BACKGROUND
recent genome-wide transcriptome studies discovered that the mammalian genome is pervasively transcribed into rna.
within the protein-coding gene regions, there are many alternative transcriptional start sites, which results in the production of not only protein-coding mrna but also non-protein-coding rnas   <cit> .
intriguingly, the recent identification and characterization of various mrnas demonstrated that there are several bifunctional mrnas.
the bifunctional rna transcripts have both protein-coding capacity and activity as functional ncrna in a context-dependent manner  <cit> .
for example, steroid receptor activator rna  acts as bifunctional transcript.
non-coding sra stimulates transcriptional activity of myod and enhances muscle cell differentiation, whereas sra protein binds to and counteracts sra ncrna function  <cit> .
p <dig> mrna interacts with mdm <dig> protein and prevents mdm <dig> function  <cit> .
the untranslated region  of several mrna transcripts can also function as a trans-acting regulatory rna that is independent of the protein-coding functions.
several mrna 3′utrs act as competing endogenous rnas  or natural microrna sponges.
examples include the 3′utr of pten mrna  <cit> , the 3′utr of zeb <dig> mrna  <cit>  and the 3′utr of hmga <dig> mrna  <cit> .
the 5′utr of mrna also acts as regulatory rna, as we have previously reported that the 5′utr of vegf mrna promotes tumor malignancy by silencing of stat <dig> mrna expression  <cit> .
skeletal muscle differentiation is a powerful system for investigating functional rnas, because it can be recapitulated in vitro and because the myogenic differentiation program is well characterized and evolutionarily conserved  <cit> .
there is increasing evidence to support that several ncrnas, such as linc-md <dig>  sine-containing ncrnas, and the 3′utr of dmpk  mrna regulate myogenesis through modulation of myogenic gene expression, such as pax <dig>  myod, myf <dig>  and mirna- <dig> and − <dig>  <cit> .
skeletal muscle differentiation occurs by a stepwise progression.
the expression of the retinoblastoma protein  is critically important to this process  <cit> .
the role of rb is multifaceted and includes the orchestration of cell cycle arrest and prevention of cell cycle reentry.
furthermore, rb also enforces a stable muscle-specific gene expression.
thus, the silencing of rb expression by sirna or gene knockout abrogates myoblast differentiation  <cit> .
in this study, we focused on insulin receptor substrate- <dig>  gene products because irs- <dig> plays critical roles in myogenic growth and differentiation as previously demonstrated by our group and others  <cit> .
we found that one of the transcriptional variants of irs- <dig> mrna was highly expressed in skeletal muscle relative to liver and adipose tissue.
we demonstrate that the irs- <dig> transcript is a bifunctional mrna.
the 5′utr of the irs- <dig> transcript repressed rb mrna expression and suppressed muscle cell differentiation.
our results suggest that the irs- <dig> transcript is a regulator of myogenic differentiation.
RESULTS
characterization of mouse irs- <dig> transcriptional variants
mouse irs- <dig> gene consists of two exons and one intron.
the exon  <dig> encodes 5′utr, irs- <dig> protein sequence and partial 3'utr, while the exon  <dig> encodes only 3′utr .
a bioinformatics analysis of the mouse irs- <dig> gene region using the ucsc genome database  <cit>  revealed  <dig> different transcriptional variants in the irs- <dig> gene region, including irs- <dig> protein-coding mrnas and non-protein coding transcripts.
each of the transcripts is derived from a distinct transcription start site and has a transcriptional termination site.
the database includes tissue- and cell type-specific expression of irs-1-related transcripts .
for instance, heart and liver express irs- <dig> mrna , while brain poorly expresses it.
fibrocytes and fibroblasts express several irs- <dig> transcripts, whereas mel cells, erythroblasts and megakaryocytes do not express any irs- <dig> transcripts.
notably, exon 2-derived transcripts  that do not encode irs- <dig> protein are highly expressed in several tissues  and cells .
fibroblasts clearly express intron 1-derived non-coding transcript  .figure  <dig>
characterization of two distinct
irs-1
transcripts.
schematic representation of two transcriptional variants of mouse irs- <dig> mrnas .
the positions of specific qpcr primer sets are shown as arrows .
expression levels of s5′-irs- <dig> mrna in the indicated mouse tissues, proliferating c2c <dig> myoblasts  and differentiating c2c <dig> myotubes .
mean ± sd, n = 4- <dig>   expression levels of fl-irs- <dig> mrna in the indicated mouse tissues, c2c <dig> myoblasts  and c2c <dig> myotubes .
mean ± sd, n = 4- <dig>   specific knockdown of fl-irs- <dig> mrna  or s5′-irs- <dig> mrna  in myoblasts  and myotubes .
mean ± sd, n =  <dig>  *p <  <dig>  versus no treatment .
effect of specific knockdown of fl-irs- <dig> mrna or s5′ irs- <dig> mrna on irs- <dig> protein expression during myogenesis.
quantification analysis and statistic were performed on three independent experiments.
mean ± sd, *p <  <dig>  versus each culture conditions  in no transfection .
n.s., not significant.
we tried to measure the expression profile of these unique transcripts in several mouse tissues and c2c <dig> muscle cell lines by quantitative rt-pcr  using specific primer sets for each transcript .
the intron 1-derived transcript  was ubiquitously expressed .
