RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) β-Exon Splicing

Article (PDF Available)inJournal of Biological Chemistry 285(44):33779-87 · October 2010with40 Reads
DOI: 10.1074/jbc.M110.124255 · Source: PubMed
Abstract
Mammalian MBNL (muscleblind-like) proteins are regulators of alternative splicing and have been implicated in myotonic dystrophy, the most common form of adult onset muscular dystrophy. MBNL3 functions as an inhibitor of muscle differentiation and is expressed in proliferating muscle precursor cells but not in differentiated skeletal muscle. Here we demonstrate that MBNL3 regulates the splicing pattern of the muscle transcription factor myocyte enhancer factor 2 (Mef2) by promoting exclusion of the alternatively spliced β-exon. Expression of the transcriptionally more active (+)β isoform of Mef2D was sufficient to overcome the inhibitory effects of MBNL3 on muscle differentiation. These data suggest that MBNL3 antagonizes muscle differentiation by disrupting Mef2 β-exon splicing. MBNL3 regulates Mef2D splicing by directly binding to intron 7 downstream of the alternatively spliced exon in the pre-mRNA. The RNA binding activity of MBNL3 requires the CX7CX4–6CX3H zinc finger domains. Using a cell culture model of myotonic dystrophy and myotonic dystrophy patient tissue, we have evidence that expression of CUG expanded RNAs can lead to an increase in MBNL3 expression and a decrease in Mef2D β-exon splicing. These studies suggest that elevating MBNL3 activity in myogenic cells could lead to muscle degeneration disorders such as myotonic dystrophy.
RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts
Myocyte Enhancer Factor 2 (Mef2)
-Exon Splicing
*
Received for publication, March 17, 2010, and in revised form, July 22, 2010 Published, JBC Papers in Press, August 13, 2010, DOI 10.1074/jbc.M110.124255
Kyung-Soon Lee
,YiCao
§
, Hanna E. Witwicka
, Susan Tom
, Stephen J. Tapscott
§
, and Edith H. Wang
‡1
From the
Department of Pharmacology, University of Washington, Seattle, Washington 98195-2780 and the
§
Division of Human
Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
Mammalian MBNL (muscleblind-like) proteins are regula-
tors of alternative splicing and have been implicated in myo-
tonic dystrophy, the most common form of adult onset muscular
dystrophy. MBNL3 functions as an inhibitor of muscle differen-
tiation and is expressed in proliferating muscle precursor cells
but not in differentiated skeletal muscle. Here we demonstrate
that MBNL3 regulates the splicing pattern of the muscle tran-
scription factor myocyte enhancer factor 2 (Mef2) by promoting
exclusion of the alternatively spliced
-exon. Expression of the
transcriptionally more active ()
isoform of Mef2D was suffi-
cient to overcome the inhibitory effects of MBNL3 on muscle
differentiation. These data suggest that MBNL3 antagonizes
muscle differentiation by disrupting Mef2
-exon splicing.
MBNL3 regulates Mef2D splicing by directly binding to
intron 7 downstream of the alternatively spliced exon in the
pre-mRNA. The RNA binding activity of MBNL3 requires the
CX
7
CX
4–6
CX
3
H zinc finger domains. Using a cell culture model
of myotonic dystrophy and myotonic dystrophy patient tissue,
we have evidence that expression of CUG expanded RNAs can
lead to an increase in MBNL3 expression and a decrease in
Mef2D
-exon splicing. These studies suggest that elevating
MBNL3 activity in myogenic cells could lead to muscle degen-
eration disorders such as myotonic dystrophy.
Myotonic dystrophy (DM)
2
is an autosomal dominant neu-
romuscular degenerative disease that is characterized by myo-
tonia (muscle hyperexcitability), skeletal muscle weakening
and wasting, cardiac conduction defects, as well as non-muscle-
related symptoms including insulin resistance, cataract forma-
tion, testicular atrophy, and mental impairment (1). There are
two genetic forms of myotonic dystrophy. Type 1 (DM1)
accounts for nearly 98% of cases and is caused by CTG repeat
expansions in the 3-UTR of the Dmpk (dystrophic myotonic
protein kinase) gene (2– 4). DM2 results from a CCTG expan-
sion in the first intron of the Znf9 (zinc finger 9) gene (5).
Because the expanded repeats occur in the noncoding region of
two completely unrelated genes, the current thinking of DM
pathogenesis revolves around the expression and accumulation
of the mutant RNA transcripts in the nuclei of diseased cells.
Mbl (muscleblind) is a gene required for terminal muscle
differentiation in Drosophila melanogaster (6). Mbl mutant flies
are unable to properly organize the Z-bands in the sarcomeric
apparatus, resulting in paralysis and embryonic lethality (6). In
vertebrates, the MBNL (muscleblind-like) proteins are encoded
by three genes: Mbnl1 (formally Mbnl/Exp42), Mbnl2 (Mbll),
and Mbnl3 (Chcr/Mbxl) (7). MBNL proteins are regulators of
alternative splicing and contain four CX
7
CX
4–6
CX
3
H zinc fin-
ger domains that confer RNA binding activity (8, 9). MBNL1 is
thought to promote muscle differentiation (10). We have
reported that MBNL3 functions in an opposing manner and
inhibits muscle formation (11, 12). More recently, MBNL2 was
discovered to participate in integrin
-3 subcellular localization
(13).
The intersection of MBNL proteins with myotonic dystro-
phy became evident when endogenous MBNL1 and MBNL2
were shown to colocalize with CUG expanded transcripts that
are genetically linked to the disease (14 –16). Subsequent stud-
ies in mouse models demonstrated that inactivation of MBNL1
resulted in splicing defects of different pre-mRNAs that could
account for the myotonia, cardiac conduction defects, and
insulin resistance characteristic of DM1 (17–19). However,
mice lacking functional MBNL1 or MBNL2 protein do not
experience the muscle weakening and wasting characteristic of
DM patients (20, 21). Therefore, it remains to be determined
what is responsible for the DM-associated skeletal muscle
degeneration, one of the major causes of patient mortality.
We have found that expression of MBNL3 in proliferating
C2C12 myoblasts inhibits transcription of muscle-specific
genes, including a number of myocyte enhancer factor 2 (Mef2)
target genes (12). Mef2 is an important family of transcription
factors that work in concert with MyoD to regulate muscle gene
transcription (22). There are four Mef2 proteins in vertebrates,
known as Mef2A, Mef2B, Mef2C, and Mef2D, each encoded by
a unique gene (23–26). All of the Mef2 proteins contain a com-
mon DNA-binding and dimerization domain, but the tran-
scripts are subject to alternative splicing within their transacti-
vation domain. Between exons 6 and 7, each Mef2 gene has a
short and highly conserved
-exon that is alternatively spliced
into the mature message (27). Inclusion of the
-exon produces
a Mef2 isoform that is more robust in activating Mef2-respon-
sive promoters (27). It has been reported that Mef2 mRNAs
containing the
-exon are expressed predominantly in striated
muscle and in the brain and that inclusion of the
-exon into
the Mef2 transcript is promoted during muscle differentiation
(27). Therefore, proper control of the activation potential and
* This work was supported, in whole or in part, by National Institutes of Health
Grant AR049042. This work was also supported by University of Washing-
ton Royalty Research Fund Grant 4176.
