Muscleblind-Like Proteins
2008; Elsevier BV; Volume: 174; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.080520
ISSN1525-2191
AutoresIan Holt, Virginie Jacquemin, Majid Fardaei, Caroline A. Sewry, Gillian Butler‐Browne, Denis Furling, J. David Brook, Glenn E. Morris,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoIn myotonic dystrophy, muscleblind-like protein 1 (MBNL1) protein binds specifically to expanded CUG or CCUG repeats, which accumulate as discrete nuclear foci, and this is thought to prevent its function in the regulation of alternative splicing of pre-mRNAs. There is strong evidence for the role of the MBNL1 gene in disease pathology, but the roles of two related genes, MBNL2 and MBNL3, are less clear. Using new monoclonal antibodies specific for each of the three gene products, we found that MBNL2 decreased during human fetal development and myoblast culture, while MBNL1 was unchanged. In Duchenne muscular dystrophy muscle, MBNL2 was elevated in immature, regenerating fibres compared with mature fibres, supporting some developmental role for MBNL2. MBNL3 was found only in C2C12 mouse myoblasts. Both MBNL1 and MBNL2 were partially sequestered by nuclear foci of expanded repeats in adult muscle and cultured cells from myotonic dystrophy patients. In adult muscle nucleoplasm, both proteins were reduced in myotonic dystrophy type 1 compared with an age-matched control. In normal human myoblast cultures, MBNL1 and MBNL2 always co-distributed but their distribution could change rapidly from nucleoplasmic to cytoplasmic. Functional differences between MBNL1 and MBNL2 have not yet been found and may prove quite subtle. The dominance of MBNL1 in mature, striated muscle would explain why ablation of the mouse mbnl1 gene alone is sufficient to cause a myotonic dystrophy. In myotonic dystrophy, muscleblind-like protein 1 (MBNL1) protein binds specifically to expanded CUG or CCUG repeats, which accumulate as discrete nuclear foci, and this is thought to prevent its function in the regulation of alternative splicing of pre-mRNAs. There is strong evidence for the role of the MBNL1 gene in disease pathology, but the roles of two related genes, MBNL2 and MBNL3, are less clear. Using new monoclonal antibodies specific for each of the three gene products, we found that MBNL2 decreased during human fetal development and myoblast culture, while MBNL1 was unchanged. In Duchenne muscular dystrophy muscle, MBNL2 was elevated in immature, regenerating fibres compared with mature fibres, supporting some developmental role for MBNL2. MBNL3 was found only in C2C12 mouse myoblasts. Both MBNL1 and MBNL2 were partially sequestered by nuclear foci of expanded repeats in adult muscle and cultured cells from myotonic dystrophy patients. In adult muscle nucleoplasm, both proteins were reduced in myotonic dystrophy type 1 compared with an age-matched control. In normal human myoblast cultures, MBNL1 and MBNL2 always co-distributed but their distribution could change rapidly from nucleoplasmic to cytoplasmic. Functional differences between MBNL1 and MBNL2 have not yet been found and may prove quite subtle. The dominance of MBNL1 in mature, striated muscle would explain why ablation of the mouse mbnl1 gene alone is sufficient to cause a myotonic dystrophy. Myotonic dystrophy type 1 (DM1) is a progressive multisystemic disorder showing considerable clinical variation between individuals. DM1 is characterized by skeletal muscle weakness, wasting and pain, as well as myotonia.1Harper PS Myotonic Dystrophy. ed 3. WB Saunders, London2004Google Scholar Other symptoms may include cardiac arrhythmias, cataracts, insulin resistance, hypogonadism, neurological problems and premature male balding.1Harper PS Myotonic Dystrophy. ed 3. 