Next Article 
Molecular and Cellular Biology, July 2000, p. 4959-4969, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The 2'-5' Oligoadenylate/RNase L/RNase L Inhibitor
Pathway Regulates Both MyoD mRNA Stability and Muscle Cell
Differentiation
C.
Bisbal,1,*
M.
Silhol,1
H.
Laubenthal,1
T.
Kaluza,1
G.
Carnac,2
L.
Milligan,1
F.
Le
Roy,1 and
T.
Salehzada1,
EP 2030 and UMR 5535 CNRS, 34293 Montpellier
Cedex 5,1 and IGH CNRS UPR 1142,
34396 Montpellier Cedex 5,2 France
Received 28 December 1999/Returned for modification 16 February
2000/Accepted 6 April 2000
 |
ABSTRACT |
The 2'-5' oligoadenylate (2-5A)/RNase L pathway is one of the
enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific
protein known as RLI (RNase L inhibitor). This system has an important
role in regulating viral infection. Additionally, variations in RNase L
activity have been observed during cell growth and differentiation but
the significance of the 2-5A/RNase L/RLI pathway in these latter
processes is not known. To determine the roles of RNase L and RLI in
muscle differentiation, C2 mouse myoblasts were transfected with sense
and antisense RLI cDNA constructs. Importantly, the overexpression of
RLI in C2 cells was associated with diminished RNase L activity, an
increased level of MyoD mRNA, and accelerated kinetics of muscle
differentiation. Inversely, transfection of the RLI antisense construct
was associated with increased RNase L activity, a diminished level of
MyoD mRNA, and delayed differentiation. In agreement with these data,
MyoD mRNA levels were also decreased in C2 cells transfected with an
inducible RNase L construct. The effect of RNase L activity on MyoD
mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is
directly involved in myoblast differentiation, probably through its
role in regulating MyoD stability. This is the first identification of
a potential mRNA target for RNase L.
| |
INTRODUCTION |
The 2'-5' oligoadenylate
(2-5A)/RNase L system is an interferon (IFN)-inducible RNA degradation
pathway which is responsible for many of the antiviral and
antiproliferative effects of IFNs (37, 41).
The 2-5A pathway is composed of at least three types of enzymatic
activities: 2-5A-synthetase, 2-5A-degrading enzymes, and RNase L. 2-5A, an oligoadenylate with 2'-5' phosphodiester bonds, activates
RNase L (53), a latent endoribonuclease. Upon activation, RNase L cleaves mRNAs 3' of UpNp sequences, thus leading to the inhibition of protein synthesis (14, 21).
The activity of RNase L was originally thought to be modulated solely
by the concentration of the 2-5A activator (11, 21). Moreover, we have previously established that RNase L activity can also
be regulated by RLI (RNase L inhibitor), a protein inhibitor (5). Overexpression of the RLI cDNA in HeLa cells results in the inhibition of the IFN-activated 2-5A pathway. RLI is induced by
viruses such as encephalomyocarditis virus (EMCV) and human immunodeficiency virus (HIV), causing an inhibition of the 2-5A/RNase L
system (27, 28). The role of the 2-5A/RNase L pathway in the
selective reduction of viral mRNA during EMCV and HIV infection has
been demonstrated elsewhere (16, 25, 27).
Variations in intracellular 2-5A and 2-5A-synthetase levels have been
observed during cell growth and differentiation even in the absence of
exogenous IFN treatment. Indeed, expression of IFN-inducible proteins,
such as 2-5A-synthetase, double-standed RNA-activated protein kinase
(PKR), and p202 (a member of the "200 family" of murine proteins)
have been observed during myoblast differentiation (4, 9,
38). Recently, Kronfeld et al. (23) established the
involvement of PKR in the regulation of myogenesis using PKR
transfected C2C12 cells. Moreover, transfection studies have provided
direct proof of the role played by 2-5A-synthetase in the proliferation
of human glioblastoma (35) and NIH 3T3 cells
(51), as well as in myeloid differentiation (36).
To more precisely assess the role of the RNase L and RLI in cell
behavior, we studied their effects on muscle cell differentiation using
C2 myoblasts. These cells can be grown in proliferative medium without
activation of the myogenic program, but muscle differentiation is
induced upon reduction of growth factors in the cell culture media.
Induction of muscle-specific genes and cell fusion, resulting in the
formation of mature muscle cells known as myotubes, occurs following
the exit of these cells from the cell cycle (33, 52). These
different muscle cell events are controlled by muscle-specific
transcription factors belonging to the MyoD family (8, 30, 40,
47). The MyoD family is composed of four myogenic basic
helix-loop-helix (bHLH) transcription factors: MyoD, myogenin, Myf5,
and MRF4. At least in vitro, MyoD appears to be the master gene of
muscle skeletal differentiation, orchestrating the onset of skeletal
muscle differentiation. Indeed, even in the presence of Myf5,
inactivation of MyoD expression by a MyoD antisense strategy leads to
an inhibition of differentiation (29).
Here we show that the timing and myogenic differentiation of C2 cells
is modulated by the level of RNase L activity. In C2 cells transfected
with an RLI antisense cDNA construction (VAS), RNase L activity is
increased. In these clones, MyoD and myogenin levels are low and the
expression of 
actin and troponin T, two genes expressed at the
differentiation stage, is delayed compared to control C2 cells
transfected with an empty vector (VV). In contrast, C2 cells
overexpressing an RLI sense cDNA construct (VS) differentiate more
rapidly, as attested by the earlier kinetic expression of -actin and
troponin T. We also find that this increased differentiation capacity
is associated with high levels of MyoD and myogenin. This is likely due
to an increased stability of MyoD mRNA in these transfectants.