1
To whom correspondence should be addressed: 1959 NE Pacific St., Box
357280, Seattle, WA 98195-7280. Fax: 206-685-3822; E-mail: ehwang@u.
washington.edu.
2
The abbreviations used are: DM, myotonic dystrophy; Mef, myocyte
enhancer factor; ZFmt, zinc finger mutant.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 44, pp. 33779 –33787, October 29, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
OCTOBER 29, 2010 VOLUME 285 NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 33779
at Univeristy of Massachusetts Medical Center/The Lamar Soutter Library, on January 5, 2011www.jbc.orgDownloaded from
expression level of the Mef2 myogenic transcription factors
plays a critical role in muscle differentiation.
We report here that MBNL3 selectively binds to Mef2D
intron 7 sequences and functions as a silencer of
-exon splic-
ing during muscle differentiation. In a cell culture model of
myotonic dystrophy and DM skeletal muscle tissue, a decrease
in Mef2D
-exon splicing was accompanied by an increase in
MBNL3 expression. Interestingly, no change in MBNL1 pro-
tein expression was observed. These data suggest that an
increase in MBNL3 activity may be a hallmark of DM muscle
and may play a role in the skeletal muscle degeneration experi-
enced by myotonic dystrophy patients.
EXPERIMENTAL PROCEDURES
Cell Culture—C2C12 cells were maintained in DMEM with
10% FBS, penicillin/streptomycin, 2 m
ML-glutamine in a
humidified incubator at 37 °C in 5% CO
2
. C2C12 control myo
-
blasts and C2C12-MBNL3 myoblasts expressing Myc-tagged
MBNL3 are stable cell lines that have been described previously
(11). The stable cell lines were cultured under the same condi-
tions as C2C12 cells but in the presence of 800
g/ml G418.
CTG5 and CTG200 cells were generously provided by the
Mahadevan lab and have been characterized previously (28).
Maintenance of the CTG5 and CTG200 cells required the addi-
tion of 800
g/ml of G418 to the culture medium. Differentia-
tion of myoblasts into myotubes was achieved by culturing
cells, which had reached 70 80% confluency, into DMEM con-
taining 2% horse serum, penicillin/streptomycin,
L-glutamine,
and ITS liquid supplement (10 ng/ml insulin, 5.5
g/ml trans-
ferrin, and 10 ng/ml selenium). The cells were maintained in
differentiation medium for up to 5 days, with medium changes
every 2 days.
Retroviral Infection—Mouse Mef2D8 (without
-exon) or
Mef2D5 (with
-exon) in pclBabe vector (gift from Dr. Stephen
Tapscott) were packaged into retrovirus by transfection into
Phoenix Ampho cells (derived from T293 cells). Viral infection
of C2C12-MBNL3 stable cells was carried out at a multiplicity
of infection of 5 with 8
g/ml polybrene. Colonies of virally
infected C2C12-MBNL3 cells were selected by culturing in
medium containing 4
g/ml of puromycin. Drug-resistant cells
were maintained in 2
g/ml puromycin and 600
g/ml G418.
Analysis of Mef2
-Exon Splicing—Total RNA was purified
from cells cultured under growth and differentiation condi-
tions using the RNeasy mini kit (Qiagen) according to the man-
ufacturer’s instructions. Total RNA was extracted from human
tissues using TRIzol according to the manufacturer’s protocol.
The RNA yield was determined by measuring A
260
. RNA sam
-
ples were subjected to RT-PCR using the StrataScript one-tube
RT-PCR system (Stratagene). The splicing of the
-exon into
endogenous mouse Mef2 transcripts was determined using the
following primers: forward, 5-gatctgcgggtcatcacttc-3; and
reverse, 5-cgagtgggtagactgggaga-3. Transcripts derived from
C2C12 cells transfected with pcDNA3.0/Mef2D(6-b-7) mini-
gene construct (kindly provided by Dr. A. Berglund) were
detected using the T7 primer as the forward primer and the
reverse primer for mouse Mef2 described above. The primers
for detecting human Mef2D were: forward, 5-tacccacagcac-
ccagctt-3; and reverse, 5-tagactgggagacccaagg-3. The
Mef2DT5 mutant minigene was constructed as follows. A
unique ClaI restriction site was introduced into the WT mini-
gene using primers 5-cacaacattggaatcgatgacacaagcctaatc-3
and 5-gattaggcttgtgtcatcgattccaatgttgtg-3. The mutant plas-
mid was linearized with ClaI, blunt-ended with DNA polymer-
ase Klenow fragment, and digested with PmlI to drop out the T5
segment of intron 7. The 7-kb fragment lacking intron 7
sequence was gel-purified and recircularized. The boundaries
of the T5 deletion was confirmed by DNA sequencing.
Immunocytochemistry—The cells were fixed with 2%
paraformaldehyde, permeabilized with 0.5% Triton X-100, and
blocked with 1% normal goat serum solution in PBS. Primary
and secondary antibodies were diluted in 1% goat serum, PBS
solution. The primary antibody against myosin heavy chain,
mAb MF20 (gift from Dr. S. Hauschka), was used at a dilution of
1:100. The primary antibody reactions were incubated over-
night at 4 °C. Fluorescein-conjugated secondary antibodies
were diluted 1:1000 and applied for2hatroom temperature.
DAPI staining was used to visualize nuclei. The samples were
mounted with Vectashield and visualized using a Nikon Eclipse
E600 microscope. Two different filters (UV-2A and FITC-
HYQ) were employed to capture the stained images.
Ribonucleoprotein Immunoprecipitation—pIRES-puro Glue
vector for expression of TAP-tag proteins was obtained from R.
Moon (University of Washington). Full-length cDNA for
mouse MBNL3 was PCR-amplified and cloned downstream of
the tandem affinity tags. Three 10-cm plates of C2C12 cells
were transfected with TAP-tag MBNL3 expression plasmid.
