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• W2041569598 abstract "The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5′ splicing site selection and formation of the 12 S isoform. The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5′ splicing site selection and formation of the 12 S isoform. serine-arginine-rich green fluorescent protein glyceraldehyde-3-phosphate dehydrogenase phosphate-buffered saline conditioned medium tetramethylrhodamine isothiocyanate amino acids fluorescein isothiocyanate The Y-box protein YB-1 is a member of the cold shock protein family, which exhibits pleiotropic functions. YB-1 specifically binds to a sequence motif termed Y-box. This motif is characterized by the presence of a core ATTGG sequence, which represents the inverted CCAAT-box. YB-1 controls the transcription of numerous genes that among others include MHC class II antigen, MDR1,MMP-2, and COL1A1 (1MacDonald G.H. Itoh-Lindstrom Y. Ting J.P. J. Biol. Chem. 1995; 270: 3527-3533Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 2Bargou R.C. Jurchott K. Wagener C. Bergmann S. Metzner S. Bommert K. Mapara M.Y. Winzer K.J. Dietel M. Dorken B. Royer H.D. Nat. Med. 1997; 3: 447-450Crossref PubMed Scopus (369) Google Scholar, 3Mertens P.R. Harendza S. Pollock A.S. Lovett D.H. J. Biol. Chem. 1997; 272: 22905-22912Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 4Norman J.T. Lindahl G.E. Shakib K. En-Nia A. Yilmaz E. Mertens P.R. J. Biol. Chem. 2001; 276: 29880-29890Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). DNA binding specificity is mediated through the evolutionarily conserved cold shock domain in conjunction with the adjacent C-terminal protein residues (5Swamynathan S.K. Nambiar A. Guntaka R.V. FASEB J. 1998; 12: 515-522Crossref PubMed Scopus (84) Google Scholar, 6Izumi H. Imamura T. Nagatani G. Ise T. Murakami T. Uramoto H. Torigoe T. Ishiguchi H. Yoshida Y. Nomoto M. Okamoto T. Uchiumi T. Kuwano M. Funa K. Kohno K. Nucleic Acids Res. 2001; 29: 1200-1207Crossref PubMed Scopus (100) Google Scholar). Interactions of YB-1 with numerous cellular and viral transcription factors including JC virus antigen (7Safak M. Gallia G.L. Ansari S.A. Khalili K. J. Virol. 1999; 73: 10146-10157Crossref PubMed Google Scholar), AP-2 (8Mertens P.R. Alfonso-Jaume M.A. Steinmann K. Lovett D.H. J. Biol. Chem. 1998; 273: 32957-32965Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), Purα (9Chen N.N. Khalili K. J. Virol. 1995; 69: 5843-5848Crossref PubMed Google Scholar), CTCF (10Chernukhin I.V. Shamsuddin S. Robinson A.F. Carne A.F. Paul A. El-Kady A.I. Lobanenkov V.V. Klenova E.M. J. Biol. Chem. 2000; 275: 29915-29921Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and p53 (11Mertens P.R. Steinmann K. Alfonso-Jaume M.A. En-Nia A. Sun Y. Lovett D.H. J. Biol. Chem. 2002; 277: 24875-24882Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12Okamoto T. Izumi H. Imamura T. Takano H. Ise T. Uchiumi T. Kuwano M. Kohno K. Oncogene. 2000; 19: 6194-6202Crossref PubMed Scopus (134) Google Scholar) have been demonstrated. These interactions may in part explain cell-specific gene regulation, that is, stimulation and repression of transcription, even of the same gene (3Mertens P.R. Harendza S. Pollock A.S. Lovett D.H. J. Biol. Chem. 1997; 272: 22905-22912Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In addition, it has been proposed that YB-1 plays a role as an architectural protein by its propensity to sequence specifically unwind DNA duplexes and stabilize single-stranded templates, thereby altering sequence recognition motifs (1MacDonald G.H. Itoh-Lindstrom Y. Ting J.P. J. Biol. Chem. 1995; 270: 3527-3533Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 4Norman J.T. Lindahl G.E. Shakib K. En-Nia A. Yilmaz E. Mertens P.R. J. Biol. Chem. 2001; 276: 29880-29890Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Mertens