of the human CD44 gene containing exons v8 to v10 andflanking introns (41) between the KpnI and HindIII sites of pRK3. pRK3 andpRK20 have been described elsewhere (10). RK-MS2 was made by inserting a137-bp SpeI-EcoRI fragment of pIII/MS2-2 carrying MS2 coat protein bindingsites (42) between nucleotides 15 and 505 of the 1,220-bp intron downstream ofthe K-SAM exon, in an RK3 derivative missing the BEK exon (deletion 1156–1412 of Fig. 1 in reference 12). pRK97 and pRK98 were made from pRK3 andpRK20, respectively, by deletion of the BEK exon’s 39ss and associated polypyri-midine sequence (deletion 1156–1233 of Fig. 1 in reference 12). pRK99 wasmade from pRK20 by deleting the BEK exon (deletion 1156–1412 of Fig. 1 inreference 12) and then replacing nucleotides 213 to 505 of the intron down-stream of the K-SAM exon with the fragment carrying coat binding sites.

Extract preparations and in vitro splicing assays. HeLa nuclear extract andcytoplasmic S100 extract were prepared as described previously (40). For prep-aration of whole-cell extracts (WCE) from 293-EBNA cells, cells resuspended inlysis buffer (20 mM Tris-HCl [pH 7.6], 400 mM KCl, 20% glycerol, 1 mMdithiothreitol, 0.2% NP-40, and a cocktail of protease inhibitors) were sonicatedand then centrifuged at 10,000 rpm for 10 min in an SS-34 rotor. The supernatant(WCE) was dialyzed against buffer D for 5 h. For preparation of WCE from

293-EBNA cells overexpressing TIA-1 (WCE/TIA-1), cells were transfected with6 mg of TIA-1-expressing vector and 14 mg of pBluescript SK(1) (Stratagene)and collected 48 h later, and WCE were prepared from them as described above.

Capped 32P-labeled pre-mRNA substrates were made by runoff in vitro tran-scription with SP6 RNA polymerase as described in reference 8. For the tropo-myosin-derived transcripts, in vitro splicing was performed as described in ref-erence 5 (25-ml final volume, using 12 ml of nuclear extract, in 60 mM KCl–1.3mM MgCl2). Splicing of the E1A-derived transcripts was performed under avariety of conditions as indicated in the figure legends. Reaction mixtures wereincubated at 30°C for 90 to 120 min, and splicing products were resolved ondenaturing 5 to 6% polyacrylamide gels, followed by autoradiography.

UV cross-linking and immunoprecipitation assays. UV cross-linking was per-formed as described previously (7) with minor modifications. RNA probes weresynthesized in vitro from pBluescript SK(1)-based plasmids containing appro-priate sequences (59ss-IAS1, 59ss-RAN, IAS1, or RAN) downstream of the T7promoter. They were uniformly labeled at high specific activity using[a-32P]UTP, and 50 fmol was used per assay. Cross-linking assay mixtures (15 ml)containing 3 ml of cell extracts, supplemented or not with 150 ng of TIA-1 orhnRNP C1, were incubated with RNA probes in 0.63 buffer D containing 2.6%polyvinyl alcohol and 0.5 mg of Escherichia coli tRNA. After a 20-min incubationat 30°C, reaction mixtures were exposed to UV light for 15 min at 4°C and thentreated with a mixture of RNases A (750 ng) and T1 (250 U) for 30 min at 37°C.For direct analysis, samples were diluted with a 23 sodium dodecyl sulfate (SDS)protein loading buffer and resolved by SDS-polyacrylamide gel electrophoresis(PAGE) on 12% polyacrylamide gels. For UV cross-linking and immunoprecipi-tation assays, the RNase-treated samples were diluted to 60 ml with IPP buffer(50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% NP-40), and 5 mg of anti-TIA-1polyclonal antibody (Santa Cruz Biotech) or 10 mg of anti-hnRNP C1 monoclo-nal antibody (a generous gift from G. Dreyfuss) was added. After incubation at4°C for 3 h, 10 ml of protein G-Sepharose beads was added and incubation wascontinued overnight. After three washes of the beads with 200 ml of IPP buffer,bound proteins were eluted in 20 ml of SDS loading buffer at 100°C for 5 min andloaded on an SDS–12% polyacrylamide gel.

To analyze the role of U1 snRNP in TIA-1 binding to the 59ss-IAS1 probe,3-ml aliquots of extracts (WCE or WCE/TIA-1) were pretreated with 0.5 mg ofa 14-nucleotide oligodeoxynucleotide complementary to the 59 end of U1snRNA, or with a nonrelated probe complementary to the T7 promoter, in thepresence of 0.5 U of RNase H, for 30 min at 30°C before cross-linking to the59ss-IAS1 probe and immunoprecipitation as described above.

Recombinant proteins and total SR preparation. Total SR proteins fromHeLa cells were purified as described in reference 51. To produce recombinantTIA-1 and hnRNP C1 in E. coli, the corresponding coding sequences from thesecond (TIA-1) or first (hnRNP C1) codon up to the stop codon were insertedin frame between the BamHI and EcoRI sites of pET28-b (Novagen). Resultingplasmids were used for production of six-His-tagged proteins. They were ex-pressed and purified under nondenaturing conditions as recommended by themanufacturer and dialyzed against buffer D for 1 h.

Transfections and RT-PCR. Transfection of SVK14 (46) and 293-EBNA cells(Invitrogen) was performed as described previously (10, 13). For cotransfections,2 mg of minigene was cotransfected with 18 mg of the appropriate expressionvector. Forty-eight hours later, RNA was harvested and analyzed by reversetranscription-PCR (RT-PCR) using reporter-specific primers. Preprotachykininprimers P1 and P2 were as follows: P1, GGAAATCGGTGCCAACG; P2, GAGAGATCTGACCATGCC. CD44 and FGFR-2 primers were as follows: P3, ATCCAGTGGATCAAGCAC, and P4, GGCAACCTAGAAGGCACAG. Twentycycles of amplification were used so as to remain in the range of exponentialamplification. PCR products were caused to migrate on agarose gels, transferredto nylon filters (Hybond N1; Amersham), and hybridized with different probes.Experiments were carried out at least in triplicate, and representative results areshown here. 32P-labeled RNA probes used were obtained by in vitro transcrip-tion of DNA fragments corresponding to (i) nucleotides 3 to 134 of the 148-nucleotide K-SAM exon; (ii) linked C1 and C2 exon sequences; and (iii) linkedexons 2, 3, and 5 of the preprotachykinin gene.