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More than 50 CTG repeats cause mild to classical adult-onset DM and 700 to greater than 3000 repeats often result in the severe congenital form of the disease. However, repeat size in muscle and other tissues can be much higher than in lymphocytes.9Anvret M Ahlberg G Grandell U Hedberg B Johnson K Edstrom L Larger expansions of the CTG repeat in muscle compared to lymphocytes from patients with myotonic dystrophy.Hum Mol Genet. 1993; 2: 1397-1400Crossref PubMed Scopus (175) Google Scholar A second form of DM (DM2) is due to a CCTG repeat in intron 1 of the ZNF9 gene on chromosome 3q21.3.10Liquori CL Ricker K Moseley ML Jacobsen JF Kress W Naylor SL Day JW Ranum LP Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9.Science. 2001; 293: 864-867Crossref PubMed Scopus (1009) Google Scholar Clinical features of DM1 and DM2 are similar but not identical. DM2 patients may show proximal rather than distal muscle involvement, and the severe congenital form occurs in DM1 only. The number of repeats in DM2 may be 10-fold greater than in DM1.10Liquori CL Ricker K Moseley ML Jacobsen JF Kress W Naylor SL Day JW Ranum LP Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9.Science. 2001; 293: 864-867Crossref PubMed Scopus (1009) Google Scholar Current evidence suggests that DM pathogenesis is due to the toxic gain of function of the mutant RNA. Transgenic mouse models with expanded CUG repeats in the 3′-UTR of the unrelated muscle-specific actin or the human DMPK transcripts develop features of DM1,11Mankodi A Logigian E Callahan L McClain C White R Henderson D Krym M Thornton CA Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat.Science. 2000; 289: 1769-1773Crossref PubMed Scopus (566) Google Scholar, 12Seznec H Agbulut O Sergeant N Savouret C Ghestem A Tabti N Willer JC Ourth L Duros C Brisson E Fouquet C Butler-Browne G Delacourte A Junien C Gourdon G Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities.Hum Mol Genet. 2001; 10: 2717-2726Crossref PubMed Scopus (194) Google Scholar suggesting that the major clinical features of DM1 are due directly to the repeat expansion. The expanded repeats in DM1 and DM2 accumulate in the nuclei as discrete foci.13Taneja KL McCurrach M Schalling M Housman D Singer RH Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues.J Cell Biol. 1995; 128: 995-1002Crossref PubMed Scopus (501) Google Scholar, 14Hamshere MG Newman EE Alwazzan M Athwal BS Brook JD Transcriptional abnormality in myotonic dystrophy affects DMPK but not neighboring genes.Proc Natl Acad Sci USA. 1997; 94: 7394-7399Crossref PubMed Scopus (118) Google Scholar, 15Davis BM McCurrach ME Taneja KL Singer RH Housman DE Expansion of a CUG trinucleotide repeat in the 3′ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts.Proc Natl Acad Sci USA. 1997; 94: 7388-7393Crossref PubMed Scopus (381) Google Scholar, 16Margolis JM Schoser BG Moseley ML Day JW Ranum LP DM2 intronic expansions: evidence for CCUG accumulation without flanking sequence or effects on ZNF9 mRNA processing or protein expression.Hum Mol Genet. 2006; 15: 1808-1815Crossref PubMed Scopus (89) Google Scholar The relationship between these ribonuclear inclusions, which may consist of double stranded hairpin loop structures,17Jasinska A Michlewski G de Mezer M Sobczak K Kozlowski P Napierala M Krzyzosiak WJ Structures of trinucleotide repeats in human transcripts and their functional implications.Nucleic Acids Res. 2003; 31: 5463-5468Crossref PubMed Scopus (64) Google Scholar, 18Kino Y Mori D Oma Y Takeshita Y Sasagawa N Ishiura S Muscleblind protein. MBNL1/EXP, binds specifically to CHHG repeats.Hum Mol Genet. 