Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612-7342
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ABSTRACT |
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We tested the
hypothesis that the -myosin heavy chain (
-MHC) 3'-untranslated
region (UTR) mediates decreased protein expression after tenotomy of
the rat soleus. We also tested the hypothesis that decreased protein
expression is the result of RNA-protein interactions within the 3'-UTR.
-MHC was chosen for study because of its critical role in the
function of postural muscles such as soleus. Adult rat soleus muscles
were directly injected with luciferase (LUC) reporter constructs
containing either the
-MHC or SV40 3'-UTR. After 48 h of
tenotomy, there was no significant effect on LUC expression in the SV40
3'-UTR group. In the
-MHC 3'-UTR group, LUC expression was 37.3 ± 4% (n = 5, P = 0.03) of that in sham
controls. Gel mobility shift assays showed that a protein factor
specifically interacts with the
-MHC 3'-UTR and that tenotomy
significantly increases the level of this interaction (25 ± 7%,
n = 5, P = 0.02). Thus the
-MHC
3'-UTR is directly involved in decreased protein expression that is
probably due to increased RNA-protein binding within the UTR.
mechanical signal transduction; ribonucleic acid binding protein; translational control; muscle atrophy
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INTRODUCTION |
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TENOTOMY IS AN
UNLOADING MODEL of muscle atrophy that results in rapid decrease
of muscle cell size and protein content (9). Tenotomy
results in a 34% decrease in wet weight after 1 wk (9), and, specifically, decreases the -myosin heavy chain (
-MHC) protein content of postural muscles such as the soleus
(12). It is well known that chronic adaptation is mediated
by changes at the level of transcription and protein degradation
(14), which do not have significant effects until about 1 wk of decreased load (3, 5). Acute
regulation, however, is less well understood, but translational control
may be one of the earliest adaptive events of unloading. Protein
synthesis can be quickly adjusted by control of initiation, elongation,
or altering message stability (5, 13,
19).
Regulation that takes place during the initiation phase of translation
occurs by altering the rate at which ribosomes attach to the
5'-untranslated region (UTR) of mRNA (18). Regulation can
also occur during the elongation phase as ribosomes move along the mRNA
to translate protein. Analysis of polysomal mRNA distribution on
sucrose density gradients suggests that protein synthesis after tenotomy is translationally regulated during the elongation phase (15). The mechanisms of such regulation are unclear, but
recent work suggests that the 3'-UTR may be important. For example, in primary cultured cardiac myocytes, -MHC is translationally regulated via its 3'-UTR after contractile arrest (7). In another
study, translation of myocyte enhancer factor 2A was shown to be
repressed via sequences in its 3'-UTR (4). One
interpretation of the role of the 3'-UTR is that highly conserved
sequences within this region may help cells respond to environmental
stresses such as hypoxia or heat shock (8,
17). Mechanically transduced changes, such as the severely
decreased load after tenotomy, could also be considered a major stress.
In this study, we investigated the possibility that the 3'-UTR of
-MHC, a major contractile protein, plays a role in the
mechanotransduction pathway that leads to atrophy after tenotomy.
Translational regulation is usually mediated by the interaction of
trans-acting protein factors with the 5'- or 3'-UTR of mRNA.
These interactions, which depend on both the mRNA primary sequence and
its secondary structure, can initiate a chain of events that leads to
altered protein expression. For example, altered protein-3'-UTR binding
has a role in regulating expression of cholesterol 7-hydroxylase in
liver (1). More importantly, Booth and Kirby
(5) have shown that proteins specifically interact with
activity-responsive elements in the cytochrome c 3'-UTR of skeletal muscle. We have tested whether a similar mechanism may be involved in the regulation of
-MHC protein expression also involving the 3'-UTR.
Our hypothesis is that the -MHC 3'-UTR mediates decreased protein
synthesis after 48 h of tenotomy in rat soleus muscle. Furthermore, we hypothesize that this regulation of protein expression is the result of specific and differential interactions of
trans-acting protein factors with the
-MHC 3'-UTR that
effectively downregulate protein synthesis after tenotomy.
