Heterogeneous Ribonucleoprotein M Is a Splicing Regulatory Protein That Can Enhance or Silence Splicing of Alternatively Spliced Exons
2007; Elsevier BV; Volume: 282; Issue: 50 Linguagem: Inglês
10.1074/jbc.m704188200
ISSN1083-351X
AutoresRuben H. Hovhannisyan, Russ P. Carstens,
Tópico(s)RNA and protein synthesis mechanisms
ResumoSplicing of fibroblast growth factor receptor 2 (FGFR2) alternative exons IIIb and IIIc is regulated by the auxiliary RNA cis-element ISE/ISS-3 that promotes splicing of exon IIIb and silencing of exon IIIc. Using RNA affinity chromatography, we have identified heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a splicing regulatory factor that binds to ISE/ISS-3 in a sequence-specific manner. Overexpression of hnRNP M promoted exon IIIc skipping in a cell line that normally includes it, and association of hnRNP M with ISE/ISS-3 was shown to contribute to this splicing regulatory function. Thus hnRNP M, along with other members of the hnRNP family of RNA-binding proteins, plays a combinatorial role in regulation of FGFR2 alternative splicing. We also determined that hnRNP M can affect the splicing of several other alternatively spliced exons. This activity of hnRNP M included the ability not only to induce exon skipping but also to promote exon inclusion. This is the first report demonstrating a role for this abundant hnRNP family member in alternative splicing in mammals and suggests that this protein may broadly contribute to the fidelity of splice site recognition and alternative splicing regulation. Splicing of fibroblast growth factor receptor 2 (FGFR2) alternative exons IIIb and IIIc is regulated by the auxiliary RNA cis-element ISE/ISS-3 that promotes splicing of exon IIIb and silencing of exon IIIc. Using RNA affinity chromatography, we have identified heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a splicing regulatory factor that binds to ISE/ISS-3 in a sequence-specific manner. Overexpression of hnRNP M promoted exon IIIc skipping in a cell line that normally includes it, and association of hnRNP M with ISE/ISS-3 was shown to contribute to this splicing regulatory function. Thus hnRNP M, along with other members of the hnRNP family of RNA-binding proteins, plays a combinatorial role in regulation of FGFR2 alternative splicing. We also determined that hnRNP M can affect the splicing of several other alternatively spliced exons. This activity of hnRNP M included the ability not only to induce exon skipping but also to promote exon inclusion. This is the first report demonstrating a role for this abundant hnRNP family member in alternative splicing in mammals and suggests that this protein may broadly contribute to the fidelity of splice site recognition and alternative splicing regulation. Alternative splicing represents an important mechanism whereby a single gene transcript can give rise to numerous spliced mRNAs, thereby greatly expanding the ribonomic and proteomic diversity that can be obtained from a limited gene number (1Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1947) Google Scholar, 2Matlin A.J. Clark F. Smith C.W. Nat. Rev. Mol. Cell Biol. 2005; 6: 386-398Crossref PubMed Scopus (959) Google Scholar). Despite increasing recognition that the majority of metazoan gene transcripts are subject to alternative splicing, the molecular mechanisms that regulate this process remain poorly understood. This is particularly true for alternatively spliced gene transcripts that display tightly regulated expression of distinct splice variants in different cell types or at different stages of development. The constitutive splicing process is directed by consensus sequences at the 5′ and 3′ splice sites as well as a branch point sequence generally located 20–40 nucleotides (nt) 2The abbreviations used are:ntnucleotide(s)FGFR2fibroblast growth factor receptor 2ISEintronic splicing enhancerISSintronic splicing silencerhnRNPheterogeneous ribonucleoproteinCELFCUG-BP and ETR-3-like factorBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolRTreverse transcriptionPPTpreprotachykinin. 