RNA CUG Repeats Sequester CUGBP1 and Alter Protein Levels and Activity of CUGBP1
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m005960200
ISSN1083-351X
AutoresNikolai A. Timchenko, Zong-Jin Cai, Alana L. Welm, Sita Reddy, Tetsuo Ashizawa, Lubov Timchenko,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoAn RNA CUG triplet repeat binding protein, CUGBP1, regulates splicing and translation of various RNAs. Expansion of RNA CUG repeats in the 3′-untranslated repeat of the mutant myotonin protein kinase (DMPK) mRNA in myotonic dystrophy (DM) is associated with alterations in binding activity of CUGBP1. To investigate whether CUGBP1 is directly affected by expansion of CUG repeats in DM tissues, we examined the intracellular status of CUGBP1 in DM patients as well as in cultured cells over expressing RNA CUG repeats. The analysis of RNA·protein complexes showed that, in control tissues, the majority of CUGBP1 is free of RNA, whereas in DM patients the majority of CUGBP1 is associated with RNA containing CUG repeats. Similarly to DM patients, overexpression of RNA CUG repeats in cultured cells results in the re-allocation of CUGBP1 from a free state to the RNA·protein complexes containing CUG repeats. CUG repeat-dependent translocation of CUGBP1 into RNA·protein complexes is associated with increased levels of CUGBP1 protein and its binding activity. Experiments with cyclohexamide-dependent block of protein synthesis showed that the half-life of CUGBP1 is increased in cells expressing CUG repeats. Alteration of CUGBP1 in DM is accompanied by alteration in translation of a transcription factor CCAAT/enhancer-binding protein β (C/EBPβ), which has been previously described to be a target of CUGBP1. Analysis of C/EBPβ isoforms in DM patients with altered levels of CUGBP1 showed that translation of a dominant negative isoform, LIP, is induced by CUGBP1. Results of this paper demonstrate that the expansion of CUG repeats in DM affects RNA-binding proteins and leads to alteration in RNA processing. An RNA CUG triplet repeat binding protein, CUGBP1, regulates splicing and translation of various RNAs. Expansion of RNA CUG repeats in the 3′-untranslated repeat of the mutant myotonin protein kinase (DMPK) mRNA in myotonic dystrophy (DM) is associated with alterations in binding activity of CUGBP1. To investigate whether CUGBP1 is directly affected by expansion of CUG repeats in DM tissues, we examined the intracellular status of CUGBP1 in DM patients as well as in cultured cells over expressing RNA CUG repeats. The analysis of RNA·protein complexes showed that, in control tissues, the majority of CUGBP1 is free of RNA, whereas in DM patients the majority of CUGBP1 is associated with RNA containing CUG repeats. Similarly to DM patients, overexpression of RNA CUG repeats in cultured cells results in the re-allocation of CUGBP1 from a free state to the RNA·protein complexes containing CUG repeats. CUG repeat-dependent translocation of CUGBP1 into RNA·protein complexes is associated with increased levels of CUGBP1 protein and its binding activity. Experiments with cyclohexamide-dependent block of protein synthesis showed that the half-life of CUGBP1 is increased in cells expressing CUG repeats. Alteration of CUGBP1 in DM is accompanied by alteration in translation of a transcription factor CCAAT/enhancer-binding protein β (C/EBPβ), which has been previously described to be a target of CUGBP1. Analysis of C/EBPβ isoforms in DM patients with altered levels of CUGBP1 showed that translation of a dominant negative isoform, LIP, is induced by CUGBP1. Results of this paper demonstrate that