Developmental regulation and evolution of muscle-specific microRNAs

Accepted Manuscript Title: Developmental regulation and evolution of muscle-specific microRNAs Author: Rie Kusakabe Kunio Inoue PII: DOI: Reference:

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Please cite this article as: Kusakabe R, Inoue K, Developmental regulation and evolution of muscle-specific microRNAs, Seminars in Cell and Developmental Biology (2015), http://dx.doi.org/10.1016/j.semcdb.2015.10.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Developmental regulation and evolution of muscle-specific microRNAs

Minami, Chuo-ku, Kobe 650-0047, Japan

Department of Biology, Graduate School of Science, Kobe University,

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b

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Evolutionary Morphology Laboratory, RIKEN, 2-2-3 Minatojima-

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Rie Kusakabea,* and Kunio Inoueb

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1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan

*Corresponding author. Evolutionary Morphology Laboratory, RIKEN,

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Minatojima-Minami, Chuo-ku, Kobe 650-0047, Japan Tel.: +81 78 306 3064; fax: +81 78 306 3370.

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E-mail: [email protected] (R. Kusakabe). ABSTRACT

MicroRNAs (miRs) are a group of small RNAs that play a major role in post-transcriptional regulation of gene expression. In animals, many of the miRs are expressed in a conserved spatiotemporal manner. Muscle tissues, the major cellular systems involved in the locomotion and physiological functions of animals, have been one of the main sites

for

verification

of

miR

targets

and

analysis

of

their

developmental functions. During the determination and differentiation of muscle cells, numerous miRs bind to and repress target mRNAs in a highly specific but redundant manner. Interspecific comparisons of the sequences and expression of miRs have suggested that miR regulation became increasingly important during the course of 1 Page 1 of 26

vertebrate evolution. However, the detailed molecular interactions that have led to the highly complex morphological structures still await investigation. In this review, we will summarize the recent findings on the functional and developmental characteristics of miRs

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that have played major roles in vertebrate myogenesis, and discuss how the evolution of miRs is related to the morphological complexity

Keywords:

MicroRNA;

target

mRNAs;

myogenesis;

vertebrates;

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evolution.

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of the vertebrates.

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1. Introduction The vertebrate body is equipped with muscular organs with a high degree of functional specialty and morphological variety. In the

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terrestrial environment, skeletal muscles in the limbs and limb girdles provide the major driving force for locomotion. Facial and tongueassociated muscles in the head enable masticatory movement and

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contribute to communication between individuals. These muscles

have their anatomical counterparts in aquatic animals such as fish.

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On the other hand, the diaphragm in the trunk is an amniote-specific muscle that plays a major role in respiration. The heart is also a organ

that

contracts

autonomically

to

produce

the

an

muscular

circulating blood flow and plays a fundamental role in the adaptation to the environment. Visceral organs such as the intestines and involuntary contraction.

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trachea are associated with smooth muscle cells that provide

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In studies on vertebrate development, muscle is one of the major cellular systems in which the molecular mechanism of

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differentiation has been actively investigated. Muscle differentiation is regulated by a relatively small number of developmental genes represented by the MyoD family of myogenic regulatory factors (MRFs) that activate the transcription of muscle specific genes [1, 2]. These transcription factors can suppress non-muscle cell fates and prepare the cells for muscle differentiation. The MRF family consists of four proteins, each of which can promote the transcription of downstream muscle-specific genes. During mammalian development, multiple MRFs are expressed in an overlapping manner at different developmental stages. MRFs thus promote muscle differentiation in a highly controlled spatiotemporal fashion, resulting in the formation of distinct muscles from multiple myogenic precursor tissues such as somites and head mesoderm. However, the detailed mechanisms by which the differentiating precursor cells give rise to the morphological complexity of muscular organs have not been fully understood. 3 Page 3 of 26

Recent

progress

in

genomic

studies

has

revealed

the

involvement of microRNAs (miRs), a group of small non-coding RNA molecules (about 22 nucleotides long), in the regulation of muscle differentiation. miRs are capable of binding to the target mRNAs by leads

to

mRNA

degradation

and/or

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either perfect or imperfect base-pairing. The effect of this binding translational

repression.

