Noncoding RNAs as epigenetic mediators of skeletal muscle regeneration.

REVIEW ARTICLE

Noncoding RNAs as epigenetic mediators of skeletal muscle regeneration Gurjeev Sohi1 and Francis Jeffrey Dilworth1,2 1 Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Canada 2 Department of Cellular and Molecular Medicine, University of Ottawa, Canada

Keywords differentiation, epigenetics, gene expression, lncRNAs, miRNAs, MyoD, myogenesis, myoMiRs, satellite cells, skeletal muscle Correspondence F. J. Dilworth, Sprott Center for Stem Cell Research, Ottawa Hospital Research Institute, 501 Smyth Rd, Mailbox 511, Ottawa, ON, Canada K1H 8L6 Fax: +1 613 739 6294 Tel.: +1 613 737 8899 ext 70339 E-mail: [email protected]

Skeletal muscle regeneration is a well-characterized biological process in which resident adult stem cells must undertake a series of cell-fate decisions to ensure efficient repair of the damaged muscle fibers while also maintaining the stem cell niche. Satellite cells, the main stem cell contributing to the repaired muscle fiber, are maintained in a quiescent state in healthy muscle. Upon injury, the satellite cells become activated, and proliferate to expand the muscle progenitor cell population before returning to the quiescent state or differentiating to become myofibers. Importantly, the determination of cell fate is controlled at the epigenetic level in response to environmental cues. In this review, we discuss our current understanding of the role played by noncoding RNAs (both miRNAs and long-noncoding RNAs) in the epigenetic control of muscle regeneration.

(Received 21 October 2014, revised 1 December 2014, accepted 2 December 2014) doi:10.1111/febs.13170

Introduction Over the last half century, RNA has emerged as a key player in regulating cellular function. Extending from its role as an essential intermediate in the conversion of genomic information to protein products, RNA is now regarded as an important mediator of gene expression and protein function [1,2]. With the recent advancement of unbiased RNA detection methods for mammalian transcriptome analysis, several studies have now established that the vast majority of transcription results in noncoding RNA sequences, with

only 2% of the genome encoding for proteins [3–8]. This has coincided with an increase in the number of scientific publications dedicated to identifying and understanding the function of nonprotein-coding RNAs (ncRNAs). The ncRNA family has emerged as a functionally diverse group of nucleotide entities that possess intrinsic abilities to serve as potential protein ligands, nucleotide base-pairing partners, molecular scaffolds and enzymes. These functions have added to the complexity with which gene expression and protein

Abbreviations Ago, argonaute; CE, core enhancer; DRR, distal regulatory region; eRNA, enhancer RNA; GW182, glycine–tryptophan repeat-containing protein of 182 kDa; IGF, insulin-like growth factor; lncRNA, long nonprotein coding RNA; mRNP, mRNA-containing ribonucleoprotein; Myog, myogenin; MRFs, myogenic regulatory factors; myomiR, muscle-specific miRNA; ncRNA, nonprotein-coding RNA; PRC2, polycomb repressive complex 2; RISC, RNA-induced silencing complex; SC, satellite cell; SR, serine/arginine-rich; Yam, YY1-associated muscle lncRNA.

FEBS Journal (2014) ª 2014 FEBS

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The role of ncRNAs in muscle regeneration

function are regulated and cellular processes influenced [9]. Specifically, two of the ncRNA classes that have received extensive attention are the miRNAs and long ncRNAs (lncRNAs). Their functional versatility has conferred on them the ability to control different aspects of cellular development, from progenitor cell maintenance to commitment and subsequently differentiation.

miRNAs The miRNA class of small ncRNAs is a group of RNA polymerase II (RNA Pol II)-derived transcripts that undergo extensive processing to achieve their characteristic length of ~ 22 nucleotides [10]. Initially, miRNAs are transcribed as primary transcripts (primiRNAs) that can be several kilobases long [11]. Within the nucleus, the RNase III enzyme Drosha cleaves the pri-mRNA transcripts to produce ~ 70 nucleotide precursor miRNAs [12]. The precursor miRNA is then exported out to the cytoplasm via Exportin-5 [13]. Subsequently, the RNase III enzyme Dicer cleaves precursor miRNAs to give rise to mature miRNAs in the cytoplasm [12]. This processing mechanism is widely used in cells because miRBase (www.mirbase.org – release 21), an online repository for published miRNA sequences, now lists ~ 1800 human miRNAs. However, we note that a subgroup of miRNAs can bypass Drosha processing during their maturation [14]. miRNAs are proposed to regulate the expression of > 60% of human genes through a mechanism in which they guide a diverse set of multi-protein RNA-induced silencing complexes (RISC) to specific target mRNAs [15]. The miRNA-associated RISC complexes consist of a combination of the argonaute (Ago) protein family, the glycine–tryptophan (GW) repeat-containing protein of 182 kDa (GW182) protein family, and other

G. Sohi and F. J. Dilworth

accessory proteins. The combinatory nature of the RISC complex components and the degree of miRNA–mRNA complementarity have been demonstrated to dictate the distinct pathway with which post-translational repression of mRNA targets can occur. Although RISC complexes containing Ago1–4 can repress translation and induce mRNA decay of mRNAs targeted through miRNA-mediated base pairing, only Ago2-containing complexes possess the cleavage activity required for inducing strand breaks in mRNAs that show perfect complementarity of base pairing with miRNAs [16,17]. Both these mRNA translational silencing mechanisms are based on targetsite complementarity localized to the 30 -UTR of the transcript. However, miRNAs also display complementarity for gene promoters. In this context, miRNAs have also been shown to mediate transcriptional activation [18] or silencing [19] through targeting of Ago/GW182-containing complexes to promoter regions. Thus, miRNAs can influence the contribution of specific genes to the proteome through multiple different mechanisms. The overall impact of mature miRNAs on muscle development can be appreciated from the study by O’Rourke et al., in which skeletal muscle-specific inactivation of Dicer during embryonic myogenesis led to decreases in muscle fiber development, reduction in muscle mass and perinatal lethality in mice [20]. A large number of miRNAs have been characterized for their ability to functionally silence transcripts in muscle (see Table 1). However, the impact of distinct RISC complexes to modulate muscle regeneration has yet to be characterized.