the expansion of CUG repeats in DM affects RNA-binding proteins and leads to alteration in RNA processing. myotonic dystrophy untranslated repeat myotonin protein kinase CCAAT/enhancer-binding protein β full-length liver activator protein liver inhibitory protein kilobase(s) base pair(s) vector DNA cyclohexamide reverse transcription-polymerase chain reaction TATA box binding protein isopropyl-1-thio-β-d-galactopyranoside high pressure liquid chromatography double-stranded RNA The mutation leading to myotonic dystrophy (DM)1 (1Harper P.S. Myotonic Dystrophy. 2nd Ed. Saunders, London/Philadelphia1989Google Scholar) is an expanded CTG trinucleotide repeat located in the 3′-untranslated region (UTR) of the myotonin protein kinase (DMPK) gene (2Aslanidis C. Jansen J. Amemiya C. Shutler G. Mahadevan M. Tsilfidis C. Chen C. Alleman J. Wormskamp N.G. Vooijs M. Buxton J. Johnson K. Sweets H.J.M. Lennon G.G. Carrano A.V. Korneluk R.G. Wieringa B. de Jong P.J. Nature. 1992; 355: 548-551Crossref PubMed Scopus (462) Google Scholar, 3Fu Y.-H. Pizzuti A. Fenwick Jr., R.G. King J. Rajnarayan S. Dunne P.W. Dubel J. Nasser G.A. Ashizawa T. de Jong P. Wieringa B. Korneluk R.G. Perryman M.B. Epstein H.F. Caskey C.T. Science. 1992; 255: 1256-1258Crossref PubMed Scopus (1289) Google Scholar). It was initially suggested that alterations in expression of DMPK protein may cause DM pathogenesis (4Fu Y.-H. Friedman D.L. Richards S. Pearlman J.A. Gibbs R.A. Pizzuti A. Ashizawa T. Perryman M.B. Scarlato G. Fenwick Jr., R.G. Caskey C.T. Science. 1993; 260: 235-238Crossref PubMed Scopus (291) Google Scholar, 5Sabourin L.A. Mahadevan M.S. Narang M. Lee D.S.C. Surh L.C. Korneluk R.G. Nat. Genet. 1993; 4: 233-238Crossref PubMed Scopus (142) Google Scholar). However, both DMPK knockout and transgenic mice overexpressing DMPK did not develop the complete DM phenotype (6Jansen G. Croenen P.J.T.A. Bachner D. Jap P.H.K. Coerwinkel M. Oerlemans F. van den Brock W. Gohlsch B. Pette D. Plomp J.J. Molenaar P.C. Nederhoff M.G.J. van Echteld C.J.A. Dekker M. Berns A. Hameister H. Wieringa B. Nat. Genet. 1996; 13: 316-324Crossref PubMed Scopus (286) Google Scholar, 7Reddy S. Smith D.B.J. Rich M.M. Leferovich J.M. Reily P. Davis B.M. Tran K. Rayburn H. Bronson R. Cros D. Balise-Gordon R.J. Housman D. Nat. Genet. 1996; 13: 325-335Crossref PubMed Scopus (288) Google Scholar). Given these observations, several new hypotheses for DM pathogenesis have been proposed. One hypothesis suggests that CTG repeats might affect transcription of gene(s) that are located upstream or downstream of these CTG elements (8Boucher C.A. King S.K. Carey N. Krahe R. Winshester C.L. Rahman S. Creavin T. Meghji P. Bailey M.E. Chartier F.L. Hum. Mol. Genet. 1995; 4: 1919-1925Crossref PubMed Scopus (177) Google Scholar, 9Jansen G. Bachner D. Coerwinkel M. Wormskamp N. Hameister H. Wieringa B. Hum. Mol. Genet. 1995; 4: 843-852Crossref PubMed Scopus (56) Google Scholar). Two genes adjacent to DMPKhave been identified and investigated (8Boucher C.A. King S.K. Carey N. Krahe R. Winshester C.L. Rahman S. Creavin T. Meghji P. Bailey M.E. Chartier F.L. Hum. Mol. Genet. 1995; 4: 1919-1925Crossref PubMed Scopus (177) Google Scholar, 9Jansen G. Bachner D. Coerwinkel M. Wormskamp N. Hameister H. Wieringa B. Hum. Mol. Genet. 1995; 4: 843-852Crossref PubMed Scopus (56) Google Scholar, 10Klesert T.R. Cho D.H. Clark J.I. Maylie J. Adelman J. Snider L. Yuen E.C. Soriano P. Tapscott S.J. Nat. Genet. 2000; 25: 105-109Crossref PubMed Scopus (199) Google Scholar). Deletion of DMAHP in mice showed that reduction of DMAHP is associated with the increased rate of cataracts but not myotonia or cardiac abnormalities (10Klesert T.R. Cho D.H. Clark J.I. Maylie J. Adelman J. Snider L. Yuen E.C. Soriano P. Tapscott S.J. Nat. Genet. 