Compared to the regulation of the gene function at the transcription

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level, gene silencing by miRs generally has a relatively milder effect; miRs can act as modulators of muscle-specific gene expression, fine-

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tuning the master regulators such as MRFs. This feature of miR function has been regarded as an ideal key modifier of muscle

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physiology and morphology, in which the backbone mechanisms for body patterning have been conserved across lineages. The evolution of the complex body structure of animals is

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associated with an expansion of genome size, whereas the number of proteins encoded by the different genomes has stayed relatively For

example,

the

nematode

Caenorhabditis

elegans

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constant.

possesses about 19,000 protein-coding genes in a genome of 100

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Mb, whereas the humans possess about 20,000 genes in a 3,200 Mb genome [3, 4]. A substantial portion of the mammalian genome does not encode amino acid sequences; it potentially harbors regulatory elements and functional RNAs such as miRs. From the phylogenetic point of view, the increasing ratio of non-coding region of the genome has been discussed as correlated with the increasing organismal complexity [5, 6]. Intriguingly, total number of miRs has drastically increased during the divergence of vertebrates; more miR genes have been identified in lineages that diverged later in evolution [5, 7, 8]. In this review, we will summarize the roles of miRs in myogenetic pathways, in which miRs function as posttranscriptional modulators. We will also discuss how the major miR function could affect morphological changes and the elaboration of muscle tissues during vertebrate evolution.

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2. Formation of skeletal and cardiac muscles Vertebrate muscle cells are categorized into three different types: skeletal, cardiac, and smooth muscle. Of these, the skeletal

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and cardiac muscle cells share some major cellular characteristics. They both consist of sarcomeres, bundles of striated muscle fibers. In addition, both cell types function via the interaction between actin

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filaments and myosin motor proteins, i.e., the power stroke. During development, the proliferation and differentiation of muscle precursor

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cells are controlled by different but overlapping transcription factors, such as the MyoD family of basic-helix-loop-helix transcription factors

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(myogenic regulatory factors: MRFs), serum response factors (SRF), and myocyte enhancer factor-2 (MEF2) [2, 9-11].

On the other hand, skeletal and cardiac muscles differ in

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terms of their protein content due to the differential usage of contractile proteins; there are multiple isoforms of contractile

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proteins which are predominantly expressed either in skeletal or cardiac muscles. Moreover, skeletal and cardiac muscles follow

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substantially different myogenetic pathways involving intermediate primordial cells. Development of skeletal muscles is initiated as the commitment of multipotent mesodermal cells into skeletal myoblasts (Fig. 1). In the post-otic (somitic) muscles, myoblasts express the MyoD family of myogenic regulatory factors as well as the paired class

homeodomain

protein

Pax3

[12].

Pax3

is

required

for

maintenance of proliferative status; therefore, the balance of activity of MyoD and Pax3 plays a pivotal role in the cellular transition of myoblasts.

After

undergoing

sufficient

rounds

of

cell

division,

myoblasts cease to proliferate and undergo fusion to form large multinucleated myoblasts. The fused myoblasts begin to transcribe a large

amount

multinucleated

of

mRNA

myoblasts

for are

contractile gradually

proteins.

organized

Then, into

the

mature

myotubes associated with sarcomeric structures. Another type of striated muscle, the cardiac muscle, contributes to contraction of the 5 Page 5 of 26

heart. Like skeletal muscles, cardiac myoblasts originate from the embryonic mesoderm. They migrate toward the forming primitive heart tube, and directly join the contractile tissue of the heart, known as the myocardium. Cardiac myoblasts do not fuse, resulting in a

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myocardium composed of spindle-shaped mononuclear cells. These cells can contract like individual cells. Within the mesoderm, cardiac

muscles have two distinct origins called the first and second heart

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fields (FHF and SHF)[13, 14]. Regardless of their origins, however, the first sign of cardiac commitment is the expression of transcription

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factors Nkx2.5 and GATA4 [15]. In mammals, shortly after birth, most cardiomyocytes become binucleated due to DNA replication

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without cell division (Fig. 1) [16]. Binucleated cardiomyocytes are associated with highly organized sarcomeres which might prevent cytokinesis, and thus exhibit the hypertrophic enlargement but no

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proliferative characteristics [17]. The multinucleated cardiomyocytes are specifically found in mammals and might be related to the lack of

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regenerative capacity of the mammalian adult heart [18-20].

