**Non-Coding RNAs in Muscle Dystrophies**

**Daniela Erriquez <sup>1</sup> , Giovanni Perini 1,2,\* and Alessandra Ferlini 3,\***


*Received: 5 August 2013; in revised form: 5 September 2013 / Accepted: 9 September 2013 / Published: 30 September 2013*

**Abstract:** ncRNAs are the most recently identified class of regulatory RNAs with vital functions in gene expression regulation and cell development. Among the variety of roles they play, their involvement in human diseases has opened new avenues of research towards the discovery and development of novel therapeutic approaches. Important data come from the field of hereditary muscle dystrophies, like Duchenne muscle dystrophy and Myotonic dystrophies, rare diseases affecting 1 in 7000–15,000 newborns and is characterized by severe to mild muscle weakness associated with cardiac involvement. Novel therapeutic approaches are now ongoing for these diseases, also based on splicing modulation. In this review we provide an overview about ncRNAs and their behavior in muscular dystrophy and explore their links with diagnosis, prognosis and treatments, highlighting the role of regulatory RNAs in these pathologies.

**Keywords:** microRNAs (miRNAs); long non-coding RNAs (lncRNAs); Duchenne muscular dystrophy (DMD); Becker muscular dystrophy (BMD); Myotonic dystrophies (DM1 and DM2); Facioscapulohumeral dystrophy (FSHD)

### **1. Introduction**

Transcription of the eukaryotic genome yields only 1%–2% of protein coding transcripts and the remainder is classified as non-coding RNAs (ncRNAs). In other words, non-coding RNAs are the main output of the global transcription process, highlighting the idea that such an intense cellular effort cannot be just simple noise. Rather, it is reasonable to speculate that this underscored transcriptome possesses specific vital functions [1–3].

In general, non-coding RNAs are divided into structural and regulatory RNAs. The first ones include ribosomal, transfer, small nuclear and small nucleolar RNAs (rRNAs, tRNAs, snRNAs and snoRNAs respectively), which have been deeply characterized at the functional level. The second ones are a very broad class of RNAs whose main categorization essentially relies on their length.

Small ncRNAs are defined as transcripts shorter than 200 nucleotides. The most functionally characterized are microRNAs (miRNAs), piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs), which are critical for the assembly and the activity of the RNA interference machinery.

RNAs longer than 200 nucleotides are named long non-coding RNAs (lncRNAs) and are a very heterogeneous group of molecules. Because there is not an official way to classify them, they can be placed in one or more categories depending on their genome localization and/or on their orientation (sense, antisense, bidirectional, intronic or intergenic lncRNAs) [4,5].

In the past years, several reports have increased our knowledge about additional levels of regulation of many physiological processes that are mediated by ncRNAs. Even more interesting, these flexible molecules have been found to be dysregulated in many pathological human disorders.

In this review, we will focus on RNAs involved in human skeletal muscle dystrophies. There is a continuous flow of new scientific reports that underpin functional links between ncRNAs and skeletal muscle biology, suggesting that these molecules can play a crucial function both in physiological muscle development and in pathological muscle disorders.

Muscular dystrophies (MDs) are strictly inherited conditions recognized as a common pathogenic mechanism of disruption/impairment of the muscle cell membrane (sarcolemma) which causes a cascade of pathogenic events, including: inflammation, cell necrosis and cell death with progressive fibrosis replacing the muscle mass. MDs represent diseases of extraordinary interest both in medical genetics and biology. The high translational value of research about MDs has recently driven scientific findings toward precise genetic diagnoses as well as novel therapies [6–9].

There are more than 30 different types of inherited dystrophies that are characterized by muscle wasting and weakness of variable distribution and severity, manifesting at any age from birth to middle years, resulting in mild to severe disability and even short life expectancy in the worse cases. Clinical and pathological features are generally the parameters to classify the most common type of MDs. The broad spectrum of MDs arises from many different genetic mutations that reflect defects not only in structural proteins, but also in signaling molecules and enzymes. Dystrophin was the first mutant structural protein shown to cause MD. Mutations in the dystrophin gene lead to two more common type of dystrophy: the severe Duchenne muscular dystrophy (DMD OMIM 300677) due to out-of-frame mutations, and the milder Becker muscular dystrophy (BMD OMIM 300376) associated with in-frame mutations. Some "exceptions to the reading frame rule" are associated with intermediate phenotypes. The genetic causes of the highly heterogeneous Limb Girdle Muscular Dystrophies (LGMDs) reside in many genes (such as α, β, γ, δ, and ε sarcoglycans) encoding for structural proteins that are part of the complex sarcolemma network and deeply involved, together with dystrophin, in force transduction [10,11].

