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Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy

Abstract

Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3′ untranslated region of the DM protein kinase gene. People with DM1 have an unusual form of insulin resistance caused by a defect in skeletal muscle. Here we demonstrate that alternative splicing of the insulin receptor (IR) pre-mRNA is aberrantly regulated in DM1 skeletal muscle tissue, resulting in predominant expression of the lower-signaling nonmuscle isoform (IR-A). IR-A also predominates in DM1 skeletal muscle cultures, which exhibit a decreased metabolic response to insulin relative to cultures from normal controls. Steady-state levels of CUG-BP, a regulator of pre-mRNA splicing proposed to mediate some aspects of DM1 pathogenesis, are increased in DM1 skeletal muscle; overexpression of CUG-BP in normal cells induces a switch to IR-A. The CUG-BP protein mediates this switch through an intronic element located upstream of the alternatively spliced exon 11, and specifically binds within this element in vitro. These results support a model in which increased expression of a splicing regulator contributes to insulin resistance in DM1 by affecting IR alternative splicing.

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Figure 1: Human IR exon 11 (36 nucleotides), which encodes the variable carboxy terminal region of the α-subunit, is predominantly included in normal but not DM1 skeletal muscle tissue to yield IR-B.
Figure 2: DM1 skeletal muscle tissues do not express markers of regeneration.
Figure 3: Aberrant-splicing phenotype of DM1 is reproduced in primary fibroblast cultured cells only after conversion to skeletal muscle.
Figure 4: DM1-converted muscle cultures exhibit decreased responsiveness to the metabolic effects of insulin.
Figure 5: Overexpression of expanded CUG transcripts or CUG-BP in NIH3T3 cells reproduces the aberrant regulation of alternative splicing observed in DM1 skeletal muscle.
Figure 6: Aberrant regulation of cTNT and IR alternative splicing in DM1 tissues and cells in culture correlates with increased steady-state levels of CUG-BP.

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Acknowledgements

We thank C. Thornton, J. Day, H. Vogel, Z. Korade-Mirnics, E. Hoffman, E. Pegoraro, S. Servidei, C. Angelini, the University of Miami Brain and Tissue Bank (N01-HD-8-3284), CHTN, and Midwestern and Southern Divisions for tissue samples; K. Hess for help with statistical analysis; D. Miller for the MyoD/NeoR amphotrophic retrovirus; M. Swanson for antibody to CUG-BP; P. Anderson for cTNT antibody; S. Hauschka and S. Tapscott for advice on converting fibroblast cultures; L. Timchenko for RNA samples and a normal fibroblast culture; N. Webster for IR minigene constructs; and A. Ladd, N. Charlet-B., C. Thornton, J. Day, N. Webster, and K. Mihindukulasuriya for comments on the manuscript. This work was supported by NIH (R01AR45653) and the Muscular Dystrophy Association (T.A.C.).

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Correspondence to Thomas A. Cooper.

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Savkur, R., Philips, A. & Cooper, T. Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 29, 40–47 (2001). https://doi.org/10.1038/ng704

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