From the Departments of Biochemistry and
§ Cardiology, Jichi Medical School, Minamikawachi-machi,
Kawachi-gun, Tochigi 329-04, Japan
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ABSTRACT |
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Muscle-specific alternative RNA splicing is an
essential step during myogenesis. In this paper, we report that a
muscle-specific transcription factor, MyoD, plays a central role in the
induction of muscle-specific alternative splicing during myogenesis.
Recently, we reported that muscle and nonmuscle isoforms of the
mitochondrial ATP synthase -subunit (F1
) were
generated by alternative splicing and that acidic stimulation promoted
this muscle-specific alternative splicing (Endo, H., Matsuda, C., and
Kagawa, Y. (1994) J. Biol. Chem. 269, 12488-12493). In
this report, mouse myoblasts are shown to express the muscle-specific
isoform of F1
after induction with low-serum medium
(differentiation medium) or acidic medium, although myotube formation
was not detected after acidic induction. RNA blot analysis revealed
that the expression levels of both MEF2 and myogenin were increased by
low-serum induction, but not by acidic induction. High expression of
MyoD mRNA was observed after both types of induction.
Overexpression of exogenous MyoD in fibroblasts showed that MyoD was
necessary for muscle-specific alternative splicing in both types of
induction. Exogenous Id, a negative regulator of MyoD, blocked
muscle-specific alternative splicing of F1
pre-mRNA
by both types of induction. In addition, MyoD induced several
muscle-specific alternative splicings, including structural protein
pre-mRNAs such as
-tropomyosin and neural-cell adhesion molecule
and transcriptional protein pre-mRNAs such as MEF2A and MEF2D. Our
analysis of the two induction systems shows a common
MyoD-dependent mechanism of muscle-specific alternative splicing in several genes, independent of MEF2 and myogenin.
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INTRODUCTION |
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Alternative pre-mRNA splicing is a fundamental process in eukaryotes and is regulated by cell-specific, tissue-specific, and developmental stage-specific pathways to generate mRNAs that differ in protein-coding potential, stability, and translation efficacy (1, 2). In vertebrates, cis-regulatory elements or trans-acting factors that affect tissue-specific alternative splicing have been identified (1-4). Several factors are responsible for alternative splicings, such as serine/arginine-rich (SR)1 proteins, U2 auxiliary factor, polypyrimidine tract-binding protein, and exon recognition factors (1, 2, 5). Understanding the regulatory cascade for tissue-specific alternative splicing during terminal differentiation would elucidate a direct trigger for the alternative splicing mechanism.
Myogenic differentiation involves both muscle-specific RNA processing and transcriptional activation of muscle-specific genes, e.g. the basic helix-loop-helix (bHLH) proteins of the MyoD family including MyoD, Myf5, MRF4, and myogenin, which promote skeletal muscle-specific gene expression (6-17) and are built up to a positive autoregulatory loop (6-12). On the other hand, Id, a helix-loop-helix protein, is a negative regulator of bHLH proteins through direct protein-protein interaction. Id is expressed at various levels in some stem cell lines, and a decrease in Id mRNA levels triggers terminal differentiation (18). Myocyte-specific enhancer factor 2 (MEF2) was originally identified as a muscle-specific DNA-binding factor induced when skeletal myoblasts differentiate into myotubes (19). There are four members of the MEF2 family of proteins (MEF2A through MEF2D), and all share a region of homology, the MADS box and the MEF2 domain (20-25). A recent study shows that the MEF2 and MyoD families act within a regulatory network that establishes differentiated phenotypes of skeletal muscle and that MEF2 factors act as coregulators to potentiate the myogenic activities of myogenic bHLH proteins (26).