2000; 25: 105-109Crossref PubMed Scopus (199) Google Scholar). An alternative hypothesis suggests that DM pathogenesis is due to expression of mutant DMPK mRNA (11Wang J. Pegoraro E. Menegazzo E. Gennarelli M. Hoop R.C. Angelini C. Hoffman E.P. Hum. Mol. Genet. 1995; 4: 599-606Crossref PubMed Scopus (167) Google Scholar). This hypothesis proposes that mutant DMPK mRNA has a dominant effect on RNA metabolism (11Wang J. Pegoraro E. Menegazzo E. Gennarelli M. Hoop R.C. Angelini C. Hoffman E.P. Hum. Mol. Genet. 1995; 4: 599-606Crossref PubMed Scopus (167) Google Scholar). Following this RNA-based hypothesis, we suggested that expanded RNA CUG repeats in DMPK mRNA may serve as binding sites for specific CUG triplet repeat RNA-binding proteins (12Timchenko L.T. Timchenko N.A. Caskey C.T. Roberts R. Hum. Mol. Genet. 1996; 5: 115-121Crossref PubMed Scopus (169) Google Scholar, 13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar, 14Caskey C.T. Swanson M.S. Timchenko L.T. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 607-614Crossref PubMed Google Scholar, 15Roberts R. Timchenko N.A. Miller J.W. Reddy S. Caskey C.T. Swanson M.S. Timchenko L.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13221-13226Crossref PubMed Scopus (137) Google Scholar, 16Lu X. Timchenko N.A. Timchenko L.T. Hum. Mol. Genet. 1999; 8: 53-60Crossref PubMed Scopus (105) Google Scholar, 17Timchenko L.T. Am. J. Hum. Genet. 1999; 64: 360-364Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). We also proposed that the dramatic increase of (CUG)n repeats in DM patients might affect expression of these RNA-binding proteins (14Caskey C.T. Swanson M.S. Timchenko L.T. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 607-614Crossref PubMed Google Scholar, 17Timchenko L.T. Am. J. Hum. Genet. 1999; 64: 360-364Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The RNA-based hypothesis for DM pathogenesis has been recently proved by Thornton and colleagues (18Mankodi A. Logigian E. Callahan L. McClain C. White R. Henderson D. Krym M. Thornton C.A. Science. 2000; 289: 1769-1773Crossref PubMed Scopus (572) Google Scholar), showing that overexpression of RNA CUG repeats in transgenic mice induces myopathy and myotonia. A number of RNA-binding proteins with specific binding activity to CUG repeats have been identified (12Timchenko L.T. Timchenko N.A. Caskey C.T. Roberts R. Hum. Mol. Genet. 1996; 5: 115-121Crossref PubMed Scopus (169) Google Scholar, 13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar, 16Lu X. Timchenko N.A. Timchenko L.T. Hum. Mol. Genet. 1999; 8: 53-60Crossref PubMed Scopus (105) Google Scholar, 19Good P.J. Chen Q. Warner S.J. Herring D.C. J. Biol. Chem. 2000; 275: 28563-28592Abstract Full Text Full Text PDF Scopus (109) Google Scholar, 20Miller J.W. Urbinati C.R. Teng-Umnuay P. Stenberg M.G. Byrne B.J. Thornton C.A. Swanson M.S. EMBO J. 2000; 19: 4439-4448Crossref PubMed Scopus (728) Google Scholar). These proteins include CUGBP1, ETR-3, and Brunol 1 and recently identified a group of expansion binding proteins that preferentially bind to double-stranded structures containing CUG repeats (20Miller J.W. Urbinati C.R. Teng-Umnuay P. Stenberg M.G. Byrne B.J. Thornton C.A. Swanson M.S. EMBO J. 2000; 19: 4439-4448Crossref PubMed Scopus (728) Google Scholar). All of these proteins are likely to be involved in DM pathogenesis, because their activities are altered in DM patients (13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar, 14Caskey C.T. Swanson M.S. Timchenko L.T. