Non-coding RNAs in muscle differentiation and musculoskeletal disease Monica Ballarino, … , Alessandro Fatica, Irene Bozzoni J Clin Invest. 2016;126(6):2021-2030. https://doi.org/10.1172/JCI84419. Review RNA is likely to be the most rediscovered macromolecule in biology. Periodically, new non-canonical functions have been ascribed to RNA, such as the ability to act as a catalytic molecule or to work independently from its coding capacity. Recent annotations show that more than half of the transcriptome encodes for RNA molecules lacking coding activity. Here we illustrate how these transcripts affect skeletal muscle differentiation and related disorders. We discuss the most recent scientific discoveries that have led to the identification of the molecular circuitries that are controlled by RNA during the differentiation process and that, when deregulated, lead to pathogenic events. These findings will provide insights that can aid in the development of new therapeutic interventions for muscle diseases. Find the latest version: https://jci.me/84419/pdf The Journal of Clinical Investigation REVIEW Non-coding RNAs in muscle differentiation and musculoskeletal disease Monica Ballarino,1,2 Mariangela Morlando,1 Alessandro Fatica,1 and Irene Bozzoni1,2,3 1Department of Biology and Biotechnology Charles Darwin and 2Institute Pasteur Fondazione Cenci-Bolognetti, Sapienza University of Rome, Rome, Italy. 3Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy. RNA is likely to be the most rediscovered macromolecule in biology. Periodically, new non-canonical functions have been ascribed to RNA, such as the ability to act as a catalytic molecule or to work independently from its coding capacity. Recent annotations show that more than half of the transcriptome encodes for RNA molecules lacking coding activity. Here we illustrate how these transcripts affect skeletal muscle differentiation and related disorders. We discuss the most recent scientific discoveries that have led to the identification of the molecular circuitries that are controlled by RNA during the differentiation process and that, when deregulated, lead to pathogenic events. These findings will provide insights that can aid in the development of new therapeutic interventions for muscle diseases. Non-coding RNAs (ncRNAs) include different families of tran- tides, while miR-133a-1 and miR-133a-2 are identical and differ scripts of both small (sncRNA) and large (lncRNA) sizes. The from miR-133b by 2 nucleotides. Moreover, they are organized in impact of these molecules in the control of cell development, dif- duplicated clusters (miR-1-1/miR-133a-1 and miR-1-2/miR-133a-2) ferentiation, and growth has now been established, and it is clear that in vertebrates also originated the miR-206/miR-133b locus. that these molecules exert their functions in both nuclear and cyto- While mice lacking only one of the two miR-1 or miR-133 copies plasmic compartments (1). Muscle differentiation has been one of displayed minor defects, mainly of cardiac type, deletion of both the most exploited and studied processes due to the availability of copies resulted in lethality (9, 10). Conversely, deletion of the suitable cellular systems that faithfully recapitulate in vivo differ- regenerative miR-206 in mice substantially delayed regeneration entiation and animal models for different muscle diseases (2–4). induced by cardiotoxin injury (11). Alterations in myogenesis may underlie many muscle disor- Figure 1 summarizes the most relevant circuits controlled by ders, including sarcopenia, cachexia, and muscular dystrophies, miRNAs in skeletal muscle cells. miRNAs can target transcription where alterations in regenerative capacity play a crucial role in factors, either promoting or inhibiting myogenesis. Conversely, disease progression and outcome. Moreover, perturbation of reg- these same transcription factors can directly control expression of ulatory circuits controlling muscle homeostasis are involved in these miRNAs through regulatory feedback loops (9, 12, 13). structural and functional changes that occur during muscle atro- Deregulation of miRNA expression is a common feature of phy and hypertrophy; therefore, the identification of new compo- several skeletal muscle disorders, and rescue of their correct nents controlling muscle differentiation and regeneration could expression in mouse models ameliorates disease phenotypes clarify the molecular pathogenic mechanisms in different diseases (11, 14, 15). miRNA profiles of different skeletal muscle disorders and potentially allow the identification of new therapeutic targets. (16–21) revealed that, despite their heterogeneity, these disorders share a common set of deregulated miRNAs, which generally MicroRNAs includes