however, we could not determine the levels of exon 2-derived transcripts , since they were not amplified by qpcr using our primer set.
characterization of two distinct irs- <dig> mrnas expression
we are interested in whether the 5′utr of mrna acts as functional rna similar to results we previously demonstrated  <cit> .
therefore, we focused on two irs- <dig> mrna variants that have different 5′utrs because of different transcription start sites .
as shown in figure 1a, one is transcribed from a +  <dig> nucleotide  transcription start site in exon  <dig>  thus it contains full-length 5′utr sequence .
the other variant is transcribed from a + 534 nt transcription start site in exon  <dig> and results in a short 5′utr sequence .
both fl- and s5′-irs- <dig> mrnas contain the same protein coding sequence  encoding exon  <dig> and should produce the same irs- <dig> protein .
we then examined the expression profile of the two irs- <dig> transcripts in several mouse tissues by qrt-pcr using a specific primer set for each transcript .
s5′-irs- <dig> mrna was ubiquitously expressed in multiple tissues, especially in adipose tissue, liver and skeletal muscle that are well known tissues highly expressing irs- <dig> protein  <cit>  .
interestingly, fl-irs- <dig> mrna was highly expressed only in skeletal muscle , which suggests that fl-irs- <dig> mrna may have a skeletal muscle-specific function.
to determine the expression profile of the two irs- <dig> mrna transcripts during myogenesis, we used mouse myoblast cell line c2c <dig> that is a well characterized model system for studying muscle differentiation in vitro.
c2c <dig> cells proliferate as myoblasts in growth medium , while confluent c2c <dig> myoblasts exit the cell cycle and fuse with one another to form multinucleated muscle cells  in differentiation medium .
the expression levels of s5′-irs- <dig> mrna were low in myoblasts and increased by  <dig> -fold in myotubes .
in contrast, fl- irs- <dig> mrna levels were relatively high in myoblasts, but decreased in differentiated myotubes .
when comparing the levels of s5′- and fl-irs- <dig> mrnas between skeletal muscle tissue and c2c <dig> myotubes, expression levels of both mrnas were similar .
in c2c <dig> myoblasts, s5′-irs- <dig> mrna levels were lower than skeletal muscle tissue, while fl-irs- <dig> mrna levels were higher .
to determine an expression relationship between the two irs- <dig> mrnas and irs- <dig> protein in c2c <dig> cell myogenic process, fl-irs- <dig> mrna or s5′-irs- <dig> mrna was specifically knocked down by targeting their 3′utr sequence encoded in exon  <dig>  or in intron  <dig>  .
the si-s5′-irs- <dig> reduced s5′-irs- <dig> mrna levels to approximately 30% of control cells without affecting fl-irs- <dig> mrna levels .
the sh-fl-irs- <dig> efficiently decreased fl- irs- <dig> mrna levels to approximately 20% compared with the sh-control and did not alter s5′-irs- <dig> mrna levels .
in untransfected  or control transfected cells , the irs- <dig> protein levels were increased during differentiation from myoblasts to myotubes .
when fl-irs- <dig> mrna was knocked down, the irs- <dig> protein levels decreased in differentiating myotubes but not in proliferating myoblasts .
in contrast, knockdown of s5′-irs- <dig> mrna clearly reduced irs- <dig> protein levels in both myoblasts and myotubes .
these results indicate that the s5′-irs- <dig> transcript participates in continuous irs- <dig> protein production during myogenesis and that fl-irs- <dig> mrna is involved in irs- <dig> protein synthesis only in differentiating myotubes.
fl-irs- <dig> transcript regulates myoblast differentiation
in myoblasts, the fl-irs- <dig> transcript was highly expressed and was not involved in irs- <dig> protein production.
therefore, we hypothesized that fl-irs- <dig> mrna functions as an ncrna with activity independent of its protein-coding function.
to address this hypothesis, we examined the effect of knockdown of fl-irs- <dig> transcript on the c2c <dig> cell phenotype.
the knockdown of fl- irs- <dig> mrna markedly enhanced differentiation into myotubes .
the results of fusion index show that the number of differentiated myotubes was increased more than 3-fold by knockdown of fl- irs- <dig> mrna compared with untransfected and sh-control-transfected cells .
the mean diameter of myotubes was also increased by approximately 130% after knockdown of fl-irs- <dig> mrna .
the expression of myosin heavy chain, which is a myogenic differentiation marker, was also enhanced by knockdown of fl-irs- <dig> mrna .
in contrast, knockdown of s5′-irs- <dig> mrna did not affect c2c <dig> cell differentiation .
these data indicate that accelerated differentiation by knockdown of fl-irs- <dig> transcript was not caused by a reduction of irs- <dig> protein, but instead, by a blockade of fl-irs- <dig> transcript function.figure  <dig>
fl-
irs-1
transcript regulates c2c <dig> differentiation.
effect of knockdown of fl-irs- <dig> mrna on differentiation of c2c <dig> myoblast.
fusion index  and a mean diameter of myotubes  are shown.
mean ± sd, n =  <dig>  *p <  <dig>  versus no treatment .
effect of knockdown of fl- irs- <dig> mrna on expression levels of myosin heavy chain in differentiating c2c <dig> cells .
it is well known that rb plays a critical role in inducing myogenic differentiation, and the introduction of rb in myoblasts accelerates differentiation to myotubes  <cit> .