RESULTS

IAS1 activation of heterologous exons. Efficient recognitionof the chicken b-tropomyosin gene’s exon 6A (1) normallyrequires a 33-nucleotide pyrimidine-rich sequence (S4) start-ing 37 nucleotides downstream from its 59ss. Can IAS1 replaceS4 in this system? Various pre-mRNA substrates (Fig. 1A)containing exons 6A and 7 were used for in vitro splicing assaysin HeLa cell nuclear extract. As expected from previous work(17), splicing (Fig. 1B) of a pre-mRNA lacking S4 (6A-D4-7,lane 7) was inefficient compared to splicing of pre-mRNAswith S4 (Tropo 6A-7, lane 2) or a purine-rich sequence (6A-P3AS-7, lane 3). When S4 was replaced with IAS1 (IAS1 down,Fig. 1A), splicing dropped to levels below those observed in the

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absence of S4 (compare lanes 6 and 7, Fig. 1B). In the IAS1down pre-mRNA, IAS1 lies 43 nucleotides downstream of theexon 6A-intron junction. In vivo, IAS1 activates splicing only ifit lies immediately downstream from the K-SAM exon’s 59ss(F. Del Gatto-Konczak, unpublished data). A further pre-mRNA was made (IAS1 up, Fig. 1A) which lacked S4 butcontained IAS1 positioned immediately downstream from theexon 6A 59ss. Splicing of IAS1 up was at least as efficient assplicing of substrates containing S4 or the purine-rich sequence(compare lanes 2, 3, and 4, Fig. 1B). Pre-mRNA RAN (Fig.1A) is a version of IAS1 up in which IAS1 has been replacedwith a random sequence incapable of activating K-SAM exonsplicing in vivo (10). Splicing of pre-mRNA RAN was signifi-cantly less efficient than splicing of substrate IAS1 up (Fig. 1B,compare lanes 4 and 5) while being slightly more efficient thansplicing of pre-mRNA 6A-D4-7 (compare lanes 5 and 7). Theseresults show that IAS1 can activate splicing of a heterologousexon in vitro, provided that it is positioned immediately down-stream of the exon’s 59ss. They also show that the randomsequence does not act as a repressor of an adjacent 59ss invitro.

In the FGFR-2 pre-mRNA, IAS1 is involved in a competi-tive splicing choice. This encouraged us to test if IAS1 canfunction when two 59ss are in competition. We used pre-mRNAs derived from the adenovirus E1A gene (Fig. 2), inwhich the unique 13S 59ss (D1) has been replaced with differ-ent pairs of competing splice sites (5). Pre-mRNA D2/D2-wtcontains two identical copies of the E1A 12S 59ss D2. Whenthis pre-mRNA is spliced in nuclear extract, the distal D2 59ssis markedly preferred to the proximal D2 59ss (Fig. 3A, lane 1).This preference for the distal 59ss is probably due to involve-ment of the nuclear cap binding complex (CBC), which acts to

FIG. 1. IAS1 activates splicing of a heterologous tropomyosin exon. (A) Schematic representations of pre-mRNAs used for in vitro splicing. In Tropo 6A-7pre-mRNA, exons 6A and 7 are separated by a 284-nucleotide intron including the S4 activating sequence. In 6A-P3AS-7 and IAS1 down, S4 has been replaced witha purine-rich sequence and with IAS1, respectively. In 6A-D4-7, the S4 sequence is deleted. In IAS1 up and RAN, IAS1 and the random RAN sequence, respectively,have been inserted immediately downstream of the 59ss. The last nucleotides of exon 6A are boxed, and the IAS1 and RAN sequences are shown in boxes. (B) In vitrosplicing assays using pre-mRNAs shown in panel A in HeLa cell nuclear extract. mRNAs obtained by splicing exons 6A and 7 together are identified, as well as excisedintrons. The space of migration between the pre-mRNAs and mRNA has been reduced. The amounts of excised introns (I) and remaining pre-mRNA (P) werequantified using a Fuji phosphorimager, and the percentage of splicing was determined as I/(I 1 P) 3 100%. The mean of three determinations is given below the lanes.nt, nucleotides.

FIG. 2. Schematic representations of pre-mRNAs with competing 59ss usedfor in vitro splicing. Part of the E1A pre-mRNA is shown, with the naturalcompeting D2 and D1 59ss. In the other pre-mRNAs, the D1 59ss has beenreplaced with a pair of competing 59ss as shown, and the major splicing reactionsobserved are indicated.

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favor use of the 59ss closer to the pre-mRNA’s cap (31). How-ever, placing IAS1 immediately downstream from the proximalsite (pre-mRNA D2/D2-IAS1, Fig. 2) induces a very strongshift of splicing toward use of this latter site (Fig. 3A, lane 2).

TIA-1 activation of 5*ss. Having established that IAS1 canactivate splicing of an adjacent 59ss in vitro, we searched forpossible effects of TIA-1 on IAS1 activity. To stand a chance ofobserving an effect on splicing in vitro of an increase in TIA-1levels, it is necessary to start with splicing extracts in which theconcentration of TIA-1 is suboptimal. For this reason, we usedS100 extract (with added SR proteins) for further experiments,as TIA-1 is less abundant in cytoplasmic S100 extracts than innuclear extracts (data not shown). In the absence of addedexogenous TIA-1, only use of the proximal D2 site linked toIAS1 was detected in S100 extract (Fig. 3B, lane 1). Splicingwas less efficient than that in nuclear extract (compare withFig. 3A, lane 2), most probably because several factors, includ-ing TIA-1, are limiting in the S100 extract. Addition of recom-binant TIA-1 (600 ng) led to a strong stimulation of splicingusing the D2-IAS1 site (Fig. 3B, lane 2). In contrast, additionof hnRNP C1, a protein which, like TIA-1, binds to U-richsequences (20), actually decreased use of the D2-IAS1 site(lane 3). While no comparable stimulation by TIA-1 of use ofa D2 site linked to the random sequence was observed (com-pare lanes 4 and 5), addition of 600 ng of TIA-1 did weaklystimulate use of both copies of the D2 59ss in the D2/D2-wtsubstrate (compare lanes 4 and 5 in Fig. 3B). This suggests thatTIA-1 can also activate 59ss linked to sequences other thanIAS1, at least in S100 extract supplemented with SR proteins,which, compared to nuclear extract, is suboptimal for splicing.Note that both D2 59ss copies are stimulated to approximatelythe same extent, and so TIA-1 is not preferentially activatingeither the proximal or the distal 59ss here. Importantly, acti-vation by TIA-1 of the D2 59ss not linked to IAS1 cannot be

detected on the pre-mRNA containing a competing D2 59ssadjacent to IAS1 (compare lanes 1 and 2, dist. D2/A mRNA).TIA-1 is thus showing a preference for the 59ss adjacent toIAS1.