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 607-614Crossref PubMed Google Scholar, 15Roberts R. Timchenko N.A. Miller J.W. Reddy S. Caskey C.T. Swanson M.S. Timchenko L.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13221-13226Crossref PubMed Scopus (137) Google Scholar, 20Miller J.W. Urbinati C.R. Teng-Umnuay P. Stenberg M.G. Byrne B.J. Thornton C.A. Swanson M.S. EMBO J. 2000; 19: 4439-4448Crossref PubMed Scopus (728) Google Scholar, 21Philips A.V Timchenko L.T. Cooper T. Science. 1998; 280: 737-741Crossref PubMed Scopus (699) Google Scholar). CUGBP1 was discovered in 1996 and has been characterized in detail (12Timchenko L.T. Timchenko N.A. Caskey C.T. Roberts R. Hum. Mol. Genet. 1996; 5: 115-121Crossref PubMed Scopus (169) Google Scholar, 13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar, 14Caskey C.T. Swanson M.S. Timchenko L.T. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 607-614Crossref PubMed Google Scholar, 15Roberts R. Timchenko N.A. Miller J.W. Reddy S. Caskey C.T. Swanson M.S. Timchenko L.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13221-13226Crossref PubMed Scopus (137) Google Scholar, 17Timchenko L.T. Am. J. Hum. Genet. 1999; 64: 360-364Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 21Philips A.V Timchenko L.T. Cooper T. Science. 1998; 280: 737-741Crossref PubMed Scopus (699) Google Scholar, 22Timchenko N.A. Welm A.L. Lu X. Timchenko L.T. Nucleic Acids Res. 1999; 27: 4517-4525Crossref PubMed Scopus (146) Google Scholar). CUGBP1 is localized in both nuclei and cytoplasm. The nuclear function of CUGBP1 seems to be associated with splicing (21Philips A.V Timchenko L.T. Cooper T. Science. 1998; 280: 737-741Crossref PubMed Scopus (699) Google Scholar). It has been demonstrated that cardiac troponin T (cTnT) is a target of CUGBP1 (21Philips A.V Timchenko L.T. Cooper T. Science. 1998; 280: 737-741Crossref PubMed Scopus (699) Google Scholar) and that splicing of cTnT is altered in hearts of DM patients. CUGBP1 is homologous to Brunol proteins that play a role in translation (19Good P.J. Chen Q. Warner S.J. Herring D.C. J. Biol. Chem. 2000; 275: 28563-28592Abstract Full Text Full Text PDF Scopus (109) Google Scholar). This suggests that CUGBP1 may play a role in translation of RNA. In agreement with this suggestion, a significant portion of CUGBP1 has been found to localize in the cytoplasm (22Timchenko N.A. Welm A.L. Lu X. Timchenko L.T. Nucleic Acids Res. 1999; 27: 4517-4525Crossref PubMed Scopus (146) Google Scholar). It has been recently shown that CUGBP1 is associated with polysomes and is involved in the regulation of translation of C(U/C)G-containing mRNA (22Timchenko N.A. Welm A.L. Lu X. Timchenko L.T. Nucleic Acids Res. 1999; 27: 4517-4525Crossref PubMed Scopus (146) Google Scholar). This function of CUGBP1 may play an important role in regulation of cell growth and proliferation by controlling activities of transcription factors and cell cycle proteins. A member of the CCAAT/Enhancer Binding Protein family, C/EBPβ, is encoded by an intronless gene, but a single mRNA of C/EBPβ can produce several isoforms with different transcriptional activities (Ref. 23Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar and see Fig. 5 A). Three major isoforms of C/EBPβ are produced by alternative translation: full-length (FL, 38 kDa), liver activator protein (LAP, 35 kDa) and liver inhibitory protein (LIP, 20 kDa). CUGBP1 has been shown to regulate translation of a dominant negative isoform of C/EBPβ, liver-enriched transcriptional inhibitory protein (LIP) (22Timchenko N.A. Welm