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.