2The abbreviations used are:ntnucleotide(s)FGFR2fibroblast growth factor receptor 2ISEintronic splicing enhancerISSintronic splicing silencerhnRNPheterogeneous ribonucleoproteinCELFCUG-BP and ETR-3-like factorBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolRTreverse transcriptionPPTpreprotachykinin. upstream of the 3′ splice site. However, these sequence determinants are not sufficient to accurately specify the actual splice sites that are used for splicing of constitutive as well as alternative exons. In recent years, numerous studies have demonstrated an important role for an additional layer of cis-acting control sequences often referred to as auxiliary cis-elements (2Matlin A.J. Clark F. Smith C.W. Nat. Rev. Mol. Cell Biol. 2005; 6: 386-398Crossref PubMed Scopus (959) Google Scholar, 3Ladd A.N. Cooper T.A. Genome Biology. 2002; http://genomebiology.com/2002/3/11/reviews/0008PubMed Google Scholar). These sequences have been identified in both exons and introns and can have either positive or negative effects on splicing of a given exon. As such they are commonly referred to as exonic splicing enhancers or silencers and intronic splicing enhancers or silencers (ISEs or ISSs). These elements function largely through the activity of RNA-binding proteins that associate with them and influence the efficiency with which nearby splice sites are recognized by the basal splicing machinery. Most alternatively spliced exons are associated with multiple auxiliary cis-elements that can positively or negatively affect its inclusion. These observations have led to models of combinatorial control, in which the splicing outcome is determined by the net effect of several cis-elements that interact with differentially expressed RNA-binding proteins in varied cellular milieus (1Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1947) Google Scholar, 2Matlin A.J. Clark F. Smith C.W. Nat. Rev. Mol. Cell Biol. 2005; 6: 386-398Crossref PubMed Scopus (959) Google Scholar, 4Smith C.W. Valcarcel J. Trends Biochem. Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar). Although several cell-type-specific factors have previously been described, differential expression of such factors alone has not proven sufficient to account for cell-type-specific splicing decisions, which also involve contributions from ubiquitously expressed regulatory factors. Thus a challenge in the field of splicing regulation has been to determine the different contributions of numerous cis-elements and RNA-binding proteins and how their combined functions yield distinct splicing patterns in different cells.Our studies have focused on alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts. Mutually exclusive splicing of two exons, IIIb and IIIc, gives rise to two functionally different receptors, FGFR2-IIIb and FGFR2-IIIc, in epithelial and mesenchymal cells, respectively. These exons encode the C-terminal half of an Ig-like domain in the receptor's extracellular domain in a region that determines ligand-binding specificity. Consequently, the two different receptor isoforms display distinct binding preferences for the FGF family of ligands (5Ornitz D.M. Xu J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1414) Google Scholar, 6Yeh B.K. Igarashi M. Eliseenkova A.V. Plotnikov A.N. Sher I. Ron D. Aaronson