3. Modulation of myogenetic pathways by microRNAs Recent progress in the genomic studies of numerous animal

species has revealed important roles of miRs in both skeletal and cardiac muscle development. miRs are encoded in the genome as longer precursor molecules (500-3,000 nucleotides) that consist of palindromic

repetitive

sequences.

miR

precursor

genes

are

transcribed by RNA polymerase II bound to the promoter sequence at the 5’ region, in a manner similar to that for protein-coding genes . The primary transcripts of miRs (pri-miRs) are processed by the RNAse III Drosha (and DGCR8/Pasha) to form 70-110 nucleotide precursor-miRs (pre-miRs), an event occurring within the nucleus [21, 22]. Pre-miRs adopt a secondary structure consisting of a double-stranded stem and a single-stranded loop region (Fig.2A). 6 Page 6 of 26

Pre-miRs are then transported to the cytoplasm, and further cleaved by another RNAseIII named Dicer to release double-stranded miRs as short as 22 bp [23]. One strand of the mature miRs is preferentially incorporated into the RNA-induced silencing complex (RISC), which is

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the ribonucleoprotein complex that also takes up the target mRNA [24, 25]. Within the RISC, miRs bind to the 3’ untranslated regions

(UTRs) of the target mRNAs, and either inhibit the translation of The

importance

of

miRs

in

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mRNAs or lead to their degradation (Fig. 2A).

myogenesis

was

first

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demonstrated by Dicer mutant mice in which Dicer was inactivated specifically in skeletal muscles [26]. Muscle-specific miRs function in

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a complicated way that involves interaction with other regulatory proteins. Both in vitro and in vivo analyses have gradually revealed target mRNAs for these miRs, as summarized in Table 1. Among the

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well-studied miRs involved in myogenic differentiation are miR-1, miR-206 and miR-133, all of which are expressed specifically in

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skeletal and/or cardiac muscles. miR-206 is a derivative of miR-1; these two miRs retain high sequence similarity, including a shared

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seed sequence (6-8 nucleotides at the 5’ end) that is critical for target specificity, and therefore regulate overlapping sets of target mRNAs (reviewed in [27, 28]). The function of miR-1/206 and miR133 in muscle differentiation is conserved between invertebrates and vertebrates [29].

The function of these three miRs in skeletal myogenesis has

been analyzed using C2C12 myoblast cells [30]. When placed in differentiation

medium,

C2C12

cells

rapidly

differentiate

into

multinucleated myoblasts that express miR-1/206 and miR-133 as well

as

various

muscle-specific

protein

genes

[30,

31].

By

overexpression and knockdown experiments, it has been revealed that miR-1/206 enhances myoblast differentiation and that miR-133 maintains myoblast proliferation [30, 32]. Identified targets of these 3

miRs

include

histone

deacetylase

4

(HDAC4),

which

is

a

transcriptional repressor of muscle gene expression, and serum 7 Page 7 of 26

response factor (SRF), which represses the proliferative status of the myoblast [30, 33]. HDAC4 is known to repress the myogenic transcription factor MEF2; thus silencing of HDAC4 by miR-1 indirectly transforms the cells to a more differentiated state. miR-1/206 and

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miR-133 also bind to and downregulate mRNAs of polypyrimidine tract binding protein (PTB) and its paralog nPTB, the important alternative splicing regulators in neuron- and muscle-specific splicing

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events [34, 35]. These proteins are expressed at low levels in C2C12

cells. The silencing by miR-1/206 and miR-133 can negatively

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modulate the activity of PTB and nPTB, thereby leading to muscle differentiation.