lncRNAs The lncRNA class of ncRNAs regroups all transcripts of > 200 nucleotides. They represent the most numer-

Table 1. MyomiRs and their validated targets in muscle MyomiR

Validated targets in muscle

miR-1

DLL1 [116], HDAC4 [76], RASGAP [117], CDK9 [117], FN [117], RHEB [117], PKCe [118], HSP60 [118], HAND2 [119], PAX3 [93], PAX7 [120], FZD7 [121], FRS2 [121], NCX1 [122], ANXA5 [122], HSP70 [123], MYLK3 [124], CALM1 [124], CALM2 [124], SRI [125], CDC42 [126], KLF4 [127], TWF1 [128], IGF1 [129], IGF1R [129], c-MET [130], BCL2 [131], MEF2A [132], PP2A Regulatory Subunit B56a [133], NOTCH3 [134] CCND2 [135], SRF [76], RhoA [136], CDC42 [136], NELFA [136], IGF1R [79], FGFR1 [137], PP2AC [137], PRDM16 [77,78,138], NFATc4 [139], KLF15 [140], CTGF [141], nPTB [142] GJA1 [143], PAX3 [93], PAX7 [92,120], c-MET [130], KSRP [144], HDAC4 [145], NOTCH3 [134], HMGB3 [146], TIMP3 [147], HSP60 [148] p21 [149] PAX7 [92], PTEN [150], FOXO1 [150] PDCD4 [151], PACS2 [151], DYRK2 [151], SOX6 [152], SK3 [153]

miR-133 miR-206 miR-208b miR-486 miR-499

2



ous and functionally diverse class of ncRNAs. Many lncRNAs share features common to mRNAs, such as 50 -capping, poly-adenylation and exon/intron splicing, yet they are distinct in that they are devoid of ORFs, which precludes their translation to proteins [21,22]. These lncRNAs can be transcribed in both the sense and antisense directions by RNA Pol II from genomic sequences found within promoters, exonic and intronic sequences of protein-coding genes [23]. The lncRNAs that lack poly(A) tails are generally transcribed by RNA Pol III [24,25]. In most cases, lncRNA transcripts are less abundant than protein-coding transcripts. Furthermore, the expression of lncRNAs is generally more tightly regulated, both spatially and temporally, compared with the expression of proteincoding genes [8,26]. The primary nucleotide sequence encoded by the lncRNA may allow for the targeting of specific genomic loci, whereas their complex secondary structure allows for the interaction with DNA, RNA and proteins to confer functionally diverse roles in the cell. Importantly, given that lncRNAs can localize to both the nucleus and the cytoplasm, lncRNAs have the potential to act on every aspect of gene expression [27,28]. This functional diversity of lncRNAs, alongside their cell-type- or development stagespecific expressional pattern, positions them as important molecular factors controlling cell identity and lineage commitment [29,30]. In the nucleus, lncRNAs can act either in cis (acting on a gene within the same locus) or trans (acting on a gene at a different locus) to alter transcription via recruitment of a variety of chromatin-modifying enzymes. As a consequence, they have an ability to regulate the chromatin environment at a particular locus or an entire chromosome [31–35]. Interestingly, genome-wide RNA immunoprecipitation sequencing revealed that thousands of lncRNAs associate with the Ezh2-containing polycomb repressive complex 2 (PRC2) [36]. The mechanism whereby lncRNAs interact with chromatin-remodeling complexes, such as PRC2, and help guide them to a particular locus in cis or trans is the subject of ongoing investigations. For instance, evidence exists of specific lncRNAs that serve as guiding scaffolds via their ability to directly bind DNA elements via RNA : DNA complementarity [33]. Moreover, the same lncRNA can often scaffold with different epigenetic complexes. Nuclear lncRNAs have also been shown to interact with the splicing factors and impact splicing events, by influencing their activity and their distribution within nuclear (speckle) domains [37,38]. Moreover, some nuclear lncRNAs interact with RNA sequences to influence their splicing [39]. The ability of nuclear lncRNAs to favor or disrupt FEBS Journal (2014) ª 2014 FEBS


chromosome interactions also enables them to influence chromosome looping [40–42]. Moreover, not only do nuclear lncRNAs act as protein scaffolds, but they can also serve as structural scaffolds helping regulate the formation of nuclear compartments such as speckles, paraspeckels and polycomb bodies [37,43,44]. In the cytoplasm, lncRNAs can hybridize to transcripts and modulate their translational control. They do so by regulating polysome loading to the mRNA transcript [45–47], Staufen1-mediated mRNA decay [48,49] and miRNA-mediated mRNA degradation [29,30,50–52]. miRNA-mediated mRNA degradation is primarily inhibited by cytoplasmic lncRNAs, referred to as competing endogenous RNAs, which serve as molecular sponges for miRNAs, thereby leading to a reduction in levels of unbound miRNAs available to target their respective mRNA targets. Although competing endogenous RNAs were first described in plants [53], subsequent studies in mammals have implicated these cytoplasmic lncRNAs in a number of cellular processes, including cell differentiation and pluripotency [29,30,50]. More recently, circular RNAs have also been demonstrated to serve as miRNA sponges. Because circular RNAs have greater stability and a lower turnover, they have the potential to offer prolonged control of miRNA sponge activity [51,52,54]. Although we continue to uncover the different functions of lncRNA, we are only beginning to elucidate their roles in regulating muscle regeneration.

