Increased
interaction
contractile
activity decreases
in the 3’-UTR of cytochrome
ZHEN YAN, STANLEY
MARC T. HAMILTON,
Department
of Integrative
Houston, Texas 77225;
University
of Liverpool,
SALMONS,
YAN LI DANG,
AND FRANK W. BOOTH
Biology, University
of Texas Medical School,
and Department
of Human Anatomy and Cell Biology,
Liverpool
L69 3BX, United Kingdom
Yan, Zhen,
Stanley
Salmons,
Yan Li Dang,
Marc
T.
Hamilton,
and Frank
W. Booth.
Increased
contractile
activity
decreases RNA-protein
interaction
in the 3’-UTR
of
cytochrome
c mRNA.
Am. J. Physiol.
271 (CeZl PhysioZ. 40):
Cll57-Cl166,
1996.-This
study was designed
to gain an
insight into mechanisms
by which cytochrome
c gene expression is enhanced
by increased
contractile
activity
in skeletal
muscle. When rat tibialis
anterior
muscles were stimulated
(10 Hz, 0.25 ms) for 0,2,6,12,
or 24 h or 2,5,9, or 13 days (n =
4 for each time point), cytochrome
c protein
(enzyme-linked
immunosorbent
assay) and mRNA
(Northern
blot analysis)
concentrations
started to increase
by 9 days, and this was
associated with concurrent
decreases in cytochrome
c mRNAprotein interaction
(RNA gel mobility
shift assay). We found
that the decreased RNA-protein
interaction
in the stimulated
muscle extract was restored by ultracentrifugation
(150,000
g, 1 h) in the supernatant
fraction.
The 150,000 g pellet
fraction
of stimulated
muscle was capable of inhibiting
the
RNA-protein
interaction
in control tibialis
anterior
muscles.
These results provide evidence of an inhibitory
factor that is
responsible
for decreasing
RNA-protein
interaction
in the
3’-untranslated
region of cytochrome
c mRNA in continuously
stimulated
muscle.
skeletal
binding;
muscle; chronic stimulation;
inhibitor;
3’untranslated
ribonucleic
region
acid-protein
contractile
activity induces in
skeletal muscle an increased oxidative capacity for ATP
production
(4, 22, 31, 38). Consequently,
the muscle
becomes more resistant
to fatigue
during
moderate
intensity
exercise. For example,
when cytochrome
c
(+ci) increased 116% in rat skeletal muscle by endurance training,
time to exhaustion
during a treadmill
run was increased by 400% (11). This functional
change
is due mainly to an increase in mitochondrial
volume
density together with increases in the amount of enzymatic activity
of mitochondrial
proteins
involved
in
oxidative metabolism
(23). It was found that mRNAs of
several mitochondrial
proteins
increased
in skeletal
muscle of living animals after elevations
in contractile
activity
(1, 26, 52, 53). However,
the mechanisms
involved
in these mitochondrial
adaptations
remain
unclear.
Cytochrome
c is a mitochondrial
protein encoded by a
nuclear gene (35) and targeted to the intermembrane
space of mitochondria
after posttranslational
modification (51). It plays an essential role in oxidative respiration by transferring
electrons between the respiratory
complexes III and IV (20) and is a possible rate-limiting
factor for electron transport
(19). Cytochrome
c protein
concentration
in rat skeletal muscle increases
after
SUSTAINED
RNA-protein
c mRNA
LOW-INTENSITY
0363-6143/96
$5.00 Copyright
treadmill
running
(15) and chronic stimulation
in vivo
(10) and is directly correlated
with the muscles’ oxidative capacity and the animal’s endurance
capacity (15).
Thus it is important
to elucidate mechanisms
underlying the control of cytochrome c gene expression.
Treadmill
running
exercise had no detectable effect
on the transcription
rates of the cytochrome c gene, as
determined
by nuclear run-on assays by Neufer and
Dohm (36). In our preliminary
experiments,
chronically
stimulated
muscles showed no increase in the activity
of a reporter
gene driven by the promoter
(-726
to
+610) of the rat somatic cytochrome
c gene (data not
shown). We therefore
began to investigate
possible
activity-responsive
elements in its 3’-untranslated
region (UTR). Because mRNAlocalization
(44) and stability (43) and protein translation
(48) may all be regulated by the interaction
between cytoplasmic
proteins
and the mRNA in the control of gene expression,
we
hypothesized
that there was a sequence-specific
cytochrome c mRNA-protein
interaction
in the cytoplasm of
skeletal muscle, which was responsible
for the regulation in response to increased contractile
activity. To test
this hypothesis, we investigated
the RNA-protein
interaction in control and chronic stimulated
rat skeletal
muscle.
METHODS
Animal
and hutian
subjects. Female Sprague-Dawley
rats
(Harlan,
125-149
g body wt) were housed in temperaturecontrolled
quarters
(21°C) with a 12:12 h light-dark
cycle and
provided
with water
and chow ad libitum.
For invasive
surgical procedures,
the rats were anesthetized
by injection
of
a mixture
of ketamine
(54 mg/ml),
xylazine
(2.2 mg/ml),
and
acepromazine
(3.5 mg/ml)
into the right gluteal
muscles (1.4
ml/kg).
All the protocols
were approved
by the Institutional
Animal
Welfare Committee
and the Committee
for the Protection of Human
Subjects at the University
of Texas, Houston
Health
Science Center.
Chronic stimulation.
A battery-operated
miniature
stimulator (24) was implanted
aseptically
in the abdominal
cavity,
and the electrodes
were implanted
under the left common
peroneal
nerve to stimulate
the muscles
in the anterior
compartment
of the hindlimb
(30). Stimulation
(pulses of 0.25
ms at 10 Hz, 24 h/day) (13,52) was commenced
at least 48 h
postoperatively.
The duration
of stimulation
is given for the
individual
experiment.
The contralateral
hindlimb
was used
as control.
PZasmid constructs.
Cytochrome
c riboprobes
used in RNA
gel mobility
shift assay (GMSA)
and ultraviolet
(UV) crosslinking
assay were made with DNA templates
from the
following
subcloned
DNAs. For probe A (5’-UTR,
see Fig. ZC),
a Xho I-Asp 718 I fragment
of 3064 base pairs (bp) from
pRC4CAT/-66
(14) was inserted
into the polylinker
region of
o 1996 the American
Physiological
Society
Cl157
Cl158
PROTEIN-MRNA
INTERACTION
pBluescript
SK( +> (Stratagene).
For probe B (coding region,
see Fig. ZC), a BamH
I-&OR
I fragment
(958 bp) from pRC4
(46) was inserted
into the polylinker
region of pBluescript
SK(+).
For probes C (3’-UTR),
D, and F (portions
of the
3’-UTR;
see Fig. ZC), a &Z II-Kpn
I fragment
(1402 bp) from
pRC4CAT/-726
(14) was inserted
into the polylinker
region
of pBluescript
SK(+).
For probe E (see Fig. ZC), an EcoR
I-Kpn I fragment
(981 bp) from pRC4CAT/-726
was inserted
into pBluescript
SK(+).
For probe G (see Fig. ZC), a 304-bp
Dra I-EcoR
I fragment
from pRC4CAT/-726
was inserted
into pBluescript
SK( +> by digestion
with EcoR I and Sma I.
For probes H and I (see Fig. ZC), two pairs of oligonucleotides
were synthesized
(Genosys).
Their
sequences
were derived
from probe F (+ 1337 to +1452),
the dividing
point being
chosen to correspond
to a consecutive
three-base
mismatch
between
the human
and rat somatic
cytochrome
c genes.
Each pair of oligonucleotides
(55 and 71 bp) had a BamH I site
at the 5’ ends and an EcoR I site at the 3’ ends. The sequence
of the sense strand of the 55-nucleotide
(nt) oligonucleotide
was 5’GATCCACCATAATTTAATTCATACACCAAATTCAGATCATGAATGGCTAACAATG3’.
The sequence of the antisense
strand of the 55-nt oligonucleotide
was 5’AATTCATTGTTAGCCATTCATGATCTGAATTTGGTGTATGAATTAAATTATGGTG3’.
