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Overexpression of CUG Triplet Repeat-binding Protein, CUGBP1, in Mice Inhibits Myogenesis

2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês

10.1074/jbc.m312923200

1083-351X

Nikolai A. Timchenko, Roma H. Patel, Polina Iakova, Zong‐Jin Cai, Ling Quan, Lubov Timchenko,

Ubiquitin and proteasome pathways

Accumulation of RNA CUG repeats in myotonic dystrophy type 1 (DM1) patients leads to the induction of a CUG-binding protein, CUGBP1, which increases translation of several proteins that are required for myogenesis. In this paper, we examine the role of overexpression of CUGBP1 in DM1 muscle pathology using transgenic mice that overexpress CUGBP1 in skeletal muscle. Our data demonstrate that the elevation of CUGBP1 in skeletal muscle causes overexpression of MEF2A and p21 to levels that are significantly higher than those in skeletal muscle of wild type animals. A similar induction of these proteins is observed in skeletal muscle of DM1 patients with increased levels of CUGBP1. Immunohistological analysis showed that the skeletal muscle from mice overexpressing CUGBP1 is characterized by a developmental delay, muscular dystrophy, and myofiber-type switch: increase of slow/oxidative fibers and the reduction of fast fibers. Examination of molecular mechanisms by which CUGBP1 up-regulates MEF2A shows that CUGBP1 increases translation of MEF2A via direct interaction with GCN repeats located within MEF2A mRNA. Our data suggest that CUGBP1-mediated overexpression of MEF2A and p21 inhibits myogenesis and contributes to the development of muscle deficiency in DM1 patients. Accumulation of RNA CUG repeats in myotonic dystrophy type 1 (DM1) patients leads to the induction of a CUG-binding protein, CUGBP1, which increases translation of several proteins that are required for myogenesis. In this paper, we examine the role of overexpression of CUGBP1 in DM1 muscle pathology using transgenic mice that overexpress CUGBP1 in skeletal muscle. Our data demonstrate that the elevation of CUGBP1 in skeletal muscle causes overexpression of MEF2A and p21 to levels that are significantly higher than those in skeletal muscle of wild type animals. A similar induction of these proteins is observed in skeletal muscle of DM1 patients with increased levels of CUGBP1. Immunohistological analysis showed that the skeletal muscle from mice overexpressing CUGBP1 is characterized by a developmental delay, muscular dystrophy, and myofiber-type switch: increase of slow/oxidative fibers and the reduction of fast fibers. Examination of molecular mechanisms by which CUGBP1 up-regulates MEF2A shows that CUGBP1 increases translation of MEF2A via direct interaction with GCN repeats located within MEF2A mRNA. Our data suggest that CUGBP1-mediated overexpression of MEF2A and p21 inhibits myogenesis and contributes to the development of muscle deficiency in DM1 patients. DM1 1The abbreviations used are: DM1, myotonic dystrophy type 1; sORF, short out of frame open reading frame; C/EBPβ, CCAAT/enhancer-binding protein β; RBD, RNA-binding domain; GFP, green fluorescent protein; H/E, hematoxylin/eosin; IF, immunofluorescence; IP, immunoprecipitation. is a multisystem disease mainly characterized by defects in skeletal muscle with the involvement of many tissues and systems such as cardiac muscle, brain, eye, and endocrine system (1Harper P.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York1995: 4227-4251Google Scholar). DM1 is caused by an expansion of CTG trinucleotide repeats within the 3′-untranslated region of the myotonin protein kinase gene (2Aslanidis C. Jansen G. Amemiya C. Shutler G. Mahadevan M. Tsilfidis C. Clen 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. deJong P.J. Nature. 