Muscle regeneration In postnatal life, primary muscle stem cells involved in injury repair are satellite cells (SCs) that reside along the muscle fiber between the sarcolemma and the basal lamina [55]. When associated with a healthy muscle fiber, a SC is maintained in a quiescent state [56]. However, upon injury, the SC becomes activated and begins to proliferate to expand the muscle progenitor pool. The SC must then make a cell-fate decision to either self-renew for maintenance of the stem cell population or undergo muscle differentiation to establish new muscle fibers [57,58]. Over the course of 28 days, SCs that survive injury-induced damage give rise to a complete regeneration of the muscle through a multistep process. These steps include: (a) maintenance of the SC quiescent state; (b) activation of SC to either self-renew or commit to the muscle-progenitor lineage; (c) expansion of the muscle progenitor population; and (d) exit of the cell cycle and terminal differentiation to form myofibers (see Fig. 1). Thus muscle regeneration represents an excellent model for studying the control of cell-fate decisions in adult tissue. The role of epigenetic enzymes in 3


ous and functionally diverse class of ncRNAs. Many lncRNAs share features common to mRNAs, such as 50 -capping, poly-adenylation and exon/intron splicing, yet they are distinct in that they are devoid of ORFs, which precludes their translation to proteins [21,22]. These lncRNAs can be transcribed in both the sense and antisense directions by RNA Pol II from genomic sequences found within promoters, exonic and intronic sequences of protein-coding genes [23]. The lncRNAs that lack poly(A) tails are generally transcribed by RNA Pol III [24,25]. In most cases, lncRNA transcripts are less abundant than protein-coding transcripts. Furthermore, the expression of lncRNAs is generally more tightly regulated, both spatially and temporally, compared with the expression of proteincoding genes [8,26]. The primary nucleotide sequence encoded by the lncRNA may allow for the targeting of specific genomic loci, whereas their complex secondary structure allows for the interaction with DNA, RNA and proteins to confer functionally diverse roles in the cell. Importantly, given that lncRNAs can localize to both the nucleus and the cytoplasm, lncRNAs have the potential to act on every aspect of gene expression [27,28]. This functional diversity of lncRNAs, alongside their cell-type- or development stagespecific expressional pattern, positions them as important molecular factors controlling cell identity and lineage commitment [29,30]. In the nucleus, lncRNAs can act either in cis (acting on a gene within the same locus) or trans (acting on a gene at a different locus) to alter transcription via recruitment of a variety of chromatin-modifying enzymes. As a consequence, they have an ability to regulate the chromatin environment at a particular locus or an entire chromosome [31–35]. Interestingly, genome-wide RNA immunoprecipitation sequencing revealed that thousands of lncRNAs associate with the Ezh2-containing polycomb repressive complex 2 (PRC2) [36]. The mechanism whereby lncRNAs interact with chromatin-remodeling complexes, such as PRC2, and help guide them to a particular locus in cis or trans is the subject of ongoing investigations. For instance, evidence exists of specific lncRNAs that serve as guiding scaffolds via their ability to directly bind DNA elements via RNA : DNA complementarity [33]. Moreover, the same lncRNA can often scaffold with different epigenetic complexes. Nuclear lncRNAs have also been shown to interact with the splicing factors and impact splicing events, by influencing their activity and their distribution within nuclear (speckle) domains [37,38]. Moreover, some nuclear lncRNAs interact with RNA sequences to influence their splicing [39]. The ability of nuclear lncRNAs to favor or disrupt FEBS Journal (2014) ª 2014 FEBS


chromosome interactions also enables them to influence chromosome looping [40–42]. Moreover, not only do nuclear lncRNAs act as protein scaffolds, but they can also serve as structural scaffolds helping regulate the formation of nuclear compartments such as speckles, paraspeckels and polycomb bodies [37,43,44]. In the cytoplasm, lncRNAs can hybridize to transcripts and modulate their translational control. They do so by regulating polysome loading to the mRNA transcript [45–47], Staufen1-mediated mRNA decay [48,49] and miRNA-mediated mRNA degradation [29,30,50–52]. miRNA-mediated mRNA degradation is primarily inhibited by cytoplasmic lncRNAs, referred to as competing endogenous RNAs, which serve as molecular sponges for miRNAs, thereby leading to a reduction in levels of unbound miRNAs available to target their respective mRNA targets. Although competing endogenous RNAs were first described in plants [53], subsequent studies in mammals have implicated these cytoplasmic lncRNAs in a number of cellular processes, including cell differentiation and pluripotency [29,30,50]. More recently, circular RNAs have also been demonstrated to serve as miRNA sponges. Because circular RNAs have greater stability and a lower turnover, they have the potential to offer prolonged control of miRNA sponge activity [51,52,54]. Although we continue to uncover the different functions of lncRNA, we are only beginning to elucidate their roles in regulating muscle regeneration.