S.A. Mohammadi M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2266-2271Crossref PubMed Scopus (146) Google Scholar). Several auxiliary cis-elements that have been shown to play a role in regulation of these alternative exons are shown in Fig. 1A. Although several abundant and ubiquitously expressed factors, including polypyrimidine tract-binding protein, TIA-1, and hnRNP A1, have been shown to bind to several of these elements and play a role in splicing regulation, the mechanism by which cell-type-specific alternative splicing of this transcript occurs has not been fully elucidated (7Carstens R.P. McKeehan W.L. Garcia-Blanco M.A. Mol. Cell Biol. 1998; 18: 2205-2217Crossref PubMed Scopus (85) Google Scholar, 8Carstens R.P. Wagner E.J. Garcia-Blanco M.A. Mol. Cell Biol. 2000; 20: 7388-7400Crossref PubMed Scopus (124) Google Scholar, 9Hovhannisyan R.H. Carstens R.P. Mol. Cell Biol. 2005; 25: 250-263Crossref PubMed Scopus (27) Google Scholar, 10Baraniak A.P. Chen J.R. Garcia-Blanco M.A. Mol. Cell Biol. 2006; 26: 1209-1222Crossref PubMed Scopus (93) Google Scholar, 11Del 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 (167) Google Scholar, 12Del Gatto-Konczak F. Olive M. Gesnel M.C. Breathnach R. Mol. Cell Biol. 1999; 19: 251-260Crossref PubMed Scopus (196) Google Scholar, 13Le Guiner C. Lejeune F. Galiana D. Kister L. Breathnach R. Stevenin J. Del Gatto-Konczak F. J. Biol. Chem. 2001; 276: 40638-40646Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 14Le Guiner C. Plet A. Galiana D. Gesnel M.C. Del Gatto-Konczak F. Breathnach R. J. Biol. Chem. 2001; 276: 43677-43687Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). We have focused our recent studies on an element we have termed ISE/ISS-3, so named because it has been shown to promote splicing of exon IIIb (as an ISE) and at the same time to repress splicing of exon IIIc (as an ISS) in epithelial cells (9Hovhannisyan R.H. Carstens R.P. Mol. Cell Biol. 2005; 25: 250-263Crossref PubMed Scopus (27) Google Scholar, 15Hovhannisyan R.H. Warzecha C.C. Carstens R.P. Nucleic Acids Res. 2006; 34: 373-385Crossref PubMed Scopus (14) Google Scholar). Thus in cells that express FGFR2-IIIb (e.g. DT3 cells) deletion of this element in transfected FGFR2 minigenes results in a partial switch from exon IIIb splicing to exon IIIc splicing, whereas the exclusive inclusion of exon IIIc in cells that express FGFR2-IIIc (e.g. AT3 or 293T cells) is unaffected by the same deletion. We have also shown that this sequence displays epithelial cell-type-specific functions in a heterologous minigene context, suggesting that it may associate with at least one regulatory factor whose expression is limited to cells expressing FGFR2-IIIb (15Hovhannisyan R.H. Warzecha C.C. Carstens R.P. Nucleic Acids Res. 2006; 34: 373-385Crossref PubMed Scopus (14) Google Scholar). In the present study we describe one of the proteins, heterogeneous ribonucleoprotein M (hnRNP M), that binds directly and specifically to the wild-type ISE/ISS-3 sequence. In a cell line that normally includes exon IIIc (293T), we determined that overexpression of hnRNP M induced exon IIIc skipping, suggesting that this factor plays a role in silencing of this exon in cells that express FGFR2 IIIb. Transfection studies using minigenes containing several unrelated alternative exons showed that hnRNP M can induce either exon skipping or exon inclusion in different contexts. Therefore, we have identified hnRNP M as a factor involved in FGFR2 splicing regulation, and we propose that this factor may also play a more general role in the regulation of diverse alternatively spliced metazoan gene transcripts.EXPERIMENTAL PROCEDURESPlasmid Construction—The plasmids and