1992; 355: 548-551Crossref PubMed Scopus (458) Google Scholar). In DM1 patients, the size of DNA CTG expansion correlates with the severity of the disease. Patients with CTG expansion containing 50-80 CTG repeats are almost asymptomatic. Individuals bearing the myotonin protein kinase gene with 100-500 CTG repeats develop a disease in adult life (classical adult form of DM1) that is characterized by a progressive muscle wasting with myotonia. The most severe form of DM1, congenital disease, affects patients before or after birth and is associated with long CTG expansions (up to 2,000 repeats). This form of disease is characterized by a delay or arrest of skeletal muscle development (3Sahgal V. Bernes S. Sahgal S. Lischwey C. Subramani V. J. Neurolog. Sci. 1983; 59: 47-55Abstract Full Text PDF PubMed Scopus (25) Google Scholar). Although there is an overlap in range of repeats between different forms of DM1, there is a clear correlation of repeat number with severity of phenotype and reduction of age of onset. Investigations of molecular alterations in DM1 suggest that the expansion of CTG repeats causes the DM1 pathology through different mechanisms, mediated at both DNA and RNA levels. It has been shown that CTG repeats reduce expression of myotonin protein kinase in cis (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 T.C. Science. 1993; 260: 235-238Crossref PubMed Scopus (291) Google Scholar), causing abnormalities in cardiac muscle (5Berul C.I. Maguire C.T. Aronovitz M.J. Greenwood J. Miller C. Gehrmann J. Housman D. Mendelsohn M.E. Reddy S. J. Clin. Invest. 1999; 103: 1-7Crossref PubMed Scopus (152) Google Scholar). CTG repeats also affect transcription of genes adjacent to myotonin protein kinase (6Thornton C.A. Wymer J.P. Simmons Z. McClain C. Moxley R.T. Nat. Genet. 1997; 16: 407-409Crossref PubMed Scopus (195) Google Scholar), leading to the development of cataracts (7Klesert 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, 8Sarkar P.S. Appukuttan B. Han J. Ito Y. Tsai W. Chai Y. Stout J.T. Reddy S. Nat. Genet. 2000; 25: 110-114Crossref PubMed Scopus (151) Google Scholar). A number of recent studies indicate that other symptoms in DM1 such as myotonia (9Mankodi M. Logigian E. Callahan L. McClain C. White R. Henderson D. Krym M. Thornton C.A. Science. 2000; 289: 1769-1772Crossref PubMed Scopus (566) Google Scholar, 10Seznec H. Agbulut O. Sergeant N. Savouret C. Ghestem A. Tabti N. Willer J.C. Ourth L. Duros E Brisson E. Fouquet C. Butler-Browne G. Delacourte A. Junien C. Gourdon G. Hum. Mol. Genet. 2001; 10: 2717-2726Crossref PubMed Scopus (194) Google Scholar), delay of skeletal muscle differentiation (11Amack J.D. Paguio A.P. Mahadevan M.S. Hum. Mol. Genet. 1999; 8: 1975-1984Crossref PubMed Scopus (117) Google Scholar, 12Timchenko N.A. Iakova P. Cai Z-J. Smith J.R. Timchenko L.T. Mol. Cell Biol. 2001; 21: 6927-6938Crossref PubMed Scopus (153) Google Scholar), and a resistance to insulin (13Savkur R.S. Philips A.V. Cooper T.A. Nat. Genet. 2001; 29: 40-47Crossref PubMed Scopus (645) Google Scholar) are mediated through an RNA-based mechanism. According to this mechanism, RNA CUG repeats cause the DM1 disease via specific RNA CUG-binding proteins (14Timchenko L.T. Am. J. Hum. Genet. 1999; 64: 360-364Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Two families of RNA CUG-binding proteins have been identified: CUGBP (RNA-binding protein, binding to RNA CUG repeats) (15Lu X. Timchenko N.A. Timchenko L.T. Hum. Mol. Genet. 