Muscle regeneration In postnatal life, primary muscle stem cells involved in injury repair are satellite cells (SCs) that reside along the muscle fiber between the sarcolemma and the basal lamina [55]. When associated with a healthy muscle fiber, a SC is maintained in a quiescent state [56]. However, upon injury, the SC becomes activated and begins to proliferate to expand the muscle progenitor pool. The SC must then make a cell-fate decision to either self-renew for maintenance of the stem cell population or undergo muscle differentiation to establish new muscle fibers [57,58]. Over the course of 28 days, SCs that survive injury-induced damage give rise to a complete regeneration of the muscle through a multistep process. These steps include: (a) maintenance of the SC quiescent state; (b) activation of SC to either self-renew or commit to the muscle-progenitor lineage; (c) expansion of the muscle progenitor population; and (d) exit of the cell cycle and terminal differentiation to form myofibers (see Fig. 1). Thus muscle regeneration represents an excellent model for studying the control of cell-fate decisions in adult tissue. The role of epigenetic enzymes in 3


activation. Interestingly, miR-489 was found to posttranscriptionally suppress the oncogene Dek, a protein that promotes the proliferative expansion of quiescent SCs [65]. More recently, another study identified miR195 and miR-497 as novel regulators of SC selfrenewal through the attenuation of cell-cycle progression in muscle progenitor cells. In this case, it was shown that miR-195 and miR-497 cause cell-cycle exit by targeting Cdc25a, Cdc25b and Ccnd2 to establish the quiescent state [66]. The lncRNA Uc.283 + A has been demonstrated to complement with the immature pri-miR-195 transcript and prevent its cleavage by Drosha, which is required for the formation of a functional miR-195 molecule [39]. Consistent with mechanisms existing to maintain quiescent cells that are poised for activation, lncRNA Uc.283 + A may turn out to be a key regulator that can quickly disrupt the availability of functional miR-195 required for maintaining the quiescence of SCs. Under such conditions, rapid degradation of pri-miR-195 would be anticipated given that Drosha-mediated cleavage occurs upon hybridization of two complementarity RNA molecules. Although it is critical for SCs to be able to maintain a quiescent state, in order to prevent the premature onset of myogenesis, it is equally important for the SCs to be primed for activation. As a consequence, quiescent SCs have been observed to transcribe Myf5, but utilize components of the miRNA-processing machinery to prevent it from being translated. Specifically, Myf5 is continuously targeted for degradation by miR-31 located within the (mRNP) granules in the cytoplasm [62]. It is only upon the dissociation of these mRNP granules from the SCs that cell quiescence is abolished and myogenesis initiated [62]. Interestingly, GW182 was also observed to colocalize with miR-31 and myf-5 at these mRNA granules [62]. The mRNA granules observed in the SCs were distinct from stress granules that have been characterized by the presence of the RNA-binding protein HuR [67,68], which happened to remain in the nucleus of quiescent SCs [62]. Additional evidence suggests that HuR continues to remain predominantly nuclear during the early phases of differentiation, subsequently translocating to the cytoplasm where it increases the stability of MyoD, myogenin (Myog) and p21 [69,70]. Although HuR stabilizes MyoD, Myog and p21, it can also exert its pro myogenic effect by destabilizing the mRNA-encoding nucleophosmin, a protein that promotes cell-cycle progression upon association with decay factor KSRP [71]. Both stabilizing and destabilizing properties of HuR are likely dependent on its associated complex subunits, which are of equal FEBS Journal (2014) ª 2014 FEBS


importance for HuR-mediated induction of muscle differentiation. Determining the potential importance of components of the mi-RISC complex in mediating translocation of miRNAs and lncRNAs would help elucidate whether specific components of the mi-RISC complex are critical towards ensuring quiescence and appropriate activation of SCs into muscle differentiation (see Fig. 2 for established roles of mi-RISC complexes in muscle). ncRNAs regulating the proliferation state of muscle stem cells Upon muscle damage, SCs become activated and undergo a first cell division between 24 and 48 h after injury [72]. During this cell division, the SC must decide between: (a) dividing asymmetrically to generate a SC and a committed muscle progenitor (ultimately maintaining the SC pool); (b) dividing symmetrically to generate two daughter SCs (ultimately expanding the SC pool); or (c) dividing symmetrically to generate two committed muscle progenitor cells (ultimately depleting the SC pool) (see Fig. 1) [57]. The population of committed muscle progenitors, defined as being Pax7+/ Myf5+/MyoD+/Myog (Step 2) then undergoes a rapid expansion during which cells divide every 8–10 h [72]. Thus, the epigenetic machinery must make the cellfate decision to either replenish the SC compartment or commit to generate a large number of progenitors committed to repair the damaged muscle fibers. This stage of the regeneration process has been well characterized for miRNA expression in both mice [73,74] and humans [75]. Assuming that miRNAs that decrease their expression during differentiation are important for maintaining the proliferative environment, 17 miRNAs are predominantly expressed in proliferating myoblasts with predicted target genes that function in a wide range of cellular processes during myogenesis [75]. Although many of the differentially expressed miRNAs have been previously investigated for their role in myogenesis, there are many whose role remains uncharacterized. An initial stage in the transition to the activated SC state is the downregulation of miR-31 and the release of the Myf5 mRNA from the mRNP granules to permit its translation for protein expression. The expression of Myf5 and MyoD in the activated SC leads to the activation of miR-133a/miR-133b [76], a pair of muscle-enriched miRNAs that are a part of the myomiR family of short ncRNAs (see Table 1). The expression of miR-133 plays a dual role in the proliferating myoblasts: (a) it prevents commitment to the alternate brown adipose cell fate by inhibiting transla5