On the other hand, post-transcriptional control is shown during
myogenesis, e.g. the transcripts of -tropomyosin (
-TM)
(3, 27-30), neural-cell adhesion molecule (N-CAM) (31-33), MEF2A
(21), and MEF2D (24, 25), which are regulated by alternative splicing in substantially different splicing patterns. The gene of
-TM contains two sets of alternatively spliced, mutually exclusive exons
whose utilization is tissue-dependent and developmentally regulated (3, 27-30); exon 6a is shared by mRNAs expressed in mouse smooth muscle and nonmuscle tissues, whereas exon 6b is present
only in skeletal muscle-specific transcripts (27, 28). N-CAM mRNA
includes the muscle-specific sequence domain (MSD) located between
exons 12 and 13 in a cassette mode. Whereas
-TM mRNA contains
exon 6a and N-CAM mRNA does not include the MSD in mouse myoblasts,
exon 6b is selected in
-TM mRNA and the MSD is included in N-CAM
mRNA when myoblasts convert to myotubes (27, 28, 32, 33). MEF2A
transcripts are ubiquitous, but accumulate preferentially in skeletal
muscle, heart, and brain (20, 21). In these tissues, MEF2A activity is
elevated because the tissue-specific isoform containing the SEEEELEL
sequence is present (21). MEF2D transcripts are widely expressed, but
alternative splicing in the two domains gives rise to a muscle-specific
isoform (24, 25). One domain is alternatively spliced in a mutually
exclusive fashion, and the other is regulated in a cassette mode and
includes the peptide TGDHLDL.
Recently, we cloned the mitochondrial ATP synthase -subunit
(F1
) gene and showed that its heart and skeletal
muscle-specific isoforms are generated by alternative splicing in the
human and the cow (34, 35). As muscle tissues require a rapid energy supply, the expression of muscle-type F1
is thought to
be an adaptation to the tissue-specific energy requirement. We also showed that this muscle-type mRNA excluding exon 9 in a cassette mode was induced cell-specifically by intracellular acidosis in human
fibrosarcoma and rhabdomyosarcoma cells (36). This acidic induction of
alternative splicing is a reversible system, indicating that the
nonmuscle-type mRNA containing exon 9 is a default type and that
de novo protein synthesis is required for muscle-specific alternative splicing. The alternative splicing of F1
is
very useful as a marker for studying muscle-specific splicing because the splicing is controlled in a simple cassette mode and the expression levels before and after induction do not change significantly.
In this report, we show the new muscle-specific alternative splicing induction system using mouse myoblasts, in which acidic stimulation induces muscle-specific splicing without expression of MEF2 and myogenin and finally without myotube formation. We identified a muscle-specific transcriptional regulatory factor involved in splicing regulation and investigated its influence on many genes. A common MyoD-dependent regulatory cascade for muscle-specific alternative splicing is described, and Id is shown to inhibit muscle-specific alternative splicing via blocking the induction of MyoD.
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MATERIALS AND METHODS |
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cDNA Cloning and Amplification of a Portion of the Mouse
F1 Genomic DNA--
The cDNA encoding mouse
F1
was isolated from a Balb/c 3T3 cDNA library using
the plaque hybridization method with the cloned PCR product of human
F1
cDNA as a probe (35). For preparation of the
probe, sense and antisense strand oligonucleotides
(5'-TTC-GAA-ATA-TGG-CAA-CTT-TGA-AAG-AT-3' and
3'-GGA-CTT-CTG-TTC-TTC-TTT-GTG-G-5', corresponding to nucleotides 96-121 and 284-305, respectively, in HeLa F1
cDNA
(GenBankTM/EMBL Data Bank accession number D16562)) were synthesized
and used as primers in the PCR amplification of cDNA from human
liver. The amplified DNA fragment (210 base pairs in length) was
subcloned into pBluescript II (Stratagene). A Balb/c 3T3 cDNA
library, constructed in the
gt10 phage vector, was a gift from Dr.
S. Tominaga (Jichi Medical school, Tochigi, Japan). Plaque
hybridization was performed using 32P-labeled human
F1
cDNA, and positive clones were isolated. The cDNA was subcloned and sequenced using the dideoxy chain
termination method (37) with Sequenase (U. S. Biochemical Corp.). The
nucleotide sequences of both strands of the cDNA were determined.