conversely, inhibition of rb expression in myoblasts abrogates differentiation into myotubes  <cit> .
thus, we hypothesized that fl-irs- <dig> transcript may affect rb expression.
fl-irs- <dig> 5′utr reduces rb mrna expression in c2c <dig> cells
we examined the expression levels of rb mrna in myoblasts and myotubes.
myoblasts expressed low levels of rb mrna and myotubes expressed higher levels .
the rb mrna expression profile had an inverse relationship with fl-irs- <dig> transcript levels .figure  <dig>
fl-
irs-1
transcript reduces rb mrna levels.
expression levels of rb mrna in c2c <dig> myoblasts  and myotubes .
partial sequence complementarity between the 5′utr of fl-irs- <dig> transcript  and rb mrna .
schematic representation of position of complementary element in the 5′utr of fl-irs- <dig> transcript.
a position of an lna-based antisense oligonucleotide  is shown.
effect of fl-irs- <dig> 5′utr on rb mrna expression levels.
c2c <dig> myoblasts were untransfected  or transfected with an rb mrna-expressing plasmid in combination with the indicated effector plasmid .
rb mrna levels were measured by qrt-pcr.
mean ± sd, n = 4- <dig>  *p <  <dig>  versus control transfectant .
effect of fl-5′-as-odn on rb mrna reduction by fl-irs- <dig> 5′utr.
c2c <dig> myoblasts were co-transfected with plasmids expressing rb mrna and the fl-irs- <dig> 5′utr rna in combination with the indicated as-odn.
mean ± sd, n = 4- <dig>  *p <  <dig>  versus control transfectant .
effect of knockdown of endogenous fl-irs- <dig> transcript on endogenous rb mrna levels.
c2c <dig> myoblasts were transfected with the indicated shrna or sirna.
mean ± sd, n =  <dig>  *p <  <dig>  versus no treatment .
effect of supplementary expression of irs- <dig> protein on rb mrna levels under fl-irs- <dig> transcript knockdown conditions.
c2c <dig> myoblasts were transfected with the sh-fl-irs- <dig> plasmid in combination with a plasmid encoding irs- <dig> protein fused to v <dig> tag .
expression levels of exogenous irs- <dig> protein were determined by immunoblot analysis using anti-v <dig> antibody.
mean ± sd, n =  <dig>  *p <  <dig>  versus control transfectant .
a bioinformatics analysis showed that there was a partial sequence-complementary element between the 5′utr of fl-irs- <dig> transcript and rb mrna .
given that several functional rna transcripts can interact with target mrnas by intermolecular base-pairing via a partially complementary element  <cit> , fl-irs- <dig> 5′utr may target rb mrna via the complementary element.
to assess whether fl- irs- <dig> 5′utr participated in rb expression, c2c <dig> myoblasts were transfected with an rb mrna-expressing plasmid in combination with a plasmid expressing the fl-irs- <dig> 5′utr fused to the luciferase gene  or a control luciferase plasmid .
overexpression of the 5′utr, but not control plasmid, markedly reduced rb mrna levels .
to demonstrate that the complementary element in the 5′utr was responsible for the reduction of rb mrna, we deleted the complementary element .
the deletion of the element fully abrogated the ability to decrease rb mrna expression .
to exclude the possibility that the deletion may affect the secondary structure of fl-irs- <dig> 5′utr, resulting in a loss of silencing activity for rb mrna, we examined effect of an lna-based antisense oligonucleotide against the complementary element .
fl-5′-as-odn, but not control as-odn, completely prevented the decrease of rb mrna expression induced by fl-irs- <dig> 5′utr .
these results indicated that fl-irs- <dig> 5′utr decreased rb mrna levels via its complementary sequence.
to verify that endogenous fl-irs- <dig> transcript participated in endogenous rb mrna expression, we knocked down the fl-irs- <dig> transcript.
the silencing of fl-irs- <dig> mrna increased rb mrna levels by more than 2-fold .
in contrast, knockdown of s5′-irs- <dig> mrna did not affect the rb mrna levels.
these results suggest that the elevation of rb mrna levels was not caused by decreasing irs- <dig> protein after knockdown of fl-irs- <dig> transcript.
the supplementary expression of exogenous irs- <dig> protein using an irs-1-expressing plasmid that encodes only the open reading frame of irs- <dig> without the 5′ and 3′-utrs did not affect the elevation of rb mrna levels .
we additionally examined the effect of knockdown of fl-irs- <dig> transcript on other myogenic regulators expression, such as myod, myogenin, myf <dig>  mrf <dig> and mef <dig>  the silencing of fl-irs- <dig> mrna did not affect the levels of all myogenic factors tested .
fl-irs- <dig> 5′utr does not reduce rb mrna expression in hepatocytes
we further tested whether the fl-irs- <dig> 5′utr-induced rb mrna reduction occurred in hepatocytes because irs- <dig> is an important factor in hepatocytes.
a cultured hepatocyte cell line was co-transfected with plasmids expressing rb mrna and the fl-irs- <dig> 5′utr.
unlike skeletal muscle cells, the overexpression of the 5′utr did not reduce rb mrna levels in hepatocytes .
in addition, knockdown of fl-irs- <dig> transcript did not alter rb mrna levels in hepatocytes .figure  <dig>
fl-
irs-1
transcript does not reduce rb mrna expression in hepatocytes.