A.L. Lu X. Timchenko L.T. Nucleic Acids Res. 1999; 27: 4517-4525Crossref PubMed Scopus (146) Google Scholar). The ability of CUGBP1 to regulate translation of C(U/C)G-containing mRNAs suggests that altered expression of CUGBP1 in DM may lead to changes in translation of C(U/C)G-containing mRNAs. In this paper, we present evidence showing that RNA CUG repeats directly affect expression and activity of CUGBP1. Experiments in tissue culture demonstrate that endogenous CUGBP1 is titrated from a free pool by overexpression of transcripts with long CUG repeats. Formation of CUGBP1·CUG RNA complexes is accompanied by increased stability of CUGBP1 protein and subsequent elevation of CUGBP1. Similar to results obtained in tissue culture, analysis of DM patients showed that protein levels of CUGBP1 are increased in DM patients and the majority of CUGBP1 is detectable within RNA·protein complexes containing CUG repeats. Sequestration of CUGBP1 in DM patients is accompanied by altered translation of CUGBP1-dependent mRNAs. Long CTG repeats (170 and 500 repeats) were synthesized as described previously (24Ordway J.M. Detloff P.J. BioTechniques. 1996; 21: 609-612Crossref PubMed Scopus (31) Google Scholar), fused with a human β-actin promoter, and cloned into the pSV2 vector. The resulting plasmid included the sequence of the human β-actin gene, consisting of 3 kb of 5′-flanking sequence followed by 78 bp of 5′-UTR and 832 bp of intron I. CTG repeat sequences were cloned between the β-actin promoter and the SV40 polyadenylation signal. Cos7 cells were grown in Dulbecco's modified Eagle's medium. For transfections, plasmid DNA (18 μg/15-cm dish) was mixed with LipofectAMINE or Lipofectin and added to cultured cells. Control transfections were performed with vector DNA (BS). The efficiency of transfection was evaluated by using plasmid DNA containing β-galactosidase. After transfection, cells with empty vector and CTG-containing plasmids were used for protein and RNA extraction. To analyze the level of RNA (CUG)170–500 expression in cultured cells, total RNA was purified from transfected cells with TRI reagent. The integrity of RNA has been verified by gel electrophoresis. To evaluate the level of CUG expression, RNA was transferred onto a Z-probe membrane and hybridized with a 32P-(CAG)8 probe. RNA containing 170 CUG repeats was detected after 12 h of exposure. RNA containing 500 CUG repeats was detected after 36 h of exposure because of lower efficiency of RNA expression from the construct containing 500 CTG repeats. No signals for CUG-containing RNAs were detected in control cells transfected with BS after 36 h of exposure. Cos7 or HT1080 cultured cells were transfected with wild type (empty) vector or with plasmid expressing 170 CUG repeats. Cyclohexamide (CHX, 10 μg/ml) was added, and proteins were isolated 2, 4, and 8 h after CHX addition. CUGBP1 levels were examined by Western assay with monoclonal antibodies to CUGBP1 (13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar). The block of protein synthesis by CHX was verified by analysis of p21 protein half-life in HT1080 cells. In experiments presented in the manuscript, the p21 half-life in both control and CUG-expressing cells was 40–60 min and agreed with our previous estimate (25Timchenko N.A Wilde M. Nakanishi M. Smith J.R. Darlington G.J. Genes Dev. 1996; 10: 804-815Crossref PubMed Scopus (346) Google Scholar). Densitometric analysis of CUGBP1 protein levels indicated that CUGBP1 half-life is ∼3 h in control cells. RNA oligomer CUG8 was labeled with [γ-32P]ATP and T4 kinase. Cytoplasmic proteins were incubated with the probe, treated with UV light, and analyzed by polyacrylamide gel electrophoresis containing SDS. Where indicated, unlabeled RNA competitor (100 ng) was added prior to protein addition. To verify the concentration of proteins used in UV cross-linking assay, the membrane was stained with Coomassie Blue after the UV cross-link analysis. All gels presented in this paper were equally loaded. Proteins from DM tissues (14Caskey C.T. Swanson M.S. Timchenko L.T. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 607-614Crossref PubMed Google Scholar, 21Philips A.V Timchenko L.T. Cooper T. Science. 1998; 280: 737-741Crossref PubMed Scopus (699) Google Scholar) were isolated as described previously (12Timchenko L.T. Timchenko N.A. Caskey C.T. Roberts R. Hum. Mol. Genet. 1996; 5: 115-121Crossref PubMed Scopus (169) Google Scholar, 13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar, 14Caskey C.T. Swanson M.S. Timchenko L.T. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 607-614Crossref PubMed Google Scholar, 15Roberts R. Timchenko N.A. Miller J.W. Reddy S. Caskey C.T. Swanson M.S. Timchenko L.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13221-13226Crossref PubMed Scopus (137) Google Scholar). Protein extracts from normal controls were purchased from CLONTECH Co. 50 or 100 μg of protein was loaded on a 10–12% polyacrylamide gel and transferred onto a nitrocellulose filter (Bio-Rad). The filter was blocked with 10% dry milk/2% bovine serum albumin prepared in TTBS buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.1% Tween-20) for 1 h at room temperature. Primary antibodies to CUGBP1 or to C/EBPβ (C-19, Santa Cruz Biotechnology) were added, and the filter was incubated for 1 h, washed, and then incubated with secondary antibody for 1 h. Immunoreactive proteins were detected using the ECL method. After detection of the protein of interest, the membrane was stripped and reprobed with anti-β-actin. For quantitative analysis, the intensity of the signals was determined on the Alpha Imager 2000 gel documentation and analysis system. Protein levels were calculated as a ratio to control protein. In the case of C/EBPβ, the ratios LAP/FL or LIP/FL were calculated. To study CUGBP1 expression in cultured cells, whole cell protein extracts were prepared from transfected cells as described (13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar) and analyzed by Western assay with monoclonal (13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar) or polyclonal antibodies against CUGBP1 (16Lu X. Timchenko N.A. Timchenko L.T. Hum. Mol. Genet. 1999; 8: 53-60Crossref PubMed Scopus (105) Google Scholar). To verify protein loading, membranes were stripped and reprobed with antibodies against β-actin. The intensity of CUGBP1 was determined on the Alpha Imager 2000 documentation and analysis system, and the amount of CUGBP1 was calculated as a ratio to β-actin. Total RNA was extracted from heart tissues and from transfected cells with TRI reagent (Molecular Research Center). mRNA was extracted with a poly(A) Quick kit (Stratagene). Normal control poly(A) and total RNA were fromCLONTECH Co. Total RNA (1 μg) and mRNA (100 ng) were used for the RT reaction with M-Mul (Stratagene) and oligo(dT) primers. The RT product (2 μl) was then used for PCR, with two sets of primers: one set for the CUGBP1 gene and another one for the TBP gene (internal control). A PCR assay (50 μl) contained 20 pmol of each primer; 50 mm KCl; 10 mm Tris-HCl, pH 9.0; 0.1% Triton X-100; 1.5 mm MgCl2; 200 μm each of dATP, dCTP, dTTP, and dGTP; 100 μCi of [α-32P]dCTP; and 5 units of Taq polymerase (Promega). Amplification