Mouse BALB/c genomic DNA was purified from liver cells according to a
previously described method (38). A portion of the mouse
F1
genomic gene was amplified using the long and
accurate PCR method at 94 °C for 1 min, followed by 40 cycles at
98 °C for 20 s and 68 °C for 15 min using a pair of specific
primers (5'-CCC-GCC-AGG-CTG-TCA-TCA-CAA-AGG-AGT-TGA-TT-3' and
3'-CAG-TCT-TCT-TTG-AAC-CAG-GTG-ACT-CAA-TGT-TTC-5', corresponding to the cDNA sequences upstream and downstream, respectively, of an
alternatively spliced exon), in a total volume of 50 µl using the
TaKaRa LA PCR kit (Takara Shuzo Co., Kyoto, Japan). This PCR fragment
(~2.8 kilobase pairs in length) was subcloned into the dideoxy-T
vector of pBluescript II SK+ and then sequenced on both
strands.
Cell Culture-- Mouse C3H10T1/2 clone 8 fibroblast cells (10T1/2 cells) were obtained from the Japanese Cancer Research Resources Bank. Mouse C2C12 cells (a myoblast cell line) and BC3H1 cells (a myoblastoid cell line) were obtained from the American Type Culture Collection. The C2C12 and 10T1/2 cells were grown in growth medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Flow Laboratories)) at 37 °C under 5% CO2. The cells were plated on 10-cm diameter tissue culture dishes. When the C2C12 cells grew to semiconfluence, the growth medium was replaced with two different media. Differentiation (low-serum) medium contained Dulbecco's modified Eagle's medium with 2% heat-inactivated horse serum (Irvine Scientific), and acidic (low-pH) medium contained Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 2.7 mM NaHCO3 for C2C12 and 10T1/2 cells or 5.4 mM NaHCO3 for BC3H1 cells. BC3H1 cells are less viable in acidic medium. Cells were harvested after 3 days.
RNA Preparation and RNA Blot Analysis--
Total RNA was
prepared from C2C12 and 10T1/2 cells using the acid guanidine method
(39). Formaldehyde gel for RNA electrophoresis was prepared as
described previously (38). cDNA for probes were labeled with
[-32P]dCTP (Amersham Pharmacia Biotech) using a random
priming kit (Amersham Pharmacia Biotech). RNA blot hybridization was
performed as described previously (38). Full-length mouse MyoD cDNA
(provided by Dr. H. Weintraub, Hutchinson Cancer Research Center) (6) was digested with XhoI (cDNA encompassing codons
1-1785). Mouse Id cDNA encompassing nucleotides 61-851 (18),
mouse myogenin cDNA encompassing nucleotides 511-766 (7), and
human MEF2A cDNA encompassing nucleotides 1234-1427 (21) were
constructed by RT-PCR. First-strand cDNA was synthesized with
Superscript IITM (Life Technologies, Inc.) and primed with
oligo(dT16) primer. PCR was carried out for 30 cycles at
94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min
in a total volume of 100 µl containing 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 11.5 mM MgCl, 200 nM each dNTP, 10 pmol of each oligonucleotide primer, 1:20
(v/v) synthesized first-strand cDNA, and 2.5 units of recombinant
Taq DNA polymerase (Takara Shuzo Co.). These cloned
cDNAs were subcloned into the EcoRV site of pBluescript
II SK+ and subsequently sequenced according to the dideoxy
termination method (37). Primer sequences used for amplification were
as follows: mouse Id, 5'-GCT-CCC-CTC-CGC-CTG-TTC-TC-3' (sense strand) and 5'-AGT-GTC-TTT-CCC-AGA-GAT-CCC-3' (antisense strand); MEF2A, 5'-CGA-GTT-GTC-ATC-CCC-CCT-TCA-A-3' (sense strand, primer G) and 5'-GTG-TTG-TAG-GCA-GTC-GGC-ATT-G-3' (antisense strand, primer H); and
myogenin, 5'-CGC-CTA-CAG-GCC-TTG-CTC-AGC-T-3' (sense strand) and
5'-GCA-ACA-GAC-ATA-TCC-TCC-ACC-3' (antisense strand).