Importantly, this effect appears to be specific since the half-life of
other mRNAs was not altered. Finally, in C2 cells which express RNase L
after induction by IPTG
(isopropyl-
-D-thiogalactopyranoside), MyoD mRNA levels
are greatly reduced. Therefore, our work identifies the
muscle-regulatory MyoD as a potential cellular mRNA target of RNase L
and explains how RNase L and RLI might control skeletal muscle cell differentiation.
| |
MATERIALS AND METHODS |
Cells and cell extracts.
Permissive C2 myoblasts (clone
C2.7) (a generous gift of A. Bonnieu, INRA, Montpellier, France) have
been previously described (33, 52). Proliferating myoblasts
were routinely maintained in Dulbecco modified Eagle medium (DMEM)
(Gibco-BRL) supplemented with 10% (vol/vol) fetal calf serum (growth
medium, GM) at low cell density (300 cells/cm2) and
subcultured twice a week. For differentiation experiments, cells were
plated at high density (104 cells/cm2 in GM),
and after 2 days the medium was changed to DMEM-2% fetal calf serum
(differentiating medium, DM). Cells were collected 24 h after
seeding (day 0) and at 24-h intervals thereafter for preparation of
cell extracts. At the indicated times, cells were resuspended in 2 volumes of radioimmunoprecipitation assay buffer (50 mM HEPES, pH 7.5;
400 mM NaCl; 1% [vol/vol] NP-40; 1 mM phenylmethylsulfonyl fluoride;
10 µg of aprotinin per ml; 150 µg of leupeptin per ml), disrupted
in a Dounce homogenizer, and centrifuged at 10,000 × g
(S10). The protein concentration in the supernatant (S10) was determined by spectrophotometry (48).
Expression vectors and transfections.
The coding sequence of
the human RLI cDNA (5) was directionally subcloned in
pcDNA3neo (Invitrogen) by standard procedures (39). RLI
antisense-pcDNA3neo (7 µg), RLI sense-pcDNA3neo (7 µg), or the
empty vector pcDNA3neo (7 µg) was transfected into C2 cells by
calcium phosphate coprecipitation (39). Stable transfectants were selected by culturing the cells in the presence of 1 mg of G418
(Gibco-BRL) per ml. The yield of transfection was approximately 80%,
so we did not isolate individual clones. The polyclonal cell population
expressing the transfected antisense RLI cDNA was named VAS, the
polyclonal cell population expressing the transfected sense RLI cDNA
was named VS, and the polyclonal cell population transfected with the
empty vector was named VV.
For conditional expression of RNase L, the LacSwich II inductible
mammalian expression system (Stratagene) was used. First the pCMVLacI
repressor vector (0.5 µg of DNA) was transfected into C2 cells with
Fugene (2 µl) according to the manufacturer's instructions
(Boehringer). After selection of stable transfectants by culturing the
cells in the presence of hygromycin (200 U/ml), the expression of the
Lac repressor expression was tested by Northern blotting
(39). After transfer, the nylon sheets were incubated with a
[32P]cDNA probe corresponding to the
XbaI-StuI fragment of the LacI repressor cDNA
radiolabeled using the multiprime procedure (Gibco-BRL). After
autoradiography on a PhosphorImager 445-SI (Molecular Dynamics), the
mRNAs were quantified by image analysis with the ImageQuant program
(Molecular Dynamics). The clone expressing the greatest quantity of Lac
repressor was then transfected by calcium phosphate coprecipitation
(39) with the pOPRSVI vector harboring the coding sequence
of the human RNase L cDNA (7 µg) (53). Stable
transfectants were selected by culturing the cells in the presence of 1 mg of G418 (Gibco-BRL) per ml. For analysis, clones were treated with 5 mM IPTG for 8 h. Cells were then collected, and each clone was analyzed for RNase L expression by the 2-5A radiocovalent binding assay
(see below). One clone (RNase L3) was selected and used in further experiments.
Western blot assay.
Proteins (100 µg) were fractionated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred electrophoretically to a nitrocellulose membrane
according to the method of Towbin et al. (45). The
nitrocellulose membranes were incubated in phosphate-buffered saline
(PBS; 140 mM NaCl, 2 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4 [pH 7.4]) supplemented with 5%
(wt/vol) skimmed milk for 30 min and then soaked overnight at 4°C in
the same buffer with one of the following antibodies: anti-Myf-5
(C-20), anti-MyoD (M-318), anti-myogenin (M-225), or anti-p21 (C-19)
(all rabbit polyclonal antibodies from Santa Cruz Biotechnology) at 0.5 µg/ml; mouse monoclonal anti--sarcomeric actin (1/500 dilution),
rabbit polyclonal antiactin (1/100 dilution), mouse monoclonal
antitropomyosin (1/400 dilution), mouse monoclonal anti-troponin T
(1/1,000 dilution), all from Sigma Immuno Chemicals; rabbit polyclonal
anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH)
(1/3,000 dilution), a generous gift of G. Cathala (IGMM,
Montpellier, France); and rabbit polyclonal anti-RLI (1/500 dilution)
(28). The filters were washed with PBS supplemented with
0.05% (vol/vol) Tween 20 and incubated for 1 h at room
temperature with donkey anti-rabbit or sheep anti-mouse immunoglobulin
antibody conjugated to horseradish peroxidase (Amersham). Specific
proteins were visualized using a chemiluminescence kit (NEN). The gels were scanned, and protein bands were quantified by image analysis with
the Intelligent Quantifier program (Bio Image Systems Corp.). The
experiments were done in triplicate.