Can this effect of TIA-1 be reproduced using other pairs ofsplice sites, particularly when one of them is the K-SAM exonDSAM 59ss? We have analyzed splicing of pre-mRNA fromother constructs, which contain as competing splice sites thestrong E1A 13S D1 59ss and the weaker DSAM 59ss. In pre-mRNA D1/DSAM-IAS1 (Fig. 2), the DSAM site is followed byIAS1. In pre-mRNA D1/DSAM-RAN, IAS1 has been replacedwith the random sequence described above. When the D1/DSAM-IAS1 pre-mRNA was spliced in S100 extract (withadded SR proteins), the D1 site was scarcely used for splicing(Fig. 4A, lane 2), while the DSAM 59ss was preferred (Fig. 4A,lane 2). However, addition of TIA-1 (300 or 600 ng) stronglyactivated use of the DSAM 59ss (approximately fourfold), with-out affecting the very weak use of the D1 59ss (lanes 3 and 4).As observed previously (Fig. 3B), addition of hnRNP C1 de-creased use of the IAS1-linked (DSAM) 59ss (Fig. 4A, lane 5).No comparable TIA-1-induced activation was observed whenthe DSAM 59ss was linked to the random sequence (comparelanes 6 and 7). However, addition of 600 ng of TIA-1 didstimulate use of the D1 59ss in the D1/DSAM-RAN substrate(compare lanes 6 and 7 in Fig. 4A). Yet despite this, no acti-vation of the D1 59ss by TIA-1 could be observed when it wasin competition with an IAS1-linked 59ss (the DSAM-IAS1 59ss)on D1/DSAM-IAS1 pre-mRNA (compare lane 2 to lanes 3 and4). TIA-1 is once again showing a preference for the 59ssadjacent to IAS1.

If TIA-1 does really have a preference for the IAS1-linked59ss, it should be able to provoke a significant switch in splicesite use under appropriate circumstances. To attempt to visu-alize such a switch, we set out to increase the use of the D1 sitefor splicing of the D1/DSAM-IAS1 pre-mRNA. On this pre-mRNA, the D1 site is distal, and use of a distal 59ss close to acap site is known to be facilitated by the CBC (31). Cap bindingprotein is less abundant in S100 extract than in nuclear extract,and its concentration in S100 extract may be suboptimal (25).We therefore performed splicing assays using the D1/DSAM-IAS1 pre-mRNA in the presence of a mixture of S100 extractand nuclear extract, trying in this way to limit starting concen-trations of TIA-1 while benefiting from significantly higherlevels of CBC. Under these conditions, the distal D1 site wasindeed efficiently used (Fig. 4B, lane 2). The competing (prox-imal) DSAM 59ss was also used for splicing, though at a lowerlevel (Fig. 4B, lane 2). However, addition of TIA-1 (200, 400,or 600 ng) strongly activated use of the DSAM 59ss (approxi-mately fourfold), whereas splicing using the D1 59ss concomi-tantly decreased (lanes 3 to 5). These results show that TIA-1can effectively provoke a significant switch in splice site use,from predominant use of the D1 site (a strong 59ss) to approx-imately equal use of the D1 and the IAS1-linked DSAM 59ss.Taken together, our results show that TIA-1 activates 59ss usein a manner dependent on the downstream intron sequence,with a preference for a downstream U-rich sequence.

Preferential binding of TIA-1 to IAS1 adjacent to a 5*ss. Weused UV cross-linking analysis to test whether TIA-1 can bindto IAS1. The first RNA probe used (59ss-IAS1) contained astrong 59ss sequence (AG/GUAAGU) linked to IAS1. Themajor protein cross-linked to this probe in HeLa cell nuclearextract was an approximately 60-kDa protein (which might bePTB or U2AF, both known to bind to pyrimidine-rich sequenc-es); several smaller proteins were also detected (Fig. 5A, lane1). A number of proteins cross-linked weakly to the 59ss-IAS1probe in the S100 extract, which contains only low amounts of

FIG. 3. Effect of IAS1 and TIA-1 on competing D2 59ss in vitro. In vitrosplicing assays were performed using pre-mRNAs shown in Fig. 2. For splicedmRNAs, the donor (D) and acceptor (A) (the E1A 39ss) splice sites used areidentified. (A) Splicing was carried out in HeLa cell nuclear extract (NE). Notethat a cryptic splicing reaction occurs with the D2/D2-wt pre-mRNA using acryptic 59ss located 88 nucleotides upstream of the distal D2 site. The crypticintron is visible on the photo, but the corresponding mRNA has not beenretained. (B) Splicing was in cytoplasmic S100 extract (9 ml) with 0.5 mg of SRproteins added (S1001SR). Lanes 1 to 3, D2/D2-IAS1 pre-mRNA spliced inextract alone (lane 1) or in extract with 600 ng of TIA-1 (lane 2) or 600 ng ofhnRNP C1 (lane 3). Lanes 4 to 6, D2/D2-wt pre-mRNA spliced in extract alone(lane 4) or with 600 ng of TIA-1 (lane 5) or 600 ng of hnRNP C1 (lane 6) added.



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TIA-1 and hnRNP C (Fig. 5A, lane 2). When the latter extractwas enriched with recombinant TIA-1 or hnRNP C1, the probecross-linked to major new proteins with net molecular massesof '45 (corresponding in size to recombinant TIA-1, lane 3)and '40 (corresponding in size to recombinant hnRNP C1,lane 4) kDa, respectively. We also tested S100 extract enrichedwith recombinant ASF/SF2 lacking its RS domain. This pro-tein, which binds with high affinity to purine-rich sequences,was not detected with the 59ss-IAS1 probe (data not shown).

To test if TIA-1 expressed in mammalian cells can also bindto IAS1, we performed similar experiments using WCE pre-pared from human 293-EBNA cells, either transfected or notwith the mouse TIA-1 expression vector pTIA-1. Transfectionled to an approximately 10-fold increase in TIA-1 levels (datanot shown). The 59ss-IAS1 probe cross-linked to several pro-teins in the untransfected, control WCE (Fig. 5A, lane 5). InWCE from cells transfected with pTIA-1, a major new proteincorresponding in size to TIA-1 was detected with the 59ss-IAS1probe (lane 6). However, when the 59ss-RAN (the randomsequence linked to a 59ss) probe was used, no difference be-tween WCE from untransfected (lane 8) and transfected (lane9) cells could be observed. Thus, these results show that bothTIA-1 produced bacterially and TIA-1 produced in a mamma-lian cell can bind specifically and efficiently to IAS1.

Somewhat different results were obtained when the probeused was IAS1 without an adjacent 59ss (Fig. 5B). This probedetects a major '40-kDa protein in HeLa cell nuclear extract(lane 1) which is hnRNP C (Fig. 5D), and a major '80-kDaprotein in S100 extract (lane 2). While we detected cross-linking of the IAS1 probe with recombinant TIA-1 (lane 3) orhnRNP C1 (lane 4) in S100 extract supplemented with theseproteins, interaction of TIA-1 with the IAS1 probe appearedweaker than that with the 59ss-IAS1 probe (compare lane 3,Fig. 5B, with lane 3, Fig. 5A). This difference between the twoprobes was also observed using WCE from cells overexpressing

TIA-1 following transfection (compare Fig. 5A and B, lane 6).As expected, we did not detect any cross-linking of TIA-1 tothe random sequence probe RAN (Fig. 5B, compare lanes 8and 9).