effect of fl-irs- <dig> 5′utr on rb mrna expression levels.
cultured hepatocyte cell lines  were untransfected  or transfected with an rb mrna-expressing plasmid in combination with the indicated effector plasmid.
rb mrna levels were measured by qrt-pcr.
mean ± sd, n =  <dig>  *p <  <dig>  versus control transfectant .
effect of knockdown of endogenous fl-irs- <dig> transcript on endogenous rb mrna levels.
cultured hepatocyte cell lines were transfected with the indicated shrna.
fl-irs-1
and rb mrna  levels were measured by qrt-pcr.
mean ± sd, n =  <dig>  *p <  <dig>  versus no treatment .
reduction of rb mrna by fl-irs- <dig> transcript did not require dicer and upf1
we then examined the molecular mechanism responsible for fl-irs- <dig> 5′utr reducing rb mrna levels.
there is evidence that several mirnas and ncrnas regulate myogenic differentiation through the dicer- and upf1-mediated target mrna decay system  <cit> .
the knockdown of dicer or upf <dig> by sirna  did not alter the suppression of rb mrna by fl-irs- <dig> 5′utr .
in addition, northern blot analysis using fl-5′-as-odn showed a small rna with approximately 20–25 nt in length or the 5′utr rna distinct from fl-irs- <dig> mrna was not detected in either myoblasts and myotubes .
these data suggest that fl-irs- <dig> transcript was not processed to mirna or intermediate 5′utr rna.
discussion
in this study, we found that fl-irs- <dig> transcript acts as a bifunctional rna.
one function is as a regulatory rna in proliferating myoblasts that reduced rb mrna expression and suppressed myoblast differentiation.
the other function is as mrna that produced irs- <dig> protein during differentiation into myotubes.
by contrast, s5′-irs- <dig> mrna was only involved in irs- <dig> protein synthesis during myogenesis.
rb plays a crucial role in the switch from proliferation to differentiation in myoblasts  <cit> .
myoblasts lacking rb lose the capacity to differentiate into myotubes  <cit> , and the overexpression of rb in myoblasts enhances differentiation into myotubes  <cit> .
it is well established that rb function is regulated by posttranslational modification  during myoblast differentiation.
although it is considered that the rb expression levels are tightly regulated in myoblasts before and during differentiation, how rb levels are regulated in myoblasts is still unclear.
in this study, the knockdown of endogenous fl-irs- <dig> transcript increased rb mrna levels and accelerated differentiation of myoblasts to myotubes.
thus, our findings suggest that fl-irs- <dig> transcript could be one of the endogenous downregulator of rb expression in myoblasts.
a functional relevance of between irs- <dig> protein and rb protein was previously reported  <cit> .
irs- <dig> inhibits transcriptional activity of rb  <cit> .
in addition, several reports indicate that the igf-1/irs- <dig> pathway suppresses rb-mediated cell cycle arrest by inducing prolonged phosphorylation of rb  <cit> .
however, at least to our best knowledge, our study is the first report showing a relationship between irs- <dig> transcript and rb mrna.
there are several ncrnas that regulate myogenesis through modulation of myogenic gene expression, such as pax <dig>  <cit> , myf <dig>  <cit> , myod  <cit> , and mirna- <dig> and − <dig>  <cit> .
several sine-containing ncrnas and mrnas interact with their target mrnas such as myf <dig> by base-pairing with partially complementary elements to induce degradation of them and regulate c2c <dig> cell myogenesis  <cit> .
in these cases, the silencing of target mrnas by the ncrnas requires base-pairing to target mrna and a dicer- and upf1-dependent mechanism  <cit> .
here, we demonstrated that the reduction of rb mrna by fl-irs- <dig> transcript did not require dicer and upf <dig>  however, the complementary element in the 5′utr of fl-irs- <dig> transcript was necessary to decrease rb mrna levels.
in addition, the reduction of rb mrna by the fl-irs- <dig> transcript was not observed in hepatocytes despite the important role of rb in hepatocyte differentiation  <cit> .
thus, skeletal muscle-specific factors may be required in rb mrna silencing.
further studies are required to clarify a molecular mechanism of fl-irs- <dig> transcript-mediated gene silencing.
until recently, it has been generally accepted that mrna functions only in protein production, and cell phenotypes depend exclusively on the function of these proteins.
unexpectedly, recent studies demonstrated that several mrnas may have bifunctional abilities in a context-dependent manner.
it is predicted that the human genome contains approximately  <dig> possible potentially new bifunctional mrnas  <cit> .
one characterized bifunctional mrna is sra during myogenic processes  <cit> .
in myogenic proliferation, the protein-coding sra transcript is abundantly expressed, and sra protein binds to and counteracts sra ncrna-mediated myod activation and suppresses differentiation  <cit> .
during myogenic differentiation, the expression of non-coding sra transcript is elevated and enhances transcriptional activity of myod and differentiation  <cit> .
these findings suggest that the effect of sra on myogenesis may result from the correct balance between non-coding and coding sra transcripts.
the present study showed that the balance between coding and non-coding irs- <dig> transcripts may be important for regulating myogenic differentiation.
beyond utrs function as cis regulatory elements that impact the translational activity and stability of their own mrnas, utrs are also trans modulator of gene expression through their target transcript inhibition as demonstrated here and by others  <cit> .
as recently suggested by the pandolfi group  <cit> , mrna transcripts can potentially perturb the interaction with target rnas and possess a biological activity independent of the translation of the protein they encode.
therefore, some of the phenotypic features of cells may depend not only on the protein function but also on the regulatory rna function that is distinct from their protein products.