1999; 8: 53-60Crossref PubMed Scopus (104) Google Scholar, 16Timchenko L.T. Timchenko N.A. Caskey C.T. Roberts R. Hum. Mol. Genet. 1996; 5: 115-121Crossref PubMed Scopus (169) Google Scholar, 17Timchenko L.T. Miller J.W. 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 (398) Google Scholar) and EXP (also known as MNBL, muscle blind) proteins (18Miller 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 (718) Google Scholar). One of the members of the CUGBP family, CUGBP1, contributes to at least three symptoms of DM1: the delay of skeletal muscle differentiation (12Timchenko N.A. Iakova P. Cai Z-J. Smith J.R. Timchenko L.T. Mol. Cell Biol. 2001; 21: 6927-6938Crossref PubMed Scopus (153) Google Scholar), myotonia (19Charlet-B N. Savcur R.S. Singh G. Philips A.V. Grice E.A. Cooper T.A. Mol. Cell. 2002; 10: 45-53Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar), and the insulin resistance (13Savkur R.S. Philips A.V. Cooper T.A. Nat. Genet. 2001; 29: 40-47Crossref PubMed Scopus (645) Google Scholar). Previous studies showed that the expansion of CUG repeats in DM1 leads to a 3-5-fold elevation of CUGBP1 binding activity and protein levels (13Savkur R.S. Philips A.V. Cooper T.A. Nat. Genet. 2001; 29: 40-47Crossref PubMed Scopus (645) Google Scholar, 19Charlet-B N. Savcur R.S. Singh G. Philips A.V. Grice E.A. Cooper T.A. Mol. Cell. 2002; 10: 45-53Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar, 20Philips A.V. Timchenko L.T. Cooper T.A. Science. 1998; 280: 737-741Crossref PubMed Scopus (695) Google Scholar, 21Timchenko N.A. Cai Z-J. Welm A.L. Reddy S. Ashizawa T. Timchenko L.T. J. Biol. Chem. 2001; 276: 7820-7826Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), perhaps by stabilizing CUGBP1 protein within RNA CUG-CUGBP1 complexes (21Timchenko N.A. Cai Z-J. Welm A.L. Reddy S. Ashizawa T. Timchenko L.T. J. Biol. Chem. 2001; 276: 7820-7826Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). CUGBP1 possesses two major biological functions: regulation of splicing and translation (12Timchenko N.A. Iakova P. Cai Z-J. Smith J.R. Timchenko L.T. Mol. Cell Biol. 2001; 21: 6927-6938Crossref PubMed Scopus (153) Google Scholar, 20Philips A.V. Timchenko L.T. Cooper T.A. Science. 1998; 280: 737-741Crossref PubMed Scopus (695) Google Scholar, 21Timchenko N.A. Cai Z-J. Welm A.L. Reddy S. Ashizawa T. Timchenko L.T. J. Biol. Chem. 2001; 276: 7820-7826Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 22Timchenko N.A. Welm A.L. Lu X. Timchenko L.T. T L. Nucleic Acids Res. 1999; 27: 4517-4525Crossref PubMed Scopus (146) Google Scholar). Translational function of CUGBP1 was investigated mainly in cultured cells. These studies showed that CUGBP1 increases translation of p21 (12Timchenko N.A. Iakova P. Cai Z-J. Smith J.R. Timchenko L.T. Mol. Cell Biol. 2001; 21: 6927-6938Crossref PubMed Scopus (153) Google Scholar) and translation of a dominant negative isoform of transcription factor CCAAT/enhancer-binding protein β, LIP (22Timchenko N.A. Welm A.L. Lu X. Timchenko L.T. T L. Nucleic Acids Res. 1999; 27: 4517-4525Crossref PubMed Scopus (146) Google Scholar). It has been shown that, in tissue culture models, CUGBP1-mediated induction of p21 is required for proper differentiation of myocytes (12Timchenko N.A. Iakova P. Cai Z-J. Smith J.R. Timchenko L.T. Mol. Cell Biol. 2001; 21: 6927-6938Crossref PubMed Scopus (153) Google Scholar). However, the role of translational function of CUGBP1 in vivo has not been addressed. To examine the role of translational activity of CUGBP1 in development of skeletal muscle deficiency in DM1 patients, we generated transgenic mice that overexpress CUGBP1 mainly in skeletal muscle and to a lesser extent in the heart. We