P.R. Alfonso-Jaume M.A. Steinmann K. Lovett D.H. J. Biol. Chem. 1998; 273: 32957-32965Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In addition to their role in regulating gene transcription, cold shock proteins exhibit a wide spectrum of activities by virtue of sequence-specific and -nonspecific RNA binding. YB-1 has been identified as the major component of messenger ribonucleoprotein particles (mRNPs) in mammalian cells, which constitute templates for the translational machinery (13Davydova E.K. Evdokimova V.M. Ovchinnikov L.P. Hershey J.W. Nucleic Acids Res. 1997; 25: 2911-2916Crossref PubMed Scopus (92) Google Scholar, 14Evdokimova V.M. Wei C.L. Sitikov A.S. Simonenko P.N. Lazarev O.A. Vasilenko K.S. Ustinov V.A. Hershey J.W. Ovchinnikov L.P. J. Biol. Chem. 1995; 270: 3186-3192Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 15Evdokimova V.M. Ovchinnikov L.P. Int. J. Biochem. Cell Biol. 1999; 31: 139-149Crossref PubMed Scopus (76) Google Scholar). At higher concentrations Y-box proteins Xenopus FRGY2 and human YB-1 act as repressors of translation in a process called mRNA masking (13Davydova E.K. Evdokimova V.M. Ovchinnikov L.P. Hershey J.W. Nucleic Acids Res. 1997; 25: 2911-2916Crossref PubMed Scopus (92) Google Scholar, 16Matsumoto K. Meric F. Wolffe A.P. J. Biol. Chem. 1996; 271: 22706-22712Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 17Minich W.B. Korneyeva N.L. Ovchinnikov L.P. FEBS Lett. 1989; 257: 257-259Crossref PubMed Scopus (21) Google Scholar, 18Minich W.B. Korneyeva N.L. Berezin Y.V. Ovchinnikov L.P. FEBS Lett. 1989; 258: 227-229Crossref PubMed Scopus (16) Google Scholar), whereas at lower YB-1 concentrations mRNA translation is activated (19Evdokimova V.M. Kovrigina E.A. Nashchekin D.V. Davydova E.K. Hershey J.W. Ovchinnikov L.P. J. Biol. Chem. 1998; 273: 3574-3581Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In this regard, specific binding of YB-1 to the 5′-cap structure may be of importance as mRNA decapping and degradation is inhibited after binding (20Evdokimova V. Ruzanov P. Imataka H. Raught B. Svitkin Y. Ovchinnikov L.P. Sonenberg N. EMBO J. 2001; 20: 5491-5502Crossref PubMed Scopus (220) Google Scholar). Sequence-specific mRNA binding by YB-1 occurs through the evolutionarily conserved cold shock domain, which contains the RNA-binding motifs RNP1 and RNP2 (21Bouvet P. Matsumoto K. Wolffe A.P. J. Biol. Chem. 1995; 270: 28297-28303Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 22Matsumoto K. Wolffe A.P. Trends Cell Biol. 1998; 8: 318-323Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Upon binding, YB-1 regulates mRNA half-lives, e.g. of the interleukin-2 mRNA during T-cell activation (23Chen C.Y. Gherzi R. Andersen J.S. Gaietta G. Jurchott K. Royer H.D. Mann M. Karin M. Genes Dev. 2000; 14: 1236-1248PubMed Google Scholar) and granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils (24Capowski E.E. Esnault S. Bhattacharya S. Malter J.S. J. Immunol. 2001; 167: 5970-5976Crossref PubMed Scopus (81) Google Scholar). Recently, a novel role of YB-1 in splicing has been proposed as YB-1 binds to the A/C-rich exon enhancer element in the CD44 gene and thereby directs alternative splicing (25Chansky H.A. Hu M. Hickstein D.D. Yang L. Cancer Res. 2001; 61: 3586-3590PubMed Google Scholar, 26Stickeler E. Fraser S.D. Honig A. Chen A.L. Berget S.M. Cooper T.A. EMBO J. 2001; 20: 3821-3830Crossref PubMed Scopus (161) Google Scholar). This A/C-rich exon enhancer element is not directly bound by serine-arginine-rich (SR)1 proteins, whereas theCD44 alternative splicing was affected through prebinding of YB-1 to this element (26Stickeler E. Fraser S.D. Honig A. Chen A.L. Berget S.M. Cooper T.A. EMBO J. 2001; 20: 3821-3830Crossref PubMed Scopus (161) Google Scholar). Yet, the mechanism by which YB-1 activates exon recognition in the CD44 gene is unknown. In addition it has been shown that