Immunofluorescence.
Each polyclonal population of
transfected C2 myoblasts and myotubes were fixed for 5 min in 3.7%
(vol/vol) formalin-PBS, followed by a 30-s extraction in 
20°C
acetone and rehydratation in PBS containing 0.5% (vol/vol) bovine
serum albumin (BSA). Expression of MyoD was analyzed using the 5.8A
mouse monoclonal antibody against MyoD diluted 1/5 (a gift of P. Dias
and P. J. Houghton [10]). Primary antibody diluted in PBS-BSA was incubated for 1 h at 37°C and then washed in PBS, followed by a 30-min
incubation with biotinylated anti-mouse antibody (1/200 dilution;
Amersham). Biotinylated antibodies were finally revealed after a 30-min
incubation with streptavidin-Texas red (1/200 dilution; Amersham). DNA
was stained with a Hoechst stain (0.1 mg/ml; Sigma). The experiments were done in triplicate.
Radiobinding and radiocovalent binding assay: affinity labeling
of RNase L with 2-5ApCp.
Transfected C2 cells were harvested at
various time points before and after differentiation, as indicated in
the legends to the figures. For radiobinding (22), cell
extracts (600 µg of protein) of VV-, VAS-, and VS-expressing cells
were incubated with 20,000 cpm of
2-5A4-3'-[32P]pCp (2-5ApCp; 3,000 Ci/mmol) on
ice for 15 min as previously described (6). The radiolabeled
RNase L was then precipitated at 20°C for 5 min by using 300 µl
of polyethylene glycol 6000 (25% [wt/vol]) after addition of 150 µl of bovine serum as a carrier. After centrifugation
(10,000 × g, 10 min), the radioactivity of the pellet
containing the 2-5ApCp bound to RNase L was measured. The experiments
were done in triplicate and the standard deviation is indicated on the plots.
For the radiocovalent binding assay (
50), the radiolabeled
2-5A
4-3'-[
32P]pCp probe was oxidized at the
3' end as previously described
(
2). Cell extracts (600 µg
of protein) from the RNase L3 clone
were incubated with the oxidized
2-5A
4-3'-[
32P]pCp probe for 30 min in ice and
for a further 20 min at room
temperature in the presence of sodium
cyanoborohydride (20 mM,
final concentration). Proteins were analyzed
by SDS-PAGE in a
10% (vol/vol) polyacrylamide gel (
24), and
the labeled proteins
were visualized after autoradiography. The gels
were scanned,
and the protein bands were quantified by image analysis
with the
Intelligent Quantifier program (Bio Image Systems Corp.). The
experiments were done in triplicate, and the standard deviation
is
indicated on the
plots.
Analysis of mRNA stability.
VV, VAS, and VS cells were
plated at 104 cells/cm2 in GM and, after 2 days, the medium was changed to DM. At day 0 (24 h after seeding,
before differentiation) or at day 4 (after the induction of
differentiation) cells were treated with actinomycin D (5 µg/ml) for
0, 30, 60, 120, or 240 min; mRNA was then prepared and analyzed by
Northern blotting (39). After transfer, the nylon sheets were incubated with different [32P]cDNA probes (as
indicated in the figure legends) synthesized by the multiprime
procedure (Gibco-BRL). After autoradiography on a PhosphorImager 445-SI
(Molecular Dynamics), the mRNAs were quantified by image analysis with
the ImageQuant program (Molecular Dynamics). Each lane was normalized
with the S26 probe (46). The experiments were done in
triplicate, and the standard deviation is indicated on the plots.
Nuclear run-on transcription assay.
Preparation of nuclei
and elongation of nascent transcripts were performed as described by
Greenberg and Ziff (15), except that heparin was added to
the incubation mixture (final concentration, 1 mg/ml) (7).
GAPDH, MyoD, Myf5, myogenin, and -actin plasmid DNA were spotted
onto nitrocellulose. Prehybridation and hybridization were performed in
4× STE (600 mM NaCl; 80 mM Tris-HCl, pH 7.5; 4 mM EDTA), 0.2%
(wt/vol) SDS, 0.1% (wt/vol) PPi, 2 mg of heparin per ml,
and 50% (vol/vol) formamide. Filters were washed in 2× SSC (1× SSC
is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS (wt/vol) for 10 min at 20°C and then in 0.2 × SSC at 65°C for 30 min. Filters
were then RNase A treated in 2× SSC for 15 min at 37°C. After
autoradiography on a PhosphorImager 445-SI (Molecular Dynamics), the
mRNAs were quantified by image analysis with the ImageQuant program
(Molecular Dynamics). The experiments were performed in triplicate, and
the standard deviation is indicated on the plots.
rRNA cleavage.