minigenes used in this study were constructed using standard cloning techniques. pI-11-FS, pI-11-FS-CXS, and pI-11-FS-CXS-IIIb Mut minigenes and their derivatives were previously described (9Hovhannisyan R.H. Carstens R.P. Mol. Cell Biol. 2005; 25: 250-263Crossref PubMed Scopus (27) Google Scholar, 16Muh S.J. Hovhannisyan R.H. Carstens R.P. J. Biol. Chem. 2002; 277: 50143-50154Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The plasmid minigene containing the R35 (28Cooper T.A. Methods Mol. Biol. 1999; 118: 391-403PubMed Google Scholar) exon was generated by PCR of the R35 (28Cooper T.A. Methods Mol. Biol. 1999; 118: 391-403PubMed Google Scholar) plasmid (kindly provided by Thomas Cooper) using primers TNT-Xba-F (5′-GGCCTCTAGAGTCGAAGTCCTCACCTGGTG-3′) and TNT-Not-R (5′-GCGCGGCCGCACTAGTTTAGAGGGGAGAGCGC-3′). This amplified a SalI to SpeI segment from the original construct that contained the exon and cTNT-derived intronic sequences (17Cooper T.A. Mol. Cell Biol. 1998; 18: 4519-4525Crossref PubMed Scopus (43) Google Scholar). This fragment was inserted into the XbaI and NotI sites of the pI-11(-H3)-PL vector (9Hovhannisyan R.H. Carstens R.P. Mol. Cell Biol. 2005; 25: 250-263Crossref PubMed Scopus (27) Google Scholar), thereby positioning the exon between two adenoviral derived flanking exons. The preprotachykinin minigene pPPT-106 (kindly provided by Gil Cote) was previously described (18Jin W. Huang E.S. Bi W. Cote G.J. J. Biol. Chem. 1999; 274: 28035-28041Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). For in vitro transcription we modified the pDP19 vector (Ambion) by inserting the sequence 5′-GAATTATCGATCTCGAG-3′ between the EcoRI and HindIII sites to generate pDP19RC-ΔEE. This modification permitted insertion of ClaI/XhoI fragments containing ISE/ISS-3 (or control sequences) under the control of the T7 promoter for in vitro transcription. To obtain the 3×WT and 3×MT inserts, the two pairs of primers were kinased and annealed: 43nt-ISE3-Sal-Xho-F (5′-TCGACGTGTGGTGATGGGCCTGCAGAGGTGAGCTGGCCGGTGTCTCTCC-3′) with 43nt-ISE3-Sal-Xho-R (5′-TCGAGGAGAGACACCGGCCAGCTCACCTCTGCAGGCCCATCACCACACG-3′), and 43nt-3xGU Mut-Sal-Xho-F (5′-TCGACGTGTGACGATGGGCCTGCAGAGACGAGCTGGCCGACGTCTCTCC-3′) with 43nt-3xGU Mut-Sal-Xho-R (5′-TCGAGGAGAGACGTCGGCCAGCTCGTCTCTGCAGGCCCATCGTCACACG-3′). The annealed oligonucleotides were ligated, digested by XhoI and SalI, and separated on polyacrylamide gels to select three tandem copies of the sequence. Thus we obtained two new constructs, one containing three repeats of wild-type 43-nt sequence from ISE/ISS-3 (3×WT) and another containing three repeats of mutant 43-nt sequence from ISE/ISS-3 in which three GU-AC substitutions occurred in each repeat (3×MT) flanked by ClaI and XhoI sites (Fig. 1C). To create the templates for pulldown assays PCR of these sequences was performed to introduce a ClaI site at the 5′-end of the sequence, and the products were re-cloned as ClaI/XhoI fragments into the DP19RC-ΔEE plasmid. Similarly, the previously described "full-length" ISE/ISS-3, 5′-AC, and BS fragments were also inserted in pDP19RC-ΔEE (Fig. 1B). To make the construct for expression of hnRNP M, pIN-ΔInt-hnRNP M, an EcoRV-AgeI fragment from the pHCM4 construct (kindly provided by Maurice Swanson) was inserted in the previously described pIRESneo3-ΔInt vector (15Hovhannisyan R.H. Warzecha C.C. Carstens R.P. Nucleic Acids Res. 2006; 34: 373-385Crossref PubMed Scopus (14) Google Scholar, 19Newman E.A. Muh S.J. Hovhannisyan R.H. Warzecha C.C. Jones R.B. McKeehan W.L. Carstens R.P. RNA (N.Y.). 