High-throughput analysis of the RNA-induced silencing complex in myotonic dystrophy type 1 patients identifies the dysregulation of miR-29c and its target ASB2

Abstract

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2.

Epigenetics of skeletal muscle-associated genes in theASB, LRRC, TMEM, andOSBPLgene families

“…ASB2 is the most well studied of the nine SkM- or SkM/heart-associated ASB genes. Its predominant isoform in SkM, ASB2β (Figure 4d), encodes a protein implicated in regulating myogenesis, decreasing SkM mass, and possibly aiding recovery of SkM after exercise and contributing to the symptoms of myotonic dystrophy type 1 [53, 60-63]. Despite the importance of the β or α isoforms of this gene to myogenesis, hematopoiesis, cardiomyocyte maturation, cardiac homeostasis, certain types of leukemia, and Notch, TGFβ, and retinoic acid signaling pathways (Table S6), there has been only one study published about epigenetic regulation of ASB2 .…”

MicroRNA-Based Therapeutic Perspectives in Myotonic Dystrophy

“…Lastly, Capella et al (2018) performed an innovative sequencing screen of RISC-associated mRNAs for identifying those displaying expression dysregulation between primary skeletal muscle fibroblasts derived from DM1 patient´s biopsies and matched controls [53]. In the second step, a bioinformatics analysis defined the miRNA/mRNA relevant interactions.…”

The Changed Transcriptome of Muscular Dystrophy and Inflammatory Myopathy: Contributions of Non-Coding RNAs to Muscle Damage and Recovery

“…The progressive muscle damage associated with muscular dystrophy leads to accumulated deposition of excessive fibrous connective tissue, and many have described reduction of miR-29 expression levels in muscular dystrophy [11,12,18,28,29]. In MD, decreased miR-29 leads to increased levels of the pro-fibrotic protein ankyrin repeat and suppressor of cytokine signaling box-containing 2 (ASB2) [24].…”