Cells were resuspended in 1 volume of buffer
(5 mM Tris-HCl, pH 7.6; 1.25% [vol/vol] glycerol; 20 mM KCl; 1.25 mM
magnesium acetate), vortexed for 1 min, and kept on ice for 10 min. The cell-buffer suspension was passed through a 1-ml tuberculin syringe and
centrifuged at 15,000 × g 2 min. The protein
concentration of the supernatant was determined by spectrophotometry
(48). Cell extracts (100 µg of protein) were incubated for
1 h at 30°C in the presence or absence of 2-5A4 (1 µM, final concentration). The total RNA was extracted, denatured, and
analyzed by electrophoresis on 1.8% agarose gel. The gels were stained
with ethidium bromide, and the RNA bands were visualized under UV light
(18-20, 49). The experiments were done in triplicate.
| |
RESULTS |
RNase L and RLI are sequentially induced during C2
differentiation.
We first studied the behavior of RLI and RNase L
during the induction of differentiation in the myogenic C2 cell line.
The cells were cultivated in either GM or growth-factor-deprived medium (DM), and cell extracts were prepared at different times. RLI was
identified by immunoblot analysis using a previously described RLI-specific polyclonal antibody (28). Because the
specificity and the affinity of RNase L for its activator, 2-5A, is
very high, and the nuclease activity of RNase L is strictly dependent
on its activation by 2-5A (12, 53), the measurement of the
binding of 2-5A to RNase L is a good indicator of the presence of
activatable RNase L. Thus, RNase L levels were followed by monitoring
its 2-5A binding activity. RLI levels did not change during the first day in DM compared to its basal level in GM. Moreover, it was induced
after 2 days in DM, with a maximal level observed at day three (Fig.
1A and B). In contrast, RNase L 2-5A
binding activity increased immediately upon culture of C2 cells in DM
but decreased by day 3 when RLI was highest (Fig. 1C).
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FIG. 1.
Increases in the RLI and RNase L proteins during the
differentiation of C2 myoblasts into myotubes. (A) C2 cells were grown
in GM and then shifted to DM on day 1. At the indicated times, cells
were harvested and lysed as described in Materials and Methods. Total
protein samples (100 µg) were analyzed for RLI protein by Western
blotting with a polyclonal RLI antiserum. (B) Densitometric analysis of
the gel shown in panel A. A value of 100% corresponds to the amount of
RLI protein in proliferating myoblasts at day 0. Error bars refer to
the standard deviation obtained in three independent experiments. (C)
C2 cells were grown in GM and then shifted to DM on day 1. At the
indicated times, cells were harvested and lysed. Proteins (600 µg)
were incubated with radiolabeled
2-5A4-3'-[32P]pCp (2-5ApCp) in a radiobinding
assay; 100% corresponds to the amount of 2-5ApCp bound to RNase L in
proliferating myoblasts. Error bars refer to the standard deviation
obtained in three independent experiments.
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Alterations in RLI levels in C2 cells modulates their
differentiation potential.
RNase L 2-5A binding activity was
induced transiently at the beginning of the differentiation process.
Concomitant with the decrease in RNase L 2-5A binding activity, RLI was
induced. The ratio between these two proteins appeared to be important
in the differentiation of C2 cells. To show the direct involvement of RLI and RNase L in the differentiation process, C2 cells were transfected with the empty pcDNA3 vector (as control cells, we used VV
cells), RLI sense-pcDNA3 (VS cells), or RLI antisense-pcDNA3 (VAS cells).
RLI and RNase L 2-5A binding activity were monitored during
differentiation as described above. The results obtained are presented
in Fig.
2. RLI and RNase L 2-5A binding
activity were equivalent
in the control (VV) and untransfected C2 cells
(data not shown).
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FIG. 2.
Increases in RLI and RNase L proteins during the
differentiation of transfected C2 cells. (A) Pooled C2 cells stably
transfected with an RLI cDNA sense construct (VS) and an RLI cDNA
antisense construction (VAS) were grown in GM and then shifted to DM on
day 1. At the indicated times, cells were lysed, and RLI expression was
assessed in protein samples (100 µg) using an RLI-specific antiserum.
(B) Densitometric analysis of the gel shown in panel A. A value of
100% corresponds to the amount of RLI protein at day 0 in
proliferating C2 control myoblasts. Error bars refer to the standard
deviation obtained in three independent experiments. Symbols:  , VS
cells;  , VAS cells. (C) The cells described in panel A were lysed,
and proteins (600 µg) were incubated with radiolabeled
2-5A4-3'-[32P]pCp (2-5ApCp) in a radiobinding
assay; 100% corresponds to the amount of 2-5ApCp bound to RNase L in
proliferating C2 control myoblasts. Error bars refer to the standard
deviation obtained in three independent experiments. Symbols: , VS
cells; , VAS cells.
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The basal level of RLI in the polyclonal RLI antisense (VAS) cell
population was 40% lower than in C2 and control cells (Fig.
2A and B),
and RNase L 2-5A binding activity was correspondingly
increased by
nearly 40% (Fig.
2C). Under differentiation conditions,
no RLI
induction was observed in these VAS transfectants, and
RNase L 2-5A
binding activity was twofold higher than that observed
in C2 and
control cells (Fig.
2C).
The basal level of RLI in the polyclonal RLI sense (VS) cell population
was twofold higher than that detected in C2 and control
cells, and
during differentiation RLI levels remained increased
(Fig.
2A and B).
