|
Advertisement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
J. Biol. Chem., Vol. 280, Issue 27, 25361-25368, July 8, 2005
The RNA-binding Protein HuR Binds to Acetylcholinesterase Transcripts and Regulates Their Expression in Differentiating Skeletal Muscle Cells*![]() ![]() ![]() ¶![]() ![]() ![]() ![]() ||**![]() ![]() ![]() ![]() ![]() **¶¶
From the
Received for publication, September 22, 2004 , and in revised form, May 3, 2005.
During myogenic differentiation, acetylcholinesterase (AChE) transcript levels are known to increase dramatically. Although this increase can be attributed in part to increased transcriptional activity, posttranscriptional mechanisms have also been implicated in the high levels of AChE mRNA in myotubes. In this study, we observed that transfection of a luciferase reporter construct containing the full-length AChE 3'-untranslated region (UTR) resulted in significantly higher (5-fold) luciferase activity in differentiated myotubes versus myoblasts. RNA-electrophoretic mobility shift assays (REMSAs) performed with a full-length AChE 3'-UTR probe and the AU-rich element revealed that the intensity of RNA-binding protein complexes increased as myogenic differentiation proceeded. Using several complementary approaches including supershift REMSA, mRNA-binding protein pull-down assays, and immunoprecipitation followed by reverse transcription-PCR, we found that the mRNA-stabilizing protein HuR interacts directly with AChE transcripts. Stable overexpression of HuR in C2C12 cells increased the expression of endogenous AChE transcripts as well as that of the luciferase reporter construct containing the AChE 3'-UTR. In vitro stability assays performed with protein extracts from these cells versus controls resulted in a slower rate of AChE mRNA decay. The down-regulation of HuR expression mediated through small interfering RNA further confirmed the role of HuR in the regulation of AChE mRNA levels. Taken together, these studies demonstrate that HuR interacts with the AChE 3'-UTR to regulate posttranscriptionally the expression of AChE mRNA during myogenic differentiation.
Neurotransmission at cholinergic synapses of the central and peripheral nervous systems is promptly terminated by the catalytic activity of the enzyme acetylcholinesterase (AChE)1 (for review see Refs. 14). In addition to this pivotal role in terminating synaptic transmission, several lines of evidence have shown that AChE assumes other functions necessary for the normal operation of the nervous system (for examples see Refs. 57). Appropriate expression of AChE is not only important in the brain, but it is also critical for normal skeletal muscle activity as exemplified by the phenotype seen in patients suffering from a myasthenic syndrome linked to a reduction of AChE at the neuromuscular junction (8, 9). Given the functional significance of AChE in the nervous system, it thus becomes important to identify the molecular and cellular mechanisms regulating its expression in developing tissues as well as in neurons and skeletal muscle cells placed under a variety of conditions known to markedly affect AChE expression.
AChE is encoded by a single gene that is alternatively spliced to produce catalytic subunits that can be assembled into different molecular forms. In skeletal muscle, the AChET transcript is predominantly expressed (1013) resulting in the synthesis of a catalytic subunit that can associate with structural subunits encoded by separate genes (14). In particular, the asymmetric forms of AChE as well as AChE transcripts themselves are preferentially expressed at the level of the neuromuscular junction (1417). In mature muscle, AChE expression is tightly controlled by nerve-evoked electrical activity as well as by nerve-derived trophic factors (14, 1821). However, AChE is also known to be regulated during myogenic differentiation, at times preceding the occurrence of nerve-muscle interactions (2224). Under these conditions, however, there is relatively little information available concerning the nature of the molecular mechanisms that control AChE expression.
In a recent study, we have shown that an increase in the transcriptional
activity of the AChE gene could partially account for the initial
increase in AChE transcript levels observed in differentiating muscle cells
grown in culture (25). In this
latter study, the contribution of E- and N-box motifs located within the first
intronic region, along with their respective transcription factors, myogenin
and GA-binding protein
Cell CultureMouse C2C12 cells (ATCC, Manassas, VA) were cultured on culture dishes (6-well and 100-mm) coated with Matrigel (Collaborative Biomedical Products, Bedford, MA) in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% fetal bovine serum, 292 ng/ml L-glutamine, and 100 units/ml penicillin-streptomycin in a humidified chamber at 37 °C with 5% CO2. Myogenic differentiation and fusion was induced by replacing the growth medium on confluent myoblasts with differentiation medium containing low serum (2% horse serum). Culture medium was changed every 48 h.