Further experiments were carried out to demonstrate thatthe '45-kDa protein detected in extracts with the 59ss-IAS1probe really was TIA-1. A variety of extracts were incubatedwith either the 59ss-IAS1 probe or the IAS1 probe before UVcross-linking. Aliquots were analyzed either before immuno-precipitation (total) or after immunoprecipitation with anti-bodies recognizing either TIA-1 or hnRNP C1. Both TIA-1and hnRNP C1 cross-linked to the 59ss-IAS1 probe in HeLacell nuclear extract (Fig. 5C, lanes 2 and 3), albeit weakly. Thiscross-linking appears to be weak because of competition forthe probe by the major 60-kDa cross-linking protein (lane 1).(Note that this competition does not necessarily take placeduring in vitro splicing assays where bona fide splicing sub-strates are used.) Thus, when the IAS1 probe is used (Fig. 5D),cross-linking to the 60-kDa protein is much less marked (lane1), while cross-linking to hnRNP C1 concomitantly increasesgreatly (lane 3). However, despite the apparently greater probeavailability, no cross-linking of the IAS1 sequence to TIA-1 isseen (lane 2). This result is in agreement with those shown inFig. 5A and B, which demonstrated that TIA-1 binds veryweakly to IAS1 which is not adjacent to a 59ss.

Using WCE from untransfected 293-EBNA cells (Fig. 5C,lanes 4 to 6), we detected efficient cross-linking of both TIA-1(lane 5) and hnRNP C1 (lane 6) to the 59ss-IAS1 probe. Fur-thermore, a strong increase of TIA-1 cross-linking was ob-served with WCE from cells transfected with pTIA-1 (comparelanes 5 and 8). Interestingly, when the IAS1 probe was used,TIA-1 was detected, and only weakly, solely in WCE from thetransfected cells (Fig. 5D, compare lanes 5 and 8), whilehnRNP C1 was detected in WCE from both untransfected(lane 6) and transfected (lane 9) cells. Taken together, the

FIG. 4. Effects of IAS1 and TIA-1 on competing D1 and DSAM 59ss in vitro. In vitro splicing assays were performed using pre-mRNAs shown in Fig. 2. For splicedmRNAs, the donor (D) and acceptor (A) (the E1A 39ss) splice sites used are identified. (A) Splicing was carried out in cytoplasmic S100 extract (9 ml) with 0.5 mg ofSR proteins added (S1001SR). Lane 1, D1/DSAM-IAS1 pre-mRNA starting material. Lanes 2 to 5, D1/DSAM-IAS1 pre-mRNA spliced in extract alone (lane 2), inextract with 300 or 600 ng of TIA-1 added (lanes 3 and 4, respectively), or in extract with 600 ng of hnRNP C1 added (lane 5). Lanes 6 to 8, D1/DSAM-RAN pre-mRNAspliced in extract alone (lane 6), extract with 600 ng of TIA-1 added (lane 7), or extract with 600 ng of hnRNP C1 added (lane 8). (B) Splicing was in a 6:4 mixtureof nuclear extract and S100 extract (NE/S100). Lane 1, D1/DSAM-IAS1 pre-mRNA starting material. Lanes 2 to 6, D1/DSAM-IAS1 pre-mRNA spliced in extract alone(lane 2); in extract with 200, 400, or 600 ng of TIA-1 added (lanes 3 to 5, respectively); or in extract with 400 ng of hnRNP C1 added (lane 6). Lanes 7 to 9,D1/DSAM-RAN pre-mRNA spliced in extract alone (lane 7) or in extract with 400 ng of TIA-1 (lane 8) or 400 ng of hnRNP C1 (lane 9) added. The radioactivitiespresent in the mRNAs were determined by a phosphorimager, corrected for their content in C residues, and used to calculate the ratio of use of D1 versus that of DSAM.



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results in Fig. 5 demonstrate that TIA-1 interacts preferentiallywith the IAS1 motif when adjacent to a 59ss. This conclusionwas confirmed by competition experiments using WCE fromtransfected cells. Interaction of TIA-1 with the labeled 59ss-IAS1 probe was strongly reduced (four- to fivefold) in thepresence of an 80-fold excess of unlabeled 59ss-IAS1 probe,while the presence of a 320-fold excess of unlabeled IAS1probe had no significant effect (data not shown).

TIA-1 binding is U1 snRNP dependent. The preferred bind-ing site for TIA-1 has been identified as a U-rich sequence byexperiments analyzing TIA-1–RNA interaction in the absenceof any other cellular proteins (14). Under these conditions,there was no indication that the U-rich sequence need beadjacent to a sequence resembling a 59ss. It seemed possible tous that the preferred binding of TIA-1 to IAS1 adjacent to a59ss in cell extracts might reflect the presence, in these extracts,of U1 snRNP and its binding to the 59ss. Is the binding ofTIA-1 to IAS1 adjacent to a 59ss in fact dependent on U1snRNP binding to the 59ss? To address this question, we incu-bated WCE from TIA-1-transfected cells with either an oligo-nucleotide complementary to the 59 14 nucleotides of U1snRNA or an “irrelevant” oligonucleotide (corresponding toone strand of the T7 promoter), in the presence of RNase H.The former oligonucleotide provokes degradation of the 59nucleotides of U1 snRNA necessary for U1 snRNP binding tothe 59ss (and so abolishes this binding). The latter oligonucle-

otide has no such effect. When WCE from TIA-1-transfectedcells were subjected to either a mock preincubation or prein-cubation with the T7 oligonucleotide before cross-linking tothe 59ss-IAS1 probe, probe cross-linking to both TIA-1 andhnRNP C1 was readily observed (Fig. 6, lanes 1 and 3). How-ever, when preincubation was done with the antisense U1snRNA oligonucleotide, cross-linking to TIA-1 was selectivelyeliminated (lane 2). This result was confirmed when aliquots ofcross-linked material were subjected to immunoprecipitationusing anti-TIA-1 antibodies (compare lanes 4 and 5). Similarresults were observed when WCE from untransfected cellswere analyzed (lanes 6 to 10), although the amount of cross-linked TIA-1 was, as expected, lower. The U1 snRNP-depen-dent cross-linking of TIA-1 to the probe could nevertheless bevisualized after immunoprecipitation of samples with anti-TIA-1 antibodies (compare lanes 9 and 10). We conclude thatTIA-1 binding to IAS1 in cell extracts is dramatically enhancedby U1 snRNP binding to an adjacent 59ss.