The sequence of the sense strand of the 71-nt
oligonucleotide
was 5’GATCCGTTTTTGTTGGACAGCCCCGATTTAAGTAAAATTGACTTGTCATAAAGTGGATATGATCTTTTTTG3’.
The sequence of the antisense
strand
of the 71-nt oligonucleotide
was 5’AATTCAAA&!AGATCATATCCACTTTATGACAAGTCAATTTTACTTAAATCGGGGCTGTCCAACAAAAACG3’.
After the sense and antisense strands had been annealed
and phosphorylated
by T4
oligonucleotide
kinase, the two double-stranded
oligonucleotides were each inserted
into the polylinker
region of pBluescript SK( +).
The cloned DNAs were digested with the following
restriction enzymes before in vitro transcription:
probe A, BgZ II;
probe B, Act I; probe C, Cla I; probe D, EcoR I; probe E, Cla I;
probe F, Dra I; probe G, EcoR I; probe H, EcoR I; and probe I,
EcoR I (see Fig. 2C) and were extracted
with phenol
and
chloroform
followed by ethanol
precipitation.
For a nonspecific competitor
in the competition
experiments,
plasmid
pRC4(Bluescript)
was digested
with Dra I before in vitro
transcription.
For ribonuclease
(RNase)
protection
analysis,
the coding
region of the cytochrome
c gene was subcloned
into pBluescript SK(+). In brief, the BamH
I-EcoR I fragment
(958 bp)
from pRC4 (46) was inserted
into pBluescript
SK(+).
The
resulting
plasmid
pRC4(Bluescript)
was digested with Act I,
blunt-ended
with Klenow,
and ligated
to form pRC4(540).
Before in vitro transcription,
pRC4(540)
was digested
with
BamH I. A 577-nt antisense
RNA probe was synthesized
with
T7 RNA polymerase,
which contains
a portion of intron 1, all
of exon 2, all of intron 2 and a portion of exon 3. The riboprobe
has 177 nt complementary
to exon 2 and 200 nt to exon 3 in
cytochrome
c mRNA.
The DNAs in the present
studies were transformed
into
JMl09
Escherichia
coZi bacteria
(Promega).
Plasmids
were
isolated
and purified
using alkaline
lysis with differential
polyethylene
glycol precipitation
and subsequent
phenol extractions
(45). DNA concentration
and purity
were determined by UV spectrophotometry
at 260 and 280 nm. All the
cytochrome
c inserts in the constructed
plasmids
were verified by restriction
mapping
and DNA sequencing
(United
States Biochemical).
Northern
analysis.
Plasmid
pRC4(Bluescript)
was used as
a template
to generate
radiolabeled
probe for Northern
blot
DURING
CHRONIC
STIMULATION
analysis
of cytochrome
c mRNA
as described
by Babij and
Booth (3).
RNA isolation.
RNAwas
extracted
according to a procedure
by Chomczynski
and Sacchi (9) with RNAzol
B (Biotecx
Laboratories)
as described earlier (53).
Radiolabeled
RNAprobes.
For RNase protection
and RNAprotein interaction
analysis,
[w~~P]CTP
(400 Wmmol;
Amersham)
and [cx-~~P]UTP
(3,000 Wmmol;
ICN)
were used,
respectively,
for in vitro transcription
with T7 and T3 RNA
polymerase
(Promega).
After transcription
at 37°C for 1 h, the
reaction
mixtures
were treated
with RNase-free
deoxyribonuclease (Promega)
for 15 min at 37°C. After extraction
with
phenol-chloroform
and precipitation
in ethanol
with 20 pg
E. coZi tRNA, the riboprobes
were quantified
and their specific
activities
determined
(45). The 5’-termini
of all the RNA
probes contained
55-106 nt transcribed
from the pBluescript
SK( +).
RNase protection
assay. Cytochrome
c mRNA was quantified by RNase protection
assay according to Gilman
(17) with
modification
(42). Extracted
RNA (10 ug) was hybridized
to a
32P-labeled
antisense
cytochrome
c RNA
probe
[SO,000
counts amin-l(cpm)*
sample-l]
at 45°C for 12-18 h and digested with RNase T2 for 1 h at 30°C. The protected
RNA
fragments
were fractionated
by electrophoresis
on a denaturing 6% polyacrylamide
gel. Appropriate
bands were excised
and their radioactivities
measured
in a scintillation
counter
(Beckman).
To estimate
cytochrome
c mRNA per unit muscle
wet weight, total RNAwas
quantified
according to Munro and
Fleck (34).
CytopZasmic
extract. Muscle
samples
were collected
and
fast-frozen
in liquid nitrogen.
Ice-cold homogenization
buffer
[25 mM tris(hydroxymethyl)aminomethane
(Tris)-phosphate,
pH 7.8, 2 mM trans-1,2-diaminocyclohexane-N,N,N’,N’tetraacetic
acid, 10% glycerol,
2 mM dithiothreitol
(DTT), 1
mM phenylmethylsulfonyl
fluoride,
1 mM EDTA,
1 mM
benzamidine,
10 ug/ml leupeptin,
10 pg/ml pepstatin,
and 1
ug/ml aprotinin]
was added (5 ml/g, 1 ml minimum),
and the
sample was homogenized
at 4°C with a Polytron
homogenizer
(Brinkman)
at 70% of maximal
intensity
(10-s burst, 3 times
with 10-s intervals).
The homogenate
was spun at 4°C for 15
min
at 15,000 g in a Sorvall
(DuPont)
centrifuge.
The
supernatant
(S15; ’ cytoplasmic
extract)
was snap-frozen
in
liquid
nitrogen
and stored at -80°C
pending
analysis.
To
further
fractionate
the muscle
extract,
S15 supernatants
were thawed on ice and ultracentrifuged
at 150,000 g (Beckman TY 65 rotor; 42,000 revolutions/min)
for 1 h at 4°C (18)
into a 150,000 g supernatant
(Sl50) and a 150,000 g pellet
fraction (Pl50). The pellet fraction (Pl50) was resuspended
in
homogenization
buffer (one-third
of the S15 fraction volume
before ultracentrifugation).
Both were snap-frozen
in liquid
nitrogen.
The protein
concentrations
of S15, Sl50, and Pl50
fractions were determined
by the Lowry method (28).
GMSA
and UV cross-linking
assays. The assays were
performed
according
to Rondon et al. (40) with modification.
For GMSA, cytoplasmic
extract (15 ug protein) was incubated
at room temperature
for 10 min with 1.5 ng 32P-labeled
RNA
probe in a reaction
mixture
containing
10% glycerol,
12 mM
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic
acid (pH
7.9), 15 mM KCl, 0.25 mM EDTA, 0.25 mM DTT, 5 mM MgC12,
and E. coZi tRNA (200 ng/pl) in a total volume of 15 pl. RNase
Tl (0.6 units; CalBiochem)
was then added and the solution
incubated
at 37°C for a further
20 min. RNA-protein
complexes were resolved on a native 5% polyacrylamide
gel in
low-ionic-strength
running
buffer (22.25 mM Tris base, 0.5
mM EDTA,
and 22.25 mM boric acid, pH 8.0). For UV
cross-linking
assay, the same procedures
as GMSA
were
followed,
but after RNase Tl treatment,
the mixture
was
PROTEIN-MRNA
INTERACTION
irradiated
in an open tube (on ice) under UV light (0.12 J) for
5 min in a Stratolinker
chamber
(Stratagene).
After the
reaction
mixture
was heated
(lOO*C)
in Laemmli
sample
loading
buffer for 3 min, the reaction
mixture
was subjected
to electrophoresis
on a 10% sodium dodecyl sulfate-polyacrylamide gel. Prestained
molecular
mass markers
(Sigma) were
run simultaneously.
Autoradiograms
were obtained
by exposure of X-OMAT
film (Kodak) to the dried gels.