YB-1 directly interacts with the translocation liposarcoma protein, which serves as an adapter molecule connecting gene transcription and RNA splicing and thereby regulates the adenovirus E1A pre-mRNA splicing (25Chansky H.A. Hu M. Hickstein D.D. Yang L. Cancer Res. 2001; 61: 3586-3590PubMed Google Scholar). In the present study a direct in vivo interaction of YB-1 with SRp30c is demonstrated, which has fundamental effects on the subcellular YB-1 localization and is of functional relevance in the alternative splicing process. A cDNA library derived from a human mesangial cell line (27Banas B. Luckow B. Moller M. Klier C. Nelson P.J. Schadde E. Brigl M. Halevy D. Holthofer H. Reinhart B. Schlondorff D. J. Am. Soc. Nephrol. 1999; 10: 2314-2322Crossref PubMed Google Scholar) was subcloned into vector MAB86 (Invitrogen). YB-1 cDNA was PCR-amplified using 5′-CATGCCATGGCAATGAGCAGCGAGGCCGAGACCC-3′ and 5′-GCACTAGTTCAGCCTCGGGAGCGGGAATTCTC-3′ as primers and inserted into restriction sites NcoI and SpeI of pDBLeu as bait. In-frame cloning and sequences were verified by full-length sequencing using the ABI PRISM sequencing reaction (Applied Biosystems). A yeast two-hybrid screen (ProquestTM, Invitrogen) (28Woods R.A. Gietz R.D. Methods Mol. Biol. 2001; 177: 85-97PubMed Google Scholar, 29Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2008) Google Scholar) was performed with a total of 2 × 106 transformants being plated on 15-cm plates of Leu−, Trp−, and His− synthetic complete medium containing 40 mm 3-amino-1,2,4-triazole (Sigma). 86 His+ clones were isolated and tested for fulfillment of all five selection criteria, including growth in media containing 60 mm 3-amino-1,2,4-triazole (3-AT, Sigma), ura+ 5-fluoro-orotic-acid (Invitrogen) metabolism and β-galactosidase expression. HeLa and HEK-293T cells were cultured in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine at 37 °C and 5% CO2. For heat-shock experiments, cells were incubated for 1 h at 42 °C in complete medium and allowed to recover for 1 h at 37 °C before analysis by immunofluorescence. Cells were transfected with purified plasmid DNA by the calcium phosphate precipitation method as described previously (30Holst B. Hastrup H. Raffetseder U. Martini L. Schwartz T.W. J. Biol. Chem. 2001; 276: 19793-19799Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Expression plasmids for YB-1 (pSG5-YB-1) and SRp30c have been described previously (1MacDonald G.H. Itoh-Lindstrom Y. Ting J.P. J. Biol. Chem. 1995; 270: 3527-3533Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 31Stoss O. Olbrich M. Hartmann A.M. Konig H. Memmott J. Andreadis A. Stamm S. J. Biol. Chem. 2001; 276: 8665-8673Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The plasmids encoding for GFP-YB-1 fusion proteins were obtained from H. D. Royer and K. Jurchat (Max-Delbrück Center, Berlin, Germany). In these plasmids full-length YB-1 cDNA was cloned into the AflII and HindIII restriction sites of vector pcDNA6/V5-His (Invitrogen) that was modified with a GFP sequence inserted at the protein C terminus. All deletion constructs of YB-1 were generated by PCR amplification of the respective cDNA sequences and subcloning into vector pcDNA6/V5-His. Sequences were verified by automatic sequencing reactions (ABI PRISM sequencing reaction, Applied Biosystems). For immunoprecipitation 2 × 106 cells were transfected with a total of 20 μg of purified plasmid DNA as indicated. 