RT-PCR Analysis and Southern Blot Analysis of the Alternative
Exon--
To identify the possible alternative exons of
F1,
-TM from exons 5 to 7, N-CAM from exons 12 and
13, MEF2A, and MEF2D (10 µg of total RNA) were used to synthesize
first-strand cDNAs as described above. PCR amplifications of
F1
,
-TM, N-CAM, and MEF2A were performed in a total
volume of 100 µl as described above by heating the DNA at 94 °C
for 30 s, 55 °C for 30 s, and 72 °C for 1 min in 25, 25, 35, and 30 cycles, respectively. PCR of MEF2D was performed (stored
at 94 °C for 9 min; subjected to 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and then stored
at 72 °C for 7 min) in a total volume 100 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, 200 µM each dNTP, 10 pmol of each
oligonucleotide primer, 1:20 (v/v) synthesized first-strand cDNA,
and 2.5 units of recombinant Taq DNA polymerase (AmpliTaq
GoldTM, Perkin-Elmer). The oligonucleotides were as follows: F1
, 5'-GTC-ATC-ACA-AAA-GAG-TTG-ATT-G-3' (sense strand,
primer A) and 5'-TAA-TGG-AGG-AAC-AGT-TTC-TTC-G-3' (antisense strand, primer B); N-CAM, 5'-GAC-TGG-CGG-CCC-TCA-ACG-GCA-A-3' (sense strand at
exon 12, primer E) and 5'-CCA-GAT-AGT-GTC-TGA-TGG-GGG-A-3' (antisense
strand at exon 13, primer F);
-TM, 5'-GCT-GGT-GAT-CCT-GGA-AGG-GG-3' (sense strand at exon 5, primer C) and 5'-GTA-TTT-GTC-CTC-TTT-GGT-GG-3' (antisense strand at exon 7, primer D); MEF2A, as described above; and
MEF2D, 5'-CAA-CGA-GCC-ACA-CGA-GAG-CCG-CAC-CAA-3' (sense strand, primer
I) and 5'-AAG-CTC-GGG-CAC-TGA-CAT-AGC-C-3' (antisense strand, primer J)
(25). Amplified PCR products of MEF2D were digested with
XmnI. The size of the PCR products was determined by 3%
agarose gel electrophoresis, and the products were stained with
ethidium bromide. One-fifth of the
-TM PCR products was fractionated
on a 3% agarose gel and transferred to a nylon membrane (Hybond
N+, Amersham Pharmacia Biotech) under denaturing
conditions. The nylon membrane was then hybridized with (probe A
5'-CTT-GAG-GGC-TTG-GTC-CAT-3') for exon 6a and (probe B
5'-TTC-CAG-GGA-TTT-CAA-GTT-GT-3') for exon 6b as probes. The
oligonucleotide probes were labeled with [
-32P]ATP
(Amersham Pharmacia Biotech) by T4 nucleotide kinase.
Construction of Expression Plasmids--
The Id cDNA coding
sequence 61-851 (18) was cloned as described above. Human
6-o-methylguanine-DNA methyltransferase (hMGMT) (provided by
Dr. H. Hayakawa, Kyushu University School of Medicine) (40) was used as
a control. MyoD, Id, and hMGMT cDNAs were ligated to the
XhoI site of pcDEBSR, which is an eukaryotic
expression vector containing a hygromycin resistance gene
(pcDEBSR
-MyoD, pcDEBSR
-Id, and
pcDEBSR
-hMGMT). To confirm the expression of MyoD
mRNA, we used RT-PCR analysis. The oligonucleotide sequences for
PCR amplification were as follows:
5'-CGG-CGG-CAG-AAT-GGC-TAC-GAC-ACC-3' (sense strand,
corresponding to MyoD cDNA codons 817-840); and oligonucleotide B,
5'-CAC-TGC-ATT-CTA-GTT-GTG-GTT-TGT-3' (antisense strand,
corresponding to an SV40 poly(A) additional site).