To determine
whether
the 150,000 g pellet fraction
from
the stimulated
tibialis
anterior
muscle
would inhibit
the
RNA-protein
interaction
in the 3’-UTR
of cytochrome
c
mRNA
(see Fig. 6), P150 (4.5 pg protein)
from either the
control
(C-P150)
or stimulated
(S-P150)
tibialis
anterior
muscles was incubated
with different
muscle extracts (15 pg
protein)
at room temperature
for 15 min before the GMSA
and UV cross-linking
assays. To test whether
heat denaturation would abolish the action of the inhibitory
factors (see
Fig. 7), the 150,000 g pellet fraction
from the stimulated
tibialis
anterior
muscle (S-Pl50)
was incubated
at 98°C for 10
min before incubating
with the tibialis
anterior
muscle S15
supernatant.
Enzyme-linked
immunosorbent
assay. Muscle samples were
collected
and homogenized
as described
before, except that
the homogenization
buffer contained
175 mM KC1 (22). The
muscle
homogenates
were freeze-thawed
three times
and
centrifuged
at 15,000 g for 15 min at 4°C. Direct competitive
enzyme-linked
immunosorbent
assays (ELISA)
were performed according
to Ausubel
et al. (2). Briefly,
purified
rat
heart cytochrome
c (Sigma,
4 ug/ml)
in phosphate-buffered
saline containing
0.05% NaN3 was adsorbed to the wells of a
microtiter
plate at room temperature
overnight.
The residual
binding
capacity
of the plate was blocked
by incubation
of
blocking
solution
(0.17 M HsB04,0.12
M NaCl, pH 8.5,0.05%
Tween 20, 1 mM EDTA, 0.25% bovine serum albumin,
0.05%
NaNa) at room temperature
for 30 min. For assaying muscle
and cytochrome
c standards
(purified
rat heart cytochrome
c;
Sigma),
a sample was mixed (1:lO) with primary
antibody
6H2.B4
(mouse anti-rat
cytochrome
c monoclonal
antibody,
0.78 ug/ml in blocking
solution)
(25) in the wells of a fresh
microtiter
plate in duplicates
and incubated
at room temperature for 30 min. The mixture
(50 ul) was transferred
to the
coated microtiter
plate and incubated
at room temperature
for 2 h. After the well was rinsed with distilled
water three
times,
50 ul of secondary
antibody
(1:5,000
in blocking
solution)
were added to each well and incubated
for 2 h at
room temperature.
To start
ELISA,
100 pl of 3,3’,5,5’tetramethylbenzidine
peroxidase
enzyme immunoassay
substrate (Bio-Rad)
were added to each well and incubated
for 10
min before the addition
of 100 ul of 1 M HzS04. The plate was
read at 450 nm in a microtiter
plate reader (Bio-Rad).
Statistics.
mRNAs
that were determined
by RNase protection from control and stimulated
muscles were compared
by a
paired Student’s
t-test (Fig. 1A). Data from the time course
experiment
(including
mRNAs determined
by Northern
analysis) were compared
by one-way analysis of variance
(Fig. 1B);
P < 0.05 was regarded
as significant.
When this significance
level was reached, a two-tailed
Dunnett
multiple-range
test
was performed
to determine
which time points were significantly different from the O-h group.
RESULTS
Cytochrome c mRNA at the 9th day of stimulation.
To
determine
whether
cytochrome
c gene expression
increases in response to increased
contractile
activity,
cytochrome
c mRNA was determined
in rat tibialis
anterior
muscle after 9 days of chronic stimulation.
DURING
CHRONIC
STIMULATION
Cl159
Cytochrome
c mRNA per unit of extracted
RNA was
31% (P < 0.05) greater in the stimulated
than in the
control muscles (Fig. 1A). Because total RNAper
gram
of wet weight increased from 1.27 t 0.07 mg/g wet wt in
control
to 2.11 -+ 0.11 mg/g wet wt in stimulated
muscles (P < 0.05), cytochrome c mRNA concentration
(cpm/g wet wt) was actually 111% higher (P < 0.01) in
the stimulated
than in the control muscles (Fig. 1A).
There was a 21% decrease in muscle wet weight after
stimulation
(0.42 +
_ 0 .02 g in control and 0.33 t 0.03 g
in stimulated
muscles; P < O.Os>, so cytochrome
c
mRNA expressed per whole muscle increased by 68% as
a result of stimulation
(P < 0.05). These results proved
that continuous
stimulation
was associated with an
increase in cytochrome
c mRNA and justified
usage of
stimulation
for additional studies of cytochrome c mRNA
expression.
Body weights during 13-day continuous
stimulation.
The body weight at the time of surgery (2 days before
the onset of stimulation)
was 151 2 1 g (n = 36). The
body weights after stimulation
for the 0-, 2-, 6-, 12-, and
24-h and 2-, 5-, 9-, and 13-day groups were 147 t 2,
160 t 3, 153 2 6, 149 t 2, 156 t 5, 163 k 3, 167 t 3,
184 t 4, and 188 k 4 g, respectively.
The body weights
for 5-, 9-, and 13-day stimulated
groups were significantly greater (P < 0.05) than for the O-h control.
Muscle wet weight during 13-day continuous stimulation. The tibialis anterior
muscle weight (ratio to the
contralateral
control)
increased
at 2-h stimulation
(1.10 t 0.04) compared with the O-h group (0.98 t 0.02;
P < 0.05; Fig. 1B) b u t remained similar to the O-h group
from 6 h to 5 days and decreased after 9 and 13 days of
stimulation
(0.79 t 0.02 and 0.59 t 0.04 for 9- and
13-day groups, respectively;
P < 0.05 for both groups;
Fig. 1B).
Cytochrome
c protein and mRNA concentrations
and
total RNA concentration
during
13-day continuous
stimulation.
Cytochrome
c protein and mRNA concentrations and total RNA concentration
were unchanged
at the 5th day of continuous
stimulation
but increased
at the 9th day. Cytochrome
c protein concentration
was
5.23 t 1.13 nmol/g wet wt in O-h contralateral
control
tibialis anterior
muscles. The change of cytochrome c
protein concentration
in the stimulated
muscle (as a
ratio to the contralateral
control) became significant
after 9 days (2.29 t 0.24; P < 0.05) and 13 days of
stimulation
(3.06 t 0.52; P < 0.05) compared with the
O-h group (1.06 -+ 0 . 12; Fig. 1B).
Cytochrome
c mRNA was quantified
by Northern
blot
analysis (Fig. 1B) and expressed as relative
units per
gram wet weight (concentration)
after calculation
with
total RNA concentration
(53). Cytochrome
c mRNA
concentration
in the stimulated
muscle (as a ratio to
the contralateral
control) increased for the g-day group
(1.95 + 0.14; P < 0.05) compared with the O-h group
(1.00 ? 0.05; Fig. 1B). These data confirm the increase
in cytochrome c mRNA measured by RNase protection
assay (Fig. 1A).
Total RNA concentration
was 1.07 t 1.10 mg/g wet
wt in O-h contralateral
control tibialis anterior muscles.
Total RNA concentration
in the stimulated
muscles (as
Cl160
PROTEIN-MRNA
INTERACTION
A
DURING
CHRONIC
STIMULATION
B
4
tRyA St,im Cpn
Base
201-
1
Cytochrome
c concentration
i
Stimulation
Control
1__1
180-
4
: -------A!
Cytochrome
c mRNA concentration
3
Stim
Con
Chanae
cpmlpg RNA
36OztlP
27S26
+31%
cpm/mg wet weight
749&39**
35!5k!53
+ll 1%
cpm/muscle
250#*
149zt24
+68%
Cvtochrome
c mRNA
Fig. 1. A: increases
in cytochrome
c mRNA concentration
in rat tibialis
anterior
(TA) muscle by indirect
continuous
stimulation.
Cytochrome
c mRNA
was quantified
by ribonuclease
protection
analysis
of total RNA from rat TA
muscles
with
an antisense
riboprobe
from rat somatic
cytochrome
c coding region.
tRNA,
tRNA
control;
Stim,
g-day stimulated
TA muscle;
Con, contralateral
control TA
muscle. Arrows
point to protected
bands. Predicted
sizes of
protected
bands
are 177 and 200 nucleotides
(nt; see
METHODS).