Fig. 2. Modes of miRNA action and the role of the mi-RISC complex in muscle differentiation. The illustration depicts the mi-RISC complex and its involvement in four major mechanisms, whereby most miRNAs function to repress their mRNA targets. These include: (1) translation repression at the initiation stage, (2) translation repression at the post-initiation stage, (3) degredation via mRNA cleavage, and (4) degredation via deadenylation. The three major components of the mi-RISC complex, Ago proteins, GW182 proteins and accessory proteins whose role in muscle differentiation have been evaluated are listed below. Ago, argonaute, GW182, glycine–tryptophan repeat-containing protein of 182 kDa.

tion of the adipogenic transcriptional regulator PRDM16 [77,78]; and (b) it blocks terminal differentiation of the muscle progenitor cells by preventing translation of SRF [76] and IGF1R [79]. Although MyoD is expressed in proliferating myoblasts, its protein level in this cellular state remains lower than that observed in cells induced to undergo terminal differentiation. This lower level of MyoD is regulated through transforming growth factor-beta signaling which acts to repress miR-29, through a mechanism where the DNA-binding transcription factor YY1 mediates recruitment of the PRC2 protein Ezh2 to the miR-29 locus [80]. The inhibition of miR-29 allows for upregulation of HDAC4 protein levels that, in turn, has been implicated in reducing MyoD gene expression, although the precise mechanism of action has not been investigated [81]. Meanwhile, it has recently been demonstrated that HDAC4 promotes SC proliferation by upregulating Pax7 expression, whereas MyoD expression remained unchanged in SC-specific HDAC4 knockout mice [82]. Moreover, HDAC4 inactivation led to a significant decrease in the MyoD target, miR133, and a concomitant increase in the adipogenic transcription regulator PRDM16 [82]. However, the precise mechanism by which transforming growth factor-beta signaling mediates the repression of miR-29 transcription through YY1 requires further clarification. We note that YY1-mediated recruitment of PRC2 to target loci has also been demonstrated to 6

restrict expression of the myomiRs (see Table 1) miR1, miR-133 and miR-206 during myoblast proliferation [83]. Moreover, YY1 binding has also been detected at the genomic loci that give rise to lncRNAs. Using genome-wide chromatin immunoprecipitation-sequence analysis, Lu et al. identified 63 potential YY1-associated muscle (Yam) lncRNAs [84]. Although most of the Yam lncRNAs remain uncharacterized, Yam-1 was shown to act in cis to upregulate neighboring gene expression leading to a block in muscle differentiation [84]. Among these neighboring genes, miR-715 expression is being activated in cis, where the miRNA is contributing to prevent terminal muscle differentiation by inhibiting translation of Wnt7b [84]. Although YY1 DNA binding has previously been shown to influence the myogenic process via direct regulation of miRNA expression [83,85], YY1–Yam-1–miR-715 appears to represents a novel circuitry in which YY1 indirectly influences muscle differentiation through expression of a lncRNA that activates expression of the miR-715 miRNA. It is important to note that not all Yam lncRNAs follow similar expression patterns during myogenesis. Whereas Yam-1 and Yam-3 promote expansion of muscle progenitor cells, Yam-2 and Yam-4 are promyogenic and display increased expression during muscle differentiation [84]. Further work is required to determine the specific mechanisms through which YY1 can mediate opposing expression patterns for FEBS Journal (2014) ª 2014 FEBS


activation. Interestingly, miR-489 was found to posttranscriptionally suppress the oncogene Dek, a protein that promotes the proliferative expansion of quiescent SCs [65]. More recently, another study identified miR195 and miR-497 as novel regulators of SC selfrenewal through the attenuation of cell-cycle progression in muscle progenitor cells. In this case, it was shown that miR-195 and miR-497 cause cell-cycle exit by targeting Cdc25a, Cdc25b and Ccnd2 to establish the quiescent state [66]. The lncRNA Uc.283 + A has been demonstrated to complement with the immature pri-miR-195 transcript and prevent its cleavage by Drosha, which is required for the formation of a functional miR-195 molecule [39]. Consistent with mechanisms existing to maintain quiescent cells that are poised for activation, lncRNA Uc.283 + A may turn out to be a key regulator that can quickly disrupt the availability of functional miR-195 required for maintaining the quiescence of SCs. Under such conditions, rapid degradation of pri-miR-195 would be anticipated given that Drosha-mediated cleavage occurs upon hybridization of two complementarity RNA molecules. Although it is critical for SCs to be able to maintain a quiescent state, in order to prevent the premature onset of myogenesis, it is equally important for the SCs to be primed for activation. As a consequence, quiescent SCs have been observed to transcribe Myf5, but utilize components of the miRNA-processing machinery to prevent it from being translated. Specifically, Myf5 is continuously targeted for degradation by miR-31 located within the (mRNP) granules in the cytoplasm [62]. It is only upon the dissociation of these mRNP granules from the SCs that cell quiescence is abolished and myogenesis initiated [62]. Interestingly, GW182 was also observed to colocalize with miR-31 and myf-5 at these mRNA granules [62]. The mRNA granules observed in the SCs were distinct from stress granules that have been characterized by the presence of the RNA-binding protein HuR [67,68], which happened to remain in the nucleus of quiescent SCs [62]. Additional evidence suggests that HuR continues to remain predominantly nuclear during the early phases of differentiation, subsequently translocating to the cytoplasm where it increases the stability of MyoD, myogenin (Myog) and p21 [69,70]. Although HuR stabilizes MyoD, Myog and p21, it can also exert its pro myogenic effect by destabilizing the mRNA-encoding nucleophosmin, a protein that promotes cell-cycle progression upon association with decay factor KSRP [71]. Both stabilizing and destabilizing properties of HuR are likely dependent on its associated complex subunits, which are of equal FEBS Journal (2014) ª 2014 FEBS


