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May 30, 2011

Misregulated Alternative Splicing in Muscle Cells Linked with Myotonic Dystrophy

  • Misregulated alternative splicing of the pre-mRNA transcript for bridging integrator-1 (BIN1, or amphiphysin 2) in muscle cells appears to play a key role in the mechanisms responsible for the muscle weakness that is a major characteristic of myotonic dystrophy, according to an international team reporting in Nature Medicine.

    BIN1 plays a role in the tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Nicolas Charlet-Berguerand, Ph.D., and colleagues, have now linked abnormally spliced BIN1 mRNA with the T tubule abnormalities and muscle weakness seen in people with both severe and less severe forms of myotonic dystrophy.

    Their studies suggest this missplicing is caused by mutant RNAs sequestering muscleblind-like-1 (MBNL1), which normally regulates the alternatively spliced form of the BIN1 mRNA that is produced in muscle cells.

    Moreover, the researchers report, reproducing this myotonic dystrophy-specific BIN1 missplicing in mice leads to the animals developing T tubule alterations and muscle weakness seen in human patients. They describe their findings in a paper titled “Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy.” Corresponding author Dr. Charlet-Berguerand is based at INSERM’s Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), at the University of Strasbourg, France.

    Myotonic dystrophy is the most common adult-onset muscular dystrophy and comprises two genetically distinct forms, the researchers explain. Myotonic dystrophy of type 1 (DM1) is caused by a CTG repeat expansion in the 3′ untrans­lated region of the dystrophia myotonica-protein kinase (DMPK) gene.  DM2, on the other hand, is caused by expansions of CCTG repeats in the first intron of the CNBP gene (also known as ZNF9). A severe congenital form of myotonic dystrophy, CDM1, is also caused by CTG repeat expansions.

    Despite this background knowledge, the cause of progressive muscle weakness which characterizes mytonic dystrophy has to date remained ill defined, the authors note. There are clues, however. Expression of RNAs containing expanded CUG or CCUG repeats interferes with the splicing of other pre-mRNAs through pathologi­cal alteration of RNA-binding proteins. In individuals with CDM1, DM1, and DM2, one of these, the splicing regulator MBNL1, is trapped within nuclear RNA aggregates formed by expanded CUG and CCUG repeats.

    Using whole genome microarrays, Dr. Charlet-Berguerand’s team has now identified significant misregulation of alternative splicing of the BIN1 pre-mRNA in primary cultures of differentiated CDM1 muscle cells. Not only was a whole exon (exon 11) of the mRNA sometimes missing in the skeletal muscles of individuals with myotonic dystrophy, but the degree of misregulated splicing correlated with disease severity. BIN1 exon 11 was completely missing in severe congenital CDM1 but only partially skipped in the milder adult forms DM1 and DM2.

    Importantly, the researchers stress, misregulation of BIN1 alternative splicing appears to be specific to myotonic dystrophy, as it wasn’t detected in muscle samples from people with amyotrophic lateral sclerosis. The misregulation also did not result from a global alteration of the splicing machinery, as muscle samples from individuals with DM1 demonstrated no apparent splicing changes in MTM1, DNM2, or of BIN1’s brain-specific exons 13 to 16.

    Experiments with specially constructed exon 11 minigenes and vectors for the expanded CUG and CCUG repeats confirmed that it was expression of the repeats that induced the BIN1 splicing alteration. In addition, depletion of MBNL1 using an siRNA-mediated approach mimicked the effect of CUG or CCUG repeats and promoted exon 11 exclusion, whereas overexpression of MBNL1 stimulated exon 11 inclusion.

    Exon 11 is a muscle-specific exon encoding a phosphoinositide-binding domain. Subsequent studies showed that the isoform of BIN1 without exon 11, which is found in myotonic dystrophy patients, has lost most of its ability to bind phosphoinositides and fails to induce the formation of tubular membrane structures.

    Reproducing the exon 11 abnormality in experimental newborn mice led to T tubule abnormalities in muscle cells and the animals also displayed a 28% reduction in muscle strength but without showing any obvious signs of muscle atrophy or degeneration or disturbances in mitochondria or sarcomere structures. Interestingly, in these animals the extent of muscle weakness also correlated with the degree of BIN1 exon 11 skipping, “suggesting a direct correlation between BIN1 splicing misregulation and muscle weakness,” the authors state

    “In conclusion, our results suggest a model in which the alternative splicing of the muscle-specific exon 11 of BIN1 mRNA is regulated by the MBNL1 splicing factor,” they write. “Sequestration of MBNL1 by expanded CUG or CCUG repeats in individuals with DM1 or DM2, respectively, leads to skipping of that exon and expression of an isoform of BIN1 unable to bind phosphatidylinositol-5-phosphate and tubulate mem­branes, which ultimately results in disorganized T tubules and altered excitation-contraction coupling.”


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