Molecular
sizes from
radiolabeled
DNA
size
markers
(pBR322/Hpa
II) are shown at left. Cytochrome
c
mRNA
quantities
are listed at bottom. *P < 0.05, **I' <
0.01 vs. control.
Values
are means
i SE from 5 animals;
cpm, counts/min.
B: changes of cytochrome
c protein
and
mRNA
concentrations
in rat TA muscle
after continuous
stimulation.
TA muscles
of left hindlimb
were stimulated
for different
time durations
(0,2,6,12,
and 24 h and 2,5,9,
and 13 days; n = 4 for each time point). Contralateral
TA
muscles were used as control. Cytochrome
c protein,
mRNA,
and total RNA concentrations
were determined
by enzymelinked
immunosorbent
assay, Northern
blot, and Munro-Fleck methods,
respectively,
as described
in METHODS. Data
were presented
as ratios of values for stimulated
muscle to
control value for each time point. "P < 0.05 for a difference
to the O-h group.
Stimulation
Control
*
___-L-----+
1
4
3
Stimulation
Control
Ti---
T &
I
Total RNA concentration
!
2
---
1
--------
&
k
0’ -4
Muscle weight
3
Stimulation
Control
a ratio to contralateral control) increased at days 9 and
13 (1.52 +- 0.13 and 1.54 t 0.15, respectively; P < 0.05
for both groups; Fig. 1B).
Cytochrome c mRNA-protein interaction. To determine whether there is RNA-protein
interaction for
cytochrome c mRNA, radiolabeled RNA probes from
different portions of the cytochromc c gene were incubated with cytoplasmic extract from control tibialis
anterior muscle in GMSA and UV cross-linking assays.
GMSA showed three RNase Tl-resistant
complexes
(Fig. 2A) for probes C, 0, F, and H (Fig. 2C), which
contained either the entire 3’ region, the 3’-UTR, or the
proximal region of the 3’-UTR. Thus the protein binding site was mapped to a segment of 50 nt (probe H,
+1339 to +1388) in the 3’-UTR. UV cross-linking
assays were used to determine the molecular size and
number of possible factors that bound to the 3’-UTR. A
2
1.
0.
I
I
2h6h
Oh
12h
24h2d
I
I 1
/
5d
9d
13d
Time
37-kDa complex was detected for every probe (Fig. 2B)
and was not efficiently competed away by specific
probes (Fig. 3B). For those Probes that showed three
RNA-protein complexes in GMSA (probes C, 0, E and
H), two additional groups of complexes were detected;
one group consisted of at least three complexes (79,83,
and 90 kDa) of -84 kDa and another of at least two
complexes (56 and 59 kDa) of -58 kDa (Fig. 2B). When
the 50-nt probe H was used, a band at -30 kDa
appeared, with concurrent decreased binding at 37 kDa
(Fig. 2B).
The complexes on the native and denaturing gels
were completely eliminated by incubation of the muscle
extract with proteinase K before the reaction, showing
that protein factors were involved in the formation of
the complexes (Fig. 3A). Addition of increasing amounts
of unlabeled probe F RNA to the reaction mixtures
PROTEIN-MRNA
INTERACTION
resulted in a concentration-dependent
reduction in the
formation of the 58 and 84-kDa complexes, which were
not reduced by a nonspecific competitor from the coding
region (Fig. 3B 1,indicating that the RNA-protein interaction in this region is sequence specific.
RNA-protein
interaction
in continuous
muscles. If the cytochrome c mRNA-protein
A
stimulated
interaction
Probe
ABCDEFGHI
FP
DURING
CHRONIC
STIMULATION
Cl161
is involved in the contractile activity-mediated
increase
of cytochrome c mRNA, the interaction should change
in response to chronic stimulation, concurrent with the
increase of cytochrome c mRNA. To test this possibility,
cytoplasmic extracts from tibialis anterior muscles that
were continuously stimulated for different durations
and radiolabeled 116-nt riboprobe F (+ 1337 to + 1452;
GenBank accession nos. K00750 and M28216) were
used in GMSA assays. A consistent decrease in RNase
Tl-resistant
complexes (RNA-protein interaction) was
observed in GMSA for the tibialis anterior muscles that
had been stimulated continuously for 9 and 13 days
compared with the contralateral control (4 of 4 samples
for each time point; Fig. 4). For E&day-stimulated
muscles, a less consistent and smaller decrease in
RNA-protein complex formation was noticeable, but no
consistent change was observed for those muscles that
had been stimulated for <5 days (Fig. 4). Similar data
were found for UV cross-linking (data not shown).
These data demonstrate that the increase in cytochrome c gene expression in response to chronic stimulation in rat skeletal muscle was concurrent with the
decreased RNA-protein interaction in the 3’-UTR of
cytochrome c mRNA.
RNA-protein
interaction
after ultracentrifugation
stimulated
muscle extracts. To determine whether
g:
FP
Dra I
c
JY
------
*r.
T EF’
l
&-
C/a I
Fig. 2. RNA-protein
interaction
in 3’untranslated
region KJTR) of
cytochrome c mRNA. Gel mobility shift assay (GMSA) and ultraviolet
WV) cross-linking
assays were performed
as described in METHODS.
A: autoradiogram
from GMSA assay with radiolabeled
riboprobes
from various portions of cytochrome
c gene (lanes A-Z). HC, high
complex; DC, diffuse complex; LC, low complex; FP, free probe. B:
autoradiogram
of W cross-linking
assay with same probes used in
GMSA (lanes A-Z). Molecular masses were estimated from prestained
markers (Sigma). C: scheme of in vitro synthesized
%“P-labeled RNA
probes. For rat somatic cytochrome
c gene (top, to scale), 5’- and
3’-flanking
regions are represented
by thick lines; introns by dashed
lines; exons by open boxes (untranslated
regions) and solid boxes
(coding region); and polyadenylation
sites by solid dots. Riboprobes
(A-Z) are represented by lines (to scale), with letters corresponding
to
lane designations
in A and B.
of
the
protein factors that interact with cytochrome c mRNA
were soluble or associated with the 150,000 g pellet
fraction, we centrifuged the cytoplasmic extracts (S15)
at 150,000 g (1 h, 4°C) to obtain the 150,000 g supernatant (S150) and the 150,000 g pellet (P150) fractions.
S150 contains mainly soluble cytoplasmic proteins and
P150 contains ribosomes and sarcoplasmic reticulum
(18, 29, 41). Radiolabeled 3’-UTR transcripts from
probe F of the cytochrome c gene (+1337 to +1452)
were incubated with different fractions (S15, S150, and
P150) from the control and stimulated tibialis anterior
muscles (Fig. 5). GMSA experiments were performed
(Fig. 5A). In the control muscle, ultracentrifugation
slightly increased the binding activity in S150 (lane 2)
compared with S15 (lane 1), which may be due to the
purification
by ultracentrifugation.
There was little
binding in P150 (lane 3). In the stimulated muscle,
however, we were able to detect RNA-protein interaction in the supernatant fraction (S150, lane 5, Fig. 5A)
of the muscle extract after ultracentrifugation.
UV
cross-linking experiments were also performed (Fig.
5B). The 58- and 84-kDa complexes were present in
both S150 (lane 2) and P150 (lane 3) in the control
muscle after ultracentrifugation.
The amount of protein cross-linked to the probe in the stimulated muscle
(58- and 84-kDa complexes) was very low in S15 (lane
4) and was increased substantially by ultracentrifugation in S150 (lane 5, Fig. 5B). There was very little
binding in P150 (lane 6) for the 37-kDa complex, which
has been shown to compete weakly by specific probes.
The restoration of RNA-protein interaction in the stimulated muscle by ultracentrifugation
was not due to the
l-h incubation at 4°C during centrifugation, a possibility that was tested by GMSA and UV cross-linking
assays in a separate experiment (data not shown).