Forty-eight hours post-transfection, formaldehyde was added
to the medium at a final concentration of 1% and incubated at
37 °C for 10 min. The cells were washed with ice-cold PBS three
times and lysed in 1 ml of lysis buffer (50 m
M Tris, pH 7.5, 1%
Nonidet P-40, 0.05% sodium deoxycholate, 0.05% SDS, 1 m
M
EDTA, 150 mM NaCl) containing protease inhibitors and
RNasin (20 units; Promega). The cells were sonicated three
times 20 s (amplitude setting was 5). The resulting lysates were
cleared by centrifugation and incubated with streptavidin-aga-
rose (Novagen) overnight at 4 °C. The beads were washed 10
times with washing buffer (50 m
M Tris, pH 7.5, 1% Nonidet
P-40, 1% sodium deoxycholate, 0.1% SDS, 1
M NaCl, 2 M urea)
and then resuspended in 100
lof50mM Tris, pH 7.5, 5 mM
EDTA, 10 mM DTT, and 1% SDS and incubated at 70 °C for 45
min to reverse the cross-linking. The released RNA was puri-
fied using TRIzol according to the manufacturer’s protocol
(Invitrogen). Isopropanol precipitation was then performed in
the presence of Glycoblue (Ambion). The precipitated RNA
was resuspended in RNase-free water and digested with DNase
I for 45 min at 37 °C. DNase I was inactivated by the addition of
2.5 m
M EDTA and a 10-min incubation at 65 °C. RNA samples
were subjected to RT-PCR using StrataScript kit (Stratagene).
T2, T4, T5, and T6 primer sets are provided in Table 1.
In Vitro Transcription of RNA Probes for UV Cross-linking
Sense and antisense oligonucleotides (Table 1) were synthe-
sized to PCR-amplify fragments that collectively spanned the
Mef2D
-exon and intron 7. PCR fragments were subcloned
into pcDNA3.0 as HindIII-NotI fragments. The resulting plas-
mids were linearized by NotI and gel-purified. Linearized plas-
mids were transcribed in the presence of [
-
32
P]UTP (specific
MBNL3 Inhibits Mef2
-Exon Splicing
33780 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 NUMBER 44 OCTOBER 29, 2010
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activity, 800 Ci/mmol) using T7 RNA polymerase to generate
uniformly radiolabeled RNA. In vitro synthesized RNAs were
electrophoresed through 6% denaturing polyacrylamide gels
(0.5 TBE) at 200 volts for 30 min. Radiolabeled RNAs of the
expected size were gel-purified and used in UV cross-linking
experiments.
Expression and Purification of His-tagged MBNL3 Proteins
The cDNA for WT-MBNL3 and zinc finger mutant were
cloned into pET28 bacterial expression vector. Protein expres-
sion was carried out in BL21 cells as follows. Overnight cultures
(5 ml) were used to inoculate 200 ml of LB 30
g/ml kana-
mycin, and the cultures were grown at 37 °C to A
590
1.0.
Protein expression was induced with 0.1 m
M isopropyl
-D-
thiogalactopyranoside, and the cells shifted to 16 °C for 16 –18
h. For protein purification, the cells were pelleted, frozen with
liquid N
2
, resuspended, and thawed in 10 ml of 0.4 M HEMG
buffer (25 m
M HEPES, pH 7.9, 0.5 mM EDTA, 12.5 mM MgCl
2
,
10% glycerol, 0.4
M KCl) containing 5 mM
-mercaptoethanol
and protease inhibitors. The cells were lysed by sonication and
centrifuged at 10,000 rpm for 15 min at 4 °C. The clarified
supernatant was incubated with nickel-agarose beads. The
bound beads were washed with 10 column volumes of 0.4
M
HEMG 10 mM imidazole followed by 10 column volumes of
25 m
M imidazole in 0.4 M HEMG. Bound proteins were eluted
with 0.4
M HEMG 200 mM imidazole, and peak fractions were
dialyzed against 0.2
M HEMG. Purified proteins were aliquoted
and stored at 80 °C.
UV Cross-linking Experiments—RNA binding reaction (10
l) containing 200 ng of purified His-MBNL3 and
32
P-labeled
RNA in 20 m
M Tris-HCl, pH 7.5, 175 mM NaCl, 5 mM MgCl
2
,
1.25 m
M
-mercaptoethanol, 12.5% glycerol, 0.2 mg/ml BSA,
and 2
g of tRNA was incubated for 30 min at room tempera-
ture. For UV cross-linking, the binding reactions were trans-
ferred to 72-well microtiter dish, precooled on ice, and exposed
to 254-nm UV light for 15 min. The cross-linked samples were
transferred to 1.5-ml tubes containing 1.5
l of 10 mg/ml
RNase A and incubated at 37 °C for 10 min. The samples were
subjected to 12% SDS-PAGE after heat denaturation. The
resulting SDS-polyacrylamide gel was dried, and cross-linked
RNA-protein complexes were detected by autoradiography.
Preparation of Whole Cell Lysates —The cells were washed
twice with PBS and resuspended in cell lysis buffer (50 m
M
Tris-HCl, pH 7.5, 400 mM NaCl, 1% Nonidet P-40, 1 mM DTT,
1m
M PMSF, 1
g/ml aprotinin, 1
g/ml leupeptin, 1
g/ml
pepstatin). Cell lysates were incubated on ice for 20 min, with
vortexing every 5 min for 15 s. Whole cell extracts were col-
lected after centrifugation at 14,000 rpm in a microcentrifuge
for 10 min at 4 °C to remove insoluble material.
Western Blotting—Protein concentration of whole cell
extracts was determined using the Bio-Rad protein assay kit
according to the manufacturer’s instructions. Fifty to one hun-
dred micrograms of total protein were loaded per lane for SDS-
polyacrylamide gel electrophoresis. The proteins were trans-
ferred onto nitrocellulose membranes and probed with the
following primary antibodies at the indicated dilutions: 1:1000
myogenin mAb F5D (Developmental Studies Hybridoma
Bank), 1:200 myosin heavy chain mAb MF20 (gift from Dr. S.
Hauschka), 1:5 mouse MBNL3 mAb P1E7, 1:1000 MBNL1
mAb MB1a (kindly provided by Dr. G. Morris), 1:10,000
anti-Mef2D (Pharmigen), 1:10,000 GAPDH mAb 6C5 (Adv.
ImmunoChem Inc.), and 1:5000
-tubulin (Abcam). The
appropriate horseradish peroxidase-conjugated secondary
antibody was used at 1:5000 to 1:10,000 dilution. Proteins of
interest were detected by chemiluminescence (GE Healthcare).
Analysis of MBNL3 Transcript Levels—Total RNA was
extracted from human tissues using TRIzol according to the
manufacturer’s protocol. The RNA yield was determined by
measuring A
260
. RNA samples were subjected to quantitative
RT-PCR using Brilliant SYBR Green QPCR Master Mix (Strat-
agene). Quantitative RT-PCRs were carried out as described
previously with the annealing temperature increased to 62 °C
(12). MBNL3 transcript was detected using the following
human specific primers: hMBNL3(F), 5-aaacgaacgctcaaatgt-
catcacttgg-3; and hMBNL3(R), 5-cctggcattgcaagaggtg-3.
