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Query: UNIPROT:P06889 (Mol)
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Myotonic dystrophy 1 (DM1) is the most common inherited neuromuscular disease in adults. The disorder, characterized by myotonia, muscle wasting and weakness, cataract, insulin resistance, and mental impairment, is caused by the expansion of an unstable CTG repeat located in the 3' untranslated region of DMPK. The repeat expansion suppresses the expression of the homeobox gene SIX5. We describe here an experimental system to identify downstream transcriptional targets of mouse Six5 in order to elucidate the role of SIX5 in the pathogenesis of DM1 and development. By overexpressing a constitutively active Six5 (VP16-Six5wt) using adenovirus-mediated gene transfer in P19 cells and subsequent expression profiling using cDNA arrays, 21 genes, whose expression level increased by the treatment, were identified as potential target genes. Genes expressed in the somites, skeletal muscles, brain and meninges comprised the majority, suggesting the role of Six5 in the development and function of mesodermal tissues and brain. We provide evidence that Igfbp5 encoding a component of IGF signaling is a direct Six5-target. Moreover, the overall expression level of Igfbp5 was decreased in Six5-deficient mouse fibroblasts, and the response of human IGFBP5 to MyoD-induced muscle conversion was altered in cells of DM1 patients. Our results not only identify Six5 as an activator that directs Igfbp5 expression but also suggest that reduced SIX5 expression in DM1 might contribute to specific aspects of the DM1 phenotype.
Hum Mol Genet 2002 May 01
PMID:Identification of transcriptional targets for Six5: implication for the pathogenesis of myotonic dystrophy type 1. 1197 64

In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.
Mol Cell 2002 Jul
PMID:Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. 1215 Sep 5

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.
Mol Cell 2002 Jul
PMID:Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. 1215 Sep 6

Friedreich ataxia is commonly caused by large expansions of a GAA triplet-repeat (GAA-TR) sequence in the first intron of the FRDA gene. We used small-pool PCR to analyze somatic variability among 7190 individual FRDA molecules from peripheral blood DNA of subjects carrying 12 different expanded alleles, ranging in size from 241 to 1105 triplets. Expanded alleles showed a length-dependent increase in somatic variability, with mutation loads ranging from 47% to 78%. We noted a strong contraction bias among long alleles (>500 triplets), which showed a 4-fold higher frequency of large contractions versus expansions. Some contractions were very large; of all somatic mutations scored, approximately 5% involved contractions of >50% of the original allele length, and 0.29% involved complete reversion to the normal/premutation length (< or =60 triplets). These observations contrast sharply with the strong expansion bias seen in expanded CTG triplet repeats in myotonic dystrophy. No somatic variability was detected in >6000 individual FRDA molecules analyzed from 15 normal alleles (8-25 triplets). A premutation allele with 44 uninterrupted GAA repeats was found to be unstable, ranging in size from 6 to 113 triplets, thus establishing the threshold for somatic instability between 26 and 44 GAA triplets. Analysis of an additional 7850 FRDA molecules from serially passaged lymphoblastoid cell lines carrying nine expanded alleles (132-933 triplets) showed very low mutation loads, ranging from 0% to 6.2%. Our data indicate that expanded GAA-TR alleles in Friedreich ataxia are highly mutable and have a natural tendency to contract in vivo, and that these properties depend on multiple factors, including DNA sequence, triplet-repeat length and unknown cell-type-specific factors.
Hum Mol Genet 2002 Sep 01
PMID:The GAA triplet-repeat sequence in Friedreich ataxia shows a high level of somatic instability in vivo, with a significant predilection for large contractions. 1218 70