TIA-1 enhances K-SAM exon splicing in vivo. As describedpreviously (10), the RK3 minigene (Fig. 7) contains a wild-typeFGFR-2 gene fragment carrying the very similarly sized alter-native K-SAM and BEK exons, together with flanking intronsequences and the upstream and downstream constitutive ex-ons C1 and C2. Transfection of epithelial SVK14 cells withRK3 leads to preferential splicing of the K-SAM exon, whiletransfection of 293-EBNA cells leads to preferential splicing of

FIG. 5. Interaction between TIA-1 and IAS1. (A and B) RNA probes as shown were incubated with various extracts either alone (2) or with 150 ng of addedrecombinant TIA-1 (1 TIA-1) or hnRNP C1 (1 C1) as indicated before UV cross-linking and SDS-PAGE analysis. NE, HeLa nuclear extract; S100, HeLa S100 extract.WCE, WCE from 293-EBNA cells. WCE/TIA-1, WCE from cells transfected with pTIA-1. After UV cross-linking and RNase treatment, equivalent aliquots wereresolved directly on an SDS-polyacrylamide gel. The positions of prestained protein standards (NOVEX) are indicated. Note that the apparent molecular mass of theadduct proteins is usually 3 to 5 kDa higher than that of the corresponding protein. (C and D) RNA probes were incubated with various extracts as in panels A andB. After UV cross-linking and RNase treatment, samples were divided into three parts. One was analyzed directly (total); the others were analyzed after immuno-precipitation with antibodies against either TIA-1 (aTIA-1) or hnRNP C1 (aC1). Analysis was performed by SDS-PAGE. The aliquots loaded on the gel for the samplesanalyzed directly (total) were one-third of the amount used for those analyzed after immunoprecipitation.



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the BEK exon. Skipping of both exons is also observed at a lowlevel. Cotransfection of 293-EBNA cells with RK3 and pcoat(a bacteriophage MS2 coat protein expression vector) resultedin BEK exon splicing (Fig. 8A and B, lanes 1), reflected by

major RT-PCR product C1BC2 (Fig. 8E). For RK3 and de-rivatives, two probes were used to identify RT-PCR products.The C1C2 probe detects all products, while the K-SAM probedetects only products containing the K-SAM exon. Cotrans-fection of RK3 with pTIA-1 resulted in splicing of the K-SAMexon to the BEK exon (Fig. 8A and B, lanes 2), reflected by themajor product C1SBC2, though some C1SC2 product is alsodetected (Fig. 8E). Importantly, cotransfection of RK3 withthe hnRNP C1 expression vector did not detectably activateK-SAM exon splicing (lanes 3), suggesting that the TIA-1 ef-fect on the K-SAM exon cannot be reproduced by just anyU-rich sequence binding protein. RK20 (Fig. 7) is a version ofRK3 in which IAS1 has been replaced with the random se-quence (10) unable to bind TIA-1. Cotransfection of RK20with pTIA-1 did not lead to K-SAM exon splicing, as RT-PCRproducts of type C1BC2 (Fig. 8E) were obtained regardless ofwhether RK20 was cotransfected with pcoat (Fig. 7A and B,lanes 4), pTIA-1 (lanes 5), or phnRNP C1 (lanes 6). TIA-1activation of K-SAM exon splicing is thus IAS1 dependent.Note that, in the absence of IAS1, TIA-1 activates splicing ofC1 to C2 somewhat (Fig. 8A, lane 5) but that this effect cannotbe detected when IAS1 is present (Fig. 8A, lane 2). This is inagreement with our in vitro splicing results, which show that,while TIA-1 can activate a variety of 59ss, when a 59ss linked toIAS1 is in competition with one not so linked, it is use of theformer which is favored by TIA-1.

TIA-1 can activate K-SAM exon splicing not only to theBEK exon but also to the C2 exon. In RK97 (Fig. 7), the BEKexon’s 39ss and associated polypyrimidine sequence have beendeleted, blocking its splicing. Cotransfection of RK97 withpcoat in 293-EBNA cells results mainly in skipping of theK-SAM exon. The major RT-PCR product obtained (Fig. 8C

FIG. 6. U1 snRNP is involved in TIA-1 binding to IAS1. WCE from 293-EBNA cells (WCE) or from transfected 293-EBNA cells (WCE/TIA-1) weremock preincubated (2) or preincubated with oligonucleotides complementary tothe 59 end of U1 snRNA (U1) or complementary to the T7 promoter (T7) beforecross-linking to the 59ss-IAS1 probe. For lanes 1 to 3 and 6 to 8, each assaymixture was analyzed directly. In addition, immunoprecipitation with anti-TIA-1antibodies was performed on the mock-preincubated samples (lanes 4 and 9) andthe samples preincubated with the oligonucleotide complementary to the 59 endof U1 snRNA (lanes 5 and 10). The aliquots loaded on the gels for the samplesanalyzed directly represent one-third of the amount used for those analyzed afterimmunopurification.

FIG. 7. Schematic representations of FGFR-2 minigenes. The parent minigene RK3 is shown, with the Rous sarcoma virus long terminal repeat promoter (RSV),the alternative exons K-SAM and BEK, the upstream and downstream constitutive exons C1 and C2, and the bovine growth hormone polyadenylation sequence (BGH).Locations of primers P3 and P4 used for RT-PCR are marked. The U-rich intron-activating sequence IAS1 is identified. RK20 is similar to RK3, except for thereplacement of IAS1 with a random sequence. In RK97, the BEK exon’s polypyrimidine sequence and 39ss have been deleted. RK98 was derived from RK97 byreplacing IAS1 with the random sequence described in the text. RK-MS2 is derived from RK3 by replacing IAS1 and some downstream sequences with bacteriophageMS2 coat binding sites (MS2). In addition, the BEK exon is deleted. RK99 is similar to RK20, except that the BEK exon is deleted and bacteriophage MS2 coat bindingsites (MS2) have been placed well downstream of the K-SAM exon’s 59ss.



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and D, lanes 1) is C1C2 (Fig. 8E); only a little splicing of theK-SAM exon is observed (product C1SC2). Cotransfection ofRK97 with pTIA-1 leads to a marked increase of K-SAM exonsplicing, as evidenced by an increase in the levels of the C1SC2product (Fig. 8C and D, lanes 2). Once again, this activation ofK-SAM splicing is IAS1 dependent, as cotransfection ofpTIA-1 with RK98, a version of RK97 in which IAS1 has beenreplaced with the random sequence (Fig. 7), has no similareffect (compare lanes 3 and 4 of Fig. 8C and D).