The primers used to amplify GAPDH transcripts were:
GAPDH(F), 5-cagagactggctcttaaaaagtgc-3; and GAPDH(R),
5-gtccaccaccctgttgctgta-3.
RESULTS
MBNL3 Inhibits Mef2
-Exon Splicing during Muscle
Differentiation—We previously compared the expression pat-
tern of C2C12 cells that stably express MBNL3 (C2C12-
MBNL3) to that of control C2C12 cells using DNA microarrays
(12). We observed that seven of 31 genes down-regulated by
MBNL3 contained potential Mef2 binding sites in their pro-
moter region. Mef2 transcripts are subject to alternative splic-
ing of exons coding for their transactivation domain (Fig. 1A)
(26). Inclusion of the alternatively spliced
-exon occurs pre-
dominantly in skeletal muscle and produces Mef2 isoforms that
are more robust in activating muscle gene transcription (27).
MBNL proteins have been shown to be regulators of alternative
splicing (30). Therefore, we asked whether MBNL3 could affect
the pattern of Mef2a and Mef2d
-exon splicing during muscle
differentiation.
In C2C12 control cells, we found that ()
transcripts were
detected at low levels under proliferation conditions but
became the predominant transcript after 2 days in differentia-
tion medium (Fig. 1B, n 4). This trend was observed for both
Mef2A and Mef2D. The increase in ()
transcripts was
accompanied by a reciprocal decrease in ()
transcript levels.
The splicing pattern observed in C2C12-MBNL3 cells was rad-
ically different, whereby nearly all the Mef2a and Mef2d RNAs
consisted of the ()
-exon isoform (Fig. 1B, n 4). The pro-
found bias against the
-exon was observed both in growth and
TABLE 1
Sequence of forward and reverse primers for Mef2D pre-mRNA
mMef2D primers Sequence
T2 (forward) 5-CGG AAG CTT GGG AGG TGG GAA TAA-3
T2 (reverse) 5-AAT GCG GCC GCG CAG AGT GCT CTT T-3
T3 (forward) 5-CGG AAG CTT CAA AGA GCA CTC TGC-3
T3 (reverse) 5-AAT GCG GCC GCA CTC AGC TCT ATG-3
T4 (forward) 5-CGG AAG CTT GTG CAT AGA GCT GAG T-3
T4 (reverse) 5-AAT GCG GCC GCT AGG CTT GTG TC-3
T5 (forward) 5-CGG AAG CTT CCA AGA CAC AAG CCT A-3
T5 (reverse) 5-CCC GCG GCC GCT CAC TTG TTT ATT C-3
T6 (forward) 5-CGG AAG CTT GGA ATA AAC AAG TGA CTC-3
T6 (reverse) 5-AAT GCG GCC GCA CAT GCA CAC ACA-3
MBNL3 Inhibits Mef2
-Exon Splicing
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differentiation media. These data suggest that MBNL3 pro-
motes exclusion of the
-exon from Mef2 transcripts during
muscle differentiation.
Mef2D()
Isoform Restores Muscle Differentiation to
C2C12-MBNL3 Cells—It has been previously shown that Mef2
proteins encoded by the ()
transcript are more potent acti-
vators of transcription (27). Therefore we wished to determine
whether the predominance of the less active Mef2( )
isoform
was responsible for the down-regulation of muscle gene expres-
sion in C2C12-MBNL3 cells. If so, we would expect that selec-
tive overexpression of the Mef2D()
protein would overcome
the muscle differentiation defects of C2C12-MBNL3 cells. To
test this hypothesis, we established C2C12-MBNL3 cells that
stably expressed either Mef2D()
, Mef2D()
, or the empty
vector (pclBabe) by retroviral infection and examined Mef2D
protein levels by Western blotting. A 2.5-fold increase was
detected in virally infected C2C12-MBNL3 cells when com-
pared with C2C12 or control infected cells (Fig. 2A, n 3). The
()
isoform of Mef2D differs from the ()
isoform by only
seven additional amino acids, and we were unable to resolve
this small difference in molecular weight by SDS-polyacryl-
amide gel electrophoresis.
Unable to distinguish the two Mef2D isoforms at the protein
level, we turned to monitoring mRNA levels by RT-PCR. As
shown in Fig. 2B, C2C12 (lane 1) and control infected C2C12-
MBNL3 (lane 2) cells primarily expressed the Mef2D()
tran-
script. Infection with the ()
retrovirus increased Mef2D()
mRNA levels 2-fold (Fig. 2B, lane 3). Interestingly, the
increase in ()
levels was accompanied by a decrease in ( )
mRNA levels; however, the underlying mechanism remains to
be determined. As expected, mRNA levels of the ()
isoform
increased more than 3-fold in cells infected with the ()
ret-
rovirus (Fig. 2B, lane 4).
Confident that the different cell populations were expressing
the desired Mef2D isoforms, we examined the differentiation
potential of each cell set. High levels of the muscle differentia-
tion markers myogenin and MHC were detected in control
C2C12 cells after 4 days of differentiation (Fig. 2C, lanes 1–3).
Induction of myogenin and MHC was restored in C2C12-
MBNL3 cells infected with the Mef2D()
expressing retrovi-
rus (Fig. 2C, lanes 10 –12). By contrast, expression of the
Mef2D()
isoform had no effect on the differentiation poten-
tial of C2C12-MBNL3 cells (Fig. 2C, lanes 7–9) such that the
level of myogenin and MHC was indistinguishable from that
observed for the differentiation-deficient C2C12-MBNL3 cells
infected with control virus (Fig. 2C, lanes 4 6).
The ability of Mef2D()
to rescue muscle differentiation
also was assessed by immunocytochemistry (Fig. 3). Consistent
with the Western blot results, detection of multinucleated
myotubes that stained positive for MHC was observed only in
control C2C12 cells and in C2C12-MBNL3 cells infected with
the ()
isoform of Mef2D (Fig. 3, E, F, K, and L). These find-
ings suggest that Mef2D()
is essential for muscle differenti-
ation and that the inhibitory effect of MBNL3 on muscle differ-
entiation is due in part to silencing of Mef2D
-exon splicing.