Plasmid Construction and Transfection StudiesThe
3'-untranslated region (UTR) of the mouse The mouse HuR coding region was amplified by PCR from mouse muscle cDNA using primers designed from the available mouse HuR sequence that spans the entire coding region. The resultant PCR product was subcloned into the pCI-neo vector (Promega) driven by the cytomegalovirus enhancer/promoter. The resulting vector (pHuR) product was sequenced and used to stably transfect C2C12 cells that were selected for G418 resistance resulting in a mouse HuR overexpressing C2C12 stable cell line pool. Plasmid DNA was prepared using the Mega-Prep procedure (Qiagen, Mississauga, Ontario, Canada). DNA pellets were resuspended in 10 mM Tris-HCl, pH 8.5. Transfections were performed with the Lipofectamine reagent kit (Invitrogen) according to the manufacturer's instructions. Briefly, C2C12 myoblasts at 80% confluency were transfected with a total of 2 µg of plasmid DNA (1.0 µg of either the reporter construct or the parental vector and 1.0 µg of the vector used for transfection efficiency). Transfection efficiency was monitored by co-transfecting the basic firefly luciferase construct (pGL3) or the basic LacZ reporter vector (30). Transfected myoblasts were collected 2448 h after transfection or were induced to differentiate and collected 4872 h later.
To determine reporter gene activity, transfected myoblasts and myotubes
were washed with cold 1x PBS, scraped, and lysed in 1x reporter
lysis buffer (Invitrogen) followed by several freeze-thaw cycles. The extracts
were centrifuged (12,000 x g for 10 min at 4 °C), and the
resulting supernatant was used for the luciferase assays (Renilla and
firefly) or for HuR siRNA and Transfection StudiesThe HuR normal and mutant siRNA duplexes (target sequence, 5'-AAAAGUCUGUUCAGCAGCAUUGG-3', and mutant sequence, 5'-AAAAGUCAAUUCAUCAGCAAUGG-3') were obtained from Dharmacon RNA Technologies. C2C12 myoblasts were transfected with siRNA duplexes designed to mouse HuR sequence using the RNAiFect transfection kit (Qiagen) according to the manufacturer's instructions. Transfected myoblasts were harvested within 2448 h, and RNA was extracted (see below) and used for reverse transcription (RT)-PCR (see below).
Adenoviral Construct and Infection StudiesAn adenoviral
construct containing the human HuD sequence (AdLS5a) was generated using the
HuD sequence subcloned from the previously described pcHuD vector
(31). AdLS5a contains the
human HuD cDNA under the regulation of the cytomegalovirus promoter and bovine
growth hormone polyadenylation sequence. In these viruses, the HuD expression
cassette replaces the early region 1, and transcription is directed leftward,
relative to the conventional human adenovirus serotype 5 map. A control virus,
Ad-lacZ, contains the Escherichia coli Confluent C2C12 myoblasts were infected with 9.5 x 107 particles of the adenoviral construct in a volume of 100 µl of culture medium, with a multiplicity of infection of 475. At this multiplicity of infection, almost all of the cells on the plate were infected.2 The cells were placed in a humidified incubator at 37 °C and 5% CO2 for 1 h before the addition of a supplementary growth medium. During this period, the cell culture plates were agitated every 15 min to ensure that the HuD adenoviral construct was in contact with all of the cells in the dish. The culture medium was changed 24 h later, and myogenic differentiation was induced with a low serum culture medium. Infected myoblasts and myotubes were washed with 1x PBS 2472 h following infection, and RNA was extracted and used for RT-PCR as described below.
