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...