found that elevation of CUGBP1 causes overexpression of its translational targets p21 and MEF2A (transcription factor, myocyte enhancer factor 2A) in skeletal muscle. These alterations cause a muscular dystrophy, an increase of slow fibers, and delay of muscle development. UV Cross-linking—RNA transcripts encoding 123 CUG repeats, the full-length wild type, and mutant MEF2A mRNAs lacking the CUGBP1 binding site were synthesized in an in vitro transcription assay (Roche Applied Science) and incubated with 10-20 μg of purified proteins as described (12Timchenko N.A. Iakova P. Cai Z-J. Smith J.R. Timchenko L.T. Mol. Cell Biol. 2001; 21: 6927-6938Crossref PubMed Scopus (153) Google Scholar). After UV cross-linking, the samples were treated with RNase A (2 units/10 μl) and separated by PAGE with 0.1% SDS. Where indicated, MEF2A riboprobe containing the (GCA)9 repeat (CUGBP1 binding site within MEF2A mRNA) or sORF riboprobe specific for C/EBPβ was used. Generation of CUGBP1 Transgenic Mice—The experiments related to transgenic mice complied with federal guidelines and institutional policies. The full-length human CUGBP1 cDNA was amplified from a parental plasmid (17Timchenko L.T. Miller J.W. 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 (398) Google Scholar) with CUGBP1-specific primers. The sequence of the forward primer is as follows: 5′-TCA AAG AAA ATG AAC GGC ACC CTG-3′. The sequence of the reverse primer fused in frame with a short sequence coding for 6 histidines is as follows: 5′-ATG GTG ATG GTG ATG ATG GTA GGG CTT GCT GTC ATT CTT CGA-3′. The CUGBP1 cDNA fused with the track of histidines on its C terminus was cloned into BS vector containing chicken β-actin promoter connected with cytomegalovirus enhancer (a gift from Dr. Schneider, Baylor College of Medicine). Transgenic construct was injected into mouse FVB inbred eggs at DNX Transgenic Sciences Co. (Princeton, NJ). Genotyping of Transgenic Mice—Progenies were genotyped by PCR with CUGBP1-specific primers. The sequence of forward primer is 5′-GCT GCA TTA GAA GCT CAG AAT GCT-3′. The reverse primer has a sequence 5′-AGG TTT CAT CTG TAT AGG GTG ATG-3′. Two PCR products of different size from both wild type (540 bp) and mutant alleles (78 bp) were synthesized simultaneously. The size of PCR product from mutant allele is smaller because the transgene lacks introns. Tissue Culture—Mouse C2C12 myoblasts and human primary myoblast cultures were grown in 10-cm dishes in myoblast growth medium at 60% of density. Differentiation was induced by a switch of growth medium to fusion medium as described (12Timchenko N.A. Iakova P. Cai Z-J. Smith J.R. Timchenko L.T. Mol. Cell Biol. 2001; 21: 6927-6938Crossref PubMed Scopus (153) Google Scholar). Differentiating myoblasts were maintained for 5 days with medium changes occurring every day. Northern Analysis—Total cellular RNA was extracted with TRI Reagent (Molecular Research Center, Inc.) from C2C12 myoblasts and myotubes at different time points of differentiation. RNA was hybridized with CUGBP1 probe as described (15Lu X. Timchenko N.A. Timchenko L.T. Hum. Mol. Genet. 