48-h posttransfection cells were harvested by trypsin digestion and centrifuged, and the cell pellet was lysed in 1 ml RIPA buffer (50 mm Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium-deoxycholate, 150 mm NaCl, 1 mm EDTA, 1 mm Na3V04, 1 mm NaF) for 15 min at 4 °C. Cell lysates were precleared for 30 min at 4 °C with 40 μl of pansorbin (Calbiochem, Darmstadt, Germany) and 4 μl of nonspecific mouse IgG1 (Dako). After centrifugation at 14,000 rpm for 10 min at 4 °C 200 μl of supernatant was incubated with the indicated antibody (monoclonal anti-GFP, Clontech, Palo Alto, CA; monoclonal M2 anti-FLAG, Sigma) at 4 °C overnight. In separate experiments RNase A (100 μg/ml, Roche Applied Science) was added to the precleared lysates for 12 h at 4 °C. To ensure complete degradation of mRNA by RNase A treatment aliquots of cell lysates were processed using the RNeasy kit (Qiagen, Hilden, Germany) and subjected to oligo(dT)-primed reverse transcription by means of superscript II (Invitrogen). The housekeeping gene GAPDH was amplified by PCR using the following primer pair: 5′-TTCCATGGCACCGTCAAGGC-3′ and 5′-TCAGGTCCACCACTGACACG-3′, yielding a 570-bp amplification product. To the precleared lysates 20 μl of pansorbin was added and incubated for 1 h at 4 °C. Pansorbin-bound antibodies and proteins were pelleted by centrifugation at 14,000 rpm and washed extensively with PBS on ice followed by two final washing steps using RIPA buffer. Pellets were resuspended in 50 μl of reducing sample buffer, boiled at 95 °C for 5 min, and pelleted, and the supernatant was subjected to SDS-PAGE. Proteins were transferred to nitrocellulose and detected by suitable primary antibodies anti-GFP (1:5000) or M2 anti-FLAG (1:500) and secondary peroxidase-linked anti-mouse antibody (Amersham Biosciences) using ECL (Amersham Biosciences) as chemiluminescent. HeLa cells were grown on coverslips in six-well plates (105 cells/well). At 60% confluence cells were transfected with a total of 2.5 μg of plasmid DNA/well as indicated in the figure legends by means of calcium phosphate precipitates and cultured for 48 h. After washing with PBS, cells were fixed with 4% paraformaldehyde in PBS for 30 min, washed in PBS-CM and incubated with 50 mm ammonium-sulfate in PBS-CM for 10 min. Cells were subsequently permeabilized in buffer A (0.1% Triton X-100 in PBS-CM) and blocked with 0.2% bovine albumin in buffer A. M2 anti-FLAG antibody (1:300 in buffer A) was added for 60 min at room temperature in a humidified chamber. After three washes with buffer A secondary TRITC-conjugated anti-mouse antibody (Dinova, Hamburg, Germany) was added for 60 min at room temperature (1:600). After extensive washes with buffer A and a final wash with PBS-CM coverslips were mounted with immumount (Shannon). Confocal laser scanning microscopy (Zeiss LSM 510 Meta, Zeiss, Germany) was performed at 488 nm for GFP fluorescence (detected at 500 nm < λFITC < 540 nm) and 543 nm for excitation of TRITC-conjugated antibody (detected at 550 nm < λTRITC < 600 nm). 15 μg of pMTE1A plasmid DNA containing the E1A minigene was transfected in HeLa cells (2 × 106 cells) combined with expression plasmids pSG5-YB-1, pCR-FLAG-30c or control plasmids. Care was taken that the total DNA amount was equalized to 50 μg in each reaction by inclusion of the respective amounts of control plasmid. Cells were harvested 48 h posttransfection and mRNA was extracted using the RNeasy minikitTM (Qiagen). cDNA synthesis was performed using oligo(dT) primers (Roche Applied Science) and Superscript II RNase H− reverse transcriptase (Invitrogen). For PCR the E1A-specific primers pMTE1A-sense 5′-ATTATCTGCCACGGAGGTGT-3′ and pMTE1A-antisense 5′-GGATAGCAGGCGCCATTTTA-3′ were used with 25 cycles of amplification (90 s at 94 °C, 120 s at 50 °C, 120 s at 72 °C). Amplification products were separated on 3% agarose gels containing ethidium bromide with band intensities being quantified by OptiQuantTM software. Band intensities of all splicing isoforms combined were set as 100%, and relative band intensities were determined. To identify YB-1-interacting proteins a yeast two-hybrid screen was set up with full-length YB-1 as bait and a cDNA library generated from a human mesangial cell line (27Banas B. Luckow B. Moller M. Klier C. Nelson P.J. Schadde E. Brigl M. Halevy D. Holthofer H. Reinhart B. Schlondorff D. J. Am. Soc. Nephrol. 