importance for HuR-mediated induction of muscle differentiation. Determining the potential importance of components of the mi-RISC complex in mediating translocation of miRNAs and lncRNAs would help elucidate whether specific components of the mi-RISC complex are critical towards ensuring quiescence and appropriate activation of SCs into muscle differentiation (see Fig. 2 for established roles of mi-RISC complexes in muscle). ncRNAs regulating the proliferation state of muscle stem cells Upon muscle damage, SCs become activated and undergo a first cell division between 24 and 48 h after injury [72]. During this cell division, the SC must decide between: (a) dividing asymmetrically to generate a SC and a committed muscle progenitor (ultimately maintaining the SC pool); (b) dividing symmetrically to generate two daughter SCs (ultimately expanding the SC pool); or (c) dividing symmetrically to generate two committed muscle progenitor cells (ultimately depleting the SC pool) (see Fig. 1) [57]. The population of committed muscle progenitors, defined as being Pax7+/ Myf5+/MyoD+/Myog (Step 2) then undergoes a rapid expansion during which cells divide every 8–10 h [72]. Thus, the epigenetic machinery must make the cellfate decision to either replenish the SC compartment or commit to generate a large number of progenitors committed to repair the damaged muscle fibers. This stage of the regeneration process has been well characterized for miRNA expression in both mice [73,74] and humans [75]. Assuming that miRNAs that decrease their expression during differentiation are important for maintaining the proliferative environment, 17 miRNAs are predominantly expressed in proliferating myoblasts with predicted target genes that function in a wide range of cellular processes during myogenesis [75]. Although many of the differentially expressed miRNAs have been previously investigated for their role in myogenesis, there are many whose role remains uncharacterized. An initial stage in the transition to the activated SC state is the downregulation of miR-31 and the release of the Myf5 mRNA from the mRNP granules to permit its translation for protein expression. The expression of Myf5 and MyoD in the activated SC leads to the activation of miR-133a/miR-133b [76], a pair of muscle-enriched miRNAs that are a part of the myomiR family of short ncRNAs (see Table 1). The expression of miR-133 plays a dual role in the proliferating myoblasts: (a) it prevents commitment to the alternate brown adipose cell fate by inhibiting transla5



Fig. 3. Feedback mechanisms between myogenic regulatory factors MRFs and myomiRs during muscle differentiation. Specifically, the figure highlights the positive and negative feedback mechanisms that exist between MRFs, MyoD and myogenin, and the following myomiRs, miR-1, miR-206 and miR-486. MyoD-mediated induction of miR-1, miR-206 and miR-486 represses a unique and common subset of downstream target genes such as HDAC4, PTEN and Foxo1a. Repressing HDAC4 leads to further derepression of MyoD, whereas PTEN and FoxO1 repression enables further activation of the IGF signaling pathway and downstream Myogenin expression. The net result is an increase in muscle differentiation. MyoD mediated direct and indirect activation of Myogenin, in turn results in the induction of miR-133 which suppresses IGF signaling pathway and its own expression via targeting IGF-1R mRNA. The net result is a decrease in muscle differentiation.

been shown to be regulated by lncRNAs. Studies from the Sartorelli laboratory have reported that the binding of MyoD and Myog in extragenic enhancer regions result in the expression of a class of lncRNAs that are termed enhancer RNAs (eRNAs) [94]. In particular, they identified eRNAs that are transcribed from the two developmentally important MyoD enhancers, the core enhancer (CE) and the distal regulatory region (DRR), located upstream of the MyoD coding sequence. Specifically, CEeRNA functioned in cis to activate expression of MyoD, whereas DRReRNA worked in trans to upregulate myogenin transcription by increasing chromatin accessibility and RNA Pol II recruitment [94]. However, the molecular mechanism whereby these lncRNAs increase chromatin accessibility and the mechanisms dictating whether CERNA and DRR RNA act in cis or trans require further elucidation. In yet another example of transcription factors being regulated by lncRNAs, a recent study by Dey et al. demonstrated that the imprinted gene encoding the H19 lncRNA can be processed to give rise to miR675-3p and miR-675-5p that promote muscle differentiation [95]. Specifically, miR-675-3p and miR-675-5p induced differentiation by downregulating the proliferation promoting Smad transcription factors [95]. Finally, lncRNAs can also serve as coactivators to increase output from MyoD target genes. In this case, the lncRNA SRA serves as a molecular scaffold for the assembly of a co-activator complex consisting of the RNA helicases p68 and p72 at MyoD-bound genes 8

to mediate transcriptional activation [96]. Although RNase treatment demonstrated that MyoD association with p68 was not dependent on interaction with SRA during differentiation, a combination of SRA, p68, p72 was required to get maximum MyoD dependent transactivation of the muscle-specific creatine kinase enhancer linked to the luciferase gene [96]. Thus, lncRNAs have been shown to act in through multiple different mechanisms to regulate the ability of muscle transcription factors to induce the muscle gene expression program. Cell-cycle exit Exiting cell cycle is a critical step for commitment towards myoblast differentiation. miRNAs play an important role in regulating the cell cycle during myoblast differentiation. For instance, in Myog-mediated induction of myoblast differentiation through cell-cycle exit, Myog upregulates transcription of miR-17–92 cluster, amongst which miR-20a targets the repression of transcription factors E2F1, E2F2 and E2F3 critical for activation of the cell cycle [86,97–99]. Myog can also upregulate Lats2 kinase, which cooperates with pRB to form repressive complexes with E2F-regulated promoters [86,100]. Activation of p38alpha MAPK signaling pathway during myoblast differentiation may also contribute to the upregulation of Lats2 kinase because MAPK signaling has been demonstrate to repress the expression of miR-31, a miRNA that has FEBS Journal (2014) ª 2014 FEBS