was performed in a Robocycler (Stratagene) under the following conditions: denaturation was at 94 °C for 90 s, annealing at 54 °C for 90 s, and extension at 72 °C for 150 s. The optimal number of cycles when PCR was linear (with heteroduplex formation) was 19 cycles. The PCR products were separated by 10% polyacrylamide gel electrophoresis. Intensities of PCR products were quantified by PhosphorImager scans (Molecular Dynamics) using the ImageQuaNT version 1.1 image analysis program (Molecular Dynamics). The CUGBP1 level was calculated as a ratio of the peak area for TBP. Primer sequences for RT-PCR were as follows: CUGBP1, 5′-CCAGACAACCAGATCTTGATGCT-3′ and 5′-AGGTTTCATCTGTATAGGGTGATG-3′; TBP, 5′-CCAGGAAATAACTCTGGCTCATAAC-3′, and 5′-AGTGAAGAACAGTCCAGACTGGCAG-3. Cytoplasm from Cos7 cells or from tissues of DM patients was fractionated by size exclusion chromatography on an SEC-400 column (BioLogic HR, Bio-Rad). Standard protein molecular weight markers were run in parallel. 300-μl fractions were collected and used for further analysis of CUGBP1 protein and its binding activity. The presence of total RNA in HPLC fractions was examined by agarose gel electrophoresis followed by EtBr staining and by slot hybridization of gel filtration fractions with 18 S rRNA-specific probe (29Welm A.L. Timchenko N.A. Darlington G.J. Mol. Cell. Biol. 1999; 19: 1695-1704Crossref PubMed Google Scholar). The position of CUG repeat-containing mRNA within the fractions was determined by slot hybridization with a CAG8 DNA probe labeled by γ-32P in a kinase reaction. The conditions for slot hybridization are as follows. 100 μl of each fraction was denatured with 50% formamide and blotted onto the membrane. The membrane was preincubated with a hybridization mixture (40% formamide: 4× SSC/5%SDS) for 1 h.32P-Labeled CAG8 probe was added and incubated for overnight under the same conditions. The membrane was washed with 2× SSC at room temperature for 2 h and exposed to x-ray film (BioMax). The binding activity of CUGBP1 was examined using gel-shift and UV cross-linking assays with CUG8 probe as described above. 5 μl of each fraction was used in these assays. Western analysis of the fractions was performed as described above. CUGBP1-stable clones were generated using an inducible LacSwitch mammalian system as described in our earlier study (25Timchenko N.A Wilde M. Nakanishi M. Smith J.R. Darlington G.J. Genes Dev. 1996; 10: 804-815Crossref PubMed Scopus (346) Google Scholar). The coding region of CUGBP1 was cloned into pOP-13 vector under a Rous sarcoma virus promoter that is regulated by Lac-Repressor. Human fibroblasts were stably transfected with Lac-Repressor and pOP-13-CUGBP1-antisense plasmids. Clones resistant to hygromycin and to G418 were selected and analyzed for the CUGBP1 expression after addition of IPTG. Several clones showed 2- to 5-fold reduction of CUGBP1 protein by expression of antisense CUGBP1 mRNA. One clone showed 6- to 8-fold reduction of CUGBP1 and was selected for further studies. The paper represents data obtained with this clone. Expression of antisense CUGBP1 was induced by addition of 10 mm IPTG, and proteins were isolated 4, 8, and 24 h after IPTG addition. CUGBP1 protein levels were determined by Western analysis with monoclonal as well as with polyclonal antibodies to CUGBP1 as described (13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar, 16Lu X. Timchenko N.A. Timchenko L.T. Hum. Mol. Genet. 