Cl162
PROTEIN-MRNA
INTERACTION
DURING
CHRONIC
STIMULATION
A
Fig. 3. Effect of proteinase
K pretreatment
and RNA
competitor
on RNA-protein
interaction.
A: UV crosslinking assay with radiolabeled RNAprobe
F (see Fig. 2)
and TA muscle cytoplasmic
extracts
pretreated
with
(+K) and without t-K) proteinase K(2.5 mg/ml at 37°C
for 30 min). Molecular
masses were estimated
from
prestained
markers
(Sigma). Same results
were obtained from 5 separate experiments.
B: competition
of
RNA-protein
complex formation
by competitor
RNA.
UV cross-linking
assay was performed
with radiolabeled probe F and control TA muscle extract and with
increasing amounts (O-1,000 molar excess) of unlabeled
specific probe F or of a nonspecific probe from the coding
region of the cytochrome
c gene. Same results
were
obtained from 3 separate experiments.
FP, free probe.
-K
kDa
11284-
II
I
Inhibition
of cytochrome c mRNA-protein
by supplementation
of stimulated
muscle
interaction
extract. The
possibility was considered that ultracentrifugation
had
removed an inhibitor associated with the 150,000 g
pellet fraction, resulting in the restoration of the binding activity in the 150,000 g supernatant of the stimulated muscle. If this were true, the observed RNAprotein interaction with control muscle extracts would
be diminished when the 150,000 g pellet fraction from
the stimulated muscle was added. To test this hypothesis, an aliquot (4.5 pg protein) of P150 from the control
(C-P1501 or stimulated (S-P1501 muscle was added to
S15 or S150 of the control or stimulated muscle and
incubated at 30°C for 15 min before the UV crossOh
CT
HCFig. 4. Changes of RNA-protein
interaction
in
the 3’-UTR of cytochrome c mRNA. GMSA assay
was performed
with radiolabeled
riboprobe
(1.5
ng) from 3’-UTR of rat somatic cytochrome
c gene
(+1337 to +1452) and muscle extracts
615, 15
ug protein) from stimulated
6) and contralateral
control (0 TA muscles. HC, high complex; DC,
diffuse complex; LC, low complex. FP, free probe.
Representative
of 4 sets of animals.
DC
t
Lc _
FP
0gEZgg
T-
kDa
84!&
3532-
*‘a
FP
FP
These data indicate that 1) the protein factors in the
skeletal muscle that interact with the 3’UTR of cytochrome c mRNA are present in both the 150,000 g
supernatant and the 150,000 g pellet fractions and 2)
the decreased RNA-protein interaction in the stimulated muscle could be restored by removing the 150,000
g pellet fraction from the stimulated muscles’ 15,000 g
supernatant.
0
0008
OLn“Z:gZOO
..
63 53 35 32-
Non-specific
competitor
Specific
competitor
+K
linking assay (Fig. 6). Addition of P150 from the control
muscle had little effect on the RNA-protein interaction
of the control muscle (lane 2). However, when P150
from the stimulated muscle was added, it inhibited
specifically the formation of the 5% and 84-kDa RNAprotein complexes (lane 3, Fig. 6); the nonspecific
37-kDa complex was unaffected. A similar inhibitory
effect was observed when P150 from the stimulated
(S-P150) muscle was added to S150 of the control
muscle CC!-S150; lane 6, Fig. 6). P150 from either the
control muscle or the stimulated muscle had little effect
on the RNA-protein interaction in the stimulated muscle
(lanes 8 and 9, respectively),
because the binding
activity in S-S15 was already low. The RNA-binding
activity was significantly higher in S150 (lane 10, Fig.
6) than in S15 (lane 7, Fig. 6) of stimulated muscle, as
noted in another set of animals (Fig. 5). When P150
from the stimulated muscle (lane 121, but not that from
the control muscle (lane U), was added, the RNAbinding activity was decreased (Fig. 6). Similar inhibitory effects were observed with GMSA(data not shown).
P150 from the stimulated rat tibialis anterior muscle
2h
CC-A
6h
CT
12h
CT
24h
CT
46h
cY--i
5d
CB
9d
cc-2
13d
CT
PROTEIN-MRNA
A
INTERACTION
DURING
CHRONIC
STIMULATION
Cl163
B
,Control , Stimulation
,
I
5:m
5: 5s
m5:
~5iizJ;;3;
, Control ,
Stimulation
I
I
In 5: 5:u-l
5;EJiTG5;i;;
8
5:
Fig. 5. Restoration of cytochrome c mRNA-protein
interaction in stimulated
muscle extract by ultracentrifugation. GMSA and W cross-linking
assays were performed as described in METHODS. A: autoradiogram
from
GMSA assay with a radiolabeled
riboprobe F (1.5 ng)
from 3’-UTR of rat somatic cytochrome c gene ( + 1337 to
+1452) and different fractions (S15, S150, and P150,15
pg protein) from 13-day stimulated
and contralateral
control TA muscles. HC, high complex; DC, diffuse
complex; LC, low complex. B: autoradiogram
of UV
cross-linking
assay with same probe and muscle fractions. Molecular masses were estimated from prestained
markers (Sigma). FP, free probe. Similar results were
obtained from 3 experiments.
kDa
112-
HC -
84 -
DC
63 -
1
53 -
LC -
3532-
FP
J
1
2
3
4
5
5
could also inhibit the interaction between SE from
human skeletal muscle (biceps) and radiolabeled 3’UTR transcripts of the rat cytochrome c mRNA (data
not shown). These observations provide evidence that
the decreased RNA-protein interaction in the 3’-UTR of
cytochrome c mRNA after chronic stimulation is due to
an inhibitory component associated with the 150,000 g
s-p150
c-p150
-
+
+
-
c-s15
-
6
6
+
+
-
, c-s150
,
+
+
-
s-s15
-
+
+
-
, s-s150
pellet fraction. Treatment with micrococcal nuclease
(1,000 U/ml, 37°C 10 min) had no effect on the inhibitory role of the 150,000 g pellet fraction from the
stimulated muscle (data not shown), indicating that
the inhibitory factor does not require a nucleic acid for
its inhibitory function. The heat-treated P150 (98°C 10
min) from the stimulated muscle (H-S-P1501 lost its
ability to decrease the RNA-protein interaction compared with the nontreated P150 from the stimulated
muscle (S-P150; Fig. 7). These results demonstrate that
the inhibitory component is heat sensitive.
DISCUSSION
kDa
11284 g; 1
32 -
FP
1
2
3
4
5
6
7
8
9
10 11 12
Fig. 6. Inhibition
of RNA-protein
interaction
in 3’-UTR of cytochrome c mRNA by 150,000 g pellet fraction from stimulated
TA
muscle. UV cross-linking
assays were performed with radiolabeled
RNA probe (1.5 ng) from 3’-UTR of rat somatic cytochrome c gene
( + 1337 to + 1452). The 150,OOOg pellet fractions (4.5 pg protein) from
stimulated
(S-P1501 or contralateral
control TA muscles (C-P1501
were added to cytoplasmic extract or 150,OOOg pellet fraction (15 pg
protein) from stimulated
(S-S15 or S-S150) or contralateral
control
TA muscles (C-S15 or C-S150) and incubated at 30°C for 15 min
before assay. Molecular
masses were estimated
from prestained
markers (Sigma). FP, free probe. Similar results were obtained from
>5 experiments.
Cytochrome c gene expression is increased by continuous stimulation of skeletal muscle. For example, cytochrome c protein concentration increased by 90 and
206% in rat skeletal muscle after 26 days (8 h/day) of
stimulation (10) and 13 days of continuous stimulation
(24 h/day; Fig. lB), respectively. Our finding that
cytochrome c mRNA concentration increases by 111%
after 9 days of continuous stimulation suggests that at
least a part of the increase in cytochrome c protein is
due to pretranslational
regulation. It is not known
whether the increased cytochrome c mRNA concentration results from increased transcription or enhanced
stability of cytochrome c mRNA.
