Two Isoforms of the T-cell Intracellular Antigen 1 (TIA-1) Splicing Factor Display Distinct Splicing Regulation Activities
2007; Elsevier BV; Volume: 282; Issue: 27 Linguagem: Inglês
10.1074/jbc.m700688200
ISSN1083-351X
AutoresJosé M. Izquierdo, Juan Valcárcel,
Tópico(s)Cancer-related molecular mechanisms research
ResumoTIA-1 (T-cell Intracellular Antigen 1) and TIAR (TIA-1-related protein) are RNA-binding proteins involved in the regulation of alternative pre-mRNA splicing and other aspects of RNA metabolism. Various isoforms of these proteins exist in mammals. For example, TIA-1 presents two major isoforms (TIA-1a and TIA-1b) generated by alternative splicing of exon 5 that differ by eleven amino acids exclusive of the TIA-1a isoform. Here we show that the relative expression of TIA-1 and TIAR isoforms varies in different human tissues and cell lines, suggesting distinct functional properties and regulated isoform expression. We report that whereas TIA-1 isoforms show similar subcellular distribution and RNA binding, TIA-1b displays enhanced splicing stimulatory activity compared with TIA-1a, both in vitro and in vivo. Interestingly, TIAR depletion from HeLa and mouse embryonic fibroblasts results in an increased ratio of TIA-1b/a expression, suggesting that TIAR regulates the relative expression of TIA-1 isoforms. Taken together, the results reveal distinct functional properties of TIA-1 isoforms and the existence of a regulatory network that controls isoform expression. TIA-1 (T-cell Intracellular Antigen 1) and TIAR (TIA-1-related protein) are RNA-binding proteins involved in the regulation of alternative pre-mRNA splicing and other aspects of RNA metabolism. Various isoforms of these proteins exist in mammals. For example, TIA-1 presents two major isoforms (TIA-1a and TIA-1b) generated by alternative splicing of exon 5 that differ by eleven amino acids exclusive of the TIA-1a isoform. Here we show that the relative expression of TIA-1 and TIAR isoforms varies in different human tissues and cell lines, suggesting distinct functional properties and regulated isoform expression. We report that whereas TIA-1 isoforms show similar subcellular distribution and RNA binding, TIA-1b displays enhanced splicing stimulatory activity compared with TIA-1a, both in vitro and in vivo. Interestingly, TIAR depletion from HeLa and mouse embryonic fibroblasts results in an increased ratio of TIA-1b/a expression, suggesting that TIAR regulates the relative expression of TIA-1 isoforms. Taken together, the results reveal distinct functional properties of TIA-1 isoforms and the existence of a regulatory network that controls isoform expression. Gene expression regulation at the post-transcriptional level is a widespread feature in the human genome. Alternative splicing is a major control mechanism by which a single mRNA precursor (pre-mRNA) can produce different mRNAs in a combinatorial, but not stochastic, manner, contributing significantly in the generation of protein isoform diversity (∼80% of alternative splicing events promote changes in the coded protein) (1Smith C.W. 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The vast majority of human introns present weak splicing signals with nonconsensus features at the splice sites, branch point, and/or polypyrimidine tract (5Matlin A.J. Clark F. Smith C.W. Nat. Rev. Mol. Cell. Biol. 2005; 6: 386-398Crossref PubMed Scopus (978) Google Scholar, 6Johnson J.M. Castle J. Garrett-Engele P. Kan Z. Loerch P.M. Armour C.D. Santos R. Schadt E.E. Stoughton R. Shoemaker D.D. Science. 