DNA Transfection into Cells--
After 10T1/2 fibroblasts and
C2C12 myoblasts were stored in growth medium at 37 °C, the cells
were transfected with 5 µg each of pcDEBSR-MyoD,
pcDEBSR
-Id, and pcDEBSR
-hMGMT using the
polycationic liposome method (DMRIE-C®, Life Technologies,
Inc.). After transfection, cells were cultured in growth medium
containing 200-300 µg/ml hygromycin B. Hygromycin-resistant clones
were isolated and amplified in growth medium.
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RESULTS |
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TGF-1 Inhibits the Induction of Muscle-specific Splicing in
Differentiation Medium, but Not in Acidic Medium--
We cloned muscle
and nonmuscle types of mouse F1
cDNA and part of the
genomic gene as described under "Materials and Methods." Two
isoforms were produced by alternative splicing of a cassette exon
corresponding to exon 9 of the human F1
gene (Fig.
1). In mouse C2C12 myoblasts, we induced
muscle-specific alternative splicing in F1
pre-mRNA
during myogenesis using differentiation medium (Fig.
2a, lane 1). This
splicing was detected 48 h after induction (Fig. 2a,
lane 1). At this time, the myotube had not yet formed (Fig.
2b, lane 1). Acidic stimulation also induced the
muscle type of F1
in the myoblasts (Fig. 2c,
lane 1), but myotubes did not form for more than 3 days. In
addition, acidic induction of muscle-specific alternative splicing in
F1
pre-mRNA appeared to be reversible in mouse
myoblasts (see below), as in human cells (36).
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Expression of Endogenous MyoD Is Necessary for Induction of
Muscle-type F1--
RNA blot analyses in myoblasts in
differentiation medium and acidic medium were performed on
muscle-specific transcription factors such as MyoD, MEF2A, myogenin,
and Id. Initially, when C2C12 myoblasts were cultured in
differentiation medium, the expression level of MyoD mRNA was
increased steadily until 72 h (Fig.
3a, lanes 1-6).
MyoD mRNA was also induced in acidic medium (Fig. 3b,
lanes 1-6). Next, the expression level of Id mRNA
rapidly decreased by placement in differentiation medium, but did not significantly decrease in acidic medium (Fig. 3, c,
lanes 1-6; and d, lanes 1-6).
Expression of myogenin and MEF2 mRNAs was induced in
differentiation medium, whereas neither of these mRNAs was expressed in acidic medium (Fig. 3, e and f). The
inhibition of myogenic differentiation by TGF-
1 treatment is thought
to be due to suppression of the gene expression and function of MyoD and myogenin (42-44). The expression of MyoD mRNA increased
moderately in differentiation medium and acidic medium containing
TGF-
1 (Fig. 3, a, lanes 7-12; and
b, lanes 7-12). Id expression rapidly decreased
in differentiation medium, but was steadily expressed in acidic medium,
even in the presence of TGF-
1 in both media, as shown in Fig. 3
(c and d). In the C2C12 myoblasts we used, the
initial expression level of MyoD mRNA (lane 1 of Fig. 3,
a and b) was slightly lower than that previously
reported (6), perhaps owing to variation in the cell line. However, our
results indicate that an increase in MyoD expression is necessary for the induction of muscle-type F1
, and the expression
levels of Id, myogenin, and MEF2 appear not to be important.
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Exogenous MyoD Induces Muscle-type Splicing of F1,
but Exogenous Id Inhibits It--
To investigate whether MyoD
expression can induce muscle-specific alternative splicing of
F1
pre-mRNA, we analyzed the pattern of
F1
mRNA associated with MyoD expression in mouse
fibroblasts (10T1/2 cells) in which MyoD was not expressed. The mouse
MyoD cDNAs were subcloned into a mammalian expression vector
(pcDEBSR
) (Fig.