CONCLUSIONS
the objective of this study was to identify a novel bifunctional rna in myogenic process.
we found that fl-irs- <dig> transcript regulates rb expression at the rna levels independent of its protein-coding function.
our findings suggest that fl-irs- <dig> transcript functions as a bifunctional rna during myogenic differentiation.

 recent genome-wide transcriptome studies discovered that the mammalian genome is pervasively transcribed into rna. within the protein-coding gene regions, there are many alternative transcriptional start sites, which results in the production of not only protein-coding mrna but also non-protein-coding rnas. intriguingly, the recent identification and characterization of various mrnas demonstrated that there are several bifunctional mrnas. the bifunctional rna transcripts have both protein-coding capacity and activity as functional ncrna in a context-dependent manner. for example, steroid receptor activator rna acts as bifunctional transcript. non-coding sra stimulates transcriptional activity of myod and enhances muscle cell differentiation, whereas sra protein binds to and counteracts sra ncrna function. p mrna interacts with mdm protein and prevents mdm function. the untranslated region of several mrna transcripts can also function as a trans-acting regulatory rna that is independent of the protein-coding functions. several mrna 3′utrs act as competing endogenous rnas or natural microrna sponges. examples include the 3′utr of pten mrna, the 3′utr of zeb mrna and the 3′utr of hmga mrna. the 5′utr of mrna also acts as regulatory rna, as we have previously reported that the 5′utr of vegf mrna promotes tumor malignancy by silencing of stat mrna expression. skeletal muscle differentiation is a powerful system for investigating functional rnas, because it can be recapitulated in vitro and because the myogenic differentiation program is well characterized and evolutionarily conserved. there is increasing evidence to support that several ncrnas, such as linc-md sine-containing ncrnas, and the 3′utr of dmpk mrna regulate myogenesis through modulation of myogenic gene expression, such as pax myod, myf and mirna- and −. skeletal muscle differentiation occurs by a stepwise progression. the expression of the retinoblastoma protein is critically important to this process. the role of rb is multifaceted and includes the orchestration of cell cycle arrest and prevention of cell cycle reentry. furthermore, rb also enforces a stable muscle-specific gene expression. thus, the silencing of rb expression by sirna or gene knockout abrogates myoblast differentiation. in this study, we focused on insulin receptor substrate- gene products because irs- plays critical roles in myogenic growth and differentiation as previously demonstrated by our group and others. we found that one of the transcriptional variants of irs- mrna was highly expressed in skeletal muscle relative to liver and adipose tissue. we demonstrate that the irs- transcript is a bifunctional mrna. the 5′utr of the irs- transcript repressed rb mrna expression and suppressed muscle cell differentiation. our results suggest that the irs- transcript is a regulator of myogenic differentiation.  characterization of mouse irs- transcriptional variants mouse irs- gene consists of two exons and one intron. the exon encodes 5′utr, irs- protein sequence and partial 3'utr, while the exon encodes only 3′utr. a bioinformatics analysis of the mouse irs- gene region using the ucsc genome database revealed different transcriptional variants in the irs- gene region, including irs- protein-coding mrnas and non-protein coding transcripts. each of the transcripts is derived from a distinct transcription start site and has a transcriptional termination site. the database includes tissue- and cell type-specific expression of irs-1-related transcripts. for instance, heart and liver express irs- mrna, while brain poorly expresses it. fibrocytes and fibroblasts express several irs- transcripts, whereas mel cells, erythroblasts and megakaryocytes do not express any irs- transcripts. notably, exon 2-derived transcripts that do not encode irs- protein are highly expressed in several tissues and cells. fibroblasts clearly express intron 1-derived non-coding transcript.figure characterization of two distinct irs-1 transcripts. schematic representation of two transcriptional variants of mouse irs- mrnas. the positions of specific qpcr primer sets are shown as arrows. expression levels of s5′-irs- mrna in the indicated mouse tissues, proliferating c2c myoblasts and differentiating c2c myotubes. mean ± sd, n = 4- expression levels of fl-irs- mrna in the indicated mouse tissues, c2c myoblasts and c2c myotubes. mean ± sd, n = 4- specific knockdown of fl-irs- mrna or s5′-irs- mrna in myoblasts and myotubes. mean ± sd, n =  *p <  versus no treatment. effect of specific knockdown of fl-irs- mrna or s5′ irs- mrna on irs- protein expression during myogenesis. quantification analysis and statistic were performed on three independent experiments. mean ± sd, *p <  versus each culture conditions in no transfection. n.s., not significant. we tried to measure the expression profile of these unique transcripts in several mouse tissues and c2c muscle cell lines by quantitative rt-pcr using specific primer sets for each transcript. the intron 1-derived transcript was ubiquitously expressed. however, we could not determine the levels of exon 2-derived transcripts, since they were not amplified by qpcr using our primer set. characterization of two distinct irs- mrnas expression we are interested in whether the 5′utr of mrna acts as functional rna similar to results we previously demonstrated. therefore, we focused on two irs- mrna variants that have different 5′utrs because of different transcription start sites. as shown in figure 1a, one is transcribed from a +  nucleotide