1999; 8: 53-60Crossref PubMed Scopus (104) Google Scholar). Levels of CUGBP1 mRNA (7 + 9 kb) were calculated as a ratio to 18 S rRNA control. Immunostaining of Mouse Tissues—Paraffin and frozen sections were prepared from vastus or plantaris muscles from wild type and mutant mice with different numbers of CUGBP1 copies of the matching age and gender. Muscle sections were stained with H/E at the Baylor College of Medicine Histology Core. For immunoanalysis, sections were blocked with 1% bovine serum albumin in phosphate-buffered saline for 1 h and incubated for 1 h with antibodies against MEF2 (C-21; 1:1,000), myoglobin (sc-8081; 1:200) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), fast myosin (MY-32; 1:100), and slow myosin heavy chain (NOQ7.54D; 1:50) (Sigma). Rhodamine-labeled anti-rabbit and anti-mouse immunoglobulins from Santa Cruz Biotechnology were used as secondary antibodies with dilution 1:200. Images were analyzed in Microscopic Core Facilities of Aging Center and Department of Molecular and Cellular Biology at Baylor College of Medicine. Isolation of Proteins and Western Blotting Analysis—Whole protein extracts from mouse tissues were prepared with radioimmune precipitation buffer. Cytoplasmic, nuclear, and whole cell extracts from cultured cells were isolated as described (12Timchenko N.A. Iakova P. Cai Z-J. Smith J.R. Timchenko L.T. Mol. Cell Biol. 2001; 21: 6927-6938Crossref PubMed Scopus (153) Google Scholar). The primary antibodies were rabbit anti-CUGBP1, MEF2 (C-21), myoglobin (sc-8081), and MyoD (myogenic transcription factor) (C-20) and mouse p21 (H164), myogenin (CF5D), fast (MY-32) and slow myosin (NOQ7.54D), and α-tubulin (TU-02). Detection was performed using the ECL kit (Pierce). Membranes were stripped and reprobed with control antibodies (α-tubulin or actin), and results were quantified by densitometer analysis. The Effect of CUGBP1 on MEF2A Levels in Cultured Cells—C2C12 cells were transfected with empty pcDNA vector and with recombinant plasmid containing CUGBP1 in pcDNA vector using FuGene 6 protocol. Protein extracts were isolated in 48 h after transfection and subjected to Western blotting with antibodies against His tag, CUGBP1, and MEF2A. CUGBP1 and MEF2A protein levels were calculated as a ratio to β-actin. To visualize the effects of CUGBP1 on MEF2A levels, C2C12 cells grown in slides were transfected with GFP as control and with CUGBP1 cloned into GFP vector. Cells were fixed and subjected to IF with antibodies against MEF2A using secondary antibodies labeled with rhodamine. CUGBP1 (green signal) and MEF2A (red signal) were analyzed at ×40 magnification. Examination of MEF2A Translation in Tissue Culture—One set of plates with C2C12 cells was co-transfected with MEF2A and CUGBP1, and another set was co-transfected with MEF2A and empty vector. One day after transfection, the growth medium was replaced with Met-free medium. In 24 h, Met-deficient medium was complemented with [35S]Met, and whole cell protein extracts were prepared in 0, 1, 2, 4, and 8 h after the addition of [35S]Met. Protein extracts were diluted with phosphate-buffered saline buffer and incubated with antibodies against MEF2A (1:100) for 4 h at 4 °C, following incubation with protein A-agarose for 1 h. Immunoprecipitates were collected by centrifugation at 6,000 rpm for 10 min, washed three times with 1 ml of phosphate-buffered saline, resuspended in the loading buffer, and analyzed by polyacrylamide gel electrophoresis. Inhibition of CUGBP1 by siRNA—Two RNA CUGBP1 primers were synthesized in Qiagen to target the following CUGBP1 sequence: 5′-AAT TTG GCT GCA CTA GCT GCT-3′. The sequence of the first primer is r(UUUGGCUGCACUAGCUGCU)d(TT). The sequence of the second primer is r(AGCAGCUAGUGCAGCCAAA)d(TT). These primers were annealed to make double-stranded siRNA-CUGBP1. Transfection of siRNA CUGBP1 was performed according to the Qiagen protocol. C2C12 cells were grown at 70% density. Enhancer R was mixed with siRNA oligonucleotide and transfection reagent in a ratio of 2:1:2.5. Protein extracts were prepared from proliferating and differentiating myoblasts transfected with siRNA. In control plates, no siRNA was added. Translation