2004; 13l: 495-507Crossref Scopus (144) Google Scholar and DM pathogenesis is not entirely clear.19Ho TH Savkur RS Poulos MG Mancini MA Swanson MS Cooper TA Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy.J Cell Sci. 2005; 118: 2923-2933Crossref PubMed Scopus (155) Google Scholar Mutant DMPK mRNA in nuclear foci of DM1 cells appears to be spliced and polyadenylated normally,15Davis BM McCurrach ME Taneja KL Singer RH Housman DE Expansion of a CUG trinucleotide repeat in the 3′ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts.Proc Natl Acad Sci USA. 1997; 94: 7388-7393Crossref PubMed Scopus (381) Google Scholar whereas DM2 foci appear to consist of spliced-out introns.16Margolis JM Schoser BG Moseley ML Day JW Ranum LP DM2 intronic expansions: evidence for CCUG accumulation without flanking sequence or effects on ZNF9 mRNA processing or protein expression.Hum Mol Genet. 2006; 15: 1808-1815Crossref PubMed Scopus (89) Google Scholar As a result of this difference, DM1 foci accumulate at the periphery of nuclear splicing "speckles" on the mRNA export pathway, whereas DM2 foci do not.20Holt I Mittal S Furling D Butler-Browne GS Brook JD Morris GE Defective mRNA in myotonic dystrophy accumulates at the periphery of nuclear splicing speckles.Genes Cells. 2007; 12: 1035-1048Crossref PubMed Scopus (74) Google Scholar The Drosophila muscleblind protein, first described as a regulatory factor required for the differentiation of photoreceptor cells and muscle Z-bands,21Begemann G Paricio N Artero R Kiss I Perez-Alonso M Mlodzik M Muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins.Development. 1997; 12: 4321-4331Google Scholar, 22Artero R Prokop A Paricio N Begemann G Pueyo I Mlodzik M Perez-Alonso M Baylies MK The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2.Dev Biol. 1998; 195: 131-143Crossref PubMed Scopus (128) Google Scholar is an RNA binding protein. There are three human homologues of the muscleblind gene, MBNL1, MBNL2, and MBNL3 on chromosomes 3q25, 13q32.2 and Xq26.2 respectively, with different RNA splice forms occurring.23Fardaei M Rogers MT Thorpe HM Larkin K Hamshere MG Harper PS Brook JD Three proteins. MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells.Hum Mol Genet. 2002; 11: 805-814Crossref PubMed Scopus (360) Google Scholar Muscleblind proteins have been shown to bind specifically to expanded dsCUG RNA but not normal size CUG repeats, in a manner proportional to the size of the triplet repeat expansion.24Miller JW Urbinati CR Teng-Umnuay P Stenberg MG Byrne BJ Thornton CA Swanson MS Recruitment of human muscleblind proteins to (CUG) (n) expansions associated with myotonic dystrophy.EMBO J. 2000; 19: 4439-4448Crossref PubMed Scopus (718) Google Scholar Transfected MBNL1, MBNL2, and MBNL3 colocalize with the expanded CUG/CCUG ribonuclear inclusions in DM cells.19Ho TH Savkur RS Poulos MG Mancini MA Swanson MS Cooper TA Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy.J Cell Sci. 2005; 118: 2923-2933Crossref PubMed Scopus (155) Google Scholar, 23Fardaei M Rogers MT Thorpe HM Larkin K Hamshere MG Harper PS Brook JD Three proteins. MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells.Hum Mol Genet. 2002; 11: 805-814Crossref PubMed Scopus (360) Google Scholar, 25Fardaei M Larkin K Brook JD Hamshere MG In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts.Nucleic Acids Res. 2001; 29: 2766-2771Crossref PubMed Google Scholar, 26Paul S Dansithong W Kim D Rossi J Webster NJ Comai L Reddy S Interaction of muscleblind. CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing.EMBO J. 2006; 25: 4271-4283Crossref PubMed Scopus (119) Google Scholar Several studies have reported the colocalization of endogenous MBNL1 with ribonuclear foci,19Ho TH Savkur RS Poulos MG Mancini MA Swanson MS Cooper TA Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy.J Cell Sci. 2005; 118: 2923-2933Crossref PubMed Scopus (155) Google Scholar, 20Holt I Mittal S Furling D Butler-Browne GS Brook JD Morris GE Defective mRNA in myotonic dystrophy accumulates at the periphery of nuclear splicing speckles.Genes Cells. 