Association of MBNL3 with Intron Sequences Downstream of
the Alternatively Spliced Mef2D
-Exon in Vivo—The rescue
experiments described in Figs. 2 and 3 do not rule out the pos-
sibility that the ()
isoform of Mef2D is overriding the muscle
differentiation defect by a mechanism that is independent of
MBNL3. Therefore, we set out to gather evidence that MBNL3
is directly involved in regulating Mef2D
-exon splicing. Regu-
lators of alternative splicing often act by directly binding to the
pre-mRNA. We carried out ribonucleoprotein immunopre-
cipitation, a variation of chromatin immunoprecipitation, fol-
lowed by RT-PCR to determine whether MBNL3 binds to the
endogenous Mef2D transcript in C2C12 cells (Fig. 4). Four dif-
ferent primer sets that spanned introns flanking the
-exon
were used for RT-PCR analysis (Fig. 4A). A single PCR product
of the expected size was detected only with the T5 primer set
using the isolated RNAs as template (Fig. 4B, lane 9, n 3).
These results indicate that MBNL3 selectively associates with a
315-nucleotide region within intron 7, downstream of the
-exon, of the Mef2D pre-mRNA in vivo.
MBNL3 Directly Binds to Intron 7 of Mef2D Pre-mRNA—To
determine whether MBNL3 directly binds to the Mef2D
pre-mRNA, we carried out UV cross-linking experiments.
Fragments that collectively spanned the Mef2D
-exon and
most of intron 7 were transcribed in vitro in the presence of
-
32
P-UTP, and the resulting uniformly radiolabeled RNA
fragments were incubated with purified recombinant His-
tagged mouse MBNL3 (Fig. 5A). The U content for the RNA
fragments was calculated and did not differ dramatically, rang-
ing from 18 to 26%. The amount of input RNA for the binding
reactions was adjusted for the small differences in U content.
Purified MBNL3 bound most efficiently to T5, a RNA fragment
centrally located within intron 7, consistent with our in vivo
ribonucleoprotein immunoprecipitation results (Fig. 5B, n
4). The remaining Mef2D RNA probes showed much lower
FIGURE 1. MBNL3 promotes exclusion of
-exon from Mef2A and Mef2D
transcript during muscle differentiation. A, a schematic diagram of Mef2
alternative splicing and the relative positions of primers used for RT-PCR are
provided. The MADS box and the Mef2 domain comprise the highly con-
served DNA-binding and dimerization domains. The remainder of the protein
represents the transactivation domain. The alternatively spliced
-exon and
two forms of the
-exon are shown. B, total RNA was isolated from control
(C2C12-control) and MBNL3 (C2C12-MBNL3) expressing C2C12 cells main-
tained for 0 or 2 days in differentiation media (DM). Splicing pattern of Mef2A
and Mef2D was examined by RT-PCR using primers that can distinguish
between the ()
and ()
isoforms, as diagrammed in A. The levels of
Timm17b were measured to control for variations in input RNA. The data
shown are representative of four independent experiments.
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-Exon Splicing
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levels of interaction. MBNL3 did not bind to an unrelated RNA
fragment synthesized from the pcDNA3.0 vector, suggesting
that its RNA binding activity displays sequence specificity (Fig.
5B, pcDNA).
RNA Binding Activity of MBNL3 Requires the CX
7
CX
4–6
-
CX
3
H Zinc Finger MotifsCX
4–15
CX
4–6
CX
3
H zinc finger do
-
mains are a feature characteristic of many cellular proteins
involved in RNA processing. The high degree of amino acid
identity within the zinc finger domains of the three mammalian
MBNL proteins strongly supports the functional importance of
these motifs. Mutation of the CX
4–15
CX
4–6
CX
3
H motifs in
tristetraprolin and MBNL1, regulators of mRNA stability (31)
and alternative splicing (30), respectively, disrupts their RNA bind-
ing ability. These findings suggest that the CX
7
CX
4–6
CX
3
H
motifs may be required for MBNL3 binding to RNA. We con-
structed a bacterial expression vector for a MBNL3 zinc finger
mutant (ZFmt) in which the third cysteine of each of the four
CX
7
CX
4–6
CX
3
H motifs was changed to a serine. When com
-
parable amounts of wild-type and mutant MBNL3, as deter-
mined by Western blotting (Fig. 5C), were examined in UV
cross-linking assays, ZFmt displayed no interaction with the
Mef2D-T5 intron 7 sequence (Fig. 5D, n 4). These results
have led us to conclude that one or
more CX
7
CX
4–6
CX
3
H domains are
required for MBNL3 RNA binding
activity.
MBNL3 Directly Regulates Mef2D
-Exon Splicing—The observation
that MBNL3 interacts with the
Mef2D transcript in vivo and in vitro
supports the idea that Mef2D is
a direct target for the splicing
function of MBNL3. To investigate
this further, we used a minigene
reporter construct that contains
exons 6,
, and 7 and the interven-
ing introns of the Mef2D gene (Fig.
6A). Minigene reporters have been
instrumental in the identification of
trans-acting factors that regulate
pre-mRNA splicing (32). We took
advantage of the T7 promoter
sequence that was transcribed and
remained attached to the 5 end of
the Mef2D minigene transcript. The
presence of this short bacterial
sequence enabled us to use the T7
primer as the forward primer and a
Mef2D-specific reverse primer to
uniquely amplify spliced products
derived from the transfected plas-
mid. When the Mef2D minigene
reporter was transiently transfected
into C2C12 cells, we found that
()
splice products represented
50% of the Mef2D transcripts
detected 24 h post-transfection (Fig.
6B). No products were amplified
when C2C12 cells were transfected with the control vector
lacking any Mef2D sequence, demonstrating the specificity of
the RT-PCR conditions for exogenous Mef2D minigene tran-
scripts (data not shown).
Next, we examined the consequences of expressing wild-type
MBNL3 or the four zinc finger mutants on
-exon splicing of
transcripts derived from the Mef2D minigene (Fig. 6B). Expres-
sion of wild-type MBNL3 led to a significant decrease in ()
transcript levels in C2C12 cells compared with cells transfected
with the empty control vector. By contrast, the zinc finger
mutant (ZFmut), which lacks the ability to bind Mef2D intron
sequences, had no detectable effect on the pattern of
-exon
splicing. We confirmed by Western blotting that wild-type
MBNL3 and ZFmut protein were expressed at comparable lev-
els in the transfected cells (Fig. 6C). These data suggest that
MBNL3 inhibits
-exon splicing by binding to Mef2D
pre-mRNA.
To investigate whether MBNL3 is directly functioning at the
T5 segment of intron 7, we examined the effect of deleting this
portion of intron 7 on
-exon splicing (Fig. 6A). In the absence
of exogenous MBNL3 expression, removal of the 315-bp intron
sequence from the minigene construct (Mef2DT5) resulted in
FIGURE 2. Mef2D()
expression overcomes muscle differentiation defect of C2C12-MBNL3 cells.