There are other muscular dystrophies, such as the Facioscapulohumeral muscular dystrophy (FSHD OMIM 158900) and Myotonic dystrophies (DM1 and DM2, see below), that are due to mutations in genes with a main regulatory function. FSHD is due to deletions in non-coding RNA which cause modification of the chromatin assembly in the 4q34 chromosomal region; Myotonic dystrophies (DMs) are related to trinucleotide (DM1) and tetranucleotide (DM2) repeat expansions that produce toxic mutant mRNA with subsequent interference of RNA-splicing mechanisms [12,13].

Many lines of evidence reveal that aberrant expression levels of non-coding RNAs can result in novel types of defects that cause remarkable changes in processes such as mRNA maturation, translation, signaling pathways or gene regulation. To date, it is clear that there is involvement of several miRNAs in the muscular dystrophies, on the contrary, very little is known about the role of long ncRNAs [14].

In this review we try to recapitulate the emerging studies about this intriguing category of molecules, summarizing what is known in muscle, both in physiological and in pathological contexts; new insights are revealing that they are important players in processes such as cellular lineage commitment, growth and differentiation of skeletal muscle. Since muscle differentiation and regeneration are key features that require to be considered when designing novel therapies, addressing the role of ncRNAs in MDs is of high clinical relevance.

#### **2. Muscle-Specific and Ubiquitously Expressed miRNAs in Skeletal Muscle**

microRNAs control the stability and/or the translational efficiency of target messenger RNAs, thus causing post-transcriptional gene silencing. Mammalian miRNAs are transcribed as long primary transcripts (pri-miRNAs) and encode one or more miRNAs. Pri-miRNAs are processed by RNase III Drosha in the nucleus to generate stem-loop structures of ~70 nucleotides (pre-miRNAs) and then exported to the cytoplasm where they are further processed by RNase Dicer to yield ~22 bp mature miRNAs. A mature miRNA, incorporated into the RNA-induced silencing complex (RISC), anneals to the 3' UTRs of its target mRNAs by its complementary strand, thus causing post-transcriptional gene silencing via translational repression or mRNA degradation. In the last years new paradigms of miRNA biogenesis are also emerging in which the processing of miRNA does not require all steps mentioned above [15].

Vertebrate skeletal muscle is derived from the somites, the first metameric structures in mammalian embryos, that progressively subdivide into embryonic compartments, thus giving rise to dermomyotome and subsequently to myotome to produce differentiated muscular tissue. The process of generating muscle—myogenesis—is highly complex and requires a broad spectrum of signaling molecules, either during embryonic development and in postnatal life, that converges on specific transcription and chromatin-remodeling factors, as well as on regulatory RNAs, to activate gene and microRNA expression program [16,17].

The fate of myogenic precursor cells is first determined by paired -homeodomain transcription factors, Pax3/Pax7, followed by regulation of highly conserved MyoD (also named MyoD1, myogenic differentiation 1), Myf5 (myogenic factor 5), MyoG (myogenin), and MRF4 factors, expressed in the skeletal muscle lineage and therefore referred as myogenic regulatory factors (MRFs). The MRFs differ in the timing and the stages of myogenesis, reflecting their different roles during muscle cell commitment and differentiation. MyoD and Myf5 are both considered markers of terminal commitment to muscle fate. Myf5 is the first MRF expressed during the formation of the myotome, followed by expression of MyoD. Specifically, in the majority of muscle progenitors, MyoD functions downstream from Pax3 and Pax7 in the genetic hierarchy of myogenic regulators, whereas Myf5, depending on the context, can also act in parallel with the Pax transcription factors [18–20]. Instead, MyoG and MRF4 act subsequently to specify the immature muscle cells (myoblasts) for terminal differentiation. Myoblasts exit from cell cycle after a defined proliferation time, to become terminally differentiated myocytes [21,22]. Muscle-specific genes such as myosin heavy chain genes (*MyHC* genes) and muscle creatine kinase (*M-CK*) are expressed in the last phase of this multi-regulated program, where mononucleated myocytes specifically fuse to each other to form multinucleated myotubes [22–28].

Dicer loss-of function studies clarified the importance of miRNAs in normal skeletal muscle development [29]. miRNAs actively take part in the proliferation and differentiation of skeletal muscle cells as an integral component of genetic regulatory circuitries.

miR-1, miR-133a/b and miR-206 are largely studied and defined muscle-specific miRNAs (myomiRs). They are regulated in muscular transcriptional networks via MRFs and via others key-regulators of the myogenic program, MEF2 (myocyte enhancer factor 2) and SRFs (serum response factors). Recently, a new regulatory pathway, the mechanistic target of rapamycin (mTOR) signaling was seen to regulate miR-1 expression and was also found responsible for MyoD stability [30–36]. It is possible to functionally define miR-133 as enhancer of myoblast proliferation while miR-1 and miR-206 as enhancers of skeletal muscle differentiation [37–40]. An up-to-date list of the identified targets of miR-1, miR-133 and miR-206, together with a plethora of specific muscular pathways they are involved in, is reported in a recent review [40] and some of these will be also discussed in the next paragraph to highlight how these important families of miRNAs contribute to determine typical deficiencies occurring in a pathological muscular context. Intriguingly, these myomiRs have been shown to behave as serum biomarkers in DMD patients. They are released into the bloodstream as a consequence of fiber damage and their power as diagnostic tools is promising since increased miRNA levels correlate with severity of the disease, significantly better than other commonly utilized markers, such as creatine kinase (CK). Moreover, their major serum stability is another aspect that may make them useful not only for diagnosis but also for monitoring the condition of affected individuals after a therapeutic treatment [41,42].