transcription start site in exon thus it contains full-length 5′utr sequence. the other variant is transcribed from a + 534 nt transcription start site in exon and results in a short 5′utr sequence. both fl- and s5′-irs- mrnas contain the same protein coding sequence encoding exon and should produce the same irs- protein. we then examined the expression profile of the two irs- transcripts in several mouse tissues by qrt-pcr using a specific primer set for each transcript. s5′-irs- mrna was ubiquitously expressed in multiple tissues, especially in adipose tissue, liver and skeletal muscle that are well known tissues highly expressing irs- protein. interestingly, fl-irs- mrna was highly expressed only in skeletal muscle, which suggests that fl-irs- mrna may have a skeletal muscle-specific function. to determine the expression profile of the two irs- mrna transcripts during myogenesis, we used mouse myoblast cell line c2c that is a well characterized model system for studying muscle differentiation in vitro. c2c cells proliferate as myoblasts in growth medium, while confluent c2c myoblasts exit the cell cycle and fuse with one another to form multinucleated muscle cells in differentiation medium. the expression levels of s5′-irs- mrna were low in myoblasts and increased by -fold in myotubes. in contrast, fl- irs- mrna levels were relatively high in myoblasts, but decreased in differentiated myotubes. when comparing the levels of s5′- and fl-irs- mrnas between skeletal muscle tissue and c2c myotubes, expression levels of both mrnas were similar. in c2c myoblasts, s5′-irs- mrna levels were lower than skeletal muscle tissue, while fl-irs- mrna levels were higher. to determine an expression relationship between the two irs- mrnas and irs- protein in c2c cell myogenic process, fl-irs- mrna or s5′-irs- mrna was specifically knocked down by targeting their 3′utr sequence encoded in exon or in intron. the si-s5′-irs- reduced s5′-irs- mrna levels to approximately 30% of control cells without affecting fl-irs- mrna levels. the sh-fl-irs- efficiently decreased fl- irs- mrna levels to approximately 20% compared with the sh-control and did not alter s5′-irs- mrna levels. in untransfected or control transfected cells, the irs- protein levels were increased during differentiation from myoblasts to myotubes. when fl-irs- mrna was knocked down, the irs- protein levels decreased in differentiating myotubes but not in proliferating myoblasts. in contrast, knockdown of s5′-irs- mrna clearly reduced irs- protein levels in both myoblasts and myotubes. these results indicate that the s5′-irs- transcript participates in continuous irs- protein production during myogenesis and that fl-irs- mrna is involved in irs- protein synthesis only in differentiating myotubes. fl-irs- transcript regulates myoblast differentiation in myoblasts, the fl-irs- transcript was highly expressed and was not involved in irs- protein production. therefore, we hypothesized that fl-irs- mrna functions as an ncrna with activity independent of its protein-coding function. to address this hypothesis, we examined the effect of knockdown of fl-irs- transcript on the c2c cell phenotype. the knockdown of fl- irs- mrna markedly enhanced differentiation into myotubes. the results of fusion index show that the number of differentiated myotubes was increased more than 3-fold by knockdown of fl- irs- mrna compared with untransfected and sh-control-transfected cells. the mean diameter of myotubes was also increased by approximately 130% after knockdown of fl-irs- mrna. the expression of myosin heavy chain, which is a myogenic differentiation marker, was also enhanced by knockdown of fl-irs- mrna. in contrast, knockdown of s5′-irs- mrna did not affect c2c cell differentiation. these data indicate that accelerated differentiation by knockdown of fl-irs- transcript was not caused by a reduction of irs- protein, but instead, by a blockade of fl-irs- transcript function.figure fl- irs-1 transcript regulates c2c differentiation. effect of knockdown of fl-irs- mrna on differentiation of c2c myoblast. fusion index and a mean diameter of myotubes are shown. mean ± sd, n =  *p <  versus no treatment. effect of knockdown of fl- irs- mrna on expression levels of myosin heavy chain in differentiating c2c cells. it is well known that rb plays a critical role in inducing myogenic differentiation, and the introduction of rb in myoblasts accelerates differentiation to myotubes. conversely, inhibition of rb expression in myoblasts abrogates differentiation into myotubes. thus, we hypothesized that fl-irs- transcript may affect rb expression. fl-irs- 5′utr reduces rb mrna expression in c2c cells we examined the expression levels of rb mrna in myoblasts and myotubes. myoblasts expressed low levels of rb mrna and myotubes expressed higher levels. the rb mrna expression profile had an inverse relationship with fl-irs- transcript levels.figure fl- irs-1 transcript reduces rb mrna levels. expression levels of rb mrna in c2c myoblasts and myotubes. partial sequence complementarity between the 5′utr of fl-irs- transcript and rb mrna. schematic representation of position of complementary element in the 5′utr of fl-irs- transcript. a position of an lna-based antisense oligonucleotide is shown. effect of fl-irs- 5′utr on rb mrna expression levels. c2c myoblasts were untransfected or transfected with an rb mrna-expressing plasmid in combination with the indicated effector plasmid. rb mrna levels were measured by qrt-pcr. mean ± sd, n = 4- *p <  versus control transfectant. effect of fl-5′-as-odn on rb mrna reduction by fl-irs- 5′utr. c2c myoblasts were co-transfected with plasmids expressing rb mrna and the fl-irs- 5′utr rna in combination with the indicated as-odn. mean ± sd, n = 4- *p <  versus control transfectant. effect of knockdown of endogenous fl-irs- transcript on endogenous rb mrna levels. c2c myoblasts