Research Article: (CTG)n repeat-mediated dysregulation of MBNL1 and MBNL2 expression during myogenesis in DM1 occurs already at the myoblast stage

Date Published: May 22, 2019

Publisher: Public Library of Science

Author(s): Laurène M. André, Remco T. P. van Cruchten, Marieke Willemse, Derick G. Wansink, Atsushi Asakura.


Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder caused by the expression of trinucleotide repeat-containing DMPK transcripts. Abnormally expanded (CUG)n repeats in these transcripts form hairpin-like structures that cause the RNA to accumulate in the cell nucleus by sequestering isoforms of the Muscleblind (MBNL) family, tissue-specific regulators of developmentally programmed, post-transcriptional processes in RNA metabolism. Through this mechanism, the function of MBNL in RNA processing becomes dominantly perturbed, which eventually leads to aberrant alternative splicing and the expression of foetal splice variants of a wide variety of proteins, including the MBNL isoforms themselves. Here, we employ a patient-derived muscle cell model for DM1 to examine in detail the expression of MBNL RNA and protein variants during myogenic differentiation. This DM1 model consists of a panel of isogenic myoblast cell lines that either contain a pathogenic DMPK allele with a congenital mutation of 2600 triplets, or lack this expanded repeat through CRISPR/Cas9-mediated gene editing. We found that the temporal expression levels of MBNL1, MBNL2 and MBNL3 RNAs are not influenced by presence of the (CTG)2600 repeat during myogenesis in vitro. However, throughout myoblast proliferation and differentiation to myotubes a disproportionate inclusion of MBNL1 exon 5 and MBNL2 exons 5 and 8 occurs in cells with the (CTG)2600 repeat. As a consequence, a reduced quantity and imbalanced collection of splice variants of MBNL1 and MBNL2 accumulates in both the cytoplasm and the nucleus of DM1 myoblasts and myotubes. We thus propose that both the quantitative and qualitative changes in the intracellular partitioning of MBNL proteins are a pivotal cause of skeletal muscle problems in DM1, starting already in muscle progenitor cells.

Partial Text

Members of the Muscleblind-like (MBNL) protein family belong to a class of tissue-specific, developmentally programmed regulators of gene expression [1,2]. They control many aspects of RNA metabolism, such as alternative splicing and alternative polyadenylation, mRNA localization, translation and stability, and microRNA processing. In humans, like in other mammals, three MBNL isoforms, MBNL1, MBNL2 and MBNL3 are expressed. MBNL1 and MBNL2 are found ubiquitously, with MBNL1 being more prominent in skeletal muscle and MBNL2 relatively abundant in brain [3–5]. Expression of MBNL3 is generally low in all tissues, with exception of liver and placenta [2,3,5,6].

As important cell type-specific regulators of RNA processing, MBNL family members must be spatiotemporally expressed with high accuracy during development. Functional loss or change in the composition of the cellular MBNL isoform population is thought to be, at least in part, responsible for the spliceopathy typically seen in DM1 [34]. In the present study, we examined MBNL expression, with a focus on MBNL1 and MBNL2, in a well-controlled human cell model for DM1 with normal expression of an expanded repeat. Our unique isogenic muscle cell panel, comprising several independent cell lines with and without a congenital DM-type mutation, enabled us to study repeat effects without possible confounding influences of genetic background or other forms of interpatient variability. Using independent methods for the identification and quantification of RNA and protein, we investigated MBNL expression in detail in proliferating myoblasts and in differentiating myotubes. Earlier, we and others had already shown that (CTG)n repeat excision in DM1 cells restores the normal cellular phenotype [23,35,36]. With regard to the progressive muscle phenotype in DM1, we conclude here that (CTG)n-repeat toxicity perturbs MBNL expression as early as in myoblasts, the muscle progenitor cells.




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