2007; 12: 1035-1048Crossref PubMed Scopus (74) Google Scholar, 24Miller JW Urbinati CR Teng-Umnuay P Stenberg MG Byrne BJ Thornton CA Swanson MS Recruitment of human muscleblind proteins to (CUG) (n) expansions associated with myotonic dystrophy.EMBO J. 2000; 19: 4439-4448Crossref PubMed Scopus (718) Google Scholar, 27Mankodi A Urbinati CR Yuan QP Moxley RT Sansone V Krym M Henderson D Schalling M Swanson MS Thornton CA In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts.Nucleic Acids Res. 2001; 29: 2766-2771Crossref PubMed Scopus (182) Google Scholar, 28Mankodi A Teng-Umnuay P Krym M Henderson D Swanson M Thornton CA Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2.Ann Neurol. 2003; 54: 760-768Crossref PubMed Scopus (140) Google Scholar, 29Jiang H Mankodi A Swanson MS Moxley RT Thornton CA Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2.Hum Mol Genet. 2001; 10: 2165-2170Crossref PubMed Scopus (355) Google Scholar, 30Mankodi A Lin X Blaxall BC Swanson M Thornton CA Nuclear RNA foci in the heart in myotonic dystrophy.Circ Res. 2005; 97: 1152-1155Crossref PubMed Scopus (89) Google Scholar, 31Lin X Miller JW Mankodi A Swanson MS Thornton CA Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy.Hum Mol Genet. 2006; 15: 2087-2097Crossref PubMed Scopus (389) Google Scholar and one study suggests that MBNL1 is required for focus formation.32Dansithong W Paul S Comai L Reddy S MBNL1 is the primary determinant of focus formation and aberrant insulin receptor splicing in DM1.J Biol Chem. 2005; 280: 5773-5780Crossref PubMed Scopus (166) Google Scholar In addition, a mouse functional knockout of MBNL1 shows DM features, such as myotonia, abnormal myofibers, cataracts and aberrant splicing of chloride channel, cardiac troponin T, and fast skeletal troponin T.33Kanadia RN Johnstone KA Mankodi A Lungu C Thornton CA Esson D Timmers AM Hauswirth WW Swanson MS A muscleblind knockout model for myotonic dystrophy.Science. 2003; 302: 1978-1980Crossref PubMed Scopus (595) Google Scholar At least some of the pathological features of DM are thought to be due to misregulated alternative splicing of RNA. Misregulated alternative splicing in DM has been reported for at least 20 gene transcripts (cited by Osborne and Thornton,34Osborne RJ Thornton CA RNA-dominant diseases.Hum Mol Genet. 2006; 15: R162-R169Crossref PubMed Scopus (185) Google Scholar), including the muscle-specific chloride channel, insulin receptor, brain microtubule-associated tau, MBNL1 and MBNL2. MBNL proteins have been shown to bind specific targets on cardiac troponin T pre-mRNA (from the TNNT2 gene) and to regulate alternative splicing by repressing exon inclusion in TNNT2 mRNA and inducing exon inclusion in insulin receptor mRNA.35Ho TH Charlet-B N Poulos MG Singh G Swanson MS Cooper TA Muscleblind proteins regulate alternative splicing.EMBO J. 2004; 23: 3103-3112Crossref PubMed Scopus (376) Google Scholar Recently, overexpression of MBNL1 in a poly(CUG) mouse model for DM has been shown to reverse myotonia and correct the mis-splicing of four pre-mRNAs.36Kanadia RN Shin J Yuan Y Beattie SG Wheeler TM Thornton CA Swanson MS Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy.Proc Natl Acad Sci USA. 2006; 103: 11748-11753Crossref PubMed Scopus (278) Google Scholar Sequestration of MBNL1 by CUG repeats is therefore strongly implicated in the pathogenesis of DM,34Osborne RJ Thornton CA RNA-dominant diseases.Hum Mol Genet. 