Musashi-2 contributes to myotonic dystrophy muscle dysfunction by promoting excessive autophagy through miR-7 biogenesis repression

Abstract

Skeletal muscle symptoms strongly contribute to mortality of myotonic dystrophy type 1 (DM1) patients. DM1 is a neuromuscular genetic disease caused by CTG repeat expansions that, upon transcription, sequester the Muscleblind-like family of proteins and dysregulate alternative splicing of hundreds of genes. However, mis-splicing does not satisfactorily explain muscle atrophy and wasting, and several other contributing factors have been suggested, including hyperactivated autophagy leading to excessive catabolism. MicroRNA ( miR ) -7 has been demonstrated to be necessary and sufficient to repress the autophagy pathway in cell models of the disease, but the origin of its low levels in DM1 was unknown. We have found that the RNA-binding protein Musashi-2 (MSI2) is upregulated in patient-derived myoblasts and biopsy samples. Because it has been previously reported that MSI2 controls miR-7 biogenesis, we tested the hypothesis that excessive MSI2 was repressing miR-7 maturation. Using gene-silencing strategies (small interfering RNAs [siRNAs] and gapmers) and the small molecule MSI2-inhibitor Ro 08-2750, we demonstrate that reducing MSI2 levels or activity boosts miR-7 expression, represses excessive autophagy, and downregulates atrophy-related genes of the UPS system. We also detect a significant upregulation of MBNL1 upon MSI2 silencing. Taken together, we propose MSI2 as a new therapeutic target to treat muscle dysfunction in DM1.

CRISPR-Cas9 editing of a TNPO3 mutation in a muscle cell model of limb-girdle muscular dystrophy type D2

“…Impaired muscle differentiation has been reported in other limb-girdle muscular dystrophies and neuromuscular disorders 36 , 37 , 38 and as a role for TNPO3. 11 To address changes in myoblast fusion and myotube diameter associated with LGMDD2, we performed immunostaining of Desmin, a muscle-specific type III intermediate filament essential for proper muscular structure and function, to quantify fusion capacity and myotube diameter ( Figures 5 A–5F).…”

Quantitative magnetic resonance imaging assessment of muscle composition in myotonic dystrophy mice

“…In recent years, however, additional molecular alterations have been found to contribute to the phenotype 5 . Pathogenic upregulation of Musashi 2 (MSI2) in DM1 cells, which inhibits miR-7 biogenesis, leads to increases in autophagy and the activity of the ubiquitin-proteasome system, two pathways with a strong contribution to the muscle impairment characteristic of DM1 [28][29][30][31][32] . Nonetheless, miR-7 levels in the diaphragm and gastrocnemius www.nature.com/scientificreports/ of HSA LR mice were similar to those of control mice 29 .…”

Small molecule inhibition of multiple RNA binding proteins underlies Musashi-2 independent phenotypes

“…Autophagy is a contributor to muscle wasting which is a symptom of myotonic dystrophy 19 . When myotonic dystrophy type 1 fibroblasts were treated with Ro, miR-7 was also upregulated, phenocopying the MSI2 regulation 19 . MSI2 was identified as an RBP interacting with the SARS-CoV-2 genome and subsequent experiments showed Ro treatment led to a modest decrease in SARS-CoV-2 nucleocapsid production, which can be used as a surrogate for viral growth 32 .…”

“…In one case, MSI2 was identified as upregulating the microRNA ( miR)-7 which prevents excessive autophagy. Autophagy is a contributor to muscle wasting which is a symptom of myotonic dystrophy 19 . When myotonic dystrophy type 1 fibroblasts were treated with Ro, miR-7 was also upregulated, phenocopying the MSI2 regulation 19 .…”

“…Recent structural and biochemical characterization concluded that it binds to the RRM1 domain of MSI2 to displace target mRNAs 12 . Ro treatment leads to reduction in disease burden in a murine AML leukemia model, inhibited replication of SARS-CoV-2, and improved muscle dysfunction in myotonic dystrophy type 1 12,18,19 . In all these contexts, the Ro-induced phenotypes were presumed to be dependent on MSI2, though most studies did not directly test MSI2 dependence.…”