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Another miR reported to promote muscle differentiation is miR-27, which is expressed in various tissues in early mouse embryos [36]. In the somites, miR-27 is expressed in an alternate manner

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with Pax3; miR-27 is accumulated in sclerotomes and myotomes, whereas Pax3 is expressed in the dermomyotomes. Antagonistic

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disruption of the miR-27 function in somite explants and in the adult satellite cells has revealed that Pax3 is the direct target of miR-27

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[36]. These findings led to the conclusion that miR-27 downregulates Pax3, which would result in a rapid shift to differentiated myoblasts. Compared to skeletal myogenesis, cardiac myogenesis utilizes

a different but overlapping set of miRs. miR-208 is a myocardiumspecific miR and plays a major role in cardiac conduction and hypertrophic growth [37]. miR-1 and miR-133 regulate differentiation of embryonic stem cells into cardiomyocytes [38]. These two miRs, when combined with miR-208 and miR-499, have the capacity to generate cardiomyocytes when expressed in the cardiac fibroblasts [39] (Fig. 1). In the developing mammalian hearts, miR-1 and miR133 are abundantly expressed. One of the targets of miR-1 has been identified

as

Hand2,

a

transcription

factor

that

promotes

cardiomyocyte expansion, based on an analysis using a reporter assay [40]. This result was confirmed in vivo by the down-regulation of Hand2 protein after overexpression of miR-1 [40]. 8 Page 8 of 26

In the neonatal development of mammalian heart, miR-590 and miR-199a have been identified as miRs that can promote cardiomyocyte proliferation [41]. These miRs induce cell cycle reentry of adult cardiomyocytes when overexpressed in vivo. Thus, the

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exogenous administration of these miRs is expected to be a potential treatment for cardiac injuries and pathologies. It is noteworthy that, unlike those in other tissues, the miR species abundantly expressed

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in the heart are very different between vertebrate taxa such as mammals vs. teleosts [42]. This might be correlated with the

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observed differences in cellular characteristics of cardiomyocytes such

4.

Duplication

of

bicistronic

miR-1/miR-133

clusters

in

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vertebrates

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as regenerative capacities [17, 18].

miR-1, miR-206 and miR-133 have been analyzed in multiple

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developmental models including mammals, birds, and teleosts [27, 43-46]. In the mammalian genomes, either one miR-1 or miR-206

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gene and one miR-133 gene are encoded on the same chromosomes, forming three miR-1(206)/miR-133 tandem gene clusters (Fig. 2B) [47].

This

cluster

organization

is

highly

conserved

among

vertebrates, although some animals such as chicks and zebrafish have one additional cluster [43, 48]. In many species, some of the clusters are transcribed as a continuous primary transcript followed by cleavage into two separate mature miRs [49, 50] (Fig. 2A). The transcription of this bicistronic precursor RNA occurs specifically in skeletal and/or heart muscles, and is upregulated by representative myogenic transcription factors, such as MRFs and MEF2, which bind to the upstream sequence of the miR-1(206)/miR-133 cluster [49]. Recent progress in genome-wide studies has allowed the comparison

of

untranslated

sequences

between

different

taxa.

Comparison of miR-1(206)/miR-133 clusters has revealed that the prototypic bicistronic cluster was established as a single compact 9 Page 9 of 26

cluster in the common ancestor of chordates (Fig. 2B)[45, 51]. Extant non-vertebrate chordates, i.e., cephalochordates and urochordates, possess a single miR-1/miR-133 gene cluster [6, 51, 52]. The urochordate Ciona intestinalis miR-1/miR-133 cluster (cin-miR-1/miR-

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133) is as short as 350 bp, with the distance between the two miRcoding regions being only ~130 bp [51]. This is far more compact than the corresponding clusters in other deuterostome species; the

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corresponding cluster length is 100 kb in sea urchins and 2.6 kb in mice [6, 53]. cin-miR-1/miR-133 is transcribed specifically in the

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embryonic cell lineage of tail muscle [51, 52]. Like its mammalian counterpart, cin-miR-1/miR-133 primary precursor is transcribed as a

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single continuous RNA, controlled by a cis-regulatory region that promotes muscle-specific expression [51]. This region contains multiple binding motifs for the MyoD family of MRFs, suggesting a