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METHODS |
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Animals
Proper animal handling protocols were conducted at all times. Age- and weight (>250 g)-matched female Sprague-Dawley rats were used 1 wk postpartum, after pups had been collected for use in other experiments. Animals were provided with food and water ad libitum.DNA Construct
The full-length
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DNA Injection
Rats were anesthetized by ether inhalation and subsequent intramuscular injection of ketamine-xylazine (0.1 ml/g). A small incision (<2 cm) was made in the hindlimb and the soleus exposed by blunt dissection. The left and right soleus muscles of each animal were injected with pGL3-SV40-3'-UTR or pGL3-Tenotomy
Rats were again anesthetized by ether inhalation and subsequent intramuscular injection of ketamine-xylazine (0.1 ml/g). To induce the atrophic process, the right calcaneus tendon was severed with a sterile scalpel. The left tendon was exposed but left intact to serve as the sham control. Wounds were sealed with Nexaband tissue sealant (Veterinary Products, Phoenix, AZ). The rats were awake and mobile by 3 h posttenotomy. Rats were killed 48 h after tenotomy. The left and right soleus muscles were carefully removed, flash frozen in liquid nitrogen, and stored atImmunohistochemistry
Tissue was mounted on cork, frozen, and serial sectioned into 10-µm slices. Sections were taken from areas near the injection site as identified by india ink marking. The sections were washed in PBS, fixed with 4% paraformaldehyde for 10 min, and incubated in primary anti-luciferase antibody (Promega) for 45 min. The sections were then incubated in BSA for 15 min to reduce nonspecific binding of the secondary antibody. Finally, sections were incubated in fluorescein-labeled secondary antibody for 30 min, washed in PBS, and mounted on glass slides. The sections were analyzed for luciferase protein expression using digital microscopy (Nikon Microphot FXA microscope fitted with Photometrics-cooled charge-coupled device camera).Tissue Processing
Each soleus was ground to a fine powder in liquid nitrogen using a mortar and pestle and placed in precooled, preweighed plastic tubes for use in luminometry experiments, the bicinchoninic acid assay (BCA; Pierce, Rockford, IL), and RNA-protein gel mobility shift assays. Cell lysis buffer containing 0.5% Nonidet P-40 (NP-40), 10 mM HEPES, 3 mM MgCl2, 40 mM KCl, 2 mM dithiothreitol (DTT), 0.5 mM phenylmethylsufonyl fluoride, and Sigma protease inhibitor cocktail (Sigma, St. Louis, MO) was added (3.5 µl/mg tissue), and the tissue was homogenized on ice at high speed (30,000 rpm) for 60 s (3 × 20 s) using a Tissue Tearor homogenizer (Biospec). To eliminate large cell debris, this homogenate was centrifuged at 3,000 rpm at 4°C for 10 min. The supernatant fraction was then transferred to a new tube and stored atLuminometry Assay for Translated Luciferase
Soleus extract was thawed and 100 µl added to a glass test tube. Luciferase (500 µl) enzyme substrate (Promega) was added to the test tube, carefully mixed, and the ensuing luminescence reaction was quantified using an EG & G Berthold Lumat LB luminometer. Light production was expressed in relative luminometry units (RLU).Total Protein Measurement
For measurement of total protein, tissue was ground and homogenized in cell lysis buffer as above. The BCA assay was then used to measure protein concentration in each sample. Briefly, 10 µl of sample was added to 200 µl of working reagent and incubated at 37°C for 30 min. The colorimetric reaction was analyzed at 550 nm and compared with a protein standard prepared from known concentrations of BSA. This information was used to assess changes in total protein concentration after tenotomy and to standardize the amount of protein used in the RNA-protein gel mobility shift assay.RNA-Protein Gel Mobility Shift Assay
Probes.
Sense and antisense [-32P]CTP-labeled RNA probes
containing the complete
-MHC,
-MHC, or SV40 3'-UTR were
transcribed in vitro from cDNA linearized vectors. Sense probes were
transcribed using SP6 RNA polymerase, and antisense probes were
transcribed using T7 RNA polymerase. Both radiolabeled probes were
transcribed by using 500-µM cold ATP, GTP, and UTP and 12-µM cold
CTP and were labeled with 25 µCi [
-32P]CTP by
incubation at 37°C for 1 h. For nonradiolabeled probes, 500-µM
cold ATP, CTP, GTP, and UTP were combined and incubated with polymerase
at 37°C for 1 h. RNase-free DNase I (1 unit) was added to digest
DNA, and the unincorporated nucleotides were removed using Sephadex
spin columns (ProbeQuantTM G-50 microcolumns; Pharmacia Biotech,
Piscataway, NJ). Radiolabeled probes were analyzed by PAGE and
radioactivity determined on a scintillation counter. Nonradiolabeled
probes were analyzed by agarose/urea gel electrophoresis. The average
riboprobe RNA concentration was ~50 µg/ml as determined spectrophotometrically by excitation at 260 and 280 nm. Probe folding
pattern was estimated using the mFold 2.3 folding program (Dr. M. Zuker, Washington Univ., St. Louis, MO) (22).