2006; 15: R162-R169Crossref PubMed Scopus (185) Google Scholar but there is also evidence that elevation of CUG-BP1 may also play a role.37Faustino NA Cooper TA Pre-mRNA splicing and human disease.Genes Dev. 2003; 17: 419-437Crossref PubMed Scopus (987) Google Scholar, 38Orengo JP Chambon P Metzger D Mosier DR Snipes GJ Cooper TA Expanded CTG repeats within the DMPK 3′ UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy.Proc Natl Acad Sci USA. 2008; 105: 2646-2651Crossref PubMed Scopus (150) Google Scholar Endogenous MBNL2 has been little studied, but it does colocalize with ribonuclear foci in sections of cortical neurons29Jiang H Mankodi A Swanson MS Moxley RT Thornton CA Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2.Hum Mol Genet. 2001; 10: 2165-2170Crossref PubMed Scopus (355) Google Scholar and heart30Mankodi A Lin X Blaxall BC Swanson M Thornton CA Nuclear RNA foci in the heart in myotonic dystrophy.Circ Res. 2005; 97: 1152-1155Crossref PubMed Scopus (89) Google Scholar from DM1 patients and is able to regulate alternative splicing.35Ho TH Charlet-B N Poulos MG Singh G Swanson MS Cooper TA Muscleblind proteins regulate alternative splicing.EMBO J. 2004; 23: 3103-3112Crossref PubMed Scopus (376) Google Scholar MBNL2 has also been reported to colocalize with integrin alpha3 mRNA at integrin-containing adhesion plaques, and it was suggested that MBNL2 may transport integrin alpha3 mRNA from the nucleus to the cytoplasm,39Adereth Y Dammai V Kose N Li R Hsu T RNA-dependent integrin alpha3 protein localization regulated by the muscleblind-like protein MLP1.Nat Cell Biol. 2005; 7: 1240-1247Crossref PubMed Scopus (108) Google Scholar but the question of whether MBNL1 has a similar role was not examined. MBNL3 mRNA was present in placenta but was absent from all other tissue tested including skeletal muscle and heart.23Fardaei M Rogers MT Thorpe HM Larkin K Hamshere MG Harper PS Brook JD Three proteins. MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells.Hum Mol Genet. 2002; 11: 805-814Crossref PubMed Scopus (360) Google Scholar MBNL3 has been shown to inhibit markers of muscle differentiation in mouse myoblast cultures.40Squillace RM Chenault DM Wang EH Inhibition of muscle differentiation by the novel muscleblind-related protein CHCR.Dev Biol. 2002; 250: 218-230Crossref PubMed Scopus (49) Google Scholar Faustino and Cooper37Faustino NA Cooper TA Pre-mRNA splicing and human disease.Genes Dev. 2003; 17: 419-437Crossref PubMed Scopus (987) Google Scholar have reviewed the arguments in favor of a contribution of CUG-BP upregulation to mis-splicing in DM1. CUG-BP is not elevated in DM2 muscle biopsies or in muscle from the DM mouse model expressing 250 CUG repeats in the 3′-UTR of the skeletal muscle actin mRNA.31Lin X Miller JW Mankodi A Swanson MS Thornton CA Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy.Hum Mol Genet. 2006; 15: 2087-2097Crossref PubMed Scopus (389) Google Scholar A recent mouse model with 960 repeats in DMPK exon 15 did show CUG-BP upregulation with associated splicing changes,38Orengo JP Chambon P Metzger D Mosier DR Snipes GJ Cooper TA Expanded CTG repeats within the DMPK 3′ UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy.Proc Natl Acad Sci USA. 2008; 105: 2646-2651Crossref PubMed Scopus (150) Google Scholar although the 960 repeats were multiple interrupted concatamers rather than typical DM1 repeats. Large CUG and CAG transfected 960-repeats form nuclear foci that bind MBNL1, but only CUG repeats caused the RNA splicing changes associated with DM1, suggesting that MBNL sequestration alone is insufficient to alter splicing.19Ho TH Savkur RS Poulos MG Mancini MA Swanson MS Cooper TA Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy.J Cell Sci. 2005; 118: 2923-2933Crossref PubMed Scopus (155) Google Scholar In contrast, Miller