myomiRs. MicroRNAs (miRNAs) play important roles in all the steps of Duchenne muscular dystrophy (DMD), the most common myogenesis. Additionally, the molecular circuits controlled by and severe muscular disease characterized by mutations in the miRNAs have been largely characterized (5). During myogenesis dystrophin gene, provides a relevant example of disease-linked an intricate relationship between miRNAs and myogenic factors miRNA activity that is involved in pathogenic circuits. Besides is established, with miRNAs acting synergistically or antagonis- serving as a structural protein that protects muscle fibers from tically. miR-1, miR-133, miR-206, miR-499, and miR-208 are the mechanical damage, dystrophin controls the switch from early to so-called myomiRs because their expression is restricted to skel- late phases of differentiation, acting as an epigenetic modulator etal and/or cardiac muscles (6–8). Notably, the major myomiRs of gene expression through a pathway involving neuronal NOS have an interesting evolutionary and genomic correlation: miR-1-1 (nNOS) and histone deacetylase 2 (HDAC2) (22, 23). In normal and miR-1-2 are identical and differ from miR-206 by 4 nucleo- muscle, dystrophin activates nNOS, which in turn nitrosylates HDAC2, leading to its release from the chromatin of specific tar- get genes to drive transcriptional activation. Several miRNA genes Conflict of interest: The authors have declared that no conflict of interest exists. were identified as targets of this pathway, including miRNAs Reference information: J Clin Invest. 2016;126(6):2021–2030. doi:10.1172/JCI84419. involved in terminal differentiation of muscle, such as miR-1 and jci.org Volume 126 Number 6 June 2016 2021 REVIEW The Journal of Clinical Investigation Figure 1. miRNA-mediated regulatory networks in myogenesis and skeletal muscle diseases. Schematic representation of the differentiation stages leading from progenitor muscle cells to terminally differentiated fibers. The most relevant regulatory circuits between miRNAs and protein factors are shown. miRNA species that are up- or downregulated in dystrophic cells are represented in red and green, respectively. Polα, polymerase α. miR-133, and the more ubiquitous miR-29 and miR-30 (17). In phic phenotype (11), while sustained expression of miR-206 pro- dystrophic muscles, the absence of dystrophin disrupts such cir- moted satellite cell differentiation and fusion, suggesting that the cuitry, leading to reduced levels of specific miRNAs, which favors strong activation of miR-206 in dystrophic muscles induces com- the onset of dystrophic pathogenic traits such as oxidative dam- pensatory circuits to promote the formation of new myofibers in age through upregulation of the miR-1 target glucose-6-phos- response to disease-induced injury. This activity is mediated at the phate dehydrogenase (G6PD) and fibrosis through deregulation molecular level through suppression of several negative regulators of the collagen mRNAs targeted by miR-29 (17, 24, 25) (Figure 1). of myogenesis, including paired box protein–7 (PAX7), Notch3, Notably, both miR-1 and miR-29, which are poorly expressed in and insulin-like growth factor–binding protein–5 (IGFBP5) (11, 17). murine and human dystrophic muscles, were recovered in exon miR-206 is also linked to amyotrophic lateral sclerosis (ALS). skipping–treated mdx mice (a murine model of DMD) and DMD Expression of miR-206 was dramatically increased in an ALS myoblasts (17). Moreover, these miRNAs were downregulated mouse model where pathological alterations are first detected in in individuals with Becker muscular dystrophy (BMD), in which muscle, particularly at the neuromuscular junction (NMJ) prior mutant dystrophin cannot bind nNOS (26). In line with these to motor neuron loss (30). miR-206 was shown to be required findings, epigenetic control mediated by HDACs was identified for efficient regeneration of neuromuscular synapses after acute as a major regulatory effector in promoting muscle regeneration nerve injury, and its genetic deletion accelerated disease progres- (22, 27) and a relevant epigenetic HDAC/myomiR network was sion and diminished survival. Therefore, miR-206 was suggested recently shown to target the transcriptional regulators BRG1- to slow ALS progression by sensing motor neuron injury and pro- associated factor 60A and 60B (BAF60A and BAF60B), ulti- moting the compensatory regeneration of neuromuscular syn- mately directing promyogenic differentiation while suppressing apses (14). Two important downstream targets are likely involved the fibro-adipogenic phenotype (28). in this pathway: HDAC4, which has been implicated in the con- miR-1 shares a seed sequence with another myomiR, miR- trol of neuromuscular gene expression