2003; 302: 2141-2144Crossref PubMed Scopus (1196) Google Scholar, 7Black D.L. Cell. 2000; 103: 367-370Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Thus, often the authentic splicing motifs are indistinguishable from cryptic sequences hidden in human pre-mRNAs, raising the question of how the spliceosomal machinery is able to define an authentic splicing signal. Additional sequence elements known as "enhancers" and "silencers" are often needed to achieve the correct splicing of exons. These regulatory signals, which are located either in exons or introns, can be near (10–100 nucleotides) or very far away (>1000 nucleotides) from the splice sites (15Blencowe B.J. Trends Biochem. Sci. 2000; 25: 106-110Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). The balance between positive and negative regulation of splice site selection likely depends on DNA sequences and changes in the levels of cellular splicing factors under physiological or pathological conditions (2Mendes Soares L.M. Valcárcel J. EMBO J. 2006; 25: 923-931Crossref PubMed Scopus (58) Google Scholar, 4López A.J. Annu. Rev. Genet. 1998; 32: 279-305Crossref PubMed Scopus (539) Google Scholar, 15Blencowe B.J. Trends Biochem. Sci. 2000; 25: 106-110Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, 16Cartegni L. Chew S.L. Krainer A.R. Nat. Rev. Genet. 2002; 3: 285-298Crossref PubMed Scopus (1761) Google Scholar, 17Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1997) Google Scholar, 18Shin C. Manley J.L. Nat. Rev. Mol. Cell. Biol. 2004; 5: 727-738Crossref PubMed Scopus (239) Google Scholar, 19Izquierdo J.M. Valcárcel J. Genes Dev. 2006; 20: 1679-1684Crossref PubMed Scopus (27) Google Scholar). These elements may represent the molecular basis to selectively and specifically regulate alternative splicing of a particular pre-mRNA in different cell types, at different stages of development, or at different stages of cell differentiation and/or proliferation in either normal or pathological conditions (3Garcia-Blanco M.A. Baraniak A.P. Lasda E.L. Nat. Biotechnol. 2004; 22: 535-546Crossref PubMed Scopus (435) Google Scholar, 4López A.J. Annu. Rev. Genet. 1998; 32: 279-305Crossref PubMed Scopus (539) Google Scholar, 10Faustino N.A. Cooper T.A. Genes Dev. 2003; 17: 419-437Crossref PubMed Scopus (992) Google Scholar, 16Cartegni L. Chew S.L. Krainer A.R. Nat. Rev. Genet. 2002; 3: 285-298Crossref PubMed Scopus (1761) Google Scholar). The RNA-binding proteins T-cell intracellular antigen 1 (TIA-1) 2The abbreviations used are: TIA-1, T-cell intracellular antigen 1; TIAR, TIA-1-related protein; NRK, normal rat kidney cell; FGFR-2, fibroblast growth factor receptor 2; HMMP13, human metalloproteinases-13; snRNP, small nuclear ribonucleoprotein; RT-PCR, reverse transcriptase-PCR; GFP, green fluorescent protein; GST, glutathione S-transferase; RRM, RNA recognition motif; siRNA, small interference RNA. and TIA-1-related protein (TIAR) (20Dember L.M. Kim N.D. Liu K.Q. Anderson P. J. Biol. Chem. 1996; 271: 2783-2788Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) contain three RNA recognition motifs (RRMs) and a glutamine-rich carboxyl-terminal domain (21Beck A.R. Medley Q.G. O'Brien S. Anderson P. Streuli M. Nucleic Acids Res. 1996; 24: 3829-3835Crossref PubMed Scopus (83) Google Scholar). 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TIAR can also enhance U6 snRNP assembly on a composite regulatory element consisting of a pseudo-5′-splice site followed by uridines, playing a role in alternative splicing of the calcitonin/CGRP gene (29Zhu H. Hasman R.A. Young K.M. Kedersha N.L. Lou H. Mol. Cell. Biol. 2003; 23: 5959-5971Crossref PubMed Scopus (67) Google Scholar). TIA-1 has also been well characterized as a translational regulator. This protein has been implicated in stress-induced translational arrest, colocalizing after stress with poly(A)+ RNA in the cytoplasmic foci known as stress granules (30Kedersha N.L. Gupta M. Li W. Miller I. Anderson P. J. Cell Biol. 1999; 147: 1431-1442Crossref PubMed Scopus (914) Google Scholar, 31Kedersha N. Anderson P. Biochem. Soc. Trans. 2002; 30: 963-969Crossref PubMed Scopus (550) Google Scholar, 32Anderson P. Kedersha N. J. Cell Biol. 2006; 172: 803-808Crossref PubMed Scopus (876) Google Scholar, 33Gilks N. Kedersha N. Ayodele M. Shen L. Stoecklin G. Dember L.M. Anderson P. Mol. 