The myotubularin-related 1 (MTMR1) gene belongs to a highly conserved family of eucaryotic phosphatases, with at least 11 members in humans. The founder member of this gene family, MTM1, is mutated in X-linked myotubular myopathy, a severe congenital disorder that affects skeletal muscle, and codes for myotubularin, a specific phosphatidylinositol 3-phosphate [PI(3)P] phosphatase. MTM1 and MTMR1 are adjacent on the X chromosome, and the corresponding proteins share 59% sequence identity. In the present study, we investigated the putative role of MTMR1 in myogenesis by analysing its expression pattern in muscle cells during differentiation and in skeletal muscle throughout development. We have identified three novel coding exons in the MTMR1 intron 2 that are conserved between mouse and human, are alternatively spliced, and give rise to six mRNA isoforms. One of the transcripts is muscle-specific and is induced during myogenesis both in vitro and in vivo, and represents the major isoform in adult skeletal muscle. We show that the two main MTMR1 protein muscular isoforms, like myotubularin, efficiently dephosphorylate PI(3)P in vitro. We have also analysed whether MTMR1 alternative splicing is affected in skeletal muscle cells derived from patients with congenital myotonic dystrophy (cDM1), in which mRNA splicing disturbances of specific genes are thought to constitute an important pathogenic mechanism. We found a striking reduction in the level of the muscle-specific isoform and the appearance of an abnormal MTMR1 transcript in differentiated cDM1 muscle cells in culture and in skeletal muscle from cDM1 patients. Our results suggest that MTMR1 plays a role in muscle formation and represents a novel target for abnormal mRNA splicing in myotonic dystrophy.
Hum Mol Genet 2002 Sep 15
PMID:Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells. 1221 58

Trinucleotide repeat (TNR) instability can cause a variety of human genetic diseases including myotonic dystrophy and Huntington's disease. Recent genetic data show that instability of the CAG/CTG repeat DNA is dependent on its length and replication origin. In yeast, the RAD27 (human FEN-1 homologue) null mutant has a high expansion frequency at the TNR loci. We demonstrate here that FEN-1 processes the 5'-flap DNA of CTG/CAG repeats, which is dependent on the length in vitro. FEN-1 protein can cleave the 5'-flap DNA containing triplet repeating sequence up to 21 repeats, but the activity decreases with increasing size of flap above 11 repeats. In addition, FEN-1 processing of 5'-flap DNA depends on sequence, which play a role in the replication origin-dependent TNR instability. Interestingly, FEN-1 can cleave the 5'-flap DNA of CTG repeats better than CAG repeats possibly through the flap-structure. Our biochemical data of FEN-1's activity with triplet repeat DNA clearly shows length dependence, and aids our understanding on the mechanism of TNR instability.
Exp Mol Med 2002 Sep 30
PMID:Human FEN-1 can process the 5'-flap DNA of CTG/CAG triplet repeat derived from human genetic diseases by length and sequence dependent manner. 1251 98

Expansion of CTG triplet repeats in the 3' untranslated region of the DMPK gene causes the autosomal dominant disorder myotonic dystrophy. Instability of CTG repeats is thought to arise from their capacity to form hairpin DNA structures. How these structures interact with various aspects of DNA metabolism has been studied intensely for Escherichia coli and Saccharomyces cerevisiae but is relatively uncharacterized in mammalian cells. To examine the stability of (CTG)(17), (CTG)(98), and (CTG)(183) repeats during homologous recombination, we placed them in the second intron of one copy of a tandemly duplicated pair of APRT genes. Cells selected for homologous recombination between the two copies of the APRT gene displayed distinctive patterns of change. Among recombinants from cells with (CTG)(98) and (CTG)(183), 5% had lost large numbers of repeats and 10% had suffered rearrangements, a frequency more than 50-fold above normal levels. Analysis of individual rearrangements confirmed the involvement of the CTG repeats. Similar changes were not observed in proliferating (CTG)(98) and (CTG)(183) cells that were not recombinant at APRT. Instead, they displayed high frequencies of small changes in repeat number. The (CTG)(17) repeats were stable in all assays. These studies indicate that homologous recombination strongly destabilizes long tracts of CTG repeats.
Mol Cell Biol 2003 May
PMID:Long CTG tracts from the myotonic dystrophy gene induce deletions and rearrangements during recombination at the APRT locus in CHO cells. 1269 16