miR-208b/miR-499, also named myomiRs because of their muscle-restricted expression, are produced from the introns of two myosin genes, *β-MHC* and *Myh7b*. They are functionally redundant and play a dominant role in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs [43].

Interestingly, many miRNAs are defined as "non-muscle specific" (or also ubiquitously expressed), because essentially they are not exclusively expressed in muscular tissue. It has been, however, demonstrated that they play key-roles in modulating important pathways involved in the regulation of muscular metabolism and cellular commitment. Many miRNAs fall into this category and we report here a few relevant examples, providing for each miRNA the context in which they were studied and highlighting their global effects on muscular metabolism (Table 1).


**Table 1.** miRNAs expressed in muscular tissue (in an exclusive manner or not) and their global effect on muscle metabolism.

Some of these miRNAs counteract the differentiation process since their activity is aimed to positively regulate the proliferation phase during muscular development.

miR-125b, one of the few down-regulated miRNAs during myogenesis, together with miR-221/222, negatively contributes to myoblast differentiation and muscle regeneration, taking part in the regulatory axis that includes mTOR and IGF-II [50–52]. Similarly, miR-155 mediates the repression of differentiation targeting MEF2A, a member of MEF2 family of transcription factors. By this negative regulation, miR-155 functions as an important regulator of muscle gene expression and myogenesis [53]. miR-221/222 instead are involved in maintenance of the proliferative state promoting cell cycle progression. They are under control of the Ras-MAPK axis and inhibit the cellcycle regulator p27 (Cdkn1b/Kip1). Their ectopic expression, indeed, lead to defects in the transition from myoblasts to myocytes and in the assembly of sarcomeres in myotubes [58].

**Figure 1.** Overview of muscle-specific and ubiquitously expressed miRNAs that contribute to myogenesis and muscle regeneration processes and their regulatory activity on the muscular specific targets/chromatin modifying enzymes/cell cycle regulators (for details see the text). The main regulatory factors that exert a fundamental role during each step of normal muscle development are also reported as well as their eventual regulatory activity on the described miRNAs.

In contrast to this set of miRNAs, many other "non-muscle specific" miRNAs exert an active role in muscle differentiation through different mechanisms: miR-24, for example, has been shown to be essential for the modulation of transforming growth factor β/bone morphogenetic protein (TGF-β/BMP) pathway, a well-known inhibitor of differentiation, although its specific muscular targets are yet unknown [44]; miR-26a is involved in TGF-β/BMP pathway, where it negatively regulates the transcription factors Smad1 and Smad4, critical components of that signaling; miR26a targets the polycomb complex member Ezh2, involved in chromatin silencing of skeletal muscle genes [45,46]; miR-27b promotes entry into differentiation program both *in vitro* and *in vivo* regenerating muscles by down-regulating Pax3 [47]; miR-29 in general is defined as an enhancer of differentiation. During myogenesis it is up-regulated by SRFs and MEF2, and in a self-regulatory manner, it suppresses YY1 and HDAC4 translation by targeting their 3'-UTRs [48,49]; miR-146a is another positive regulator of myogenesis, since it modulates the activity of NUMB protein, which promotes satellite cell differentiation towards muscle cells by inhibiting Notch signaling [55,56]; miR-181 is involved in skeletal muscle differentiation and regeneration after injury and one of its targets is Hox-A11, which in turn represses transcription of MyoD [54]; miR-214 was identified in zebrafish as regulating the muscle development. Here it is expressed in skeletal muscle cell progenitors and was shown to specify muscle cell type during somitogenesis by modulating the response of muscle progenitors to Hedgehog proteins signaling [57]. Its involvement in muscle is also confirmed in C2C12 myoblasts and in skeletal myofibers of mouse where it promotes cell cycle exit and thus differentiation, targeting proto-oncogene N-*Ras* and the repressor of myogenesis Ezh2 respectively [62,63]; miR-322/424 and -503 promote myogenesis interfering with the progression through the cell cycle [59]; while miR-486 was reported to positively regulate myoblast differentiation targeting phosphatase and tensin homolog (PTEN) and Foxo1a, which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling and down-regulate the transcription factor Pax7, required only for muscle satellite cell biogenesis and speci fication of the myogenic precursor lineage [60,61]. All these data clearly show the vast scenario of functions in which miRNAs are involved and their specific activities they play in the skeletal muscle physiology (Figure 1).