Consequently, although there was an increase
in RNase L 2-5A binding
activity during differentiation, this
level was lower than that
observed in the control and RLI antisense
(VAS) cells (Fig.
2C).
We checked the RLI mRNA level in these different transfectants. In
control (VV) cells, RLI mRNA was induced during C2 differentiation
(Fig.
3A). In contrast, the level of RLI
mRNA was significantly
lower in the polyclonal RLI antisense (VAS) cell
population, indicating
that the transfected antisense cDNA exercised
its inhibitory effect
at the mRNA level (Fig.
3B). In the polyclonal
RLI sense (VS)
cell population, expression of the endogenous RLI mRNA
(3.5 kb)
(
3) and the transfected RLI cDNA containing only
the coding
sequence of RLI (1.8 kb) were observed (Fig.
3C). In these
latter
cells, expression of the endogenous RLI mRNA was induced during
C2 differentiation in a manner similar to that observed in control
cells. In contrast, expression of the transfected RLI cDNA was
stable
during differentiation.
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FIG. 3.
RLI mRNA levels increase during the differentiation of
transfected C2 cells. Pooled stable control C2 cells transfected by an
empty pcDNA3 vector (VV) (A), pooled stable C2 cells transfected with
an RLI cDNA antisense construction (VAS) (B), and pooled stable C2
cells transfected with an RLI cDNA sense construction (VS) (C) were
grown in GM and then shifted to DM on day 1. At the indicated times,
cells were harvested, and mRNAs were extracted and analyzed by Northern
blot using the murine RLI [32P]cDNA probe (VV, VAS, and
VS [ ]). The VS samples were also probed with a human RLI
[32P]cDNA fragment () as indicated. Densitometric
analysis of the gels are presented. A value of 100% corresponds to the
amount of RLI mRNA in proliferating C2 control myoblasts at day 0. Error bars refer to standard deviation obtained in three independent
experiments.
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These different levels of RLI mRNA and protein resulted in a wide range
of RNase L 2-5A binding activities, as expected (Fig.
2). To determine
whether the nuclease activity of RNase L was
also modified in these
different transfectants, the nuclease activity
of RNase L was monitored
in vitro by studying the cleavage of
rRNA. Activation of RNase L by
2-5A gives rise to a specific pattern
of degradation of rRNA (
42,
49). As illustrated in Fig.
4A
and
B, the cleavage of rRNA in control (VV) cells, after activation
of
RNase L, was visible at days 2 and 3. The nuclease activity
of RNase L
was visible in the RLI antisense (VAS) cell population
even in the
absence of exogenous 2-5A (Fig.
4C and D). It is likely
that because
the level of RLI was so low in these cells, the basal
level of 2-5A in
the cell was sufficient to activate RNase L.
In contrast, in the
polyclonal RLI sense (VS) cell population,
no cleavage of rRNA was
observed even after the addition of exogenous
2-5A (Fig.
4E and F).
Thus, the levels of RLI and RNase L activity
in these polyclonal cell
populations varied greatly both before
and during differentiation.
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FIG. 4.
Variations in RNase L activity following transfection
with RLI sense and antisense constructs. Pooled stable control C2 cells
transfected with an empty pcDNA3 vector (VV, panels A and B), the RLI
antisense cDNA (VAS, panels C and D) and the RLI sense cDNA (VS, panels
E and F) were seeded in GM and then shifted to DM (day 1). Cells were
harvested at the indicated times. Proteins (100 µg) were incubated
for 60 min at 30°C in the absence (A, C, and E) or presence (B, D,
and F) of 2-5A4 (1 µM, final concentration). rRNAs were
analyzed on 1.8% (wt/vol) agarose gels. Intact 28S and 18S rRNA
migration is indicated at the left of each gel. Major rRNA degradation
products are indicated by arrows.
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To establish whether variations in the levels of RLI and RNase L
activity affect myogenic differentiation, the appearance
of
muscle-specific proteins and muscle-specific transcription
factors was
studied in these three polyclonal cell populations
(VV, VAS, and VS).
Initially, the differentiation process was
monitored by studying the
expression of housekeeping and myogenesis-specific
proteins by Western
blot. As illustrated in Fig.
5, no
differences
were observed for constitutively expressed ubiquitous
proteins
such as GAPDH, actin, and tropomyosin in the three C2
transfected
populations. The same result was observed for p21, a
protein which
is not myoblast specific but whose expression is
regulated during
the cell cycle. This protein was strongly induced to
the same
level in the three transfected populations at day 2 (Fig.
5).
In contrast, when we studied the structural proteins, there was
clearly
a difference in the kinetics of expression of proteins
whose expression
is regulated during myogenesis.
-Actin expression
was observed 1 day
later in RLI antisense (VAS) cells compared
to control (VV) cells,
while its level was maximal at an earlier
time point in RLI sense (VS)
cells (Fig.
5). Troponin T levels
were 3.5 times lower in RLI antisense
(VAS) cells compared to
control (VV) cells at day 2. Notably, its
expression was noted
as early as 1 day following differentiation in RLI
sense (VS)
cells (Fig.
5). Nevertheless, while the kinetics of
expression
of
-actin and troponin T differed in these populations,
their
maximal levels of expression were equivalent.
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FIG. 5.