A, C2C12-MBNL3 cells that constitutively express MBNL3 were infected with no virus (C2C12), the empty
control retrovirus (pclBabe), or retroviruses that express either the ()
or ()
isoforms of Mef2D. After
puromycin selection for virally infected cells, drug-resistant cells were pooled, and whole cell lysates were
prepared. Mef2D protein level was determined by Western blotting. GAPDH levels were used to control for
variations in protein load. The positions of molecular mass markers (in kDa) are shown on the left. B, total RNA
was isolated for RT-PCR analysis from C2C12 and different virally infected C2C12-MBNL3 cells. Positions of PCR
products for ()
and ()
isoforms of Mef2D are indicated. Timm17b message levels were measured to
control for input RNA. C, control C2C12 cells and virally infected drug-resistant cell pools were maintained in
differentiation media (DM) for the indicated number of days. Whole cell lysates were prepared, and the ability
of cells to execute the muscle differentiation program was determined by following two myogenic markers,
myogenin (Myog) and MHC, by Western blotting. Tubulin protein levels were used to control for total protein
load. The protein standards are indicated on the left. The data presented was observed in three independent
experiments.
MBNL3 Inhibits Mef2
-Exon Splicing
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an increase in Mef2D()
transcript levels when examined in
C2C12 cells (Fig. 6D). These data suggest that we had removed
a cis-element required for repressing
-exon splicing. Is
MBNL3 a trans-factor that functions at the T5 splicing repres-
sor element? To address this question, we examined the ability
of MBNL3 to silence
-exon splicing of pre-mRNA derived
from the WT and T5 Mef2D minigene constructs (Fig. 6D).
Expression of MBNL3 with the WT Mef2D minigene signifi-
cantly decreased the level of ()
transcripts generated. With
the T5 minigene, which lacks the MBNL3 binding site in
intron 7, MBNL3 expression exhibited no significant effect
on
-exon splicing in C2C12 cells. It was determined by West-
ern blotting that comparable levels of MBNL3 were expressed
in the presence of the WT and T5 minigene constructs (Fig.
6E). Thus, we have mapped a silencer for
-exon splicing within
intron 7 of Mef2D pre-mRNA and provide evidence that
MBNL3 is a trans-acting factor that operates at this cis-element
to promote exclusion of the
-exon from the mature Mef2D
transcript.
Mef2D
-Exon Splicing Is Disrupted in a Cell Culture Model
of Myotonic Dystrophy—MBNL1-dependent changes in the
splicing pattern of transcripts for the insulin receptor, cardiac
troponin T, and ClC-1 chloride channel have been shown to
contribute to the insulin resistance, cardiac conduction defects,
and myotonia, characteristic of myotonic dystrophy, respec-
tively (17–20). These findings led us to ask whether mis-splic-
ing of Mef2D
-exon could be contributing to DM-associated
skeletal muscle degeneration. We observed that in CTG200
cells, which represent a pool of clones that express the full-
length DMPK 3-UTR containing 200 CTG repeats down-
stream of the GFP coding region (28), the level of Mef2D()
mRNA was nearly undetectable under all conditions (Fig. 7A).
This is in stark contrast to the increase in ()
transcript levels
that was detected in the parental C2C12 cells and in the differ-
entiated CTG5 pool of cells, which express only five CTG
repeats in the DMPK 3-UTR (Fig. 7A). In addition to abnormal
Mef2D splicing, defects in myogenesis were observed in
CTG200 cells, with induction of the myogenic markers myo-
FIGURE 3. Restoration of myotube formation by Mef2D()
expression
in C2C12-MBNL3 cells. C2C12-MBNL3 cells (A–C), clonal pools of C2C12-
MBNL3 cells expressing the indicated Mef2D isoform (D–I), and control C2C12
cells (J–L) were maintained in differentiation media for 0 or 5 days. The cells
were fixed and analyzed by immunocytochemistry using an anti-myosin
heavy chain mAb MF20 (green). The nuclei were visualized by DAPI staining
(blue). Scale bars,50
m.
FIGURE 4. MBNL3 associates with intron sequences downstream of
Mef2D
-exon in vivo. A, schematic diagram of introns and exons surround-
ing the alternatively spliced Mef2D
-exon is provided. The relative positions
of RT-PCR products for different intron primer sets are shown. B, C2C12 cells
transiently transfected with TAP-tag MBNL3 expression plasmid (lane 3, 6, 9,
and 12) were cross-linked with formaldehyde. Whole cell lysates were pre-
pared, sonicated, and precipitated with streptavidin-agarose. The presence
of Mef2D intron sequences in TAP-MBNL3-bound RNAs was determined by
RT-PCR. The cells transfected with empty control vector (lanes 2, 5, 8, and 11)
were subjected to the same protocol in parallel. Results representative of
three independent experiments are shown. Lanes 1, 4, 7, and 12 (Input), RT-
PCR signal for each primer set using C2C12 total RNA as template.
FIGURE 5. MBNL3 directly binds to intron 7 of Mef2D pre-mRNA in vitro.
A, the relative positions of uniformly radiolabeled RNA segments of Mef2D
pre-mRNA transcribed in vitro for UV cross-linking experiments are shown.
MBNL3 bound efficiently to the region indicated by the thick black line in vivo.
B, bacterially expressed His-tagged MBNL3 was purified by nickel-agarose
affinity chromatography. Purified MBNL3 was incubated with the indicated
radiolabeled RNA fragments. Protein-RNA interactions were covalently cross-
linked. The resulting stable complexes were separated on SDS-polyacryl-
amide, and protein bound to the radiolabeled probe was detected by auto-
radiography (lower panel). An aliquot of each RNA probe is shown (upper
panel). The data presented are representative of four independent experi-
ments. C, comparable amounts of purified WT MBNL3 and ZFmt were sub-
jected to SDS-PAGE followed by Western blotting using the anti-MBNL3 mAb
P1E7. The positions of molecular mass markers (in kDa) are shown on the left.
D, RNA binding of WT and ZFmt MBNL3 proteins to the T5 fragment of Mef2D
pre-mRNA was examined by UV cross-linking (n 7) as described in B.
MBNL3 Inhibits Mef2
-Exon Splicing
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genin and MHC dramatically compromised (Fig. 7 B). The phe-
notype of CTG200 cells is similar to that of C2C12-MBNL3
cells that constitutively express MBNL3. These data suggested
that MBNL3 may be contributing to the skeletal muscle pathol-
ogy of DM1 and could be overexpressed in the diseased cells.
The expression level of the splicing regulator CUGBP is
increased in DM patient tissue and in some mouse models of
DM (33, 34). These findings prompted us to examine the
expression levels of MBNL3 and MBNL1 in our cell culture
model. We generated a monoclonal antibody, mAb P1E7, that
specifically recognizes endogenous mouse MBNL3 in C2C12
whole cell lysates and does not cross-react with mouse
MBNL1.