2006; 12: 1129-1141Crossref PubMed Scopus (41) Google Scholar). Generation of N-terminal FLAG-tagged hnRNP M was carried out by inserting the same EcoRV-AgeI fragment into the FLAG expression vector pINX-N-FF-B. The latter construct was derived by insertion of sequences encoding two tandem copies of the FLAG tag upstream of the multicloning site of pIRESneo3ΔInt (sequence available on request). All plasmid constructs were prepared with Qiagen MidiPrep kits. Sequences were confirmed by the University of Pennsylvania Sequencing Facility.UV Cross-linking—To prepare RNA probes for UV-cross-linking experiments we performed in vitro transcription with T7 RNA polymerase (Ambion) after digestion of the pDP-based plasmids with XhoI using the manufacturer's recommendations. The specific activity of the 169-nt RNA transcript was 2.7 × 1013 cpm/μmol. 500,000 cpm, or 19 fmol, of [32P]UTP-radiolabeled RNA substrates were incubated at 30 °C with 12–20 μg of KATO III or 293T cell nuclear extracts in a volume of 20 μl for 20 min in 1.5-ml Eppendorf tubes. The nuclear extracts from 293T cell transiently expressing the FLAG-tagged plasmids were generated by transfection of 5 μg of plasmid in 60-mm plates using TransIT-293 Transfection Reagent (Mirus) in accordance with manufacturer recommendations. 48 h after transfection, nuclear extracts were prepared following a previously described protocol (20Tsai A. Carstens R.P. Nat. Protoc. 2006; 1: 2820-2827Crossref PubMed Scopus (41) Google Scholar). After incubation, the tubes were opened, placed on ice, and UV-irradiated in a Stratalinker 2400 (Stratagene) at a distance of 5 cm with an energy of 6000 μJ × 100. After addition of 2 μl of a stock RNase mix (10 mg/ml RNase A and 10 units/μl RNase T1) the samples were incubated at 37 °C for 60 min. Reactions were stopped with 4× LDS sample buffer (Invitrogen) and loaded onto 10% NuPAGE Bis-Tris gel (Invitrogen). The gel was run at constant 200 V for 60–70 min, then fixed, dried, and visualized by radiography.Immunoprecipitation of UV Cross-linked Proteins—Immunoprecipitation of UV-cross-linked proteins was carried out following the RNase step of the UV cross-linking protocol. Precipitation of endogenous hnRNP M from KATO III nuclear extracts was performed by first pre-clearing the UV-cross-linked extracts by addition to 30 μl of pre-washed Protein G-agarose (Roche Applied Science) in 450 μl of NET-2 buffer (50 mm Tris-Cl, pH 7.6, 150 mm NaCl, 0.01% Nonidet P-40, 1× protease inhibitor (Sigma)) at 4 °C and rotation for 30 min. The pre-cleared supernatant was then harvested after centrifugation at 1000 × g for 1 min. 1.5 μl of hnRNP M antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the precleared extract and rotated at 4 °C for 60 min and then added to another 30 μl of pre-washed protein G-agarose and incubated for an additional 60 min. The beads were then washed three times with 500 μl of NET-2 buffer, and the precipitated proteins were eluted by addition of 20 μl of 2× sample loading buffer and boiling for 5 min. The mix was transferred to a Bio-Rad microspin column, centrifuged at 500 × g for 5 min, electrophoresed on 10% NuPAGE Bis-Tris gels, and analyzed by Western blotting. Immunoprecipitation of FLAG-tagged hnRNP M from transiently transfected 293T cell nuclear extracts was carried out by addition of the UV-cross-linked extracts to 40 μl of pre-washed anti-FLAG M2-agarose slurry (Sigma) in 400 μl of cold NET-2 buffer. After rotation at 4 °C for 60 min, the beads were washed twice with 400 μl of NET-2 buffer and eluted by incubating for 30 min with rotation at 4 °C with 10 μg of 3× FLAG peptide in 60 μl of NET-2 buffer. The eluted proteins were harvested using Bio-Rad microspin columns and analyzed by Western as described below.Affinity Purification—To obtain microgram quantities of unlabeled RNA for affinity chromatography