Kinetics of protein expression in differentiation C2
cells. Pooled stable control C2 cells transfected with an empty pcDNA3
vector (VV), the RLI sense cDNA (VS), and the RLI antisense cDNA (VAS)
were seeded in GM and then shifted to DM (day 1). Cells were harvested
at the indicated times. Proteins (100 µg) were analyzed by Western
blotting with the different antisera as indicated.
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Regarding the bHLH factors, Myf5 levels were comparable in RLI sense
(VS) cells, control (VV) cells, and RLI antisense (VAS)
cells in GM.
After the shift to DM, Myf5 induction was delayed
by 1 day in RLI
antisense (VAS) cells. A more striking difference
was observed for MyoD
and myogenin. The level of MyoD was greatly
increased in RLI sense (VS)
cells compared to control (VV) cells.
In RLI antisense (VAS) cells,
MyoD protein was almost undetectable
even after the shift to DM, but it
was observed at day 2, 1 day
after maximal levels were detected in
control (VV) and RLI sense
(VS) cells. This difference in MyoD protein
expression was confirmed
by immunofluoresence using another antibody
(Fig.
6). The MyoD
protein was
overexpressed in RLI sense (VS) cells cultured either
in GM or in DM.
Additionally, MyoD protein was practically undetectable
in RLI
antisense (VAS) cells cultured in GM.
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FIG. 6.
Decreased expression of MyoD in VAS cells and
overexpression of MyoD in VS cells. Each polyclonal population of
transfected C2 (VV, VAS, and VS) myoblasts grown in GM and myotubes
differentiated in DM were fixed as described in Materials and Methods.
Expression of MyoD was analyzed using the 5.8A mouse monoclonal
antibody against MyoD. DNA was stained with Hoechst stain (0.1 mg/ml).
|
|
Similary, the myogenin level was high in RLI sense (VS) cells. Myogenin
was already present in RLI sense (VS) cells before
differentiation
(days 0 and 1) and reached five- and twofold-higher
levels than that
detected in RLI antisense (VAS) and control (VV)
cells, respectively.
In RLI antisense (VAS) cells, the myogenin
level was very low and
remained low even after the beginning of
differentiation (Fig.
5).
Therefore, changes in MyoD and myogenin
levels, as well as a modulation
of the differentiation capacity,
were major consequences of varying RLI
levels in C2
cells.
MyoD mRNA stability is dependent on the modification of RNase L
activity.
The 2-5A/RNase L system is an RNA degradation pathway.
Variations in MyoD and myogenin proteins could be due to a degradation of their mRNAs by RNase L. To test this hypothesis, we measured the
stability of different mRNAs in transfected C2 cells. At day 0, under GM conditions, and at day 4, after the shift to DM when RNase L
activity decreases, all transfected C2 cells were treated with
actinomycin D in order to measure the half-life of the mRNAs. Cells
were collected at different times after the addition of actinomycin D,
and mRNAs were extracted and analyzed by Northern blotting (Fig.
7). We found that while the half-life of
actin, GAPDH, S26, -actin, Myf5, and myogenin mRNAs were equivalent in all three polyclonal populations, the half-life of the MyoD mRNA was
modified. In GM conditions, the half-life of the MyoD mRNA in RLI
antisense (VAS) cells could not be determined because the mRNA was not
detectable. In control (VV) cells, the half-life of MyoD mRNA was
approximatively 90 min, a result similar to the results reported in P2
cells by Thayer et al. (43). In RLI sense (VS) cells, MyoD
mRNA was stabilized, with a half-life of approximately 200 min (Fig.
8A). After the decrease in RNase L
activity, at day 4, MyoD mRNA was detectable in RLI antisense (VAS)
cells, and its half-life was approximately 50 min (Fig. 8B).
Collectively, these data demonstrate that MyoD mRNA stability was
associated with RNase L activity.
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|
FIG. 7.
MyoD mRNA stability is altered by the expression of RLI.
Each polyclonal population of transfected C2 (VV, VAS, and VS)
myoblasts grown in GM and myotubes grown in DM were treated with
actinomycin D (5 µg/ml). At the indicated times, cells were harvested
and the mRNAs were extracted and analyzed by Northern blotting with the
different [32P]cDNA probes. mRNAs were visualized on a
PhosphorImager 445-SI (Molecular Dynamics).
|
|
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 8.
The half-life of the MyoD mRNA is altered by the
expression of RLI. Each polyclonal population of transfected C2 (VV,
VAS, and VS) myoblasts (GM) and myotubes (DM) were treated with
actinomycin D (5 µg/ml), and the mRNAs were analyzed as described in
Fig. 7. After visualization on a PhosphorImager 445-SI (Molecular
Dynamics), MyoD mRNAs were quantified by image analysis with the
ImageQuant program (Molecular Dynamics). Each lane was normalized with
the S26 probe. Densitometric analysis of the gel is presented. A value
of 100% corresponds to the amount of MyoD mRNA at time zero in each
polyclonal population of transfected C2 (VV, VAS, and VS) myoblasts
(GM) (A) (, control VV cells;  , RLI sense VS cells) or myotubes
(DM) (B) (, control VV cells;  , RLI sense VS cells;  , RLI
antisense cells). Error bars refer to the standard deviation obtained
in three independent experiments.
|
|
Given that only the half-life of the MyoD mRNA appeared to be affected,
it was important to assure that the difference observed
in MyoD mRNA
levels were really due to posttranscriptional regulation.