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direct regulation by MRFs – a feature also conserved in the mammalian miR-1/miR-133 cluster [49, 51]. Ciona

miR-1/miR-133

gene,

including

associated

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The

transcriptional regulatory sequences, is encoded in the opposite

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strand of the 2 kb intron of the mind bomb (mib) gene (Fig. 2B)[51]. This relative location of the miR-1/miR-133 and mib genes is conserved

in

a

wide

variety

of

chordates,

including

the

cephalochordates, lampreys, sharks, teleosts and mammals – in these animals, the miR-1/miR-133 cluster is embedded in the same (homologous) location with respect to the amino acid sequence of mib [6, 45](Fig. 2B). This observation supports the idea that the regulatory machinery for the muscle-specific synthesis of the paired miR-1/miR-133 molecules was established before the divergence of chordates, and has been maintained in urochordates and the vertebrate lineages. Recently, miR-1(206)/133 clusters have been identified in the genomic sequences of vertebrate species diverged early in evolution, such as the cyclostome lampreys and the chondrichthyans [45, 48]. Fig. 2B summarizes the deduced evolutionary pathway which is partly 10 Page 10 of 26

based on the observation from the Japanese lamprey Lethenteron japonicum

(http://jlampreygenome.imcb.a-star.edu.sg.)

[54].

The

lamprey and the chondrichthyans possess three independent scaffolds containing miR-1 and miR-133 [45, 48]. No miR-206 gene has been Collectively,

these

findings

allow

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to

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found either in the lamprey or the shark genomes. propose

the

evolutionary pathway of vertebrate miR-1/206 and miR-133 genes

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(Fig. 2B). A prototypic miR-1/miR-133 gene cluster, embedded in the mib intron, was established in the ancestor of chordates. In the

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common ancestor of vertebrates, this cluster was triplicated (Fig. 2B). This triple cluster organization has persisted throughout vertebrate

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evolution, in which one of them was retained in the mib intron, as is also the case in the cyclostome and chondrichthyan lineages (to be published elsewhere). In the lineage of osteichthyes, one of the

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clusters is converted into the miR-206/miR-133 cluster. This cluster is associated with an upstream regulatory sequence for skeletal muscle-

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specific transcriptional regulation, resulting in robust expression of miR-206 in the skeletal muscle but not in the cardiac muscle. Thus,

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the emergence of miR-206 seems to be paralleled with the evolution of jawed vertebrates, and its function in the skeletal myogenetic program has been maintained in osteichthyan lineages. 5. The function of microRNAs in subsets of skeletal muscles – Is there correlation with the morphological changes? A series of recent evidences imply that the acquisition of miR-206 might have contributed to morphological elaboration of the skeletal musculature of vertebrates, as represented by the muscles in the paired

appendages

[45].

In

both

amniotes

and

teleosts,

the

appendicular muscles differentiate much later than the trunk (body wall) skeletal muscles. Precursors for limb/fin muscles are derived from somites in the proximity of limb/fin buds of the early embryo [55, 56]. These muscle precursors undergo delamination from the 11 Page 11 of 26

epithelial somites, and migrate toward the buds. When the myoblasts arrive inside the mesenchyme of the bud, they start to express MRF and differentiate. One of the major regulatory players in this process is the paired-type transcription factor Pax3. At the onset of myoblast

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delamination, Pax3 expression is restricted to the ventral edges of the demomyotome and later to the migratory muscle precursors which

develop into various musculature components, including the fin/limb

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muscles. Pax3 is indispensable for the formation of skeletal muscles

in the paired appendages; Pax3 mutant Splotch (Sp) mice lack shoulder-associated

muscles,

tongue

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certain subsets of the skeletal muscles, such as the limb muscles, muscles

and

diaphragm,

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whereas the other muscles are formed normally [57]. In the migratory muscle precursors, Pax3 inhibits myogenic differentiation and maintains the proliferative status of myoblasts.