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We specifically addressed the contribution of TIA-1a and TIA-1b isoforms in the regulation of alternative splicing. The results show that TIA-1b displays enhanced splicing regulatory activity compared with TIA-1a. Furthermore, the expression of TIA-1 and TIAR isoforms is often restricted to certain human tissues and cell lines, suggesting that expression of these proteins and their isoforms is tightly regulated to provide specific functions in splicing regulation. Consistent with this idea, we observe that TIAR influences the relative levels between TIA-1 isoforms. Cell Cultures—HeLa, normal rat kidney (NRK), and embryonic kidney 293 cells as well as mouse embryonic fibroblast wild type knock-out for either TIA-1 or TIAR (35Piecyk M. Wax S. Beck A.R. Kedersha N. Gupta M. Maritim B. Chen S. Gueydan C. Kruys V. Streuli M. Anderson P. EMBO J. 2000; 19: 4154-4163Crossref PubMed Scopus (424) Google Scholar) were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with penicillin-streptomycin and 10% heat-inactivated fetal calf serum (Invitrogen). Constructs—Plasmids containing human TIA-1a cDNA were generated by PCR using Pfu Turbo DNA polymerase (Stratagene) (23Förch P. Puig O. Kedersha N. Martinez C. Granneman S. Seraphin B. Anderson P. Valcárcel J. Mol. Cell. 2000; 6: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). The WT and mutant U-20C Fas minigenes were generated as described previously (23Förch P. Puig O. Kedersha N. Martinez C. Granneman S. Seraphin B. Anderson P. Valcárcel J. Mol. Cell. 2000; 6: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 26Izquierdo J.M. Majós N. Bonnal S. Martínez C. Castelo R. Guigó R. Bilbao D. Valcárcel J. Mol. Cell. 2005; 19: 475-484Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). The FGFR-2 minigene RK97 was kindly provided by Dr. R. Breathnach (22Del Gatto-Konczak F. Bourgeois C.F. Le Guiner C. Kister L. Gesnel M.C. Stevenin J. Breathnach R. Mol. Cell. Biol. 2000; 20: 6287-6299Crossref PubMed Scopus (170) Google Scholar). The constructs used to overexpress TIA-1a and TIA-1b cDNAs were fused in-frame to the carboxyl-terminal part of GFP using vector pEGFP-C1 (Clontech). The sequence of all constructs was verified by automated DNA sequencing. Recombinant Proteins—Recombinant TIA-1a and TIA-1b proteins were expressed in Escherichia coli and purified from bacterial lysates, as glutathione S-transferase fusions (GST), using glutathione-Sepharose (Sigma) as described (23Förch P. Puig O. Kedersha N. Martinez C. Granneman S. Seraphin B. Anderson P. Valcárcel J. Mol. Cell. 2000; 6: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). The recombinant proteins were stored in buffer D (20 mm Hepes, pH 7.6, 20% glycerol, 0.2 mm EDTA, 1 mm dithiothreitol, and 0.01% NP-40) plus 0.1 m KCl at –70 °C until used. The purified fusion proteins were analyzed on a 10% SDS-polyacrylamide gel by staining with Coomassie Blue and Western blot. Protein Analysis—Whole-cell extracts were prepared by resuspending the cells in lysis buffer (50 mm Tris-HCl, pH 8.0, 140 mm NaCl, 1.5 mm MgCl2, 0.5% Nonidet P-40 plus a mixture of protease inhibitors), freeze-thawing three times, and centrifugation at 10,000 rpm for 5 min in a microfuge at 4 °C. Resulting supernatants were recovered and stored at –70 °C. Protein concentration was determined with the Bradford reagent (Bio-Rad protein assay) using bovine serum albumin as standard. For Western blot analysis, equal amounts of protein from the supernatants were loaded on 10% SDS-PAGE gels and Western blots carried out using nylon membranes and the following antibodies: anti-TIA-1 (C-20; Santa Cruz Biotechnology), anti-TIAR (C-18; Santa Cruz Biotechnology), anti-GFP (Clontech), anti-α-tubulin (Sigma), and anti-U2AF65 (MC3) (52Gama-Carvalho M. Krauss R.D. Chiang L. Valcárcel J. Green