4a). As a control, we used
human hMGMT cDNA subcloned into pcDEBSR
(40). MyoD-
and hMGMT-expressing permanent transformants from 10T1/2 cells were
selected, cloned, and named 10T-MyoD and 10T-hMGMT cells, respectively.
After they had grown to semiconfluence in growth medium, 10T-MyoD
cells, 10T-hMGMT cells, and wild-type 10T1/2 cells were exposed to
differentiation medium or acidic medium for 72 h. Exogenous MyoD
mRNA was expressed in 10T-MyoD cells cultured in growth medium,
differentiation medium, and acidic medium (Fig. 4b,
lanes 7-9). Exogenous MyoD mRNA was not expressed in
wild-type 10T1/2 cells or in 10T-hMGMT cells (Fig. 4b,
lanes 1-6). When 10T-MyoD cells were cultured in
differentiation medium or acidic medium, muscle-type F1
mRNA was induced (Fig. 4c, lanes 8 and
9). 10T-MyoD cells converted into multinucleate myotubes in
differentiation medium, but not in acidic medium (data not shown). In
wild-type 10T1/2 cells and in 10T-hMGMT cells, muscle-type F1
mRNA was undetectable or appeared only as faint
bands, and myotubes were not induced.
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Muscle-specific Exon Selection Requires de Novo Protein Synthesis
and Activated Protein Kinase C--
Alternative splicing in the
F1 pre-mRNA would require a common intracellular
signal cascade in the two induction systems at the late stage of
muscle-specific alternative splicing. As acidic induction is a
reversible splicing regulation system (Fig. 6), we can identify which course requires
a regulatory factor for alternative splicing in mouse C2C12 myoblasts
and BC3H1 cells. When these cells were cultured with acidic medium,
muscle-specific F1
mRNA was induced. This induction
was inhibited by cycloheximide, a protein synthesis inhibitor (Fig. 6,
a and c). On the other hand, the reverse switch
from muscle-type to nonmuscle-type F1
mRNA was not
inhibited by cycloheximide (Fig. 6, b and c). In these cells, muscle-specific exon exclusion of F1
pre-mRNA required de novo protein synthesis of such a
regulatory factor. Next, we tested the protein kinase C inhibitor H-7
and HA1004 in the system. As shown in Fig. 6 (e and
f), H-7 inhibited muscle-specific exon exclusion in BC3H1
cells in acidic medium, but did not inhibit the reverse course. HA1004,
an analogue of H-7, was used as a control. These results suggest that
activated protein kinase C is directly involved in muscle-specific exon
selection. Considering that both the protein synthesis inhibitor and
the protein kinase C inhibitor suppress only the induction of exon
exclusion, it is likely that activated protein kinase C is involved in
the regulation of alternative splicing via activation or expression of
a trans-acting protein factor.
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Muscle-specific Alternative Splicing Is Induced Coordinately by
MyoD--
Some genes have transcripts produced by muscle-specific
alternative splicing. Structural proteins such as -TM and N-CAM and transcription factors such as MEF2A and MEF2D show alternative splicing
accompanied by myotube formation and transcriptional activation of
muscle-specific genes, although the modes of these splicings are
different.
-TM and MEF2D mRNAs are regulated in mutually
exclusive modes, and F1
, N-CAM, and MEF2A mRNA are
regulated in cassette modes. We proved that these muscle-specific
alternative splicings were simultaneously induced in C2C12 myoblasts
cultured with acidic medium (data not shown) as well as during
myogenesis (20, 21, 24, 25, 27, 28, 32, 33). Then, using RT-PCR in
10T-MyoD cells, we investigated whether these alternative splicings
require MyoD expression in the early stage of muscle-specific splicing
induction.