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Among the miRs generated from the precursor gene clusters described above, miR-206 is distinctive in that its expression is robust expression

is

directly

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and specific to skeletal muscles during embryogenesis. miR-206 regulated

by

MRFs,

suggesting

a

tight

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correlation with the muscle differentiation status [58]. In addition to the difference in skeletal/cardiac specificity, miR-206 and miR-1 exhibit differences in expression within the skeletal musculature. In chicks, miR-206 is expressed in the developing fore- and hindlimb musculature as well as in somites, whereas miR-1 is undetectable in the limb musculatures [59, 60]. A similar expression pattern is observed in the teleost embryos: in the medaka, miR-206, but not miR-1, is expressed in the developing muscles in the pectoral fin, an evolutionary counterpart of the tetrapod forelimbs [45]. Recent findings suggest the functional involvement of miR206 in this limb/fin-specific myogenetic program. Studies in chicks and mice revealed that miR-206 binds to and downregulates Pax3 mRNA [59, 61]. Pax3 3’UTR contains two conserved binding sites for miR-1/miR-206 [61]. Binding of miR-206 silences Pax3 and leads to myoblast differentiation. Both miR-1 and miR-206 are regulated by 12 Page 12 of 26

MyoD when they are transcribed as primary precursors. This fact suggests that MyoD downregulates Pax3 by activating synthesis of miR-1 and miR-206. Thus, miR-206 is involved the switching from proliferation to differentiation status of migratory muscle precursors

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that give rise to the fin/limb muscles. Moreover, the downregulation of Pax3 by miR-206 is adjusted

to varying degrees in the different muscle tissues. Studies by Boutet

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et al. (2012) demonstrated that the activity of miR-206 on Pax3

mRNA differs among myogenic precursor populations, because of the Pax3

3’UTR

resulting from alternative

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different length of the

polyadenylation (APA; Fig. 3B) [62]. The 3’UTR sequence of Pax3 different

lengths

of

3’UTR.

The

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contains multiple polyadenylation signals (PAS) which generate longer

form

of

Pax3

3’UTR,

polyadenylated at the downstream PAS (PAS2 in Fig. 3B), contains

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both the two binding sites for miR-206. On the other hand, the shorter form of Pax3 polyadenylated at PAS1 is devoid of miR-206

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binding site and thus resistant to the silencing by miR-206 (Fig. 3B). Quantitative analysis of Pax3 mRNA isoforms has revealed that, in the

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stem cells taken from the adult limb muscles, the Pax3 mRNA with long 3’UTR was more abundantly expressed than that with short 3’UTR. However, in the muscle stem cells from diaphragm, short 3’UTR was predominant, which was also the case for the mRNA extracted from the embryonic limb bud [62]. Collectively, these findings indicate that the skeletal muscle precursors found in different muscle tissues contain Pax3 mRNAs with different susceptibility to the silencing by miR-206. This insight is consistent with the fact that protein level of Pax3 declines only gradually after the onset of robust expression of miR-206 during mouse development, implying that the miRs have relatively mild silencing effects ([62]; also mentioned in [63]). Collectively, these insights show that miR-206 is involved in the modulation of myogenetic pathways in distinct ways in different skeletal muscles. Emergence of miR-206 during vertebrate evolution 13 Page 13 of 26

seems to correlate with the morphological elaboration of skeletal muscles – the absence of the miR-206 gene in the lampreys and sharks, as well as in invertebrates, implies that miR-206 is a novel miR associated with the complex morphology and function of skeletal

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muscles.

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6. Perspectives

As described above, miRs are emerging as modifiers of

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myogenetic pathways in a broad range of vertebrate taxa. During the myogenesis, a specific set of miRs is synthesized in muscle cells, a

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process regulated by the muscle-specific transcription factors that bind to the upstream regions of the miR genes. Among these, miR1/206 and miR-133 are expressed specifically in muscle cells,

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throughout the myoblast determination and differentiation processes. By silencing mRNAs of multiple developmental genes, these miRs

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contribute to the fine-tuning of muscle differentiation in different body parts, rather than functioning as an on/off switch for the muscle

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fate of the cells. Accordingly, the phylogenetic analyses have shown that these miRs emerged in parallel with the evolution of the complex musculature of vertebrates.