M.R. Carmo-Fonseca M. J. Cell Biol. 1997; 137: 975-987Crossref PubMed Scopus (111) Google Scholar). The blots were developed using ECL reagent (Amersham Biosciences) according to the manufacturer's instructions. Immobilon INSTA-Blot™ membranes containing samples of several human tissues (brain, heart, small intestine, kidney, liver, lung, skeletal muscle, testes, spleen, pancreas, and ovary) and cell lines (HeLa, Jurkat, Daudi, 293, Rh 30, A375, T98G, HCT-116, and Hep-G2) were obtained from Calbiochem. Total RNAs from human tissues were obtained from Ambion and Clontech. Fluorescence Microscopy—After transfection (24 h), the cells were analyzed by fluorescence microscopy using a confocal microscope (ZEISS) equipped with a GFP excitation filter. Digital image acquisition and processing were performed with a ZEISS camera and ZEISS software package, respectively. Preparation of HeLa Nuclear Extracts and Psoralen-mediated Cross-linking Assays—HeLa nuclear extract was prepared as described previously by Dignam et al. (53Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Psoralen-mediated cross-linking assays were carried out as reported previously (26Izquierdo J.M. Majós N. Bonnal S. Martínez C. Castelo R. Guigó R. Bilbao D. Valcárcel J. Mol. Cell. 2005; 19: 475-484Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Band Shift Assays—5′-32P-end-labeled synthetic msl-2 RNA oligo containing 10 nucleotides of exonic sequence and 36 nucleotides of intronic sequence or 32P-full-labeled Fas RNA probe containing 23 nucleotides of human Fas exon 6 and 35 nucleotides of human Fas intron 6 5′-splice site were incubated with different molar amounts of GST-TIA-1a or GST-TIA-1b (from 10–8 to 10–6 m) in a total volume of 15 μl of buffer D with 0.1 m KCl in the presence of tRNA (200 ng/μl) for 30 min on ice. After incubation, the samples were resolved by electrophoresis on non-denaturing 5% polyacrylamide gels, dried, and exposed to film (26Izquierdo J.M. Majós N. Bonnal S. Martínez C. Castelo R. Guigó R. Bilbao D. Valcárcel J. Mol. Cell. 2005; 19: 475-484Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). siRNA Duplex Preparation and Transfections of siRNAs—The sequences of the 21-nt RNA oligonucleotides used for targeting TIA-1 and TIAR were synthesized and annealed as described previously (26Izquierdo J.M. Majós N. Bonnal S. Martínez C. Castelo R. Guigó R. Bilbao D. Valcárcel J. Mol. Cell. 2005; 19: 475-484Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 41Izquierdo J.M. Biochem. Biophys. Res. Commun. 2006; 348: 703-711Crossref PubMed Scopus (25) Google Scholar). HeLa cells were transfected at 30% confluency with 5 μg of siRNAs by using Lipofectin (Invitrogen). Cells were then incubated 72 h before protein extract preparation and RNA purification as described (26Izquierdo J.M. Majós N. Bonnal S. Martínez C. Castelo R. Guigó R. Bilbao D. Valcárcel J. Mol. Cell. 2005; 19: 475-484Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 41Izquierdo J.M. Biochem. Biophys. Res. Commun. 2006; 348: 703-711Crossref PubMed Scopus (25) Google Scholar). DNA Transfections, RNA Isolation, and RT-PCR Analysis—Exponentially grown HeLa and 293 cells were transfected at 50–70% confluency with 0.5 μg of WT/mutant (U-20C) Fas or FGFR-2 minigenes, respectively, and 1.5 μg of GFP-reporter plasmids using Lipofectin (Invitrogen) following the manufacturer's instructions for adherent cell lines. Cells were then incubated 24–48 h before protein and RNA purifications. Cytoplasmic RNAs were prepared using the RNeasy kit (Qiagen), quantified by OD at 260 nm, and analyzed by RT-PCR. After 25 cycles, the products were analyzed on 2% agarose gels. Control experiments with different input amounts of RNA indicated that the amplification was quantitative under these conditions (23Förch P. Puig O. Kedersha N. Martinez C. Granneman S. Seraphin B. Anderson P. Valcárcel J. Mol. Cell. 2000; 