et al24Miller JW Urbinati CR Teng-Umnuay P Stenberg MG Byrne BJ Thornton CA Swanson MS Recruitment of human muscleblind proteins to (CUG) (n) expansions associated with myotonic dystrophy.EMBO J. 2000; 19: 4439-4448Crossref PubMed Scopus (718) Google Scholar found that (CAG)54 bound very little MBNL1 in vitro, compared with (CUG)54, but in neither study is the CUG repeat sequence typical of DM1. On the relationship between nuclear foci, clinical severity and repeat size, two individuals with around 60 CUG repeats had no foci and no clinical signs of muscle disease, whereas two DM1 patients with around 75 repeats had "infrequent" foci and mild, late-onset muscle weakness.27Mankodi A Urbinati CR Yuan QP Moxley RT Sansone V Krym M Henderson D Schalling M Swanson MS Thornton CA In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts.Nucleic Acids Res. 2001; 29: 2766-2771Crossref PubMed Scopus (182) Google Scholar However, with repeat sizes >100, no correlation with the number or size of nuclear foci was observed.27Mankodi A Urbinati CR Yuan QP Moxley RT Sansone V Krym M Henderson D Schalling M Swanson MS Thornton CA In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts.Nucleic Acids Res. 2001; 29: 2766-2771Crossref PubMed Scopus (182) Google Scholar Ebralidze et al41Ebralidze A Wang Y Petkova V Ebralidse K Junghans RP RNA leaching of transcription factors disrupts transcription in myotonic dystrophy.Science. 2004; 303: 383-387Crossref PubMed Scopus (130) Google Scholar suggested an alternative or additional mechanism, showing that expanded CUG repeats sequester transcription factors, such as Sp1, STAT1, STAT3, and RAR-gamma, from chromatin and that this can result in a decreased level of the muscle chloride channel, which is reversible by overexpression of Sp1. In the present study, we have developed and characterized new monoclonal antibodies against the three human MBNL proteins. We have shown that MBNL2 decreases during myoblast differentiation and human muscle development, leaving MBNL1 as the major isoform in mature muscle. Human MBNL1 cDNA was amplified from pEGFP/MBNL125Fardaei M Larkin K Brook JD Hamshere MG In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts.Nucleic Acids Res. 2001; 29: 2766-2771Crossref PubMed Google Scholar using primers 5′-GCGGATCCCGTCACACCAATTCGGGA-3′ and 5′-GCGTCGACGTCAGATGTTCGGCAGATATTATGG-3′ with BamHI and SalI sites (underlined) for cloning into pET21b (as described previously20Holt I Mittal S Furling D Butler-Browne GS Brook JD Morris GE Defective mRNA in myotonic dystrophy accumulates at the periphery of nuclear splicing speckles.Genes Cells. 2007; 12: 1035-1048Crossref PubMed Scopus (74) Google Scholar). The amplified sequence of MBNL1 for recombinant protein production contained exons 1, 2, 3, 4, and 6 of MBNL1 variant 1 (Accession NM021038) (exon numbering from Pascual et al,42Pascual M Vicente M Monferrer L Artero R The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing.Differentiation. 2006; 74: 65-80Crossref PubMed Scopus (180) Google Scholar). MBNL2 cDNA was amplified from pEGFP/MBNL223Fardaei M Rogers MT Thorpe HM Larkin K Hamshere MG Harper PS Brook JD Three proteins. MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells.Hum Mol Genet. 2002; 11: 805-814Crossref PubMed Scopus (360) Google Scholar using primers 5′-GCGAATTCCATGGCTTTGAACGTTGC-3′ and 5′-GCGTCGACGATCCGGTGGATCCGC-3′ with EcoRI and SalI sites (underlined) for cloning into pET21b. (Note: the reverse primer codes for part of the pEGFP plasmid sequence). This cDNA is MBNL2 variant 2 (Accession AF061261) containing exons 1, 2, 3 and part of exon 4 (exon numbering from Pascual et al,42Pascual M Vicente M Monferrer L Artero R The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing.Differentiation. 