There have been numerous evidences suggesting that miR-

1/206 and miR-133 are involved in the diseased state related to myogenic regulation. For example, miR-206 in mdx mice, which is deficient in dystrophin, is expressed in an excess amount in the diaphragm but is only moderately upregulated in the hindlimb muscles [64]. This fact might reflect the pathological mechanisms underlying symptoms in the different skeletal muscle tissues in vivo. Future comparisons of miRs expressed in diseased muscles will lead to further understanding of disease mechanisms that have not been fully explained by a mutation in a single protein-coding gene. Most of the other known miRs that are expressed in muscles are also expressed in non-muscle cells, suggesting that these miRs 14 Page 14 of 26

might function in the fine-tuning of muscle vs. non-muscle cell characteristics in various tissues. In animal development, muscle tissues form in tight connection with surrounding non-muscle tissues, such as cartilage, bones, tendons and motor neurons. Future

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investigation of the miR functions extended over these tissues will provide key insights into the developmental mechanisms of the

mesodermal organs, which exhibit an enormous degree of complexity

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and morpholgical variety in vertebrates.

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Acknowledgments

We express our gratitude to the members of Inoue Laboratory and

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Sakamoto Laboratory at Kobe University for the valuable discussions, and to Drs. Shigehiro Kuraku and Shigeru Kuratani at RIKEN for their

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constant encouragement and discussions. This work was supported by Grants-in-Aid for Scientific Research from JSPS to R.K. (21770257

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and 25440108) and K.I. (20310115), and in part by a Shiseido Female Researcher Science Grant (R.K.), the research grant of Astellas Foundation for Research on Metabolic Disorders (R.K.) and the Asahi Glass Foundation (K.I.). REFERENCES

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FIGURE LEGENDS

Fig. 1. Regulation of skeletal and cardiac myogenesis.

A flowchart

M

showing the stepwise progression of muscle cell development starting with multipotent mesodermal progenitors. MiRs mentioned in the text

d

are indicated by red letters.

Ac ce pt e

Fig. 2. Structure and duplication of miR-1/miR-133 bicistronic gene cluster. (A) The schematic structure of miR-1 and miR-133 loci on the chordate genomes. miR-1 and miR-133 are tandemly located on the genome, on the opposite strand of one of the mind bomb (mib) intron.

This

locus

is

transcribed

into

pri-miR-1/miR-133

RNA

containing both microRNA. Then the precursor is processed into two separate microRNAs by the activity of Drosha and Dicer enzymes. Processed and single-stranded microRNA can bind to the 3’UTR of the target mRNA. (B) A comparison of the miR-1(206)/miR-133 gene cluster

organization

and

the

deduced

evolutionary

history

in

vertebrates plus urochordates. Gray arrows indicate the orientation of mib intron in 5’ to 3’ direction. Approximate times of evolutionary events are shown by ‘mya’ (million years ago) [65][66][67]. Fig. 3. Differential gene regulation achieved by miR-1/206 and miR22 Page 22 of 26

133 in vertebrate muscles. (A) Skeletal muscles in the trunk (yellow) express all miR-1, miR-133 and miR-206, whereas cardiac muscle (red) lacks miR-206 expression. Skeletal muscles in the limbs (pink)express miR-206 but not miR-1. (B) miR-206 is expressed

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abundantly in the limb and diaphragm muscles (which undergo migratory mode of development, require Pax3). Furthermore, Pax3

receives APA in different skeletal muscle. In diagphragm, Pax3 mRNA

cr

is polyadenylated at PAS1 and thus has no binding site for miR-206.

In the limbs, Pax3 mRNA is polyadenylated at downstream PAS2 and

us

thus is bound by miR-206.