Native Gels. To determine gross RNA-protein interactions, soleus muscle homogenate was combined with the appropriate volume of incubation buffer (0.5% NP-40, 10 mM HEPES, 3 mM MgCl2, 40 mM KCl, and 2 mM DTT), RNase inhibitor (50 units), and radiolabeled sense or antisense probe (40,000 cpm) for a final volume of 21 µl (55 mg protein/ml). The mixture was incubated for 12 min at room temperature to allow RNA-protein hybridization to occur. To verify that shifted bands were the result of protein interaction with RNA, some samples were combined with proteinase K (50 or 100 µg/ml) and then incubated at 37°C for 30 min before hybridization with radiolabeled probe. After hybridization, 10 µl of loading buffer (8% sucrose, 0.025% bromphenol blue, 0.025% xylene cyanol, and 1× Tris base EDTA boric acid) was added and the mixture electrophoresed on a 5% polyacrylamide gel. The samples were run for ~4 h at 4°C at 35-40 mA.
For competition experiments, soleus muscle homogenate was combined with the appropriate volume of incubation buffer and RNase inhibitor (50 units) as above. Then, before radiolabeled sense probe was added, nonradiolabeled sense probes (10×, 50×, or 100× vs. RNA concentration of the radiolabeled probe) were added to the mixture. After mixing and incubation for 1-3 min, radiolabeled sense probe was added (40,000 cpm) for a final volume of 21 µl (55 mg protein/ml). The mixture was incubated for 12 min at room temperature to allow RNA-protein hybridization to occur. RNA-protein complex formation was assayed by nondenaturing PAGE as above.Denaturing gels. For denaturing gels, samples were prepared as above (see Native Gels). After hybridization, the RNA-protein complexes were covalently cross-linked for 9 min (8,400 µJ), 3 cm from the source. Four microliters of 5× SDS loading buffer (95% formamide, 0.025% xylene cyanol, 0.025% bromphenol blue, 0.5 mM EDTA, and 0.025% SDS) were added and samples were then heated at 95°C for 4 min. Samples were loaded onto a 7% SDS polyacrylamide separating gel with a 4% stacking gel. Gels were run for about 3 h at room temperature at 35-40 mA.
All RNA-protein interactions were visualized by exposure of gel to Kodak Biomax MS radiosensitive film (Eastman Kodak, Rochester, NY) and analyzed by densitometry (Molecular Dynamics, Sunnyvale, CA).Statistics
Data are presented as means ± SE, where n equals the total number of rats used in a given experiment. Unless otherwise noted, paired Student's t-test was used on raw data to determine statistical significance at the P < 0.05 level. ![]() |
RESULTS |
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3'-UTR Reporter Constructs Are Expressed After Direct Injection In Vivo
Soleus muscle successfully incorporated and expressed luciferase protein after transfection by direct injection of the construct shown in Fig. 1A. Transfected muscle (Fig. 2A) stained positively for luciferase protein indicating successful local transfection. We found relatively homogenous transfection in an area local to the injection site and variable transfection at more distant locations. Untransfected muscle (Fig. 2B) showed no staining, confirming that confirms that secondary antibody does not bind the tissue in the absence of luciferase protein.
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Tenotomy Does Not Alter Total Protein Concentration by 48 h
To assess the gross short-term effects of tenotomy, the BCA assay was used to determine total protein concentration before and after tenotomy. As expected, total protein concentration in tenotomized soleus was unchanged at 104 ± 5% (n = 9, P = 0.5) of the concentration measured in sham controls. Thus 48 h posttenotomy is too early for gross changes in protein content to be detected.-MHC 3'-UTR Mediates Decreased Luciferase Protein Expression
After Tenotomy
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The Sense and Antisense -MHC 3'-UTRs Are Homologous in Shape
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A Trans-Acting Protein Factor Interacts With the
-MHC 3'-UTR in a Sequence-Specific Manner
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The mobility pattern of sense probe plus extract was completely
different from that observed with antisense probe plus extract (Fig.
5A, lane 8). This difference indicates that the
interaction seen in lane 5 is sequence specific.
The binding is also completely different when the same muscle extract
is hybridized with the radiolabeled -MHC 3'-UTR probe (Fig.
5B, lane 4). Furthermore, no band
appears at 500 nt when the radiolabeled SV40 3'-UTR sense probe is
hybridized with extract (Fig. 5C, lanes
8-11). Thus the SV40 3'-UTR probe does not interact with the
same protein factor as the
-MHC 3'-UTR probe.
Competition with cold sense probe was used as a further test of binding
specificity. Hybridization of sense probe to muscle extract was allowed
to occur in the presence of 10× or 50× nonradiolabeled homologous
sense probe. The interaction at ~500 nt slightly decreased in the
10× cold probe lane (Fig. 5A, lane 6), and the
free probe band increased. In the 50× cold probe lane, the 500 nt
RNA-protein interaction was almost completely competed away, and the
free probe band at 163 nt returned to levels approaching those observed in the probe alone lane (Fig. 5A, lane 7).