We therefore
performed nuclear run-on experiments to assess the
transcription rate
of these genes in the different transfectants
(Fig.
9). Importantly, transcription levels of
MyoD mRNA were
comparable in GM and DM and were roughly equal in all
three populations.
The transcription of GAPDH mRNA was approximately
equivalent in
the three populations of cells both before and after
differentiation.
There was a small increase in Myf5 mRNA transcription
in RLI sense
(VS) cells and RLI antisense (VAS) cells in GM and DM
compared
to control (VV) cells.
-Actin and myogenin mRNA
transcription
were already detectable in RLI sense (VS) cells before
differentiation
and increased during differentiation.
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 9.
Nuclear run-on transcription analysis in RLI-transfected
C2 cells. (A) Each polyclonal population of transfected C2 (VV, VAS,
and VS) myoblasts grown in GM and myotubes grown in DM was harvested,
and the nuclei were extracted as described in Materials and Methods. In
vitro-elongated RNAs were hybridized to a blot of DNAs corresponding to
MyoD, Myf5, myogenin, -actin, and GAPDH. (B) After visualization on
a PhosphorImager 445-SI (Molecular Dynamics), the mRNAs were quantified
by image analysis with the ImageQuant program (Molecular Dynamics). A
densitometric analysis of the autoradiography shown in panel A is
presented. Error bars refer to the standard deviation obtained in three
independent experiments.
|
|
Therefore, the difference observed in the level of MyoD mRNA was due to
a modification of its mRNA stability, whereas the
difference observed
in myogenin mRNA levels was likely due to
a modification of its
transcription.
Decreased level of MyoD mRNA in C2 cells overexpressing RNase
L.
To more precisely study the role played by RNase L in MyoD
expression, we modified RNase L activity in C2 cells using another method. C2 cells were transfected with a vector from which RNase L was
expressed upon induction with IPTG. First, C2 cells were transfected
with a vector allowing constitutive expression of the Lac repressor.
Clonal populations of these cells were tested for expression of the Lac
repressor and then transfected with a vector wherein RNase L expression
was under the control of the Lac operator. Under these conditions, the
Lac repressor binds to the Lac operator, blocking transcription of
RNase L. Synthetic inducers such as IPTG, which bind to the Lac
repressor, effectively decrease the affinity of the repressor for the
operator, resulting in the transcription of RNase L.
In a selected C2 clone (RNase L3), RNase L expression was induced with
different concentrations of IPTG (3 and 5 mM), and
the RNase L 2-5A
binding activity was studied (Fig.
10).
IPTG induced
RNase L expression, as assessed by an induction in 2-5A
binding
activity and nuclease activity (Fig.
10A and B). As in the VAS
clone, cleavage of rRNAs was observed even in the absence of 2-5A.
In
this clone, the level of MyoD mRNA was greatly decreased (Fig.
10C),
whereas Myf5, S26, and actin mRNA levels were not affected.
Moreover,
in this clone, there was a direct correlation between
the level of
RNase L and the level of MyoD mRNA.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 10.
MyoD mRNA levels are decreased in a C2 clone
overexpressing RNase L. (A) The RNase L3 clone was treated with
increasing concentrations of IPTG during 8 h. Cells were then
harvested and analyzed for RNase L 2-5A binding activity with the 2-5A
radiocovalent binding assay. A densitometric analysis of the gel is
shown. A value of 100% corresponds to the amount of 2-5A binding
activity in proliferating RNase L3 clone myoblasts in the absence of
IPTG. Error bars refer to the standard deviation obtained in three
independent experiments. (B) RNase L rRNAs cleavage activity was
assessed in the absence or presence of 2-5A (1 µM) as described in
Fig. 4. (C) mRNAs were analyzed by Northern blotting using the
indicated [32P]cDNA probes. Bands were visualized on a
PhosphorImager 445-SI (Molecular Dynamics). A densitometric analysis of
the gels is shown (, actin mRNA; , Myf5 mRNA;  , S26 mRNA; ,
MyoD mRNA). A value of 100% corresponds to the amount of each mRNA in
proliferating RNase L3 clone myoblasts in the absence of IPTG. Error
bars refer to the standard deviation obtained in three independent
experiments.
|
|
| |
DISCUSSION |
The role performed by RLI in the control of RNase L activity
during the differentiation of cultured C2 myoblasts to myotubes is
essential. To more precisely study the role of RLI and RNase L during
muscle differentiation, the level of expression of these two proteins
was modulated. Upon expression of RLI antisense and sense cDNA
constructs in C2 cells, RNase L activity was increased (VAS cells) and
decreased (VS cells), respectively. RNase L activity was also increased
upon conditional expression of RNase L.
The data presented here indicate that RNase L activity increases during
the differentiation of cultured C2 myoblasts to myotubes. RNase L
levels were maximal at day 2 following differentiation induction and
then decreased concomitantly with the expression of its inhibitor RLI
(Fig. 1, 3, and 4). Nevertheless, the decrease in RNase L activity
could not be due solely to the induction of RLI since RNase L activity
also decreased in cells where RLI expression was severely diminished by
an RLI antisense construct (Fig. 2, 3, and 4). We were unable to
determine whether RNase L is regulated at the transcriptional level
because we failed to detect RNase L mRNA in C2 cells. This is in
agreement with prior studies showing that the level of RNase L mRNA is
very low in the absence of IFN treatment (53).