1999; 10: 2314-2322Crossref PubMed Google Scholar) as prey. Of 2 × 106 colonies screened 86 fulfilled all five selection criteria (phenotypes: His+, 3ATR, β-galactosidase, Ura+, and 5-fluoro-orotic-acid+), and of these two colonies encoded for full-length splicing factor SRp30c. To confirm the result of the two-hybrid screen co-immunoprecipitation studies were performed with both proteins co-expressed as tagged fusion proteins in HEK-293T cells. GFP was fused to the C terminus of YB-1 and FLAG was fused to the N terminus of SRp30c (Fig.1A, GFP: <1 in lane 1; GFP-YB-1: <2 in lane 2, FLAG-SRp30c: <3 inlane 3). Cell lysates were subsequently used for co-immunoprecipitation studies with monoclonal antibodies directed against the respective tags. As can be seen in Fig. 1AFLAG-tagged SRp30c protein was co-immunoprecipitated with anti-GFP antibody when cells were co-transfected with GFP-YB-1 (lane 5, indicated by <4); however, not in cells expressing GFP protein alone (lane 4). Conversely, GFP-YB-1 was detected with immunoprecipitated FLAG-SRp30c (lane 8, indicated with <5); however, not in control reactions with expressed FLAG protein (Fig.1A, lane 7). The co-immunoprecipitations were nearly quantitative and suggested a direct association of YB-1 and SRp30c. To exclude the possibility that the contact of both proteins is mediated by indirect association via mRNA binding, co-immunoprecipitation studies were repeated following RNase A treatment. RNase A treatment did not change the amount of immunoprecipitated protein, indicating direct YB-1/SRp30c partnering (Fig. 1B, compare lanes 6 and 12). In the latter experiment control reactions were set up to test for consistent and complete degradation of messenger RNA by reverse transcription and amplification of the housekeeping gene GAPDH. As can be seen in Fig. 1C untreated cell extracts contained abundant amounts of GAPDH mRNA (lanes 2–4), whereas no GAPDH mRNA was present in RNase A-treated extracts (lanes 5–7). As these findings indicate interaction of introduced tagged proteins FLAG-SRp30c/GFP-YB-1 additional experiments were performed to also confirm an interaction of endogenous YB-1 with FLAG-SRp30c. For this purpose HEK-293T cells were transiently transfected with either FLAG or FLAG-SRp30c expression plasmids, and co-immunoprecipitation studies were performed by means of a polyclonal anti-YB-1 antibody (32Mertens P.R. Alfonso-Jaume M.A. Steinmann K. Lovett D.H. J. Am. Soc. Nephrol. 1999; 10: 2480-2487Crossref PubMed Google Scholar). As can be seen in Fig. 1D FLAG-SRp30c was co-immunoprecipitated with endogenous YB-1 protein (lane 3). To identify YB-1 protein domains that confer the interaction with SRp30c a panel of GFP-YB-1 deletion constructs (depicted in Fig. 2B) were co-expressed with full-length SRp30c protein in HEK-293T cells, and co-immunoprecipitations were performed using anti-FLAG antibody. As shown in Fig. 2A, the N-terminal protein domains of YB-1 containing the evolutionarily conserved cold shock domain (GFP-YB-1 Δ1, aa 21–147) are sufficient for association with FLAG-tagged SRp30c (Fig. 2A, lanes 1 and 2, indicated by <1). In comparison to full-length GFP-YB-1 wild type a similarly high percentage of expressed GFP-YB-1 Δ1 (Fig.2A, lane 3) was co-immunoprecipitated by anti-FLAG antibody (Fig. 2A, lane 4, indicated by <2). After removal of the YB-1 N-terminal protein domains aa 1–147 (GFP-YB-1 Δ3) only a weak interaction with SRp30c could be detected (Fig. 2A, lanes 7 and 8, indicated by <4). This low-level binding affinity was mapped to aa 260–317 of the C terminus (GFP-YB-1 Δ4), whereas GFP-YB-1 Δ2 comprised of aa 147–225 was not co-immunoprecipitated. Taken together, these results demonstrate that YB-1 strongly interacts with SRp30c via aa 21–147, whereas a low affinity binding domain resides in the C terminus (aa 260–317). Next