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DISCUSSION |
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In this paper, we characterized an in vivo model system
for inducing muscle-specific alternative splicing in mouse myoblasts employing mouse F1 pre-mRNA as a useful marker
during muscle differentiation (Fig. 1). We showed that acidic
stimulation induces muscle-specific alternative splicing in
F1
pre-mRNA and in other muscle-specific
pre-mRNAs in mouse myoblasts (C2C12 cells) and does not induce gene
expression of MEF2 and myogenin and myotube formation. Analyzing the
difference between the two induction systems (one is a low-serum
induction system, and the other is an acidic one), we showed that MyoD
is an essential factor for muscle-specific alternative splicing in
several mouse genes.
Muscle-specific alternative splicing events were shown to occur prior
to myotube formation (Fig. 2). Myogenin is indispensable for myotube
formation (45, 46), and MEF2 is important in the action of MyoD (26)
and in the expression of myogenin (47, 48). Thus, the absence of
myotube formation after acidic induction can be attributed to low
expression levels of myogenin and MEF2 in mouse myoblasts (Fig. 3). On
the other hand, actin, a member of the cytoskeletal protein family,
does not polymerize at low pH (49). Because the pH value of the acidic
medium was 6.6, myoblasts could not form myotubes, even if myogenin was
expressed in the cells. In addition, acidic stimulation was shown to
induce muscle-specific alternative splicings in mouse N-CAM, -TM,
MEF2A, and MEF2D pre-mRNAs (Fig. 7), with different patterns of
alternative splicing: those of
-TM and MEF2D were mutually exclusive
types, and those of F1
, N-CAM, and MEF2A were cassette
types. Therefore, it is likely that a common regulatory cascade for
muscle-specific alternative splicing exists after acidic stimulation.
We showed that the mechanism of muscle-specific alternative splicing in many genes is independent of the gene expression of MEF2 and myogenin and is different from the cascade for myotube formation.
The other striking difference between these two induction systems was
in their response to TGF-1 treatment. TGF-
1 inhibited muscle-specific alternative splicing in C2C12 cells cultured in differentiation medium, but not in acidic medium (Fig. 2). TGF-
1 can
block myogenic differentiation by inhibiting the increase in expression
of MyoD and myogenin (42-44), so it is likely that acidic induction
skips the action point of TGF-
1 or stimulates a signal cascade
downstream of the action points of MyoD and TGF-
1.
To induce and maintain terminal differentiation in mouse myoblasts, a certain trigger is needed to promote the autoregulation of MyoD, MEF2, and myogenin, and the expression and activities of MyoD and myogenin must overcome threshold values (14). MyoD induces MEF2 gene expression (50), and then both cooperatively act to increase myogenin gene expression (47, 48, 51) in myogenic differentiation. Under acidic conditions, the induced expression of MyoD prepares the splicing regulatory apparatus beyond the threshold, but does not induce transcriptional activation of the MEF2 gene. Functions of MyoD include 1) promotion of muscle-specific gene expression, such as structural proteins and transcriptional factors; 2) permanent cell cycle arrest via gene expression of the cyclin-dependent kinase inhibitor p21; and 3) preparation of a splicing regulatory apparatus. The thresholds of MyoD action in these functions vary.
A previous report showed that myogenin can induce muscle-specific
alternative splicing of - and
-tropomyosin pre-mRNAs in differentiation medium in fibroblasts (52). However, overexpression of
myogenin leads to activation of MyoD expression because of its positive
autoregulatory loop (51, 52). In contrast, the acidic induction
described here induced only MyoD expression, but did not induce
myogenin and MEF2 expression. Considering the previous report and our
results, overexpression of myogenin induced MyoD, and then
muscle-specific alternative splicings of
- and
-tropomyosins
would be induced.
In addition, Id, a negative regulator of MyoD that is highly expressed
under high-serum conditions, can block the MyoD-dependent gene expression and the activation of the positive loop of
muscle-specific transcription factors by preventing the formation of
MyoD heterodimer containing E12/E47, other bHLH proteins. Therefore,
when MyoD is overexpressed in fibroblasts under high-serum conditions,
the transfected cells cannot convert to myotubes. On the other hand, overexpression of Id in myoblasts prevents muscle-specific alternative splicings of F1 pre-mRNA and other pre-mRNAs
under acidic or low-serum conditions by blocking the positive feedback
of MyoD expression (Fig. 6). These data indicated that MyoD is
necessary for muscle-specific alternative splicing.