been demonstrated to directly target the 30 -UTR of the Lats2 kinase [101]. Indeed, activation of p38 MAPK signaling is critical for the onset of myoblast differentiation, in part through induction of cell-cycle exit. It has previously been demonstrated to downregulate cyclin D1 by antagonizing the JNK pathway, leading to cell-cycle exit [102]. Recently, p38 signaling has been shown to be required for the transcription of miR-1/ miR-133a clusters [103]. Interestingly, miR-1 directly suppresses cyclin D1 via initiating its mRNA degradation, whereas miR-133 indirectly suppresses cyclin D1 by targeting the transcription factor Sp1 [103,104]. Given the evidence for Myog to bind to miR-1 and miR-133 promoters, it would be interesting to evaluate how p38 signaling, Myog and miRNAs converge to induce cell-cycle exit leading to the onset of myoblast differentiation. Muscle gene expression The identity of mature muscle fibers is partially defined by the complement of myosin proteins expressed in the cell [105]. Work from the Olson group has shown that miRNAs present in the introns of myosin genes permit the coexpression of genes that help influence muscle function [106]. In this context, miR-208a and miR-208b are processed from the intronic RNA of the Myh6 and Myh7 transcripts respectively, whereas miR-499 is processed from the intronic RNA of the Myh7b gene. Specifically, they demonstrated that expression of the slow muscle fiber myosin genes Myh7 and Myh7b genes help reinforce the slow muscle gene expression program through the expression of miR-208b and miR-499 [106]. This reinforcement of the slow-muscle gene expression program is at least partially due to translational inhibition of the transcriptional repressors Sox6, Purb, Sp3 and HP1b whose 30 -UTR contain target sites for miR-499 and/or miR-208 [106]. By contrast, the fast muscle fiber phenotype has been shown to be activated through the function of a specific linc-RNA, termed linc-MYH, which is transcribed from an enhancer within the Myh locus [107]. The linc-MYH transcript has been shown to upregulate expression of fast fibertype genes while downregulating expression of slow fiber-type genes [107]. However, the mechanism through which linc-MYH is functioning to regulate expression of these fiber-type specific genes has not yet been established. In yet another example of how ncRNA can influence the set of genes expressed in muscle cells, the Malat-1 lncRNA acts as a regulator of alternative splicing for a specific set of pre-mRNAs by interacting FEBS Journal (2014) ª 2014 FEBS


with and influencing distribution of the Serine/Arginine-rich (SR) family of splicing factors in nuclear speckle domains [37]. In the skeletal muscle cells, Malat1 is upregulated during differentiation and may potentially serve as a cue for differentiation as a knockdown of Malat1-suppressed myoblast proliferation due to cell-growth arrest in the G0/G1 phase [108]. Recently, miR-9 has been demonstrated to target Malat-1 for degradation in the nucleus, however, its impact on muscle differentiation remains to be investigated [109]. The miR-9-mediated downregulation of Malat-1 adds to the function of miRNA to serve as post-transcriptional regulators within the nuclear speckles. Investigating the novel roles of microRNAs in regulating levels of other nuclear lncRNAs involved in myogenesis would provide further insight into the functional interplay between these two types of ncRNAs in collectively controlling this robust cellular process (see Fig. 4 for established roles of lncRNAs during muscle differentiation).

Conclusions Research over the past decade has revealed an essential role for ncRNAs in the regulation of muscle regeneration. These studies have established miRNAmediated translation inhibition as a key mechanism modulating the fate of SCs during muscle regeneration. This well-characterized mechanism of miRNA function, combined with the availability of bioinformatic tools to identify target genes, has provided ‘lowhanging fruit’ for understanding how miRNAs expressed in muscle are contributing to regeneration. However, we expect that our understanding of the mechanisms through which ncRNAs contribute to muscle regeneration is in its infancy. Studies in yeast and Caenorhabditis elegans have suggested important roles for miRNAs and their related short dsRNA-containing Ago complexes in a broad range of cellular processes [110]. Many of these processes appear to be conserved in mammalian systems, including targeting of Ago complexes to specific genomic loci for transcriptional activation [111], transcriptional repression [112], facilitation of DNA repair [113] and the modulation of alternative splicing [114]. In addition, we lack a good understanding of the molecular mechanisms through which most of the lncRNAs contribute to altered gene expression – both in cis or in trans – during muscle regeneration. The development of technologies such as CRISPR [115] that allow for efficient deletion of specific genetic loci should facilitate studies to examine the effects of individual ncRNAs on the muscle regeneration process. Characterization of these 9


these lncRNAs and how specific Yam RNAs contribute to expansion of the muscle progenitor cell population. ncRNAs Regulating the differentiation state of muscle stem cells As the muscle progenitor population continues to expand, a subset of these cells turn off Pax7 and Myf5 expression, and begin to express the muscle-specific transcription factor Myog. The expression of Myog (Pax7/Myf5/MyoD+/Myog+) activates a gene expression program that induces terminal muscle differentiation through cell-cycle exit and cell fusion to form multinucleated myofibers that reconstitute the damaged muscle [86,87]. Importantly, the expression of Myog represents a point of no return that commits the muscle progenitor to exit cell cycle [86,87]. This stage of the regeneration process has been the most well-characterized because of the availability of a greater number of cells, and our ability to control the timing of differentiation using alternate culture conditions. Regulation of the muscle transcriptional machinery The activation of pro-myogenic signals is sufficient to permit MyoD-bound target genes to be expressed [88]. However, muscle-gene expression is facilitated by increased MyoD accumulation in the differentiating cells. The suppression of transforming growth factorbeta signaling in differentiating progenitors allows for strong expression of miR-29, which reduces levels of the repressive HDAC4 and results in a subsequent increase in transcription of the MyoD gene [81]. We note that MyoD protein levels have also been shown to increase in the absence of miR-203b that targets the 30 -UTR of MyoD in the fish species Nile tilapia [89]. It remains to be determined whether this regulation of MyoD transcripts by miR-203b is conserved in mammals, and how levels of this miRNA change during differentiation. Among MyoD target genes activated during differentiation, Myog expression is a key step in the commitment of myogenic progenitors to differentiate because the two myogenic regulatory factors work synergistically to allow for high-level expression of the muscle gene expression program. Among pro-myogenic signaling pathways, activation of insulin-like growth factor (IGF) signaling has also been demonstrated to play a critical role in driving muscle differentiation in part via the up regulation of Myog [79]. FEBS Journal (2014) ª 2014 FEBS