However, cold heterologous (-3'-UTR) probe does not compete away the
interaction at 500 nt (Fig. 5D, lanes 6-8).
This indicates that a specific RNA-protein interaction does occur
between the
-MHC mRNA 3'-UTR and a protein binding factor.
Samples were incubated with proteinase K to digest the proteins to
verify that the band observed at 500 nt was the result of mRNA
interaction with a protein and not the result of nucleic acid complex
formation or double-stranded RNA. As shown in Fig. 5B
(lanes 2 and 3), incubating extract with
proteinase K for 30 min at 37°C (50 or 100 µg/ml) eliminates the
shifted band. This confirms that it is indeed a protein that is binding
to the sense -MHC 3'-UTR riboprobe.
Specific Protein Binding Factor Interaction With the -MHC 3'-UTR
Is Increased After Tenotomy
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DISCUSSION |
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Our findings support the hypothesis that a
trans-acting protein factor interacts with the -MHC
3'-UTR to induce decreased protein expression within 48 h of
unloading. This mechanism offers muscle a way to reduce mass quickly to
minimize energy demands.
Translational Control of Protein Synthesis After Tenotomy
The results of the present investigation showed that tenotomy is a negative regulator of protein expression in vivo and acts via a 3'-UTR-dependent mechanism. The decrease in luciferase expression after tenotomy may be attributed to translational control in the form of altered translational rates or altered mRNA stability. We think that this 3'-UTR-mediated effect is the result of translational block and not decreased mRNA stability. This is consistent with other studies on muscle. For example, analysis of polysomal mRNA distribution on sucrose density gradients showed that atrophy after tenotomy is translationally regulated during the elongation phase (15). Furthermore, in our laboratory, primary cultured skeletal myocytes were transfected with the same pGL3-SV40-3'-UTR and PGL3-Though our data showed that no change can be detected in total protein
levels after 48 h of tenotomy, it is well established that
tenotomy triggers a cascade that eventually leads to atrophy (9). However, we did show that expression of luciferase
from a transfected construct changed after 48 h of tenotomy.
Remembering that the effects of protein degradation are not significant
until about 1 wk of unloading, this suggests that expression of
endogenous -MHC protein would be translationally blocked after
tenotomy. Furthermore, the 25% decrease in total MHC levels observed
by Jakubiec-Puka et al. (12) after 2 days of
tenotomy could be explained entirely by decreased de novo translation
of MHC protein in the unloaded muscle.
RNA-Protein Interaction Within the -MHC 3'-UTR
Our discovery of this mechanism is novel for -MHC in vivo, but
similar findings have been reported in vitro. Yan et al.
(21) showed an increase in contractile activity of
skeletal muscle leads to a decrease in RNA-protein interaction in the
cytochrome c 3'-UTR. In addition, other data from our
laboratory showed that decreased contractile activity leads to
increased binding of proteins with the
-MHC 3'-UTR in cardiac
myocytes (10). Moreover, our interpretation fits well with
a model offered by Ku and Thomason (15) that suggests that
translational block occurs as a result of ribosomes stalled on the mRNA
strand. Protein interaction with the 3'-UTR could cause such a stall by
physically blocking ribosome movement, by altering the structure of the
mRNA, or by simply masking the mRNA (20). It is possible
that protein interaction with the 3'-UTR also increases nuclease
activity that effectively destabilizes the mRNA.
Model of Translational Control After Unloading
We have shown that tenotomy results in decreased protein expression mediated by the
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Our study confirms earlier work that suggests that the effects of
unloading are translationally controlled (5,
14) and proves that translational control, including
alterations in mRNA stability, can be directly mediated by the -MHC
mRNA 3'-UTR.
-MHC is critical for proper function of postural muscle
and is the primary protein affected during unloading-related atrophy. The mechanism suggested here would allow a muscle to regulate
-MHC
protein expression quickly and reversibly.
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ACKNOWLEDGEMENTS |
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We thank to Dr. G. Goldspink for providing the -MHC 3'-UTR, Dr.
G. Nikcevic for expert help in creating the luciferase constructs and
careful reading of the manuscript, and Dr. R. J. Solaro and David
Montgomery for critical reading of the work.
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FOOTNOTES |
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This research was supported by National Institutes of Health Grants HL-40880 (B. Russell) and AR-08405 (W. Ashley).
Address for reprint requests and other correspondence: B. Russell, Dept. of Physiology and Biophysics, Univ. of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-7342 (E-mail: Russell{at}uic.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 10 June 1999; accepted in final form 27 January 2000.
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