3
mAb P1E7 was used to monitor MBNL3 expression
levels in CTG5 and CTG200 cells under growth and differenti-
ation conditions. By Western blotting, we found that MBNL3
was present at higher levels (1.5–2.0-fold) in C2C12 cells
expressing the expanded DMPK 3-UTR responsible for myo-
tonic dystrophy (Fig. 7C). The elevated levels of MBNL3 were
observed when the cells were cultured in either growth or dif-
ferentiation medium. A similar analysis for MBNL1 indicated
no change and even a slight decrease in MBNL1 upon expres-
sion of the expanded CUG repeat transcript (Fig. 7C). These
findings suggest that MBNL3 differs from MBNL1 and may be
actively involved in the pathogenesis of myotonic dystrophy.
MBNL3 Expression Levels and Mef2D
-Exon Splicing Are
Altered in DM1 Skeletal Muscle—Whereas CTG200 cells dis-
play many of the properties of myotonic dystrophy cells, they
are not equivalent to diseased cells. In addition, the inability to
detect differentiation-dependent
-exon splicing in CTG200
cells in culture could be due to the fact that the cells fail to
execute the muscle differentiation program and thus do not
undergo the exon switch in Mef2 splicing. Therefore, we exam-
ined MBNL3 expression levels and Mef2D
-exon splicing in
differentiated tissues obtained from myotonic dystrophy
patients (Fig. 8). Total RNA was prepared from skeletal and
cardiac muscle derived from DM and control matched patients.
MBNL3 expression was monitored by quantitative RT-PCR
and found to be 7-fold higher in DM skeletal muscle (Fig. 8A).
A smaller increase (4-fold) in MBNL3 RNA levels was
detected in DM heart tissue. The elevated expression of
MBNL3 in the DM tissue prompted us to examine the pattern
of Mef2D
-exon splicing. In normal skeletal muscle, nearly all
the Mef2D transcript detected contained the
-exon (Fig. 8B).
By contrast, very low levels of ()
transcript were expressed in
DM-derived skeletal muscle. The pattern of Mef2D
-exon
splicing did not differ between normal and DM cardiac muscle,
despite the observed increase in MBNL3 expression. These
data further support our hypothesis that MBNL3 expression is
up-regulated in myotonic dystrophy and disrupts Mef2D
-exon splicing, which contributes to the skeletal muscle weak-
ening and wasting characteristic of the disease.
DISCUSSION
The MBNL proteins are regulators of alternative splicing and
have been implicated in the splicing defects associated with the
adult onset muscular dystrophy known as myotonic dystrophy.
The majority of studies to date have focused on MBNL1, the
founding member of the mammalian MBNL protein family.
Inactivation of MBNL1 leads to misregulated splicing of the
cardiac troponin T and muscle-specific chloride channel,
which has been correlated with the cardiac conduction defects
and myotonia observed in DM (19, 20, 35). Our studies with
MBNL3 have demonstrated that MBNL3 can influence the
splicing pattern of the Mef2 muscle transcription factor family,
promoting exclusion of the
-exon from the mature message.
The resulting transcripts encode a transcriptionally less active
isoform of the Mef2 proteins. The activation of
-exon splicing
occurs as proliferating myoblasts differentiate into multinucle-
ated myotubes (27, 28). During muscle differentiation, the pro-
tein levels of MBNL3 decreased as formation of the Mef2()
transcripts increased. This inverse correlation supports our
hypothesis that MBNL3 functions as an inhibitor of Mef2
-exon splicing. To our knowledge, these findings are the first
demonstration that an MBNL protein regulates the activity of a
muscle transcription factor that acts during the early stages of
myogenesis.
The ability of the splicing machinery to determine which
splice sites to use is dictated by cis-acting elements within exons
and introns that either enhance or silence the usage of adjacent
splice sites. The two major classes of cis-regulatory elements are
3
K.-S. Lee, D. T. Campogan, K. Lewis, E. A. Wayner, and E. H. Wang, submitted
for publication.
FIGURE 6.
-Exon splicing pattern of Mef2D minigene constructs in C2C12
cells. A, schematic diagrams of WT and mutant (T5) Mef2D minigene con-
structs consisting of exons 6,
, and 7 and the intervening introns are shown.
B,WTMef2D minigene construct was cotransfected into C2C12 cells with
either pcDNA3.0FLAG vector (), an expression plasmid for the ZFmt, or the
WT-MBNL3 expression plasmid (WT). Approximately 24 h post-transfection,
total RNA was isolated, and the
-exon splicing pattern was determined by
RT-PCR. Amplified products were loaded onto native polyacrylamide gels and
detected by ethidium bromide staining. The intensity of ()
and ()
splice
products was determined using National Institutes of Health Image J soft-
ware and used to calculate the percentage of total Mef2D transcript for each
isoform. Percentages from three independent experiments (n 5) are pro-
vided. C, level of WT and ZFmt MBNL3 protein expressed from the transfected
expression vector was determined by Western blotting. Tubulin protein lev-
els were measured to control for variations in protein load. D,WTorT5
Mef2D minigene was transfected into C2C12 cells in the absence () or pres-
ence () of WT-MBNL3 expression plasmid. The samples and resulting data
were analyzed as described in B. E, expression levels of MBNL3 protein were
determined by Western blotting. Tubulin protein levels were used to monitor
for differences in protein loading. Statistically significant differences are indi-
cated by the asterisks, and the p values are provided.
MBNL3 Inhibits Mef2
-Exon Splicing
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exonic splicing enhancers and exonic splicing silencers. These
elements function by recruiting factors that interact favorably
or interfere with the splicing machinery (36, 37). The discovery
that MBNL3 association with intron sequences in the Mef2D
transcript is required for inhibiting
-exon splicing suggests
that MBNL3 may represent one such trans-acting factor. We
have preliminary tandem affinity purification results suggesting
that heterogeneous nuclear ribonucleoproteins are potential
MBNL3 binding proteins.
4
Hetero-
geneous nuclear ribonucleoproteins
often interact with exonic splicing
silencers to inhibit splicing (38, 39).
It will be of interest to determine
whether the interaction of MBNL3
with heterogeneous nuclear ribo-
nucleoproteins is required for inhi-
bition of Mef2D
-exon splicing
during muscle differentiation.