6: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 26Izquierdo J.M. Majós N. Bonnal S. Martínez C. Castelo R. Guigó R. Bilbao D. Valcárcel J. Mol. Cell. 2005; 19: 475-484Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). The oligonucleotide sequences used in RT-PCR analysis were: hTIA-1.sense (5′-GCCCAAGACTCTATACGTCGGTAACC-3′) and hTIA-1.antisense (5′-GGTGCAAAAGCAGCTTTTATATCTTC-3′) for human TIA-1 gene; hTIAR.sense (5′-ATGATGGAAGACGACGGGCAGCCCCGGACTC-3′) and hTIAR. antisense (5′-TCTGGACTCAAATCCCCAACAAACACATGG-3′) for human TIAR gene; hβ-actin.sense (5′-AAAGACCTGTACGCCAACAC-3′) and hβ-actin.antisense (5′-GTCATACTCCTGCTTGCTGA-3′) for human β-actin gene; mTIA-1.sense (5′-GCCCAAGACTCTATACGTCGG-3′) and mTIA-1.antisense (5′-GGTGCAAACGCTGCTTTGATG-3′) for mouse TIA-1 gene; mTIAR.sense (5′-ATGGAAGACGACGGACAGCCC-3′) and mTIAR.antisense (5′-AATTTCTGGACTCAAATCCCC-3′) for mouse TIAR gene. PT1 and PT2 as well as P3 and P4 oligos to analyze alternatively spliced products from WT/mutant Fas and FGFR-2 minigenes were described previously (22Del Gatto-Konczak F. Bourgeois C.F. Le Guiner C. Kister L. Gesnel M.C. Stevenin J. Breathnach R. Mol. Cell. Biol. 2000; 20: 6287-6299Crossref PubMed Scopus (170) Google Scholar, 23Förch P. Puig O. Kedersha N. Martinez C. Granneman S. Seraphin B. Anderson P. Valcárcel J. Mol. Cell. 2000; 6: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 26Izquierdo J.M. Majós N. Bonnal S. Martínez C. Castelo R. Guigó R. Bilbao D. Valcárcel J. Mol. Cell. 2005; 19: 475-484Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Human TIA-1 isoforms are generated from a unique gene by exon 5 alternative splicing (Fig. 1A). Exon 5 inclusion generates TIA-1 isoform a, whereas exon skipping produces the shorter b isoform (Fig. 1A). The eleven-amino acid peptide encoded by exon 5 is located between RRMs 1 and 2 (Fig. 1B). Antibodies specific for TIA-1 or TIAR proteins were used to evaluate their relative levels of expression in human tissues and cell lines. Tissue-specific differences in expression were observed for both proteins as well as differences between their respective patterns of expression (Fig. 2); when the resolution of the blot was sufficient to distinguish different isoforms as species of different mobility, their relative expression also varied. For example, whereas the levels of TIA-1 expression in brain tissue are low compared with kidney, testes, or ovaries, the opposite trend is observed for TIAR (Fig. 2, A and B). To more clearly document changes in isoform expression, reverse transcriptase and polymerase chain reaction (RT-PCR) analyses were performed. Differences in TIA-1a versus b ratios were observed (compare for example kidney versus testis, lanes 5 and 9 of the upper panel in Fig. 2C). Although changes in mRNA abundance generally correlated with protein levels for TIA-1 (compare Fig. 2A and the upper panel of 2C), this was not always the case for TIAR, suggesting the operation of additional mechanisms of translational or protein stability control. The analysis of cell lines provided additional evidence for differences in isoform expression (Fig. 2, D and E). For example, isoform a was predominant in most cell lines expressing TIA-1, whereas isoform b was more abundant in the T cell Jurkat cell line (Fig. 2D). TIAR isoform a was predominant in Hep-G2 cells and isoform b in HCT-116, whereas the other cell lines tested expressed the two isoforms in roughly a 1:1 ratio. Changes in isoform expression ratio are also detectable in tissue samples (Fig. 2, A–C). Taken together, these observations suggest that TIA-1 and TIAR proteins and their isoforms are differentially expressed in different cell types. To further document these observations and their potential functional implications, expression of these splicing regulators was analyzed in HeLa and NRK cells in parallel with the patterns of alternative sp
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