Artificial recruitment of TIA-1 obviates the IAS1 require-ment. Our results suggest that IAS1 serves as a binding site torecruit TIA-1 close to the 59ss. If so, artificial recruitment ofTIA-1 downstream from the 59ss might activate splicing of aK-SAM exon not linked to IAS1. In RK-MS2 (Fig. 7), whichhas the BEK exon deleted, nucleotides 15 to 505 (which in-clude IAS1) of the intron downstream from the K-SAM exonhave been replaced with a tandem copy of the bacteriophageMS2 coat protein operator. Proteins can thus be recruited to

FIG. 8. TIA-1 activation of the K-SAM exon requires IAS1. Cells were cotransfected with minigenes and expression vectors for bacteriophage MS2 coat protein,TIA-1, or hnRNP C1 as shown. RT-PCR was carried out on transfected cell RNA using primers P3 and P4 shown in Fig. 7, and products were subjected to Southernanalysis. Hybridization was performed first to a probe corresponding to the K-SAM exon (B and D), and then the same blot was dehybridized and rehybridized to aprobe made up of exons C1 and C2 (A and C). RT-PCR products are identified using names corresponding to structures shown in panel E.



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RK-MS2 pre-mRNA downstream from the K-SAM exon asfusions with coat protein. When RK-MS2 is transfected to-gether with the empty expression vector pCI-neo or pcoat, theK-SAM exon is skipped. RT-PCR products detected with theC1C2 probe (Fig. 9A, lanes 1 and 3) reflect splicing of exon C1to exon C2 (product C1C2). This result is expected, as IAS1 isrequired for efficient K-SAM exon splicing. Cotransfection ofpTIA-1 with RK-MS2 does not detectably induce K-SAM exoninclusion: note that none of the RT-PCR products detectedwith the C1C2 probe (Fig. 9A, lane 2) hybridize with theK-SAM exon probe (Fig. 9B, lane 2). However, when RK-MS2is cotransfected with an expression vector for a TIA-1–coatfusion protein, some inclusion of the K-SAM exon is induced:RT-PCR products which hybridize to the K-SAM probe andcorrespond to C1SC2 can be detected (Fig. 9A and B, lanes 4).No activation of K-SAM exon splicing was observed whenRK-MS2 was cotransfected with expression vectors for hnRNP

C1-coat fusions (Fig. 9A and B, lanes 5) or hnRNP A1-coatfusions (lanes 6).

The K-SAM exon inclusion induced by TIA-coat with RK-MS2 pre-mRNA is less than that induced by TIA-1 with pre-mRNAs containing IAS1 (compare Fig. 8 and 9), althoughapproximately equal amounts of the two proteins are madefollowing transfection of their expression vectors (data notshown). The TIA-coat fusion bound to the MS2 operator maybe presented to the splicing apparatus suboptimally comparedto TIA-1 in its natural position. Activation by the TIA-coatfusion is, as expected, position dependent. In RK99 (Fig. 7),the operator is placed 213 bp downstream from the 59ss, andnot 15 bp downstream as in RK-MS2. (Note that RK99 doesnot contain IAS1, which has been replaced with the randomsequence of RK20.) When RK99 is transfected together withthe TIA-coat expression vector, no activation of K-SAM exonsplicing is observed (Fig. 9C and D). In fact, TIA-coat expres-

FIG. 9. TIA-1 activates splicing if recruited close to the 59ss. Cells were cotransfected with minigenes and the empty expression vector pCI-neo or expression vectorsfor bacteriophage MS2 coat protein or TIA-1 or the following fusions with coat protein: TIA-1–coat fusion (TIA-coat), hnRNP C1-coat fusion (C1-coat), and hnRNPA1-coat fusion (A1-coat). RT-PCR was carried out on transfected cell RNA using primers P3 and P4 shown in Fig. 7, and products were subjected to Southern analysis.Hybridization was performed first to a probe corresponding to the K-SAM exon (B and D), followed by dehybridization and rehybridization to a probe made up ofexons C1 and C2 (A and C). RT-PCR products are identified using names corresponding to structures shown in Fig. 8E.



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sion now represses K-SAM exon splicing (compare lanes 1 and2).

TIA-1’s effect on other exons in vivo. To test whether TIA-1can influence alternative splicing of other exons in vivo, weused several minigenes reflecting well-documented cases ofalternative exon splicing. Minigenes were cotransfected intocells with different expression vectors, and splicing patternswere investigated by RT-PCR analysis of transfected cell RNAusing minigene-specific primers. Normally, splicing of prepro-tachykinin pre-mRNA involves preferential skipping of op-tional exon 4 (23, 37). In 293-EBNA cells cotransfected with a

preprotachykinin minigene (containing exons 2 to 7) and theempty expression vector pCI-neo, inclusion of exon 4 is inef-ficient (34%) as judged by RT-PCR analysis (Fig. 10A, lane 1).When the minigene is cotransfected with the expression vectorpTIA-1, exon 4 inclusion increases to 63% (lane 2). However,a similar increase also occurs (to 51%, lane 3) following co-transfection with the hnRNP C1 expression vector phnRNPC1.

Exons v8, v9, and v10 of the CD44 gene’s pre-mRNA arespliced to generate mRNA for the epithelial cell form of CD44(9). A minigene was made in which these exons and their

FIG. 10. Effects of TIA-1 and hnRNP C1 on splicing in vivo. (A) The preprotachykinin minigene was cotransfected into SVK14 cells with pCI-neo (lane 1), pTIA-1(lane 2), or phnRNP C1 (lane 3). RT-PCR was carried out on transfected cell RNA using primers P1 and P2, and products were subjected to Southern analysis withhybridization to a probe made up of exons 2 to 5. (B) A hybrid FGFR-2–CD44 minigene was cotransfected into 293-EBNA cells with pCI-neo (lane 1), pTIA-1 (lane2), or phnRNP C1 (lane 3). RT-PCR was carried out on transfected cell RNA using primers P3 and P4 as marked, and products were subjected to Southern analysiswith hybridization to a probe made up of exons C1 and C2 of the FGFR-2 gene. The 6.9-kb ClaI-SmaI fragment of the human CD44 gene used is identified by arrowsand contains alternative exons v8, v9, and v10, as well as an additional alternative exon (50) represented by a black box. Note that exon v9 has two alternative 59ss (50).On the minigene map, the three major splicing events seen in 293-EBNA cells transfected with the minigene and pCI-neo are illustrated. The corresponding RT-PCRproducts are illustrated below the map. RSV, Rous sarcoma virus long terminal repeat; BGH, bovine growth hormone polyadenylation signal. Radioactivities presentin bands were determined by phosphorimager and used to calculate splicing percentages.