in an in Vitro Cell-free System—MEF2A plasmid was identified by screening of a human cardiac cDNA library with a GCA triplet repeat probe. GCA deletion mutant was generated by using QuikChange™ site-directed mutagenesis kit (Stratagene). The sequence of forward mutagenic primer is 5′-TGG CGG CGG CTG GAA GCC CGA TGG GGT CAT-3′. Rabbit reticulocyte lysate was programmed with wild type and mutant MEF2A mRNAs, and increased amounts of purified CUGBP1 were added to the incubation mixture. Translational products were subjected to SDS-PAGE and autoradiography. Interaction of CUGBP1 and MEF2A mRNA in Vivo—C2C12 myoblasts were grown in the myoblast medium until 80% density and then subjected to differentiation in the fusion medium during 5 days. Cytoplasmic protein extracts were collected from C2C12 differentiating myoblasts in cytoplasmic buffer (20 mm Tris-HCl, pH 7.5, 30 mm KCl, 5 mm EDTA, and 5 mm MgCl2) containing RNase inhibitors. CUGBP1 was immunoprecipitated with monoclonal antibodies 3B1. RNA was extracted by phenol-chloroform and reverse-transcribed with AML using oligo(dT) primer. PCR was performed with primers specific to mouse MEF2A mRNA. The sequence of the forward primer was 5′-GATGTTGAGCACTACAGACCTCA-3′, and the sequence of the reverse primer was 5′-CTCCCCTGTGGACAGTCTGAGCA-3′. The size of the PCR product with MEF2A primers was 743 bp. Myogenic Conversion of Fibroblasts into Myoblasts—Mouse fibroblasts 10T1/2 were grown onto four-chamber slides in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells grown at 60% of density were transfected with plasmids expressing MyoD, wild type and deletion mutant MEF2A, and the full-length or truncated CUGBP1. The amount of plasmid DNA was 0.2 μg per chamber and remained constant in all experiments. When one plasmid was used instead of two or three, the amount of DNA was adjusted with pcDNA vector. Transfection was performed with FuGene. In 2 days after transfection, fibroblast growth medium was replaced with myoblast fusion medium. Cells were grown for 5 days with medium changed every day. Cells were fixed with 4% formaldehyde for 30 min with two changes of fresh formaldehyde. Slides were blocked in 1% bovine serum albumin in phosphate-buffered saline containing goat serum (dilution 1:50) for 2 h. After blocking, slides were incubated with mouse antibodies against fast myosin (Sigma; dilution 1:50) and rabbit antibodies against MyoD (Santa Cruz Biotechnology; 1:50). Secondary anti-rabbit antibodies labeled with rhodamine or anti-mouse antibodies labeled with fluorescein isothiocyanate (Santa Cruz Biotechnology) were used at a dilution of 1:200. Generation of CUGBP1 Transgenic Mice—The full-length CUGBP1 coding region was placed under a modified β-actin promoter, which is mainly active in skeletal muscle and to a lesser extent in the heart (23Araki K. Araki M. Miyazaki J. Vassalli P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 160-164Crossref PubMed Scopus (258) Google Scholar), and fused on the C terminus to an oligonucleotide coding for 6 His amino acids. The resulting transgenic construct was injected into inbred eggs of FVB mice, and pups were genotyped by PCR with CUGBP1-specific primers. The expression of transgenic protein (His-CUGBP1) was verified by Western blotting with antibodies to His tag (Fig. 1, A and B). We first examined expression of His-CUGBP1 in different tissues of CUGBP1 transgenic mice. In agreement with previous publications (23Araki K. Araki M. Miyazaki J. Vassalli P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 160-164Crossref PubMed Scopus (258) Google Scholar), β-actin promoter fused with cytomegalovirus enhancer