were transfected with the indicated shrna or sirna. mean ± sd, n =  *p <  versus no treatment. effect of supplementary expression of irs- protein on rb mrna levels under fl-irs- transcript knockdown conditions. c2c myoblasts were transfected with the sh-fl-irs- plasmid in combination with a plasmid encoding irs- protein fused to v tag. expression levels of exogenous irs- protein were determined by immunoblot analysis using anti-v antibody. mean ± sd, n =  *p <  versus control transfectant. a bioinformatics analysis showed that there was a partial sequence-complementary element between the 5′utr of fl-irs- transcript and rb mrna. given that several functional rna transcripts can interact with target mrnas by intermolecular base-pairing via a partially complementary element, fl-irs- 5′utr may target rb mrna via the complementary element. to assess whether fl- irs- 5′utr participated in rb expression, c2c myoblasts were transfected with an rb mrna-expressing plasmid in combination with a plasmid expressing the fl-irs- 5′utr fused to the luciferase gene or a control luciferase plasmid. overexpression of the 5′utr, but not control plasmid, markedly reduced rb mrna levels. to demonstrate that the complementary element in the 5′utr was responsible for the reduction of rb mrna, we deleted the complementary element. the deletion of the element fully abrogated the ability to decrease rb mrna expression. to exclude the possibility that the deletion may affect the secondary structure of fl-irs- 5′utr, resulting in a loss of silencing activity for rb mrna, we examined effect of an lna-based antisense oligonucleotide against the complementary element. fl-5′-as-odn, but not control as-odn, completely prevented the decrease of rb mrna expression induced by fl-irs- 5′utr. these results indicated that fl-irs- 5′utr decreased rb mrna levels via its complementary sequence. to verify that endogenous fl-irs- transcript participated in endogenous rb mrna expression, we knocked down the fl-irs- transcript. the silencing of fl-irs- mrna increased rb mrna levels by more than 2-fold. in contrast, knockdown of s5′-irs- mrna did not affect the rb mrna levels. these results suggest that the elevation of rb mrna levels was not caused by decreasing irs- protein after knockdown of fl-irs- transcript. the supplementary expression of exogenous irs- protein using an irs-1-expressing plasmid that encodes only the open reading frame of irs- without the 5′ and 3′-utrs did not affect the elevation of rb mrna levels. we additionally examined the effect of knockdown of fl-irs- transcript on other myogenic regulators expression, such as myod, myogenin, myf mrf and mef the silencing of fl-irs- mrna did not affect the levels of all myogenic factors tested. fl-irs- 5′utr does not reduce rb mrna expression in hepatocytes we further tested whether the fl-irs- 5′utr-induced rb mrna reduction occurred in hepatocytes because irs- is an important factor in hepatocytes. a cultured hepatocyte cell line was co-transfected with plasmids expressing rb mrna and the fl-irs- 5′utr. unlike skeletal muscle cells, the overexpression of the 5′utr did not reduce rb mrna levels in hepatocytes. in addition, knockdown of fl-irs- transcript did not alter rb mrna levels in hepatocytes.figure fl- irs-1 transcript does not reduce rb mrna expression in hepatocytes. effect of fl-irs- 5′utr on rb mrna expression levels. cultured hepatocyte cell lines were untransfected or transfected with an rb mrna-expressing plasmid in combination with the indicated effector plasmid. rb mrna levels were measured by qrt-pcr. mean ± sd, n =  *p <  versus control transfectant. effect of knockdown of endogenous fl-irs- transcript on endogenous rb mrna levels. cultured hepatocyte cell lines were transfected with the indicated shrna. fl-irs-1 and rb mrna levels were measured by qrt-pcr. mean ± sd, n =  *p <  versus no treatment. reduction of rb mrna by fl-irs- transcript did not require dicer and upf1 we then examined the molecular mechanism responsible for fl-irs- 5′utr reducing rb mrna levels. there is evidence that several mirnas and ncrnas regulate myogenic differentiation through the dicer- and upf1-mediated target mrna decay system. the knockdown of dicer or upf by sirna did not alter the suppression of rb mrna by fl-irs- 5′utr. in addition, northern blot analysis using fl-5′-as-odn showed a small rna with approximately 20–25 nt in length or the 5′utr rna distinct from fl-irs- mrna was not detected in either myoblasts and myotubes. these data suggest that fl-irs- transcript was not processed to mirna or intermediate 5′utr rna. discussion in this study, we found that fl-irs- transcript acts as a bifunctional rna. one function is as a regulatory rna in proliferating myoblasts that reduced rb mrna expression and suppressed myoblast differentiation. the other function is as mrna that produced irs- protein during differentiation into myotubes. by contrast, s5′-irs- mrna was only involved in irs- protein synthesis during myogenesis. rb plays a crucial role in the switch from proliferation to differentiation in myoblasts. myoblasts lacking rb lose the capacity to differentiate into myotubes, and the overexpression of rb in myoblasts enhances differentiation into myotubes. it is well established that rb function is regulated by posttranslational modification during myoblast differentiation. although it is considered that the rb expression levels are tightly regulated in myoblasts before and during differentiation, how rb levels are regulated in myoblasts is still unclear. in this study, the knockdown of endogenous fl-irs- transcript increased rb mrna levels and accelerated differentiation of myoblasts to myotubes. thus, our findings suggest that fl-irs- transcript could be one of the endogenous downregulator of rb expression in myoblasts. a functional relevance of between irs- protein and rb protein was