we set out to determine the subcellular localization of YB-1 and SRp30c. For SRp30c a nuclear localization has previously been described (33Denegri M. Chiodi I. Corioni M. Cobianchi F. Riva S. Biamonti G. Mol. Biol. Cell. 2001; 12: 3502-3514Crossref PubMed Scopus (126) Google Scholar), whereas YB-1 has been reported to localize both to the nucleus and the cytoplasm, depending on the cell origin and transformation (2Bargou R.C. Jurchott K. Wagener C. Bergmann S. Metzner S. Bommert K. Mapara M.Y. Winzer K.J. Dietel M. Dorken B. Royer H.D. Nat. Med. 1997; 3: 447-450Crossref PubMed Scopus (369) Google Scholar, 6Izumi H. Imamura T. Nagatani G. Ise T. Murakami T. Uramoto H. Torigoe T. Ishiguchi H. Yoshida Y. Nomoto M. Okamoto T. Uchiumi T. Kuwano M. Funa K. Kohno K. Nucleic Acids Res. 2001; 29: 1200-1207Crossref PubMed Scopus (100) Google Scholar, 34Stein U. Jurchott K. Walther W. Bergmann S. Schlag P.M. Royer H.D. J. Biol. Chem. 2001; 276: 28562-28569Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Transfections of HeLa cells were performed with expression plasmids for GFP-YB-1, FLAG-SRp30c, and tag control plasmids. In HeLa cells expressing GFP-YB-1 alone, fluorescence was predominantly localized in the cytoplasm (Fig. 3a,panel B), whereas GFP was detected diffusely in all cellular compartments (Fig. 3a, panel A). In contrast, FLAG-SRp30c protein was localized in a speckled pattern in the nucleus (Fig. 3a, panel C). A dramatic change of the subcellular GFP-YB-1 localization was detected with combined expression of both proteins, GFP-YB-1 and FLAG-SRp30c. Under these conditions GFP-YB-1 co-localized with SRp30c (Fig. 3a, panel D), while the subcellular distribution of GFP was unchanged after co-transfection with FLAG-SRp30c (Fig. 3a, panel C). The nuclear localization of both proteins in most cells was not in a speckled pattern. These findings indicate that FLAG-SRp30c confers shuttling of YB-1 to the nuclear compartment. Transient transfections of HeLa cells were repeated with expression plasmids for FLAG or FLAG-SRp30c, and the subcellular distribution of endogenous YB-1 was assessed by immunohistochemistry using a specific anti-YB-1 antibody (32Mertens P.R. Alfonso-Jaume M.A. Steinmann K. Lovett D.H. J. Am. Soc. Nephrol. 1999; 10: 2480-2487Crossref PubMed Google Scholar). These experiments were designed to exclude any contribution of the GFP tag to the nuclear shuttling of GFP-YB-1 protein. Here, a similar shift of endogenous YB-1 from the cytoplasm to the nuclear compartment was apparent with introduced FLAG-SRp30c but not FLAG alone (Fig. 3b). To exclude a cell-specific effect of this protein shuttling analogous experiments were performed with HEK-293T cells yielding similar results (data not shown). Since the N-terminal YB-1 protein domains were mapped as high affinity interacting domains with FLAG-SRp30c, the corresponding GFP-YB-1 deletion construct, GFP-YB-1 Δ1, was tested for co-localization with SRp30c. As can be seen in Fig.4A (images c andd) construct GFP-YB-1 Δ1 co-localized with SRp30c in the nucleus, whereas construct GFP-YB-1 Δ3 lacking aa 1–147 localized in the cytoplasm when co-expressed with SRp30c (Fig. 4B,images c and d). These results support the notion that the N-terminal YB-1 protein domains are responsible for the interaction with SRp30c. As the nuclear co-localization of YB-1 and SRp30c is dependent on the cellular SRp30c content, we subsequently addressed the question whether a shift of nuclear SRp30c into stress-induced Sam68 nuclear bodies (33Denegri M. Chiodi I. Corioni M. Cobianchi F. Riva S. Biamonti G. Mol. Biol. Cell. 2001; 12: 3502-3514Crossref PubMed Scopus (126) Google Scholar) by heat shock treatment affects the subcellular YB-1 localization. As can be seen in Fig.5, the localization of SRp30c (Fig. 5,panel A) and GFP-YB-1 (Fig. 5, panel B) remained unchanged after 1 h heat shock treatment at 