2006; 74: 65-80Crossref PubMed Scopus (180) Google Scholar). The MBNL2 cDNA used for recombinant protein production is present in all three of the MBNL2 variants described in42Pascual M Vicente M Monferrer L Artero R The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing.Differentiation. 2006; 74: 65-80Crossref PubMed Scopus (180) Google Scholar). MBNL3 cDNA was digested directly from pEGFP/MBNL324Miller JW Urbinati CR Teng-Umnuay P Stenberg MG Byrne BJ Thornton CA Swanson MS Recruitment of human muscleblind proteins to (CUG) (n) expansions associated with myotonic dystrophy.EMBO J. 2000; 19: 4439-4448Crossref PubMed Scopus (718) Google Scholar using EcoRI and SalI and cloned into pET21b. This cDNA is MBNL3 variant 5 (AY372211).42Pascual M Vicente M Monferrer L Artero R The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing.Differentiation. 2006; 74: 65-80Crossref PubMed Scopus (180) Google Scholar Therefore, this sequence will be present in MBNL3 variants 1, 5 and 6; but may not necessarily be present in MBNL3 variants 2, 3 and 4. After transformation of E. coli BL21(DE3) with the pET constructs and induction with IPTG, bacterial pellets were washed by sonication in TNE buffer and recombinant protein extracted by sonication in 6 mol/L urea in PBS. MBNL1 and MBNL2 were purified by His-tag column chromatography in 6 mol/L urea. The recombinant proteins were used as immunogens for production of monoclonal antibodies using the hybridoma method.43Nguyen TM Morris GE A rapid method for generating large numbers of high-affinity monoclonal antibodies from a single mouse.in: Walker JM The Protein Protocols Handbook. 2nd Edition. Humana Press, Totowa NJ2002: 1129-1138Google Scholar Resulting hybridoma culture supernatants were screened initially by enzyme-linked immunosorbent assay and Western blot against the recombinant protein. Enzyme-linked immunosorbent assay-positive supernatants were further screened by western blotting of human muscle extracts and for colocalization with transfected pEGFP/MBNL1, 2 or 3 in COS-7 cells. Hybridomas were cloned twice by limiting dilution and Ig subclasses were determined using an isotyping kit (Zymed Labs Inc., San Francisco). Monoclonal antibody MB1a (20 and Results) recognizes the linker between two zinc finger domains (exon 342Pascual M Vicente M Monferrer L Artero R The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing.Differentiation. 2006; 74: 65-80Crossref PubMed Scopus (180) Google Scholar) since it failed to bind recombinant MBNL1 lacking only this region (data not shown). Cell and muscle samples were extracted in 1% SDS buffer and boiled for 3 minutes. Proteins (30 μg) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) using 10% polyacrylamide gels and transferred to nitrocellulose membranes (BioRad). After blocking non-specific sites, membranes were incubated with primary antibodies: the monoclonal antibodies against MBNL were used at a dilution of 1/100 of the culture supernatant and a monoclonal antibody against emerin was used as a loading control for cell culture extract.44Manilal S Nguyen TM Sewry CA Morris GE The Emery-Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein.Hum Mol Genet. 1996; 5: 801-808Crossref PubMed Scopus (319) Google Scholar Antibody reacting bands were visualized following development with peroxidase-labeled goat anti-mouse Ig and a chemiluminescent detection system (SuperSignal, Pierce). The sum of the peak areas of major protein bands on a stain gel were used to estimate relative protein content of muscle extracts. Biopsies for Western blot were obtained from quadriceps muscles during autopsies, in accordance with French legislation on ethical rules.
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