previously reported. irs- inhibits transcriptional activity of rb. in addition, several reports indicate that the igf-1/irs- pathway suppresses rb-mediated cell cycle arrest by inducing prolonged phosphorylation of rb. however, at least to our best knowledge, our study is the first report showing a relationship between irs- transcript and rb mrna. there are several ncrnas that regulate myogenesis through modulation of myogenic gene expression, such as pax, myf, myod, and mirna- and −. several sine-containing ncrnas and mrnas interact with their target mrnas such as myf by base-pairing with partially complementary elements to induce degradation of them and regulate c2c cell myogenesis. in these cases, the silencing of target mrnas by the ncrnas requires base-pairing to target mrna and a dicer- and upf1-dependent mechanism. here, we demonstrated that the reduction of rb mrna by fl-irs- transcript did not require dicer and upf however, the complementary element in the 5′utr of fl-irs- transcript was necessary to decrease rb mrna levels. in addition, the reduction of rb mrna by the fl-irs- transcript was not observed in hepatocytes despite the important role of rb in hepatocyte differentiation. thus, skeletal muscle-specific factors may be required in rb mrna silencing. further studies are required to clarify a molecular mechanism of fl-irs- transcript-mediated gene silencing. until recently, it has been generally accepted that mrna functions only in protein production, and cell phenotypes depend exclusively on the function of these proteins. unexpectedly, recent studies demonstrated that several mrnas may have bifunctional abilities in a context-dependent manner. it is predicted that the human genome contains approximately possible potentially new bifunctional mrnas. one characterized bifunctional mrna is sra during myogenic processes. in myogenic proliferation, the protein-coding sra transcript is abundantly expressed, and sra protein binds to and counteracts sra ncrna-mediated myod activation and suppresses differentiation. during myogenic differentiation, the expression of non-coding sra transcript is elevated and enhances transcriptional activity of myod and differentiation. these findings suggest that the effect of sra on myogenesis may result from the correct balance between non-coding and coding sra transcripts. the present study showed that the balance between coding and non-coding irs- transcripts may be important for regulating myogenic differentiation. beyond utrs function as cis regulatory elements that impact the translational activity and stability of their own mrnas, utrs are also trans modulator of gene expression through their target transcript inhibition as demonstrated here and by others. as recently suggested by the pandolfi group, mrna transcripts can potentially perturb the interaction with target rnas and possess a biological activity independent of the translation of the protein they encode. therefore, some of the phenotypic features of cells may depend not only on the protein function but also on the regulatory rna function that is distinct from their protein products.  the objective of this study was to identify a novel bifunctional rna in myogenic process. we found that fl-irs- transcript regulates rb expression at the rna levels independent of its protein-coding function. our findings suggest that fl-irs- transcript functions as a bifunctional rna during myogenic differentiation. 
the overexpression of the 5′utr markedly reduced rb mrna expression, and this reduction was fully dependent on the complementary element and was not compensated by irs- protein. conversely, knockdown of fl-irs- mrna increased rb mrna expression and enhanced myoblast differentiation into myotubes. the fl-irs- 5′utr contains a partial complementary sequence to rb mrna, which is a critical factor for myogenic differentiation. in cultured skeletal muscle c2c cells, the fl-irs- transcript functioned as a bifunctional mrna. fl-irs- mrna was highly expressed only in skeletal muscle tissue. in this study, we identified a novel bifunctional rna that is transcribed from insulin receptor substrate- gene with full-length 5′utr sequence. the fl-irs- transcript produced irs- protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory rna in proliferating myoblasts. our findings suggest that the fl-irs- transcript regulates myogenic differentiation as a regulatory rna in myoblasts. bifunctional rnainsulin receptor substrate-1myogenic differentiationrbissue-copyright-statement© the author 2015
 there is evidence that several messenger rnas are bifunctional rnas, i.e. rna transcript carrying both protein-coding capacity and activity as functional non-coding rna via 5′ and 3′ untranslated regions.  in this study, we identified a novel bifunctional rna that is transcribed from insulin receptor substrate- gene with full-length 5′utr sequence. fl-irs- mrna was highly expressed only in skeletal muscle tissue. in cultured skeletal muscle c2c cells, the fl-irs- transcript functioned as a bifunctional mrna. the fl-irs- transcript produced irs- protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory rna in proliferating myoblasts. the fl-irs- 5′utr contains a partial complementary sequence to rb mrna, which is a critical factor for myogenic differentiation. the overexpression of the 5′utr markedly reduced rb mrna expression, and this reduction was fully dependent on the complementary element and was not compensated by irs- protein. conversely, knockdown of fl-irs- mrna increased rb mrna expression and enhanced myoblast differentiation into myotubes.  our findings suggest that the fl-irs- transcript regulates myogenic differentiation as a regulatory rna in myoblasts. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bifunctional rnainsulin receptor substrate-1myogenic differentiationrbissue-copyright-statement© the author 2015 