1999; 8: 53-60Crossref PubMed Scopus (105) Google Scholar). C/EBPβ isoforms were analyzed by Western blotting with polyclonal (C-19, Santa Cruz Biotechnology) antibodies. Ratios of LIP/LAP and LAP/FL isoforms were calculated by densitometry. CUGBP1 has been shown to interact with CUG repeats, and its activity is altered in DM patients (13Timchenko L.T. Miller J. Timchenko N.A. DeVore D.R. Datar K.V. Lin L. Roberts R. Caskey C.T. Swanson M.S. Nucleic Acids Res. 1996; 24: 4407-4414Crossref PubMed Scopus (399) Google Scholar, 15Roberts R. Timchenko N.A. Miller J.W. Reddy S. Caskey C.T. Swanson M.S. Timchenko L.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13221-13226Crossref PubMed Scopus (137) Google Scholar, 21Philips A.V Timchenko L.T. Cooper T. Science. 1998; 280: 737-741Crossref PubMed Scopus (699) Google Scholar). To examine whether CUG repeats can directly affect CUGBP1, we initially investigated the status of CUGBP1 in DM cells containing the mutant DMPK transcripts with a large expansion of CUG repeats (15Roberts R. Timchenko N.A. Miller J.W. Reddy S. Caskey C.T. Swanson M.S. Timchenko L.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13221-13226Crossref PubMed Scopus (137) Google Scholar, 21Philips A.V Timchenko L.T. Cooper T. Science. 1998; 280: 737-741Crossref PubMed Scopus (699) Google Scholar) and in control tissues with a normal size of CUG repeats within DMPK mRNA. Cardiac proteins were fractionated by size exclusion chromatography and analyzed by gel-shift assay with a CUG8 probe. Normal control hearts contain a majority of CUGBP1 located in fractions with molecular mass corresponding to the size of free CUGBP1 (51 kDa). With the sensitivity of our gel-shift assays, little or no CUGBP1 is detectable in high molecular mass fractions that contain RNA (Fig.1 A). Additionally, we performed slot hybridization with a short CAG8 probe (see “Experimental Procedures”) that specifically hybridizes to CUG repeats. Under conditions of our experiments, RNA CUG repeats were not detected in any size exclusion fractions from control hearts. On the contrary, gel-shift analysis of fractions from DM tissue showed a remarkably different profile. We found that the majority of CUGBP1 is observed in high molecular weight fractions of DM extracts. Free CUGBP1 is detectable in low molecular weight fractions only after long exposure (data not shown). Slot hybridization of the DM fractions with the CAG8 probe revealed the presence of CUG repeat-containing RNAs that are colocalized with CUGBP1. The detection of CUG repeat-containing RNAs in DM cells and not in control cells is consistent with CUG repeat expansion in the DMPK gene. Under conditions of hybridization, short CUG repeats or interrupted CUG repeats are not detectable. Western analysis of size exclusion chromatography fractions confirmed that CUGBP1 is present in high molecular mass complexes in cardiac DM cells (Fig. 1 B). Reprobing the membranes with antibodies to α-actin showed a similar position of α-actin in fractions from DM and normal hearts, indicating that global differences in the fractionation procedure did not take place. It is necessary to note that the sensitivity of Western analysis after gel filtration was significantly reduced, and we were able to detect CUGBP1 only in fractions with higher amounts of the protein, whereas gel-shift detected CUGBP1 in several fractions. The above results show that the majority of CUGBP1 is associated with RNA in DM heart extracts, whereas in control samples, CUGBP1 is not bound to RNA. Furthermore, RNA·CUGBP1 complexes in DM extracts colocalized with CUG-containing RNAs. Under sensitivity of slot hybridization with the short CAG probe (24 nucleotides), normal DMPK transcripts with six CUG repeats were not detectable.
Referência(s)