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flanking introns were placed between two constitutively splicedexons, C1 and C2, of the human FGFR-2 gene (Fig. 10B).When this minigene was transfected into the epithelial cell lineSVK14, exons v8, v9, and v10 were efficiently spliced to theflanking exons C1 and C2 (data not shown). However, whenthe minigene was transfected into 293-EBNA cells togetherwith the control vector pCI-neo, RT-PCR analysis revealed anumber of products (Fig. 10B, lane 1). The identity of some ofthese products was investigated by hybridization to a probecomposed of v8, v9, and v10 sequences and by sequencing ofsubcloned fragments (data not shown). Unlike in SVK14 cells,product C1V8V9V10C2 is not the major product, representingonly 12% of all products. More abundant products correspondeither to skipping of all CD44 exons (product C1C2) or tosplicing of exons v8, v9, and v10, with use of an alternative 59ssfor exon v9 and inclusion of an additional exon (represented bya black box) between v9 and v10 (product C1V8V9*V10C2).(This splicing possibility has already been described for themouse CD44 gene [50]). The identity of other products was notestablished. When the CD44 minigene was transfected into293-EBNA cells together with pTIA-1, the C1C2 product dis-appeared and the RT-PCR products (lane 2) shifted in favor ofthe C1V8V9V10C2 product, which now represents 45% of allproducts. A similar increase of this product (to 34% of allproducts, lane 3) also occurs following cotransfection with thehnRNP C1 expression vector.

In summary, for both the preprotachykinin and CD44 pre-mRNAs, TIA-1 overexpression and hnRNP C1 overexpressionhave qualitatively similar effects. The smaller effect of phnRNPC1 transfection could be explained if the transfection-inducedincrease in hnRNP C1 levels is lower than that in TIA-1 levels.On the other hand, our in vitro analysis has shown that TIA-1can activate 59ss not linked to IAS1, albeit less effectively thanthose so linked. This could also contribute to the increasedeffect of TIA-1 relative to that of hnRNP C1. TIA-1 overex-pression does not lead to the activation of splicing of all exons,however, as TIA-1 had no detectable effect (data not shown)on splicing of another alternative exon, the poorly splicedEIIIb exon (24) of the rat fibronectin gene.

The similar effects on preprotachykinin and CD44 pre-mRNAs of TIA-1 and hnRNP C1 overexpression suggest thatincreasing the level of any protein which binds to U-rich se-quences may suffice to perturb splicing in these cases. Onepossible mechanism could involve competition for pyrimidine-rich binding sites with a protein such as PTB. Insofar as PTBis known to repress splicing of a variety of exons (48a), limitingits access to the pre-mRNA could favor exon inclusion. Itshould be recalled that, in contrast to the preprotachykinin andCD44 exons, for the K-SAM exon, TIA-1 overexpressionmarkedly stimulates splicing, while hnRNP C1 overexpressionhas no detectable effect. This suggests that TIA-1’s stimulationof K-SAM exon splicing cannot be attributed to the same typeof effect as that exerted on either preprotachykinin or CD44exon splicing.

DISCUSSION

The activating element IAS1 of the FGFR-2 gene partici-pates with a variety of other elements in controlling splicing ofthe K-SAM exon (12). IAS1 lies immediately downstreamfrom the K-SAM exon and indeed activates splicing only whenso positioned. Our results demonstrate that (i) IAS1 can func-tion independently of other FGFR-2 elements to activate het-erologous 59ss, (ii) TIA-1 binds to IAS1 in cell extracts if a 59ssis adjacent to it, (iii) this binding is dependent on intact U1snRNA, and (iv) TIA-1 activates use of 59ss. The extent of

activation observed depends on the sequence downstreamfrom the 59ss. 59ss adjacent to IAS1 are preferentially acti-vated, and increasing TIA-1 levels can provoke a switch to useof such sites. Replacing IAS1 downstream from the K-SAMexon with a binding site for the bacteriophage MS2 coat pro-tein allows activation of this exon’s splicing by a TIA-1–coatfusion. Activation occurs only if the fusion binds close to the59ss, indicating that TIA-1 needs to be close to the 59ss toactivate. Our results identify TIA-1 as a novel splicing activatorand suggest that TIA-1 activates splicing by binding close to a59ss, for a direct or indirect interaction with U1 snRNP boundto the 59ss. Does TIA-1 bind transiently to U1 snRNP beforeinteraction of the resulting complex with the splice site? Wehave no evidence in favor of this. Though TIA-1 and U1snRNP do coimmunoprecipitate weakly, we have not been ableto rule out nonspecific RNA bridging as an explanation (ourunpublished data). An alternative possibility is that TIA-1 andU1 snRNP associate in situ at the 59ss region: their association,dependent on the 59ss and the adjacent IAS1, would last onlythe time needed for splicing. Clearly, further experiments arerequired to define in detail the nature and the chronology ofthe interactions between TIA-1, U1 snRNP, and the 59ss andflanking IAS1 sequence and to clarify whether TIA-1 enhancesU1 snRNP binding to the 59ss or acts at some later step. It isinteresting to note, however, that incubation of the IAS1 upand RAN tropomyosin pre-mRNAs (Fig. 1) in nuclear extractunder conditions allowing only formation of early (E) com-plexes (absence of ATP and Mg21) specifically promotes U1snRNP-dependent protection of the 59ss linked to IAS1 (C. F.Bourgeois, L. Kister, and J. Stevenin, unpublished data). Thissuggests that IAS1 acts to facilitate the U1 snRNP-59ss bindingstep.

We were led to investigate TIA-1 by recent work on yeast U1snRNP (39, 53). Yeast U1 snRNP is more complex than mam-malian U1 snRNP, containing in addition to the proteins foundin mammalian U1 snRNP a number of specific proteins includ-ing Nam8p (21). When yeast U1 snRNP binds to a 59ss, Nam8pis positioned so as to contact intron sequences downstreamfrom the splice site (39, 53). Nam8p is required for splicingwhen the 59ss is noncanonical, as for example in the MER2pre-mRNA (36). Nam8p is also required in yeast strains lack-ing the nuclear CBC (16) and is presumably indispensable tofacilitate 59ss recognition in the absence of CBC activation.While Nam8p activity is maximal if sequences downstream ofthe 59ss are U rich (39), this is not a requirement, as sequencesimmediately downstream of the MER2 59ss, for example, arenot particularly U rich. Nam8p, with its three RNA bindingdomains, can presumably interact with a variety of RNA se-quences, albeit with different affinities, and activate splicing viathem to different extents.