Sequence-specific RNA-protein interaction in the 3’UTR is reportedly involved in posttranscriptional
regulation, through alteration of mRNA stability and translatability, and correct coding of certain amino acids (8,
12, 27, 32, 37, 49). In the present study, we detected
specific RNA-protein complexes from interactions between cytoplasmic proteins and a proximal region (50
nt) in the 3’-UTR of cytochrome c mRNA (Fig. 2B).
Because the poly(A) tail was not included in the riboprobes in this research, it is clear that the protein
Cl164
PROTEIN-MRNA
INTERACTION
DURING
factors are not poly(A)-binding
proteins, which have
been reported to influence mRNA stability (5).
At least two possibilities exist as to the sequence
basis for the RNA-protein interaction in the 3’-UTR of
cytochrome c mRNA. First, cytoplasmic truns-factors
recognize a specific secondary structure on the mRNA.
There are precedents for this possibility. For example,
stem-loop structures have been found in the 3’-UTRs of
transferrin receptor and type I deiodinase mRNAs (6,
33) that mediate posttranscriptional
regulation. Second, cytoplasmic trans-factors interact with a specific
RNA&-element
rather than a secondary structure; for
example, cytoplasmic proteins bind to AUUUA motifs
in the 3’UTR of cytokine mRNAs and regulate their
stability (16, 54). We suspect that the cytochrome c
mRNA-protein
interaction is not due to the AUUUA
motif in the 50-nt region, because there are five AUUUA
motifs outside the 50-nt region that did not show
specific RNA-protein
interaction
(Fig. 2, A and B).
However, the possibility still exists that the AUUUA
and its flanking nucleotides in the 50-nt region function
as a trans-acting factor binding site or sites. Our
finding that the RNA binding activity was detectable in
all seven species (rat, mouse, human, rabbit, pig, dog,
A
B
H-S-PI50
-
-
+
H-S-P150
-
-
+
S-P1 50
c-s15
-
+
-
S-Pi50
-
+
-
+
+
+
c-s1 5
+
+
+
1
I
kDa
HC-
84DC
631
53-
LC -
FP
FP
Fig. 7. Loss, by heat denaturation,
of inhibitory
function of 15O.OOOp
pellet fraction-from
stimulated
TA muscle on‘RNA-protein
intera;
tion in 3’-UTR of cytochrome
c mRNA. GMSA and UV cross-linking
assays were performed
as described in METHODS. A: autoradiogram
from GMSAassay
with radiolabeled riboprobe F (1.5 ng) from 3’-UTR
of rat somatic cytochrome
c gene (+1337 to +1452). Cytoplasmic
extract (15 pg protein) from control TA muscles (C-X5)
was incubated at 30°C for 15 min with or without
150,OOOg pellet fraction (4.5
pg protein) from stimulated
TAmuscle that had either been heated at
98°C for 10 min (H-S-P1501 or not (S-P150). HC, high complex; DC,
diffuse complex; LC, low complex; FP, free probe. B: autoradiogram
of
W cross-linking
assay with same probe and muscle fractions.
Molecular masses were estimated from prestained
markers (Sigma).
FP, free probe. One of two experiments
with similar results.
CHRONIC
STIMULATION
and chicken; data not shown) tested with the 116-nt rat
riboprobe is suggestive of a degree of conservation that
would be consistent with a functionally important role
of this region of cytochrome c mRNA.
Another finding that supports a potential functional
role for RNA-protein interaction is that it decreases
coincidentally with increases in cytochrome c protein
and mRNA during continuous electrical stimulation of
the skeletal muscle. Continuous stimulation for 9 days
diminished specific RNA-protein
interaction
in the
3’-UTR of cytochrome c mRNA in rat tibialis anterior
muscle. Concurrently, cytochrome c protein and mRNA
concentrations first increased at 9 days of continuous
stimulation in the same muscles. However, at 5 days of
continuous stimulation, none of these three changes
had occurred. This finding further supports the hypothesis that chronic stimulation increases cytochrome c
gene expression through decreased RNA-protein interaction in the 3’-UTR.
Our finding that cytochrome c protein concentration
in rat tibialis anterior muscle increased significantly
after 9 and 13 days is in agreement with the delayed
induction of other mitochondrial enzymes and proteins
during chronic stimulation in both rabbit (13, 21, 39,
47,521 and rat (50). An increase of cytochrome c mRNA
concentration was also observed at day 9 of stimulation, indicating that the increased cytochrome c gene
expression due to chronic stimulation is at least partly
due to an increase in the mRNA level. However, the
increase in cytochrome c protein concentration (stimulation to control ratio of 2.29 + 0.24 and 3.06 t 0.52 for
9- and 13-day groups, respectively) was more than the
increase in the mRNA concentration (stimulation to
control ratio of 1.95 t 0.14 and 1.78 t 0.43 for 9- and
13-day groups, respectively). The possibility of translational or posttranslational
control, referred to by Williams et al. (521, therefore remains.
The initial attempt to use ultracentrifugation
to
locate the protein factors that interact with cytochrome
c mRNA provided information that they are present in
both the 150,OOOg supernatant and the 150,OOOg pellet
fractions in control tibialis anterior muscles. Although
the RNA-protein interaction complexes of the 150,OOOg
pellet fraction from control muscle were detected in
both the GMSA and W cross-linking experiments, we
believe that most of the RNA-binding factors are soluble
proteins in the 150,000 g supernatant fraction because
it took three times as much muscle to obtain an amount
of 150,000 g pellet protein that yielded equal or less
RNA-protein interaction. We also included the stimulated muscle extracts in the above ultracentrifugation
experiment, which generated unexpected and important data, i.e., removal of the 150,000 g pellet fraction
restored the decreased RNA-protein interaction in the
stimulated muscle. These results argue against a disappearance of proteins binding to the RNA as a cause of
decreased RNA-protein interaction after chronic stimulation. They also indicate that the decreased binding is
not due to degradation of the binding factors during
preparation of the muscle extract.
PROTEIN-MRNA
INTERACTION
There are at least three possible explanations
for the
restoration
of RNA-protein
interaction
in the stimulated muscle by ultracentrifugation.
First, chronic
stimulation
could have increased
nuclease
activity
associated with the 150,000 g pellet fraction, leading to
increased cytochrome c mRNA degradation.
Ultracentrifugation removed the nuclease activity from the 150,000
g supernatant
and resulted
in less degradation
of
cytochrome
c riboprobe
during
the GMSA and UV
cross-linking
assays. This possibility
was not consistent with the observation
that cytochrome
c mRNA
concentration
increased
rather
than decreased
after
stimulation.
In addition,
the UV cross-linking
assay
showed decreased formation
of complexes at 84 and 58
kDa, but not at 37-kDa,
in the stimulated
muscle,
suggesting that this is not the result of an increase in
nonspecific nuclease activity. Second, incubation
of the
stimulated
muscle extract during the l-h ultracentrifugation could have resulted in increased affinity
of the
binding factors. This hypothesis was eliminated
by the
observation
that incubation
of the muscle extracts at
4°C for 1 h without ultracentrifugation
had no effect on
binding pattern or intensity (data not shown). Third, a
factor, or factors, associated with the 150,000 g pellet
fraction could have inhibited
the RNA-protein
interaction in the stimulated
muscle. This hypothesis
was
tested by addition
of the 150,000 g pellet fractions
to
the cytoplasmic
extracts from the control muscle. The
experiments,
which showed that the 150,000 g pellet
fraction from the stimulated
but not the control muscle
was able to inhibit
the RNA-protein
interaction,
provide evidence for the induction
or activation
of such an
inhibitor
of RNA-protein
interaction.
The mechanism
underlying
the inhibition
is not known, but it seems
that the inhibitory
factor does not bind directly to the
cytochrome c mRNA to compete for the binding because
the 150,OOOg pellet fraction from the stimulated
muscle
showed no binding activity (Fig. 6). Thus an inhibitor
of
RNA-protein
interaction
in the 3’-UTR of cytochrome c
mRNA is induced by continuous
stimulation.
