Myotonic dystrophy (DM1) is an autosomal dominant neuromuscular disorder associated with a (CTG) n expansion in the 3¢-untranslated region of the DM1 protein kinase (DMPK) gene. To explain disease pathogenesis, the RNA dominance model proposes that the DM1 mutation produces a gain-of-function at the RNA level in which CUG repeats form RNA hairpins that sequester nuclear factors required for proper muscle development and maintenance. Here, we identify the triplet repeat expansion (EXP) RNAbinding proteins as candidate sequestered factors. As predicted by the RNA dominance model, binding of the EXP proteins is speci®c for dsCUG RNAs and proportional to the size of the triplet repeat expansion. Remarkably, the EXP proteins are homologous to the Drosophila muscleblind proteins required for terminal differentiation of muscle and photoreceptor cells. EXP expression is also activated during mammalian myoblast differentiation, but the EXP proteins accumulate in nuclear foci in DM1 cells. We propose that DM1 disease is caused by aberrant recruitment of the EXP proteins to the DMPK transcript (CUG) n expansion.

In myotonic dystrophy (DM), expression of RNA containing expanded CUG or CCUG repeats leads to misregulated alternative splicing of pre-mRNA. The repeat-bearing transcripts accumulate in nuclear foci, together with proteins in the muscleblind family, MBNL1 and MBNL2. In transgenic mice that express expanded CUG repeats, we show that the splicing defect selectively targets a group of exons that share a common temporal pattern of developmental regulation. These exons undergo a synchronized splicing switch between post-natal day 2 and 20 in wild-type mice. During this post-natal interval, MBNL1 protein translocates from a predominantly cytoplasmic to nuclear distribution. In the absence of MBNL1, these physiological splicing transitions do not occur. The splicing defect induced by expanded CUG repeats in mature muscle fibers is closely reproduced by deficiency of MBNL1 but not by deficiency of MBNL2. A parallel situation exists in human DM type 1 and type 2. MBNL1 is depleted from the muscle nucleoplasm because of sequestration in nuclear foci, and the associated splicing defects are remarkably similar to those observed in MBNL1 knockout mice. These results indicate that MBNL1 participates in the post-natal remodeling of skeletal muscle by controlling a key set of developmentally regulated splicing switches. Sequestration of MBNL1, and failure to maintain these splicing transitions, has a pivotal role in the pathogenesis of muscle disease in DM.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease that is associated with a (CTG)n repeat expansion in the 3'-untranslated region of the myotonin protein kinase (Mt-PK) gene. This study reports the isolation and characterization of a (CUG)n triplet repeat pre-mRNA/mRNA binding protein that may play an important role in DM pathogenesis. Two HeLa cell proteins, CUG-BP1 and CUG-BP2, have been purified based upon their ability to bind specifically to (CUG)8 oligonucleotides in vitro. While CUG-BP1 is the major (CUG)8-binding activity in normal cells, nuclear CUG-BP2 binding activity increases in DM cells. Both CUG-BP1 and CUG-BP2 have been identified as isoforms of a novel heterogeneous nuclear ribonucleoprotein (hnRNP), hNab50. The CUG-BP/hNab50 protein is localized predominantly in the nucleus and is associated with polyadenylated RNAs in vivo. In vitro RNA-binding/photocrosslinking studies demonstrate that CUG-BP/hNab50 binds to RNAs containing the Mt-PK 3'-UTR. We propose that the (CUG)n repeat region in Mt-PK mRNA is a binding site for CUG-BP/hNab50 in vivo, and triplet repeat expansion leads to sequestration of this hnRNP on mutant Mt-PK transcripts.

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG) n repeats in the 3-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP͞hNab50) that possibly plays a role in mRNA processing and͞or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP͞hNab50), are altered in homozygous DM patient and that CUG-BP͞hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP͞hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP͞ hNab50. Nuclear concentration of the hypophosphorylated CUG-BP͞hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP͞hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP͞hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP͞hNab50, thus facilitating nuclear localization of CUG-BP͞hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP͞hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.Myotonic dystrophy (DM), the most common form of adult muscular dystrophy, although primarily a muscle disorder, also affects other organs including the heart, eye, testes, lungs, and brain (1). The primary genetic defect in DM is a (CTG) n trinucleotide repeat expansion in the 3Ј-untranslated region of the myotonin-protein kinase (DMPK) gene (2-7). The length of this expansion correlates with the severity of the clinical symptoms (8-10). The mechanism by which the CTG repeat expansion induces the DM phenotype remains puzzling, particularly because the CTG repeat is located in the 3Ј-untranslated region. In homozygous knockout mice, where DMPK is absent, only muscle weakness and myopathy are observed (11,12). Cardiomyopathy develops in mice overexpressing the DMPK gene (12), but disease symptoms in other organs, including skeletal muscle myotonia are not observed. These data show that alteration of DMPK expression is only a part of DM mechanism suggesting that other genes are involved.In pursuit of alternative mechanisms for the pathogenesis of DM, we identified novel proteins that bind specifically to triplet repeats (13) in DNA and others that bind to triplet repeat...

Myotonic dystrophy (DM) is associated with a (CTG) (n) triplet repeat expansion in the 3'-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Using electron microscopy, we visualized large RNAs containing up to 130 CUG repeats and studied the binding of purified CUG-binding protein (CUG-BP) to these RNAs. Electron microscopic examination revealed perfect double-stranded (ds)RNA segments whose lengths were that expected for duplex RNA. The RNA dominant mutation model for DM pathogenesis predicts that the expansion mutation acts at the RNA level by forming long dsRNAs that sequester certain RNA-binding proteins. To test this model, we examined the subcellular distribution and RNA-binding properties of CUG-BP. While previous studies have demonstrated that mutant DMPK transcripts accumu-late in nuclear foci, the localization pattern of CUG-BP in both normal and DM cells was similar. Although CUG-BP in nuclear extracts preferentially photocrosslinked to DMPK transcripts, this binding was not proportional to (CUG) (n) repeat size. Moreover, CUG-BP localized to the base of the RNA hairpin and not along the stem, as visualized by electron micro-scopy. These results provide the first visual evidence that the DM expansion forms an RNA hairpin structure and suggest that CUG-BP is unlikely to be a sequestered factor.

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