TIA-1 is a distant mammalian relative of Nam8p, and ourresults identify functional similarities between the two pro-teins. Both proteins can activate 59ss use, and their activity ismaximal if downstream intron sequences are U rich. There is,nevertheless, a clear difference between Nam8p and TIA-1: thelatter is not an integral part of mammalian U1 snRNP. How-ever, oligonucleotide-directed degradation of the 59 extremityof U1 snRNA virtually abolishes TIA-1 binding to IAS1 in cellextracts. This implies that TIA-1 binding to IAS1 requires U1snRNP binding to an adjacent 59ss and suggests that there maynot be a fundamental difference between TIA-1 and Nam8p tobe found here. It has been proposed that splicing of a givenvertebrate intron may require assembly at the correspondingexon’s 59ss of a complex containing core U1 snRNP and aparticular subset of the relatives of the yeast U1 snRNP-spe-cific proteins mentioned above (15). The subset would be cho-



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sen in view of the individual characteristics of the 59ss in ques-tion. One way of activating a weak 59ss would be to associate itwith an intronic sequence recognized by TIA-1, to allow as-sembly of a complex containing core U1 snRNP and TIA-1. Itshould be possible to modulate the extent of such activation.Strong activation could be achieved by using an intronic se-quence like IAS1 similar to the optimal U-rich sequence forTIA-1 binding. Weaker activation would ensue if the intronicsequence bound TIA-1 with less affinity (TIA-1 can probablybind, like Nam8p, to a variety of RNA sequences with differentaffinities). If so, it would be possible to use TIA-1 to modulatesplice site choice in alternative splicing. The results of our invitro analysis (Fig. 3 and 4) are in agreement with this model:TIA-1 can activate 59ss not linked to IAS1, but when two 59ssare in competition, one linked to IAS1 and the other not,TIA-1 markedly favors use of the former at the expense of thelatter. Our data show that TIA-1 overexpression can have aprofound effect on alternative splicing, both in vivo and invitro. Although our results are based on overexpression dataalone and it may be difficult to define the true physiologicalrole of a protein with certainty from them, our additionalobservation that TIA-1 binding to IAS1 in cell extracts is U1snRNP dependent does provide a further link to a defined partof the cellular splicing machinery. Taken together, our obser-vations strongly suggest that one physiological role for TIA-1 isin regulation of splicing. Clearly, it will be important to identifyexons which require TIA-1 for their splicing.

Which splice sites represent important targets for TIA-1activation in vivo? It is interesting to recall that if Nam8p canapparently activate a variety of 59ss (as implied by its require-ment in strains lacking nuclear CBC [see above]), Nam8p ac-tivity is normally required at only a few, specific, noncanonical59ss. Indeed, Nam8p is not needed for vegetative growth. It ispossible that in a similar fashion TIA-1 is normally required invivo only for use of a subset of 59ss. One candidate exon is theK-SAM exon. Splicing of this exon is repressed by an ESS.IAS1 is required for K-SAM exon splicing, but only to over-come the activity of this ESS (12). Our results show that TIA-1has the characteristics required for a protein activating K-SAMexon splicing naturally: it binds to IAS1 and activates theK-SAM exon 59ss in vitro and favors K-SAM inclusion in vivo.While this does not prove that TIA-1 is the actual proteinwhich activates K-SAM exon splicing in vivo, it does makeTIA-1 (or, if not TIA-1, a protein with very similar properties)a very good candidate.

We have detected no significant difference in levels of TIA-1between SVK14 cells which splice the K-SAM exon normallyand 293-EBNA cells which do not (F. Del Gatto-Konczak,unpublished data). While this excludes a simple model fortissue-specific K-SAM exon splicing based on tissue-specificexpression of TIA-1, it is not in contradiction with the pro-posed role of TIA-1 in activating K-SAM exon splicing viaIAS1. Thus, unlike K-SAM exon splicing, action of IAS1 isdefinitely not tissue specific per se: IAS1 will fully activatesplicing of a heterologous fibronectin exon both in SVK14 cellsand in HeLa cells, which normally do not splice the K-SAMexon (F. Del Gatto-Konczak, unpublished data). Thus, theprotein which acts via IAS1 is not expected to have a markedtissue-specific distribution or activity.

How then could tissue-specific splicing of the K-SAM exonbe achieved? Control of the K-SAM exon is complex. The exoncontains an ESS, which functions by binding hnRNP A1 (13).Its 59ss is weak, and splicing of the exon requires not only IAS1but also two additional intron-activating sequences, IAS2 andIAS3 (6, 12). It may be that each of these elements shows a lowdegree of tissue specificity individually but that when their

effects are added together the sum is enough to confer tissuespecificity. A similar model invoking the necessary cooperationamong a variety of regulatory elements, none of which is ab-solutely tissue specific, has been proposed previously for splic-ing of a neuron-specific exon (35). We favor, however, analternative model in which TIA-1 bound to IAS1 and hnRNPA1 bound to the ESS exert opposing influences on the 59ss andpoise the K-SAM exon on the brink of splicing, without anytissue specificity. Then, additional tissue-specific activation,possibly via IAS2 and IAS3, would suffice to tip the balance infavor of K-SAM exon splicing. Such additional activation needperhaps be only mild, the competing BEK exon being itselfrepressed in cells which splice the K-SAM exon (6, 12, 19).

Other speculative targets for TIA-1 action are pre-mRNAscoding for proteins implicated in apoptosis. Expression of sev-eral key proteins in apoptosis is regulated by alternative splic-ing (for a review, see reference 26). TIA-1 itself has beenlinked to apoptosis. The serine-threonine kinase FAST is ac-tivated during Fas-mediated apoptosis in Jurkat cells and phos-phorylates TIA-1 prior to the onset of DNA fragmentation(48). The TIA-1-related protein TIAR is very similar to TIA-1and likely to exert the same type of activity as TIA-1. Indeed,overexpression of TIAR in cells by transfection leads to thesame effects on splicing as does overexpression of TIA-1 (C. LeGuiner, unpublished data). TIAR is translocated from thenucleus to the cytoplasm during Fas-mediated apoptosis (45).In the mouse, TIAR is essential for primordial germ cell de-velopment, as it appears to be necessary for cell survival (2). Itis tempting to speculate that proapoptotic stimuli modify theactivities of TIA-1 and TIAR, leading to changes in the splicingpatterns of key pre-mRNAs. It may prove possible to test thishypothesis by inactivation of the TIA-1 and TIAR genes inchicken cells, using an approach similar to that used recently toinactivate the gene coding for another splicing factor, ASF/SF2(49).

ACKNOWLEDGMENTS

We thank G. Dreyfuss, P. Grabowski, R. Hynes, and J. Marie forkindly providing materials. We also thank G. Hildwein for excellenttechnical assistance and the staff of the IGBMC facilities for theirassistance.

This work was supported by funds from the INSERM, the CNRS,the Hopitaux Universitaires de Nantes et de Strasbourg, the Associa-tion pour la Recherche sur le Cancer, and the Ligue Nationale contrele Cancer, Comite Departemental de Loire-Atlantique. C.F.B. wassupported by fellowships from the Association pour la Recherche surle Cancer and the Ligue Nationale contre le Cancer.

F.D.G.-K. and C.F.B. contributed equally to the work.

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