The activation of this signaling pathway is enabled during differentiation, in part by the timely repression of miR-125b, which has been demonstrated to target IGF-II [90]. Interestingly, a negative feedback loop exists in which subsequent upregulation of Myog leads to upregulation of another miRNA, miR-133, which downregulates expression of the IGF-I receptor – an event that eventually attenuates the Myog-driven early muscle differentiation program to permit myofiber maturation [79]. In addition to activating Myog expression, MyoD also upregulates the expression of miR-378, a miRNA that targets the 30 -UTR of the inhibitory transcription factor MyoR that competes with MyoD for binding at target genes [91]. Thus the miR-378-mediated inhibition of MyoR translation allows for increased expression of MyoD target genes. These MyoD target genes include a series of important ncRNAs that are activated simultaneously to facilitate myotube formation. Among this group, MyoD turns on a series of muscle-enriched miRNAs that have been termed the myomiRs. This group includes miR-1, miR-206, miR-486 and miR-133 (see Fig. 3). Important to the differentiation process, the SC regulatory transcription factors Pax7 and Pax3 need to be turned off. Several of the myomiRs play an essential role in this repression, where miR-1 and miR-206 target the 30 -UTRs of both Pax3 and Pax7 for translational repression, and miR-486 can also block translation of Pax7 [92,93]. This myomiR-mediated reduction of Pax7 is essential to differentiation as expression of a miRNA-resistant form of Pax7 in differentiating muscle progenitors is sufficient to inhibit the differentiation process [92]. In addition, miR-1 expression is able to target the 30 -UTR of the transcriptional repressor YY1 to prevent protein translation. This reduction in YY1 levels is speculated to relieve PRC2-mediated repression of miR-206 and miR-133 as well [83]. Transcription factor levels in muscle cells are also controlled by the expression of lncRNAs. In the case of the pro-myogenic transcription factors Mef2C and MAML1, the 30 -UTR of their mRNA is a target for inhibition by the miRNAs miR-135, and miR-133 that continue to be expressed in differentiating muscle progenitors. To evade this miRNA-mediated block in Mef2C and MAML1 mRNA translation, the differentiating muscle cells express the lncRNA linc-MD1 [29]. This lncRNA contains a sequence complementary to miR-133 and miR-135 that acts as a molecular ‘sponge’ to sequester the miRNA that would otherwise block terminal differentiation [29]. Thus linc-MD1 acts to promote differentiation by preventing miRNA-mediated repression of pro-myogenic transcription factors. Recently, the expression of MyoD and Myog has also 7


expression. Only then will we begin to appreciate the full extent to which miRNAs and lncRNAs are contributing to the global changes occurring as SCs undergo changes in cell fate to repair injured muscle.

Acknowledgements We would like to thank Yuefeng Li for helpful discussions. Work in the Dilworth laboratory is funded by the Canadian Institutes of Health Research and Muscular Dystrophy Canada. FJD is a Canada Research Chair in Epigenetic Regulation of Transcription.

Author contributions G.S. and F.J.D. wrote the manuscript.

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Noncoding RNAs in the regulation of skeletal muscle biology in health and disease.

Noncoding RNAs and epigenetic mechanisms during X-chromosome inactivation.

Epigenetic regulation in hepatocellular carcinoma requires long noncoding RNAs.

Epigenetic modifications and noncoding RNAs in cardiac hypertrophy and failure.

Exploiting Long Noncoding RNAs as Pharmacological Targets to Modulate Epigenetic Diseases.

Towards understanding skeletal muscle regeneration.

Glycolysis in skeletal muscle regeneration.

Long Noncoding RNAs as Biomarkers in Cancer.

Long noncoding RNAs as metazoan developmental regulators.

Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

Long noncoding RNAs, emerging players in muscle differentiation and disease.

LncFunNet: an integrated computational framework for identification of functional long noncoding RNAs in mouse skeletal muscle cells.

Cellular players in skeletal muscle regeneration.

Adhesion in skeletal muscle during regeneration.

Myotube formation in skeletal muscle regeneration.

Satellite Cells and Skeletal Muscle Regeneration.

Neuraminidase-1 mediates skeletal muscle regeneration.

Possible mediators of functional hyperaemia in skeletal muscle.

Noncoding RNAs as regulators of cardiomyocyte proliferation and death.

Therapeutic applications of noncoding RNAs.

Ancestral vinclozolin exposure alters the epigenetic transgenerational inheritance of sperm small noncoding RNAs.

Noncoding RNAs in human saliva as potential disease biomarkers.

Noncoding RNAs as novel biomarkers in prostate cancer.

Noncoding RNAs as therapeutics for acetaminophen-induced liver injury.