directs the expression of His-CUGBP1 primarily in skeletal muscle and to a lesser extent in cardiac muscle (Fig. 1A). Within the sensitivity of Western blot analysis, we did not detect His-CUGBP1 in brain, spleen, and kidney (Fig. 1A). We have generated five lines of CUGBP1 transgenic animals (Table I). The analysis of His-CUGBP1 in skeletal muscle of these animals showed that the expression of His-CUGBP1 is increased to different levels (Fig. 1B). In addition, we found that the expression of His-CUGBP1 leads to a 2-3-fold elevation of the endogenous CUGBP1 levels (Fig. 1C). This estimate was done using protein extracts from vastus muscles of 3-5 mice of each line and 17 wild type mice. The mechanisms by which the transgenic His-CUGBP1 protein induces protein levels of endogenous CUGBP1 are unknown. Our data also showed that CUGBP1 immunoreactive proteins migrate as several bands, perhaps because of different levels in phosphorylation (24Roberts 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). To examine whether these immunoreactive bands are isoforms of His-CUGBP1, we immunoprecipitated His-CUGBP1 with polyclonal antibodies to His and performed a two-dimensional gel electrophoresis followed by Western blotting with monoclonal antibodies to CUGBP1. Fig. 1D shows that His-CUGBP1 migrates as several spots located between pH 6.0 and 8.0. Acidic isoforms of His-CUGBP1 represent phosphorylated forms of CUGBP1, since a treatment of His-IPs with CIP shifts these isoforms to the alkali region of the strip. Given the elevation of both His-CUGBP1 and endogenous CUGBP1, as well as phosphorylated isoforms of CUGBP1, CUGBP1 levels were calculated as a ratio of all of these isoforms in transgenic animals to endo-CUGBP1 in wild type animals. These calculations showed that an increase of total levels of CUGBP1 correlates with the number of transgenic copies (see Table I). All analyzed animals showed a relatively narrow rate of CUGBP1 overexpression (from 2- to 10-fold), perhaps due to negative effect of CUGBP1 elevation on mouse development (Fig. 2).Table 1The loss of body weight correlates with the levels of CUGBP1 inductionMouse lineTransgene copiesInduction of CUGBP1 proteinaThe induction of CUGBP1 levels in skeletal muscle was calculated as a ratio of (His-CUGBP1 + endogenous CUGBP1) in transgenic mice to endogenous CUGBP1 in wild type littermates.Body weight, percentage of wild type, newborns%CUGBP1-20bThree litters (21 mice) were weighted, p < 0.001.55-673-77CUGBP1-29cFour litters (28 mice) were weighted, p < 0.001.22-382-88CUGBP1-32dThree litters (26 mice) were weighted, p < 0.002.1395-100CUGBP1-36eFour litters (32 mice) were weighted, p < 0.001.88-1055-60CUGBP1-49fFour litters (30 mice) were weighted, p < 0.002.64-672-74a The induction of CUGBP1 levels in skeletal muscle was calculated as a ratio of (His-CUGBP1 + endogenous CUGBP1) in transgenic mice to endogenous CUGBP1 in wild type littermates.b Three litters (21 mice) were weighted, p < 0.001.c Four litters (28 mice) were weighted, p < 0.001.d Three litters (26 mice) were weighted, p < 0.002.e Four litters (32 mice) were weighted, p < 0.001.f Four litters (30 mice) were weighted, p < 0.002. Open table in a new tab Fig. 2CUGBP1 transgenic mice. A, effect of CUGBP1 transgene on mouse weight. A picture of wild type and two transgenic mice at the age of 5 days is shown. Heterozygous transgenic animals with 5-6-fold induction of CUGBP1 (TR1) survive but are underweight. A hemizygous transgenic animal (TR2) with an 8-fold increase of CUGBP1 died at 7 days of age. A bar graph shows a summary of body weight for four litters of line 20 (p < 0.001) and three