MBNL1 is expressed predomi-
nantly in skeletal and cardiac mus-
cle, whereas MBNL3 is most abun-
dant in the placenta and is present at
very low levels in muscle tissue (10,
16, 40). The discovery that MBNL3
is an inhibitor of muscle differentia-
tion led us to propose that MBNL3
and MBNL1 have opposing func-
tions during myogenesis. We found
that MBNL3 directly interacts
with and alters the splicing pattern
of Mef2 pre-mRNA. Mammalian
MBNL1, MBNL2, and MBNL3 have
similar effects on the splicing pat-
tern of cardiac troponin T and insu-
lin receptor pre-mRNA (30). These
results raise the question of whether the effect of MBNL3 on
Mef2 splicing will be unique to this muscleblind family member
and whether MBNL proteins have unique functions at select
pre-mRNAs during muscle differentiation.
MBNL3 and MBNL1 each contain four CX
7
CX
4–6
CX
3
H
zinc finger domains and differ from Drosophila Mbl, which
contains only two CX
7
CX
4–6
CX
3
H motifs (10, 11, 16). The zinc
fingers are highly conserved and are required for MBNL1 and
MBNL3 RNA binding activity (41). In addition to the zinc fin-
gers, MBNL3 also contains a proline-rich region located
between the second and third CX
7
CX
4–6
CX
3
H motifs, which is
conserved to varying degrees among the other MBNL proteins
(7). Proline-rich regions have been extensively associated with
protein-protein interactions (42). Differences in the position-
ing and spacing of the proline residues implicate different bind-
ing partners for the different MBNL proteins, which could
account for the opposing functions of MBNL1 and MBNL3 in
muscle differentiation.
Present in MBNL1 and MBNL2 but not in MBNL3 are ala-
nine-rich regions. The functional relevance of these domains in
MBNL1 and MBNL2 remains unknown. An alanine-rich region
is required for dimerization of the RNA-binding protein AUFL
and thus contributes to the binding affinity of AUFL for target
RNA (43). Intriguingly, MBNL1 has been suggested to interact
with RNA as a multimer (44).
The current model for the pathogenesis of myotonic dystro-
phy involves the sequestration of MBNL proteins by the repeat
expanded mutant RNAs that accumulate in the nuclei of DM1
4
H. E. Witwicka and E. H. Wang, unpublished data.
FIGURE 7. Increased MBNL3 protein levels accompany defects in Mef2D
-exon splicing and muscle gene
expression in a cell culture model for myotonic dystrophy. A, C2C12 clonal cell pools that express GFP fused
to the full-length DMPK 3-UTR containing either 5 (CTG5) or 200 (CTG200) CTG repeats were kindly provided by
Dr. Mani Mahadevan. These cells and control C2C12 cells were exposed to differentiation conditions, and the
splicing pattern of Mef2D was examined by RT-PCR and polyacrylamide gel electrophoresis. B, expression of
the muscle differentiation markers MHC and myogenin (Myog) were determined by Western blotting of whole
cell lysates prepared from the indicated cell lines. GAPDH protein levels were monitored to control for total
protein load. The positions of molecular mass markers (in kDa) are shown on the left. C, elevated levels of
MBNL3 but not MBNL1 in C2C12 cells expressing CUG200 repeat transcripts. Whole cell lysates were prepared
from C2C12 cells that expressed DMPK 3-UTR containing either 5 (CTG5) or 200 (CTG200) CTG repeats. The cells
were maintained in either growth (GM) or differentiation (DM) medium before the extracts were prepared.
Approximately 100
g of lysate was loaded onto SDS-polyacrylamide gels, transferred to nitrocellulose, and
subjected to Western blotting with the MBNL3 mAb P1E7 and MBNL1 mAb MB1a. Tubulin levels were used to
control for protein loading. The protein levels of MBNL3 and MBNL1 were determined using National Institutes
of Health Image J software and used to calculate the relative levels, shown below each lane, under growth and
differentiation medim conditions. The data presented were reproducibly observed in three independent
experiments.
FIGURE 8. MBNL3 expression and Mef2D
-exon splicing in muscle of DM
patients. A, MBNL3 transcript levels. Total RNA was isolated from psoas (skel-
etal) and heart (cardiac) muscle tissue obtained from two normal and two
age/gender-matched DM patients. The level of MBNL3 expression was deter-
mined by quantitative RT-PCR. GAPDH transcript levels were measured to
control for RNA input. Relative MBNL3 mRNA levels were calculated using the
conventional 2
⫺⌬⌬C
t
method (29). The p values for significant differences are
provided and indicated by the asterisks. B, Mef2D
-exon splicing. Mef2D()
and ()
transcript levels in total RNA prepared from normal and DM patient
tissues were determined by RT-PCR using human specific primers. Amplified
products were separated on polyacrylamide and visualized by ethidium bro-
mide staining. Relative intensity of ()
and ()
splice products was deter-
mined using National Institutes of Health Image J software and used to cal-
culate the percentage of total Mef2D transcripts for each isoform (n 3).
Significant differences are indicated by the p value.
MBNL3 Inhibits Mef2
-Exon Splicing
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and DM2 cells (45). The inactivation of MBNL1 leads to splicing
defects and the formation of fetal transcripts that encode for pro-
teins that are functionally inadequate in the adult. Although
MBNL3, when overexpressed as a GFP fusion protein, colocalized
with CUG expanded transcripts in DM cells, the same behavior
has not been demonstrated for the low levels of endogenous
MBNL3 present in differentiated skeletal muscle. Western blot
analysis of C2C12 cells expressing transcripts with 5 (CTG5) or
200 (CTG200) CUG repeats and skeletal muscle derived from DM
patients revealed that MBNL3 protein levels were elevated under
diseased conditions. We propose that the MBNL proteins are not
equivalent in their contribution to DM pathogenesis. MBNL3
expression levels are increased in DM skeletal muscle, similar to
what has been observed for the splicing factor CUGBP and thus
may not be subject to inactivation by sequestration. We are cur-
rently performing immunocytochemistry experiments using
MBNL3 mAb P1E7 to investigate the expression level and local-
ization of MBNL3 in normal and DM cells.
There are many examples of members of the same protein
family having functionally distinct and even opposing cellular
roles. The use of positive and negative regulators provides
greater flexibility in controlling a biological pathway. However,
upsetting the delicate balance between these stop and go signals
can lead to the development of human disease. Most likely both
the inactivation of MBNL1 and the up-regulation of MBNL3
are contributing to the pathogenesis of myotonic dystrophy.
Acknowledgments—We thank Keri Lewis for excellent technical
assistance. We also thank S. Hauschka for generously providing the
myosin heavy chain mAb MF20, M. Mahadevan for the CTG5 and
CTG200 cell lines, and A. Berglund for the Mef2D minigene plasmid.
The normal and DM human tissue samples were obtained from the
NICHD, National Institutes of Health Brain and Tissue Bank for
Developmental Disorders. We are especially grateful to S. Kloet for
critical reading of the manuscript and to other members of the Wang
lab for valuable discussions.
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