Target genes

miR-1

HDAC4 nPTB/PTB Hand2

M

microRNA

an

Table 1 MicroRNA functions in muscle cells

Pax3

Ac ce pt e

miR-27

d

Delta-like 1 (Dll1)

miR-133

SRF

Function Muscle differentiation Muscle muscle-specific splicing events

Balance between cardiac differentiatio Promoting muscle lineages

Muscle differentiation in somitic muscl Myoblast proliferation Blocking muscle differentiation

nPTB/PTB

Muscle muscle-specific splicing events

miR-199a and miR-590

Homer1, Hopx, Clic5

Cardiomyocyte proliferation

miR-206

HDAC4

Muscle differentiation

DNA polymerase alpha 1 (Pola 1)

Muscle differentiation

nPTB/PTB

Muscle-specific splicing events

Pax3

Regulation of apoptosis

Thrap1, myostatin

Cardiac hypertrophy

miR-208

23 Page 23 of 26

Fig.  1

ed

Myoblast  

miR-­‐1   miR-­‐206   Pax3   miR-­‐133   Pax7   miR-­‐27   SRF  

Differen*a*on  

cr

HDAC4  

M

Ac*ve   prolifera*on  

an

MyoD   (MRF)   SRF  

us

Primordial   cardiovascular   precursor  

Skeletal  muscle   precursor   Determina*on  

ip t

Mul*potent   mesodermal   progenitors  

Non-­‐muscle   mesodermal   precursors  

Nkx2.5   Gata4   SRF  

Heart  field  progenitors    (FHF  and  SHF)   miR-­‐1   miR-­‐133   miR-­‐208   miR-­‐499  

Hand2  

Ac

Matura*on  

Contrac*le  proteins   •  Muscle  ac*n   •  Myosin  heavy  &  light  chains   •  Muscle  crea*ne  kinase  

ce

Fused  myoblast  

pt

Reinforce  terminal   differen*a*on   cTnT   Cardiomyocyte   miR-­‐590   miR-­‐199a  

Myotube  

Binucleated   cardiomyocyte  

Cardiomyocyte   prolifera*on  

Page 24 of 26

Fig.  2

A

mind  bomb  intron

Conserved  miR-­‐1/ miR-­‐133  cluster  

miR-­‐133  

ip t

miR-­‐1  

Nucleus

Primary  precursor  RNA   of  miR-­‐1/miR-­‐133  

cr

AAAAAA

us

Diges*on  by  Drosha   pre-­‐miR-­‐1  and  pre-­‐miR-­‐133   Nuclear   transport

an

Loop  clipped  by  Dicer   Incorpora*on  into  miRISC  

miR-­‐1  

miR-­‐133  

Cyclostomes   (lamprey)  

Ac

Urochordate   (ascidian)  

ce

miR-­‐1  

AAAAAA

miR-­‐133  

miR-­‐1  

miR-­‐133  

miR-­‐133  

miR-­‐1  

miR-­‐133  

miR-­‐133  

miR-­‐1  

miR-­‐133  

pt

miR-­‐1  

miR-­‐1  

ed

B

M

Silencing  of  target   mRNA  

Cytoplasm

Chondrichthyes   (chimaera)  

Teleosts   (Medaka)  

Mammals   (human,  mouse)  

-­‐  Divergence  of   Osteichtyes  (~400mya)   -­‐  Emergence  of  jawed   vertebrates(~430  mya)   -­‐  miR-­‐206  emerged   -­‐  Conserved  3  clusters   Emergence  of  the   common  ancestor  of   chordates  (~550  mya)  

miR  clusters   duplicated  into  3   Page 25 of 26

A

Skeletal  muscles  in  the  trunk   miR-­‐1                (+)   miR-­‐133        (+)   miR-­‐206        (+)  

cr

Skeletal  muscles  in  the  limbs   miR-­‐1                (-­‐)   miR-­‐133        (+)   miR-­‐206        (+)  

an M PAS1 AAAAAA PAS1

Ac

ce

Long  3’UTR  

ed

Short  3’UTR  

pt

Pax3  mRNA  

us

Cardiac  muscle   miR-­‐1                (+)   miR-­‐133        (+)   miR-­‐206        (-­‐)  

B

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Fig.  3

PAS2 AAAAAA

Transla*on   Degrada*on  

miR-­‐206  

Alterna*ve  polyadenyla*on  (APA)  of  Pax3   Short  3’UTR  –  abundant  in  diaphragm   Long  3’UTR  –  abundant  in  limb  muscles  

Page 26 of 26