The over- or underexpression of RLI resulted in a modification of the
kinetics of appearance of myotube-specific proteins such as -actin
and troponin T (Fig. 5). The expression of these two proteins was
delayed in RLI antisense (VAS) cells (conditions where RNase L activity
was increased), whereas they were expressed earlier in RLI sense (VS)
cells (RLI was overexpressed, and RNase L activity was decreased). The
expression of such specific proteins is a consequence of an entire
differentiation program which is based on the expression of
muscle-specific transcription factors. The most striking variation
between the different transfectants was the expression of two of these
specific transcription factors: MyoD and myogenin (Fig. 5, 6, and 7).
Myogenin mRNA and protein were detectable even under nondifferentiating
conditions (GM) in RLI sense (VS) cells. In these cells, the increase
in myogenin mRNA was due to augmented transcription since the half-life
of the mRNA was not changed (Fig. 7 and 9). In contrast, the stability
of MyoD mRNA was significantly altered in cells with modified RNase L
activity (Fig. 7, 8, 9, and 10). MyoD is a muscle-specific
transcription factor that can activate downstream myogenic structural
genes and myogenic conversion in many different cell types
(32). MyoD also activates its own transcription, as well as
the transcription of myogenin. This activation is direct and does not
involve any other intermediates (17, 43). Therefore, the
modification of MyoD expression by posttranscriptional regulation
affects the level of myogenin mRNA transcripts. In cells with a high
level of MyoD (VS cells), we observed a small increase in MyoD
transcription and an important increase in myogenin transcription,
whereas in cells where the level of MyoD was low (VAS cells), MyoD
transcription was slightly decreased and myogenin transcription was
very low (Fig. 9). Consequently, the expression of other structural
genes such as -actin and troponin T which are controlled, even
indirectly, by MyoD and myogenin was also modified. Finally, it is
interesting to note that all three populations of transfectants (VV,
VAS, and VS), as well as the inductible RNase L3 clone, expressed
comparable levels of Myf5, providing additional evidence that Myf5 and
MyoD are not fully interchangeable. Previously, Montarras et al. showed that MyoD but not Myf5 is essential for the differentiation of C2 cells
(29).
Although we observed a direct correlation between the level of RNase L
activity and MyoD expression, we could not completely exclude the
possibility that MyoD is not a direct target of RNase L but rather is
indirectly regulated by another RNase which is controlled by RNase L. Likewise, although we have not found that RLI changes the activity on
other known RNases (5), it is possible that RLI regulates
also the activity of an as-yet-undefined RNase.
RNase L is not the only IFN-induced protein that is increased during
myoblasts differentiation. 2-5A-synthetase is induced in rat and mouse
myoblasts (4). PKR is induced after the shift of mouse C2C12
myoblasts to differentiating conditions. Transfection of PKR in C2C12
cells results in the increased expression of MyoD and myogenin, and the
differentiation process is induced (23). Another protein,
p202, is induced later in the differentiation of C2C12 cells. In this
case, the overexpression of p202 results in a reduced level of MyoD by
affecting its expression at the transcriptional level and inhibits MyoD
and myogenin transcriptional activity (9). These proteins,
all activated during muscle differentiation, operate at different
levels and by different pathways, but they all seem to regulate MyoD.
PKR induces MyoD and myogenin by an unknown mechanism, whereas p202 and
RNase L act at the transcriptional and posttranscriptional levels,
respectively. The activation of these proteins seems to be sequential
and may explain why previous studies, assessing the role of IFN during
differentiation, appear to be conflicting. Some studies describe an
inhibitory effect of IFN on myogenesis (26, 31), whereas
others report a positive effect (1, 13, 44).
Here we have demonstrated that RNase L and RLI are directly involved in
muscle cell regulation and have established MyoD as a potential target
of the RNase L pathway. It now remains to be determined how RNase L
affects MyoD mRNA stability in the presence of different cellular
mRNAs. It is highly unlikely that MyoD is the only mRNA which is
regulated by RNase L.
| |
ACKNOWLEDGMENTS |
We thank A. Bonnieu (INRA, Montpellier, France) for the gift of
C2 cells and MyoD, Myf5, myogenin, -actin plasmids; G. Cathala (IGMM, Montpellier, France) for polyclonal antibody against GAPDH and
fruitful discussions; and P. Dias and P. Houghton for the gift of 5.8A
mouse monoclonal antibody against MyoD. We are also very grateful to N. Taylor (IGMM, Montpellier, France) for critical reading of the manuscript.
This work was done in B. Lebleu's laboratory and was supported by the
Association pour la Recherche Contre le Cancer (ARC) research grants
(9492 and 9298). F. Le Roy is a recipient of a doctoral fellowship from
the ARC, and L. Milligan is a recipient of a doctoral fellowship from
the Fondation pour la Recherche Médicale.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: IGH CNRS UPR
1142, 141 route de la Cardonille, 34396 Montpellier Cedex 5, France.
Phone: 33-4-67-61-36-58. Fax: 33-4-67-04-02-45. E-mail:
bisbal{at}jones.igm.cnrs-mop.fr.
Present address: IGH CNRS UPR 1142, 34396 Montpellier Cedex 5, France.
| |
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Molecular and Cellular Biology, July 2000, p. 4959-4969, Vol. 20, No. 14
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