in vitro transcription reactions were performed in a 50-μl volume containing 0.1 μg/μl linearized DNA template, 2 units/μl T7 RNA polymerase (Ambion), 7.5 mm NTPs, 0.1 unit/μl inorganic pyrophosphatase (Sigma), 0.4 unit/μl SUPERase-In (Ambion) in buffer containing 80 mm Hepes-KCl, pH 7.5, 24 mm MgCl2, 2 mm spermidine, and 40 mm dithiothreitol. The reaction mix was incubated overnight at 37 °C and RNA was gel-purified from 10% denaturing acrylamide gels. Pulldown assays were performed similar to previously described protocols (21Buratti E. Dork T. Zuccato E. Pagani F. Romano M. Baralle F.E. EMBO J. 2001; 20: 1774-1784Crossref PubMed Scopus (494) Google Scholar, 22Caputi M. Freund M. Kammler S. Asang C. Schaal H. J. Virol. 2004; 78: 6517-6526Crossref PubMed Scopus (75) Google Scholar), with the following modifications. A 200-μl reaction mixture containing 1 nmol of RNA, 100 mm NaOAc, pH 5.0, and 20 mm sodium m-periodate (Sigma) was incubated in the dark at room temperature for 1 h. The oxidized RNA was precipitated with ethanol and resuspended in 500 μl of 100 mm NaOAc, pH 5.0. 200 μl of adipic acid dehydrazide bead slurry (100 μl of packed beads, Sigma) were washed twice with 1 ml of 100 mm NaOAc, pH 5.0. The resuspended RNA was added to the beads and rotated overnight at 4 °C. The beads with bound RNA were then washed twice with 1 ml of 2 m NaCl and three times with 1 ml of Buffer D (23Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9143) Google Scholar) freshly supplemented with 0.5 mm dithiothreitol and 1× phenylmethylsulfonyl fluoride. A 1-ml final volume of a mix containing 500 μl of KATO III nuclear extract in the presence of 1.6 mm MgCl2, and 5 mg/ml heparin was added to the washed beads. After rotating for 30 min at room temperature the beads were washed four times with 1 ml of buffer D supplemented with 2 mm MgCl2 and 75 μg/ml heparin. The proteins were eluted by addition of 60 μl of 1.5× SDS loading buffer and heating at 90 °C for 5 min. Beads and buffer were transferred to Handee spin columns (Pierce) and centrifuged at 1000 × g for 1 min. Eluted samples were loaded on 4–12% NuPAGE Bis-Tris gel (Invitrogen). The gel was stained using either SilverSNAP (Pierce) or Colloidal Blue staining kit (Invitrogen), and the bands of interest were excised and submitted for analysis using tandem mass spectrometry performed by the University of Pennsylvania Proteomics Core Facility.Western Blotting—To detect antibodies against hnRNP M we used the standard technique described previously (24Geng J. Carstens R.P. Protein Expr. Purif. 2006; 48: 142-150Crossref PubMed Scopus (10) Google Scholar), with modifications. The nitrocellulose filter was blocked with 2.5% Carnation milk overnight at 4 °C. The primary antibody was used at a 1:200 dilution for mouse monoclonal hnRNP M1–4 (Santa Cruz Biotechnology) or 1:1,000 dilution for monoclonal anti-FLAG M2 (Sigma). The secondary anti-mouse Ig, horseradish peroxidase-linked whole antibody (Amersham Biosciences) was used at a 1:10,000 dilution.Cell Culture, Transfection, RNA Purification, and RT-PCR Analysis—These procedures were performed as previously described (9Hovhannisyan R.H. Carstens R.P. Mol. Cell Biol. 2005; 25: 250-263Crossref PubMed Scopus (27) Google Scholar). For transient co-transfections, 293T cells were co-transfected in 12-well plates with 2 μg of total amount of plasmid (1 μg of a minigene and either 0.3, 0.5, or 1.0 μg of pIN-ΔInt-hnRNP M, normalized to 1 μg with empty vector using Lipofectamine 2000 (Invitrogen)). RNA was harvested 48 h after transfection. RNA from stably transfected AT3 and DT3 cells was prepared after 2 weeks of growth in selective media. Quantification was performed using a