The MEF2 family is differentially controlled at the transcriptional and post-transcriptional levels during muscle differentiation. As shown in Figs. 3 and 7, the gene expression of MEF2A is not induced, but muscle-specific splicings of MEF2A and MEF2D are induced in acidic stimulation. MEF2A and MEF2D accumulate preferentially in skeletal muscle, heart, and brain (20, 21, 24, 25), and these tissue-specific isoforms correlate exactly with the presence of endogenous MEF2 activity. This finding indicates that tissue-specific alternative domains of MEF2A and MEF2D play a key role in the regulation of MEF2 activities in vivo (21, 24, 25). The regulatory system of muscle-specific alternative splicing should be prepared prior to the transactivation of MEF2 function, so it is likely that post-transcriptional control of alternative RNA splicing contributes to muscle differentiation via the regulation of MEF2A and MEF2D function.
Acidic stimulation appears to trigger a common mechanism for
muscle-specific alternative splicing in the presence of MyoD. As a
result of the inhibition of muscle-specific exclusion of an
alternatively spliced exon in F1 pre-mRNA by
cycloheximide and the protein kinase inhibitor H-7, it is suggested
that de novo protein synthesis of an intracellular protein
factor and activated protein kinase C directly regulates this splicing.
The final targets of the splicing cascade would be the splicing factors involved with spliceosome-containing trans-acting factors,
such as SR proteins, which are required in the early stages of
spliceosome assembly for splice site selection. Although each splicing
machine involved in the various types of muscle-specific alternative
splicing should have its own splicing factors, muscle-specific
alternative splicings were coordinately induced by such protein
factors.
We compared the acidic induction system for muscle-specific alternative splicing in mouse myoblasts and MyoD-transfected fibroblasts with the usual low-serum stimulation system induced in differentiation medium. The striking difference is that myogenin and MEF2 are not required for muscle-specific splicing regulation. MyoD is required for muscle-specific alternative splicings in many genes induced by either low-serum or acidic stimulation at an early stage of myogenesis. Thus, we identified a common signal cascade for muscle-specific alternative splicing different from that of myotube formation (Fig. 8). In future investigations, we must find a direct effector for the formation of muscle-specific spliceosomes during myogenesis.
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ACKNOWLEDGEMENTS |
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We thank Dr. H. Weintraub for providing mouse
MyoD cDNA, Dr. S. Tominaga for the mouse BALB/c 3T3 cDNA
library, Dr. H. Hayakawa for pcDEB-hyg and human
6-o-methylguanine-DNA methyltransferase, and Dr. Y. Takebe
(National Institute of Infectious Disease) for the SR
promoter.
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FOOTNOTES |
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* This work was supported by grant-in-aids from the Ministry of Education, Science, and Culture of Japan and the Cell Science Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequences reported in this paper have been submitted to the GenBankTM/EMBL/DDBJ Data Bank with accession numbers D21073 and U43893.
¶ To whom correspondence should be addressed. Tel.: 81-285-44-2111 (ext. 3151); Fax: 81-285-44-1827; E-mail: hendo{at}jichi.ac.jp.
Present address: National Institute of Bioscience and Human
Technology, Agency of Industrial Science, Tsukuba, Japan.
1
The abbreviations used are: SR,
serine/arginine-rich; bHLH, basic helix-loop-helix; MEF2,
myocyte-specific enhancer factor 2; -TM,
-tropomyosin; N-CAM,
neural-cell adhesion molecule; MSD, muscle-specific domain;
F1
, ATP synthase
-subunit; RT-PCR, reverse
transcription-polymerase chain reaction; hMGMT, human 6-o-methylguanine-DNA methyltransferase; TGF-
,
transforming growth factor
.
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REFERENCES |
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