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG expansion in the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase gene (DMPK). Transcripts from this altered gene harbor large CUG expansions that are retained in the nucleus of DM1 cells and form foci. It is believed that the formation of these foci is closely linked to DM1 muscle pathogenesis. Here we investigated the possibility of using a nuclear-retained hammerhead ribozyme expressed from a modified tRNAmeti promoter to target and cleave mutant transcripts of DMPK. Accessible ribozyme target sites were identified in the 3'UTR of the DMPK mRNA and a hammerhead ribozyme was designed to cut the most accessible site. Utilizing this system, we have achieved 50 and 63% reductions, respectively, of the normal and CUG expanded repeat-containing transcripts. We also observed a significant reduction in the number of DMPK mRNA-containing nuclear foci in human DM1 myoblasts. Reduction of mutant DMPK mRNA and nuclear foci also corroborates with partial restoration of insulin receptor isoform B expression in DM1 myoblasts. These studies demonstrate for the first time intracellular ribozyme-mediated cleavage of nuclear-retained mutant DMPK mRNAs, providing a potential gene therapy agent for the treatment of myotonic dystrophy.
Mol Ther 2003 May
PMID:Hammerhead ribozyme-mediated destruction of nuclear foci in myotonic dystrophy myoblasts. 1271 10

Transcripts of the myotonic dystrophy protein kinase (DMPK) gene, a member of the Rho kinase family, are subject to cell-type specific alternative splicing. An imbalance in the splice isoform profile of DMPK may play a role in the pathogenesis of DM1, a severe multisystemic disorder. Here, we report how structural subdomains determine biochemical properties and subcellular distribution of DMPK isoforms. A newly developed kinase assay revealed that DMPK is a Lys/Arg-directed kinase. Individual DMPK isoforms displayed comparable transphosphorylation activity and sequence preference for peptide substrates. However, DMPK autophosphorylation and phosphorylation of MYPT1 (as putative in vivo target of DMPK), were dependent on presence of an alternatively spliced VSGGG motif and the nature of the C terminus. In-gel effects of the VSGGG motif on the migration behavior of full-length kinase provide evidence for a model in which this motif mediates 3-D-conformational changes in DMPK isoforms. Finally, different C termini endow DMPK with the ability to bind to either endoplasmic reticulum or mitochondria or to adopt a cytosolic location. Our results suggest that DMPK isoforms have cell-type and location dependent substrate specificities with a role in organellar and cytoarchitectural dynamics.
Mol Cell Biol 2003 Aug
PMID:Alternative splicing controls myotonic dystrophy protein kinase structure, enzymatic activity, and subcellular localization. 1289 25

At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x<y. S-DNAs formed many different species of slipped isomers, as indicated by its multiple electrophoretic species. In contrast, SI-DNAs formed distinct structures, as indicated by the limited electrophoretic species for all possible repeat length pairings. Sister SI-DNAs with an excess of CAG repeats always migrated slower than their sister SI-DNAs with an excess of CTG repeats. Strikingly, both the propensity to form slipped structures and the pattern of S-DNAs, but not SI-DNAs, varied for similar lengths of CTG/CAG repeats between the DM1 and SCA1 loci, highlighting a role for flanking cis-elements in S-DNA but not SI-DNA formation. Slipped structures bound structure and nucleotide-specific anti-DNA antibodies. Binding of anti-B-DNA antibodies was reduced for both S-DNAs and SI-DNAs relative to their linear forms. SI-DNAs bound anti-Z-DNA antibodies, while both S and SI-DNAs bound anti-cruciform antibodies, revealing shared characteristics between the corresponding DNA structures and slipped DNAs. Such features of the repeats may be recognized by cellular proteins known to bind such structures.
J Mol Biol 2003 Sep 19
PMID:Slipped (CTG).(CAG) repeats of the myotonic dystrophy locus: surface probing with anti-DNA antibodies. 1296 69


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