Molecular Dynamics PhosphorImager. Primers pI-11(-H3)-F and PIP11-R (16Muh S.J. Hovhannisyan R.H. Carstens R.P. J. Biol. Chem. 2002; 277: 50143-50154Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) were used to assay for inclusion or skipping of exon IIIc and exon R35 (28Cooper T.A. Methods Mol. Biol. 1999; 118: 391-403PubMed Google Scholar) in pI-11-FS, pI-11-FS-CXS-IIIb-Mut, and pI-11-R35 (28Cooper T.A. Methods Mol. Biol. 1999; 118: 391-403PubMed Google Scholar) constructs and T7 (5′-TAATACGACTCACTATAGGG-3′) and Rat-PPT-Ex5-R (5′-GTGAGAGATCTGACCATGCC-3′) primers for assay of inclusion or skipping of PPT exon 4 in the pPPT-106 construct. Molar amounts of final products where alternatively spliced exons were either included or skipped were normalized using exon included/exon excluded ratios: 1:2.05 for pI-11-FS and pI-11-FS-CXS-IIIb Mut constructs; 1:1.21 for pI-XN-R35 (28Cooper T.A. Methods Mol. Biol. 1999; 118: 391-403PubMed Google Scholar); and 1:1.23 for pPPT-106.RESULTSIdentification of Sequences in the 5′ Half of ISE/ISS-3 That Are Sufficient to Promote Exon IIIb Inclusion and Exon IIIc Skipping in Cells That Express FGFR2-IIIb—Using stable transfection minigene assays, we previously carried out extensive mutational analysis of an 85-nt sequence constituting ISE/ISS-3 and determined that mutations or deletions within the 5′-end of the sequence were more detrimental to its function than alterations at the 3′-end (Fig. 1B) (9Hovhannisyan R.H. Carstens R.P. Mol. Cell Biol. 2005; 25: 250-263Crossref PubMed Scopus (27) Google Scholar, 15Hovhannisyan R.H. Warzecha C.C. Carstens R.P. Nucleic Acids Res. 2006; 34: 373-385Crossref PubMed Scopus (14) Google Scholar). These experiments were carried out using an FGFR2 minigene, pI-11-FS-CXS, that contains both exons IIIb and IIIc and their flanking introns, but in which ISE/ISS-3 is deleted and replaced by two convenient restriction enzymes (Fig. 2A). Transfection of the DT3 cell line, which expresses endogenous FGFR2-IIIb, with pI-11-FS-CXS demonstrated predominant splicing of exon IIIc (Fig. 2B, lanes 1–3). Analysis of results with these minigenes, performed by RT-PCR using primers complementary to sequences in the exons flanking exons IIIb and IIIc, revealed a product of 386 or 383 bp depending on whether exon IIIb or exon IIIc was spliced, respectively. Because this size difference is difficult to distinguish, we digest these RT-PCR products separately with AvaI or HincII, which digest products containing exon IIIb or IIIc, respectively. Comparison of the fraction of products digested with these enzymes can therefore be used to determine the percentage of products that contain exon IIIb or IIIc. Transfection of a minigene in which ISE/ISS-3 has been restored to its normal position results in predominant splicing of exon IIIb and skipping of exon IIIc (Fig. 2B, lanes 4–6). Insertion of three tandem copies of the 43 nt that comprise the 5′ half of ISE/ISS-3 (3×WT) (Fig. 1C) likewise restores predominant exon IIIb inclusion (Fig. 2B, lanes 7–9). However, insertion of the same tandem copies containing the GU to AC mutations shown in Fig. 1C (3×MT) yields predominant exon IIIc splicing in transfected DT3 cells (Fig. 2B, lanes 10–12). Importantly, these results demonstrating the function of ISE/ISS-3 in promoting exon IIIb inclusion and exon IIIc skipping are only observed in cells that express endogenous FGFR2-IIIb. Transfection of these minigenes into cells that express endogenous FGFR2-IIIc (e.g. AT3 and 293T) results in exclusive splicing of exon IIIc, and this outcome is observed in either the presence or absence of ISE/ISS-3 (data not shown).FIGURE 2Tandem repeats of the 5′ half of ISE/ISS-3 mediate