To determine
whether an RNA or protein component
in the inhibitor
was intimately
involved
in its function,
the 150,000 g pellet fraction of the stimulated
muscle
was pretested in two ways. Pretreatment
with micrococcal nuclease did not affect the inhibitory
function (data
not shown). This implies that the inhibitor
does not
contain an RNA component or that, if it does, the RNA
component
is not required
for its inhibitory
function.
Pretreatment
with heat denaturation
abolished
the
inhibitory
function, indicating
that the inhibitor
is heat
sensitive and could be a protein. These are some initial
steps in characterizing
the inhibitory
factor. Further
characterization
will require it to be isolated.
In summary, the present study demonstrates
that a
proximal
region in the 3’-UTR of cytochrome c mRNA
interacts
with cytoplasmic
proteins
in rat skeletal
muscle. We further report that this RNA-protein
interaction decreases after continuous
muscle stimulation.
The RNA-protein
interaction
activity can be restored in
stimulated
muscle extracts by removal of the 150,000 g
pellet and decreased in control muscle extracts by the
DURING
CHRONIC
STIMULATION
C 1165
addition of the stimulated
muscle 150,000 g pellet. The
concurrent
increase in cytochrome c mRNA and protein
concentrations
(24) and decrease in the RNA-protein
interaction
in the 3’-UTR of cytochrome c mRNAin
the
g-day stimulated
muscle suggests an inhibitory
role for
the RNA-protein
interaction
in cytochrome
c gene
expression. This is the first report, to our knowledge, of
an inducible
inhibitory
factor that decreases RNAprotein interaction.
The association
of the inhibitory
factor with the 150,000 g pellet fraction is particularly
interesting
and suggests the intriguing
possibility that
cytochrome c mRNAs undergoing
translation
in stimulated muscles, a process that involves mRNA binding to
ribosomes
on the rough endoplasmic
reticulum,
are
subject to a compartmentalized
regulatory
influence.
We thank Richard C. Scarpulla for the gift of pRC4 and pRC4CATs
and for helpful discussions.
We thank Brian Black, David and Julie
Butler, Janet Mar, and Ann-Bin
Shuy for help and expert advice.
Stimulators,
redesigned in collaboration
with David Hitchings
and
his colleagues at the University
of Staffordshire,
were assembled by
Michelle Hastings.
We also thank Dr. Ronald Jemmerson
for the gift
of cytochrome c antibody.
This work was supported
by National
Institute
of Arthritis
and
Musculoskeletal
and Skin Diseases AR-19393.
Address
for reprint
requests:
F. W. Booth, Dept. of Integrative
Biology, Univ. of Texas Medical School, 6431 Fannin, 4.100 MSB,
Houston, TX 77030.
Received
1 December
1995; accepted in final form 4 April
1996.
REFERENCES
1. Annex, B. H., W. E. Kraus,
G. L. Dohm, and R. S. Williams.
Mitochondrial
biogenesis in striated muscles: rapid induction of
citrate synthase mRNA by nerve stimulation.
Am. J. PhysioZ. 260
(CeZZ PhysioZ. 29): C266-C270,1991.
2. Ausubel,
F. M., R. Brent, R. E. Kingston,
D. D. Moore, J. G.
Seidman,
J. A. Smith,
and K. Struhl.
Current Protocols in
MoZecuZar BioZogy. New York: Wiley, 1991, vol. 2, p. 11.2.1-11.2.7.
3. Babij, P, and F. W. Booth. cx-Actin and cytochrome c mRNAs in
atrophied
adult rat skeletal muscle. Am. J. PhysioZ. 254 (CeZZ
Physiol. 23): C651-C656,
1988.
K. M:, G. H. Klinkerfuss,
R. L. Terjung,
PA. Mole,
4, Baldwin,
and J. 0. Holloszy.
Respiratory
capacity of white,
red, and
intermediate
muscle: adaptative
response
to exercise. Am. J.
PhysioZ. 222: 373-378,1972.
P., S. W. Peltz, and J. Ross. The poly(A)-poly(A)5. Bernstein,
binding
protein
complex is a major determinant
of mRNA
stability in vitro. MOL. CeZZ. BioZ. 9: 659-670, 1989.
6. Berry, M. J., L. Banu, Y. Y. Chen, S. J. Mandel, J. D. Kieffer,
J. W. Harney,
and P. R. Larsen.
Recognition
of UGA as a
selenocysteine
codon in type I deiodinase requires sequences in
the 3’ untranslated
region. Nature Land. 353: 273-276,199l.
7. Carson,
J. A., Z. Yan, F. W. Booth,
M. E. Coleman,
R. J.
Schwartz,
and C. S. Stump.
Regulation
of skeletal
cx-actin
promoter
in young chickens
during hypertrophy
caused by
stretch overload. Am. J. Physiol. 268 (CeZZ Physiol. 37): C918c924,1995.
8. Casey, J. L., D. M. Koeller, V. C. Ramin, R. D. Klausner,
and
J. B. Harford.
Iron regulation
of transferrin
receptor mRNA
levels requires
iron-responsive
elements and a rapid turnover
determinant
in the 3’ untranslated
region of the mRNA. EMBO
J. 8: 3693-3699,1989.
9. Chomczynski,
P., and N. Sacchi. Single-step
method of RNA
isolation
by acid guanidinium
thiocyanate-phenol-chloroform
extraction. Anal. Biochem. 162: 156-159,1987.
Chronic electrical stimulation
10. Ciske, P E., and J. A. Faulkner.
of nongrafted
and grafted skeletal
muscles
in rats. J. AppZ.
Physiol. 59: 1434-1439,1985.
K. J., L. Packer,
and G. A. Brooks.
Biochemical
11. Davies,
adaptation
of mitochondria,
muscle, whole-animal
respiration
to
Cl166
PROTEIN-MRNA
INTERACTION
endurance
training.
Arch. Biochem.
Biophys.
209: 539-554,
1981.
12. Dodson,
R. E., and D. J. Shapiro.
An estrogen-inducible
protein binds specifically
to a sequence in the 3’ untranslated
region of estrogen-stabilized
vitellogenin
mRNA. MOL. CeZZ. BioZ.
14: 3130-3138,1994.
13. Eisenberg,
B. R., and S. Salmons.
The reorganization
of
subcellular
structure
in muscle undergoing
fast-to-slow
type
transformation.
A stereological
study. Cell Tissue Res. 220:
449-471,198l.
14. Evans, M. J., and R. C. Scarpulla.
Both upstream
and intron
sequence elements are required for elevated expression
of the rat
somatic cytochrome
c gene in COS-1 cells. MOL. Cell. BioZ. 8:
35-41,1988.
15. Fitts, R. H., F. W. Booth, W. W. Winder,
and J. 0. Holloszy.
Skeletal muscle respiratory
capacity, endurance,
and glycogen
utilization. Am. J. Physiol. 228: 1029-1033,1975.
16. Gillis,
P., and J. S. Malter.
The adenosine-uridine
binding
factor recognizes the AU-rich
elements of cytokine, lymphokine,
and oncogene mRNAs. J. BioZ. Chem. 266: 3172-3177,199l.
17. Gilman,
M. Current Protocols in Molecular Biology. New York:
Wiley, 1989, p. 4.7.1-4.7.6.
M., and C. Baglioni.
Degradation
of unstable inter18. Gorospe,
leukin-lol mRNAin
rabbit reticulocyte
cell-free system. Localization of an instability
determinant
to a cluster of AUUUA motifs.
J. BioZ. Chem. 269: 11845-11851,1994.
The role of cytochrome c
19. Gupte, S. S., and C. R. Hackenbrock.
diffusion in mitochondrial
electron transport.
J. BioZ. Chem. 263:
5248-5253,1988.
20. Hatefi, Y. The mitochondrial
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
electron transport
and oxidative
phosphorylation
system. Annu. Rev. Biochem. 54: 1015-1069,
1985.
Henriksson,
J., M. M. Chi, C. S. Hintz,
D. A. Young, K. K.
Kaiser,
S. Salmons,
and 0. H. Lowry.
Chronic stimulation
of
mammalian
muscle: changes in enzymes of six metabolic pathways. Am. J. Physiol. 251 (CeZZ Physiol, 20): C614-C632,
1986.