litters of wild type mice (p < 0.002) with a mean of 10 animals/litter weighted at 10 and 20 days of age. B, CUGBP1 transgenic mice with high copies of CUGBP1 transgene die in utero. The picture of wild type and CUGBP1 transgenic mouse (at 20 days of embryonic development) with 10-fold elevation of CUGBP1 (line 36) is shown. C, skeletal muscles of CUGBP1 transgenic mice are severely damaged. Cross-sections through hind limbs of wild type and mutant littermates (line 36) are shown. Note that the size of femur is unchanged, but the layer of muscles is significantly reduced. D, His-CUGBP1 from skeletal muscle of CUGBP1 transgenic mice interacts with CUG repeats. Total skeletal muscle protein extracts were incubated with the CUG123 probe and analyzed by UV cross-link assay. After UV cross-link, the membrane was stained with Coomassie (below). Positions of His-CUGBP1 (line 32) and endogenous CUGBP1 are shown. The bottom (His-IP) shows UV cross-link with His immunoprecipitates from WT and CUGBP1 transgenic mice. E, His-CUGBP1 increases translation of p21 and LIP in a cell-free translation system. His-CUGBP1 was precipitated from skeletal muscle with His tag Abs and added into reticulocyte lysate programmed with p21 mRNA (top) or with C/EBPβ mRNA. After translation, p21 or C/EBPβ was immunoprecipitated and examined by gel electrophoresis. A diagram shows the structure of C/EBPβ mRNA and the position of the CUGBP1 binding site.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A Severity of Developmental Delay Correlates with Levels of CUGBP1 Elevation—It has been previously shown that transgenic mice overexpressing the human DM1 region with greater than 300 CTG repeats developed myotonia and are growth-retarded (10Seznec H. Agbulut O. Sergeant N. Savouret C. Ghestem A. Tabti N. Willer J.C. Ourth L. Duros E Brisson E. Fouquet C. Butler-Browne G. Delacourte A. Junien C. Gourdon G. Hum. Mol. Genet. 2001; 10: 2717-2726Crossref PubMed Scopus (194) Google Scholar). Given the accumulation of CUGBP1 in these mice, we performed gross analysis of CUGBP1 transgenic mice from different lines. This analysis showed that mutant mice, in which CUGBP1 is elevated to 2-3-fold (lines 29 and 32), have slight reduction of weight at birth but survive and develop normally. However, transgenic mice, in which CUGBP1 is 4-6-fold elevated, were growth-retarded and significantly underweight (Fig. 2, A and B). Analysis of three or four litters from different lines confirmed that weight of newborn CUGBP1 transgenic mice is ∼5-30% less than their wild type littermates (Table I) and that the reduction of weight depends on the levels of CUGBP1 protein. Animals with 8-10-fold elevation of CUGBP1 die in utero and are severely underdeveloped (Fig. 2B). This suggests that the overexpression of CUGBP1 in vivo might have severe impact on mouse development. Given the strongest expression of CUGBP1 transgene in skeletal muscle (Fig. 1C), we performed cross-sections through hind limb and femur of newborn wild type and mutant (line 36) littermates and found that whereas the size of femur in mutant mice is unchanged, the hind limb muscles look significantly reduced (Fig. 2C). Therefore, we further focused our studies on the histological analysis of skeletal muscle of CUGBP1 transgenic mice and on molecular pathways by which overexpression of CUGBP1 causes the disorder in skeletal muscle. His-CUGBP1 Precipitated from Skeletal Muscle of CUGBP1 Transgenic Mice Is Able to Regulate Translation of mRNAs—In vitro studies have demonstrated that CUGBP1 is a key regulator of translation of p21 and C/EBPβ mRNAs (12Timchenko N.A. Iakova P. Cai Z-J. Smith J.R. Ti