cell-type-specific activation of exon IIIb and silencing of exon IIIc. A, schematic of the pI-11-FS-CXS minigene. Boxes represent exons, and solid lines represent introns. Alternatively spliced exons with their sizes (in nt) are indicated as shaded boxes. The ClaI and XhoI sites in the parental vector replace ISE/ISS-3, and this plasmid version is thus designated "No insert." U and D indicate heterologous adenoviral exons upstream and downstream of exons IIIb and IIIc, respectively. The 85-nt full-length ISE/ISS-3, 3×WT, or 3×MT sequences were inserted as indicated between the ClaI and XhoI sites. B, results of RT-PCR of RNA from DT3 cells stably transfected with the indicated minigenes. U, undigested product; A, product digested with AvaI; H, product digested with HincII, shown above each lane. M, molecular weight markers. The RT-PCR products that contain either exon IIIb or exon IIIc (U-B/C-D) or exclude these exons (U-D) are schematized at the right. The percent exon IIIb inclusion is indicated beneath results for each minigene. Quantification represents the percentage of products that include exon IIIb (the band remaining after HincII digestion) divided by the sum of these products and products, including exon IIIc (the band remaining after AvaI digestion).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Identification of hnRNP M as an ISE/ISS-3-binding Protein—Given the important role of ISE/ISS-3 in mediating cell-type-specific splicing of FGFR2 exon IIIb in lieu of exon IIIc, we carried out biochemical assays to identify factors that bound specifically to this regulatory element. To characterize protein factors that bind ISE/ISS-3 we initially carried out in vitro UV-cross-linking studies using radiolabeled RNAs containing the three tandem copies of the 5′ half of ISE/ISS-3 (3×WT), the same tandem copies containing mutations (3×MT), or unrelated control RNA sequences. For our studies, we used nuclear extracts prepared from a cell line, KATO III, that also expresses FGFR2-IIIb as such extracts are expected to express the full complement of factors required for expression of this isoform, including potential cell-type-specific factors (9Hovhannisyan R.H. Carstens R.P. Mol. Cell Biol. 2005; 25: 250-263Crossref PubMed Scopus (27) Google Scholar). After UV-cross-linking and RNase digestion, SDS-PAGE analysis was carried out to identify the molecular masses of proteins that cross-linked to the 3×WT probe, but not to the 3×MT control. As shown in Fig. 3A, several proteins that cross-linked specifically to the 3×WT RNA were identified. These included bands of ∼110, 80, 70, 55, 50, and 20 kDa. To identify these proteins, we performed RNA affinity chromatography by covalently linking the same RNAs to adipic acid dihydrazide-agarose beads. After binding with KATO III nuclear extracts, the beads were washed, and bound proteins were eluted and analyzed on silver-stained SDS-PAGE gels. Although a number of proteins bound both the wild-type and mutant RNAs, several bands corresponding in size to those seen by cross-linking were noted to be more abundant in eluates from the wild-type ISE/ISS-3 RNA compared with the mutant or bead-alone controls. Among the latter were several proteins in the 70- to 80-kDa size range. This region of the gel was excised and submitted for tandem mass spectrometry (Fig. 3B). To further characterize those proteins that specifically bound the wild-type sequence, the same region of the gel containing proteins bound to the mutant control RNA was also excised and analyzed. Mass spectrometry analysis identified hnRNP M as the protein that matched the greatest number of peptides from
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