Holloszy,
J. 0. Biochemical
adaptations
in muscle. Effects of
exercise on mitochondrial
oxygen uptake and respiratory
enzyme
activity in skeletal muscle. J. BioZ. Chem. 242: 2278-2282,
1967.
Holloszy,
J. O., and E. F. Coyle. Adaptations
of skeletal muscle
to endurance exercise and their metabolic consequences.
J. AppZ.
PhysioZ. 56: 831-838, 1984.
Jarvis,
J. C., and S. Salmons.
A family of neuromuscular
stimulators
with optical transcutaneous
control. J, Med. Eng.
TechnoZ. 15: 53-57, 1991.
Jemmerson,
R., C. Mueller,
and D. Flaa. Differences
in heavy
chain amino acid sequences affecting the specificity of antibodies
for variants of cytochrome c. MOL. Immunol. 30: 1107-1114,1993.
Kraus,
W. E., T. S. Bernard,
and R. S. Williams.
Interactions
between sustained
contractile
activity and P-adrenergic
receptors in regulation of gene expression
in skeletal muscles. Am. J.
Physiol. 256 (CeZZ PhysioZ. 25): C506-C514,
1989.
Lake, R. A., D. Wotten,
and M. J. Owen. A 3’ transcriptional
enhancer regulates tissue-specific
expression
of the human CD2
gene. EMBO J. 9: 3129-3136,199O.
Layne,
E. Spectrophotometric
and turbidometric
methods for
measuring
proteins. Methods Enzymol. 3: 448-450,1957.
Lehninger,
A. L. Biochemistry
(2nd ed.). New York: Worth,
1975, p. 381.
Mayne, C. N., T. Mokrush,
J. C. Jarvis,
S. J. Gilroy, and S.
Salmons.
Stimulation-induced
expression
of slow muscle myosin in a fast muscle of the rat. Evidence of an unrestricted
adaptive capacity. FEBS Lett. 327: 297-300,1993.
Mole, P. A., L. B. Oscai, and J. 0. Holloszy.
Adaptation
of
muscle to exercise. Increase in levels of palmityl CoA synthetase,
carnitine palmityltransferase,
and palmityl CoA dehydrogenase,
and in the capacity of oxidize fatty acids. J. CZin. Invest. 50:
DURING
ferrin
STIMULATION
receptor
mRNA
stability
in the cytoplasm.
CeZZ 53: 815-
825,1988.
34. Munro,
35.
36.
37.
38.
H. N., and A. Fleck. Analysis of tissues and body fluids
for nitrogenous
constituents.
In: MammaLian
Protein MetaboZism, edited by H. N. Munro. New York: Academic, 1969, vol. III,
p. 424-525.
Nagley,
P, and A. W. Linnane.
Mitochondrial
DNA deficient
petite mutants
of yeast. Biochem. Biophys. Res. Commun. 39:
989-996,197O.
Neufer,
P. D., and G. L. Dohm.
Exercise induces a transient
increase in transcription
of the GLUT-4 gene in skeletal muscle.
Am. J. Physiol. 265 (CeZZ PhysioZ. 34): C1597-C1603,1993.
Ostareck-Lederer,
A., D. H. Ostareck,
N. Standart,
and
B. J. Thiele. Translation
of 15-lipoxygenase
mRNA is inhibited
by a protein
that binds to a repeated
sequence in the 3’
untranslated
region. EMBO J. 13: 1476-1481,1994.
Pette, D., M. E. Smith, H. W. Staudte,
and G. Vrbova.
Effects
of long-term
electrical
stimulation
on some contractile
and
metabolic characteristics
of fast rabbit muscles. Pfluegers Arch.
338:257-272,1973.
39. Reichmann,
H., H. Hoppler,
0. Mathieu-Costello,
F. von
Bergen,
and D. Pette. Biochemical and ultrastructural
changes
of skeletal muscle mitochondria
after chronic electrical stimulation in rabbits. Pfluegers Arch. 404: l-9, 1985.
40. Rondon,
I. J., L. A. MacMillian,
B. S. Beckman,
M. A.
Goldberg,
T. Schneider,
H. F. Bunn,
and J. S. Malter.
Hypoxia upregulates
the activity of a novel erythropoietin
mRNA
binding protein. J. BioZ. Chem. 266: 16594-16598,199l.
41. Ross, J., and G. Kobs. H4 histone messenger RNA decay in
cell-free
extracts
initiates
at or near the 3’ terminus
and
proceeds 3’ to 5’. J. Mol. BioZ. 188: 579-593,1986.
42. Saccomanno,
C. F., M. Bordonaro,
J. S. Chen, and J. L.
Nordstrom.
A faster ribonuclease
protection
assay. Biotechniques 13: 847~849,1992.
43. Sachs, A. B. Messenger RNA degradation in eukaryotes.
CeZZ 74:
413-421,1993.
44. St. Johnston,
D. The intracellular
localization
of messenger
RNAs. CeZZ 81: 161-170,1995.
45. Sambrook,
J., E. F. Fritsch,
and T. Maniatis.
MoZecuZar
Cloning: A Laboratory
Manual, Cold Spring Harbor, NY: Cold
Spring Harbor, 1989, p. 1.40; p. El&E19.
46. Scarpulla,
R. C., K. M. Agne, and R. Wu. Isolation
and
structure
of a rat cytochrome
c gene. J. BioZ. Chem. 256:
6480-6486,1981.
47. Seedorf,
U., E. Leberer,
48.
49.
50.
51.
52.
53.
2323-2330,197l.
32. Moseley, P. L., E. S. Wallen,
J. D. McCafferty,
S. Flanagan,
and J. A. Kern.
Heat stress regulates
the human 70-kDa
heat-shock
gene through
the 3’-untranslated
region. Am. J.
PhysioZ. 264 (Lung CeZZ. Mol. Physiol. 8): L533-L537,
1993.
33. Miillner,
E. W., and L. C. Kuhn.
A stem-loop
in the 3’
untranslated
region mediates iron-dependent
regulation of trans-
CHRONIC
54.
B. J. Kirschbaum,
and D. Pette.
Neural control of gene expression
in skeletal muscle. Effects of
chronic stimulation
on lactate dehydrogenase
isoenzymes
and
citrate synthase. Biochem. J. 239: 115-120,1986.
Sonenberg,
N. mRNA translation:
influence of the 5’ and 3’
untranslated
regions. Curr. Opin. Genet. Dev. 4: 310-315,1994.
Standart,
N., and M. Dale. Regulated polyadenylation
of clam
maternal mRNAs in vitro. Dev. Genet. 14: 492-499,1993.
Takahashi,
M., D. T. McCurdy,
D.A. Essig, and D.A. Hood.
Delta-aminolaevulinate
synthase
expression
in muscle after
contractions
and recovery. Biochem. J. 291: 219-223,1993.
Taniuchi,
H., M. Basile, M. Taniuchi,
and D. Veloso. Evidence for formation
of two thioether
bonds to link heme to
apocytochrome
c by partially purified cytochrome c synthetase.
J.
BioZ. Chem. 258: 10963-10966,1983.
Williams,
R. S., M. Garcia-Moll,
J. Mellor, S. Salmons,
and
W. Harlan.
Adaptation
of skeletal muscle to increased contractile activity. Expression
of nuclear genes encoding mitochondrial
proteins. J. BioZ. Chem. 262: 2764-2767,
1987.
Yan, Z., S. Salmons,
J. Jarvis,
and F. W. Booth.
Increased
muscle carnitine palmitoyltransferase
II mRNA after increased
contractile
activity. Am. J. PhysioZ. 268 (EndocrinoZ. Metab. 31):
E277-E281,1995.
You, Y., C.-Y. A. Chen, and A.-B. Shyu.
U-rich
sequencebinding proteins (URBPs)
interacting
with a 20nucleotide
Urich sequence in the 3’ untranslated
region of c-fos mRNA may be
involved in the first step of c-fos mRNA degradation.
MOL. CeZZ.
BioZ. 12: 2931-2940,1992.