42 °C when both proteins were overexpressed separately. Remarkably, the nuclear co-localization was no longer present with FLAG-SRp30c and GFP-YB-1 overexpressed in combination when heat shock treatment was performed. Under these conditions GFP-YB-1 was detected in the cytoplasm (Fig. 5,panel C), whereas FLAG-SRp30c remained in the nuclear compartment. Next we tested for the functional relevance of the YB-1/SRp30c interaction in the alternative splicing process. The adenovirus E1A pre-mRNA minigene was chosen as a model system in which concentration-dependent changes of the splicing pattern by SR proteins has been described (35Caceres J.F. Stamm S. Helfman D.M. Krainer A.R. Science. 1994; 265: 1706-1709Crossref PubMed Scopus (561) Google Scholar, 36Screaton G.R. Caceres J.F. Mayeda A. Bell M.V. Plebanski M. Jackson D.G. Bell J.I. Krainer A.R. EMBO J. 1995; 14: 4336-4349Crossref PubMed Scopus (245) Google Scholar). In vivosplicing was monitored by reverse transcription PCR analysis using the pMTE1A-sense and pMTE1A-antisense primers with mRNA collected from HeLa cells that were transiently transfected with the E1A-minigene and a combination of SRp30c expression vector and/or increasing amounts of YB-1 expression vector. Care was taken to ensure that equal DNA amounts were introduced in all transfections. The E1A minigene contains three different 5′ splicing sites resulting in three major isoforms 13 S, 12 S, and 9 S (Fig. 6A). As can be seen in Fig. 6B, co-transfection of increasing amounts of YB-1 resulted in the preferential formation of the 12 S isoform, whereas the 9 S isoform decreased in a concentration-dependent manner (quantification depicted in Fig. 6C). In contrast to this finding overexpression of SRp30c alone lead to the preponderance of 13 S transcripts with concomitant decreased appearance of the 9 S isoform (Fig. 6B, lane 5), as has been described previously (33Denegri M. Chiodi I. Corioni M. Cobianchi F. Riva S. Biamonti G. Mol. Biol. Cell. 2001; 12: 3502-3514Crossref PubMed Scopus (126) Google Scholar, 36Screaton G.R. Caceres J.F. Mayeda A. Bell M.V. Plebanski M. Jackson D.G. Bell J.I. Krainer A.R. EMBO J. 1995; 14: 4336-4349Crossref PubMed Scopus (245) Google Scholar). When both expression plasmids were introduced in combination, that is SRp30c at a fixed and YB-1 at increasing concentrations, the relative intensity of the 12 S isoform was increased, whereas 13 S transcripts were slightly decreased at lower YB-1 concentrations. At the same time isoform 9 S was decreased (Fig. 5D). Taken together these changes in splicing indicate YB-1-dependent selection of splice site selection that is a shift from the 13 S toward the more distal 12 S splicing site. Thus, YB-1 influences alternative splice site selection of the adenovirus E1A pre-mRNA in vivo and interferes with the SRp30c-dependent splicing pattern in a concentration-dependent manner. Here we report the identification of SRp30c as an interacting protein of YB-1 by a two-hybrid screen against a human mesangial cell cDNA library. The identified SRp30c clones encoded for full-length protein. Co-immunoprecipitation studies with tagged endogenous proteins expressed in HEK-293T and HeLa cells confirm this protein-protein interaction. Although both proteins, SRp30c and YB-1, have been shown to specifically bind to mRNA sequences (26Stickeler E. Fraser S.D. Honig A. Chen A.L. Berget S.M. Cooper T.A. EMBO J. 2001; 20: 3821-3830Crossref PubMed Scopus (161) Google Scholar, 37Young P.J. DiDonato C.J. Hu D. Kothary R. Androphy E.J. Lorson C.L. Hum. Mol. Genet. 2002; 11: 577-587Crossref PubMed Google Scholar" @default.
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• W2041569598 title "Splicing Factor SRp30c Interaction with Y-box Protein-1 Confers Nuclear YB-1 Shuttling and Alternative Splice Site Selection" @default.
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