RNA Extraction and RT-PCRTotal RNA was extracted from myoblasts and differentiating myotubes using 1.0 ml of TRIzol reagent (Invitrogen) according to the manufacturer's instructions and as described previously (25, 28). Precipitated RNA was resuspended in RNase-free water and stored at 80 °C until used. RNA from each sample was quantified using the Amersham Biosciences Gene Quant II RNA/DNA spectrophotometer and adjusted to a final concentration of 50 ng/µl. Reverse transcription of 4 µl of RNA (200 ng) was performed using the Omniscript reverse transcription kit (Qiagen) as recommended by the manufacturer. Briefly, the RNA was reverse transcribed in a final volume of 20 µl of RT master mix, 0.5 mM concentration of each dNTP, 10 units of RNase inhibitor (Applied Biosystems, Foster City, CA), 1 µM random hexamers (Applied Biosystems), and 4 units of Omniscript reverse transcriptase. RT was carried out at 37 °C for 60 min. Negative controls consisted of the same RT mixture in which the RNA was replaced with RNase-free water. All of the samples were prepared using the same RT master mix. PCR was used to amplify the cDNAs corresponding to AChE, HuR, and S12 ribosomal protein (S12 hereafter) mRNAs. The primers for AChE, S12 (used as an internal control), and HuR (5'-primer CAGAGGTCATCAAAGATGC and 3'-primer ATCCCACTCATGTGATCTAC) were synthesized based on available sequences and amplified products of 670, 368, and 394 bp, respectively (16, 17, 25, 3638). PCR was performed using HotStarTaq DNA polymerase (Qiagen) according to the manufacturer's instructions. All of the samples were prepared using the same PCR master mix. PCR cycling parameters consisted of an initial activation step at 95 °C for 15 min followed by denaturation at 94 °C for 1 min; annealing at 70 °C (for AChE), 54 °C (for S12), and 57.5 °C (for HuR) for 1 min each; and extension at 70 °C (for AChE) for 2 min and 72 °C (for S12 and HuR) for 1 min followed by a 10-min elongation step at 72 °C. PCR amplification was stopped during the linear range of amplification (16, 17). Typically, the cycle numbers were 35 cycles for AChE, 24 cycles for S12, and 24 cycles for HuR. PCR products were visualized on ethidium bromide-stained 1.5% agarose gels and quantified using the fluorescent dye VistraGreen (Amersham Biosciences) in 1.5% agarose gels. Quantification was performed using the Storm PhosphorImager and the accompanying ImageQuant software (Amersham Biosciences). The values obtained for AChE and HuR were standardized relative to the corresponding level of S12 in the same sample. In Vitro Stability AssayPoly(A)+ RNA was isolated from a total RNA extract obtained from mouse brain using Oligotex mRNA mini kit (Qiagen) according to the manufacturer's instructions. Cytoplasmic protein fractions were prepared as described previously (39) from C2C12 cells stably transfected with the HuR cDNA (pHuR cells) or from cells stably transfected with the empty vector (pCI-neo) as control. Briefly, C2C12 cell pellets were homogenized in MOPS buffer (10 mM MOPS-NaOH, pH 7.2, 200 mM sodium chloride, 2.5 mM magnesium acetate) with 100 µM dithiothreitol, 100 µM phenylmethylsulfonyl fluoride, and 1 Complete Mini, EDTA-free Protease Inhibitor Cocktail tablet as per the manufacturer's recommendations (Roche Applied Science). Homogenates were centrifuged (10,000 x g for 15 min at 4 °C), and the resulting supernatants were stored until further use. After optimization of protein and RNA concentrations, 2.5 ng/µl poly(A)+ RNA were incubated with 0.2 µg/µl protein extracts in MOPS buffer containing 1 mM ATP, 0.1 mM spermine, 2 mM dithiothreitol, and 1 unit/µl SUPERa-sin (Ambion, Austin, TX) in a final volume of 180 µl and incubated at 37 °C. At intervals of 0, 15, and 30 min, aliquots of 40 µl were removed, and the reaction stopped by adding 200 µl of ice-cold phenol/chloroform. De-proteination and precipitation of the RNA was then carried out in the presence of yeast tRNA (10 µg) as a carrier. AChE mRNA was detected by RT-PCR as described above.
In Vitro TranscriptioncDNAs encoding for the AChE
full-length 3'-UTR, the ARE region alone, and the AChE 3'-UTR
minus the ARE were obtained by PCR amplification of the
pGL33'-UTR plasmid as described previously
(19,
28). This cDNA was used as the
template for the in vitro transcription of the 3'-UTR. In
vitro transcription was performed with the T7 in vitro
transcription system (Promega) as recommended by the manufacturer. Briefly 0.5
µg of cDNA, 5 µCi of [ Electrophoretic Mobility Shift Assay and SupershiftRNA-based electrophoretic mobility shift assays (REMSAs) and supershift assays were performed with total protein extracts obtained from myoblasts and myotubes. The cells were washed with 1x PBS, scraped, and lysed in 500 µl of homogenization buffer (0.3 M sucrose, 60 mM NaCl, 15 mM Tris, pH 8.0, 10 mM EDTA) supplemented with protease inhibitors (mini-complete protease inhibitor, Roche Applied Science) followed by several freeze-thaw cycles. The samples were centrifuged (15,000 x g for 15 min at 4 °C), and the resulting supernatants were stored at 80 °C in 20100-µg aliquots to be used for REMSAs and other assays. The total amount of protein present in the extracts was determined with the Coomassie protein assay kit (Pierce). REMSAs were performed as described previously (28). In brief, 20 µg of protein extract were incubated with 2 x 105 cpm of 32P-labeled AChE 3'-UTR or the ARE region in the presence of yeast tRNA (0.2 µg/µl) and heparin (2.5 mg/ml) for 20 min at room temperature. The mixture was separated by 5% native PAGE with 0.5x Tris borate-EDTA running buffer. The gels were subsequently dried and exposed to film at 80 °C. Competition assays were performed by incubating an excess of cold probe with the protein for 10 min prior to the incubation with the radiolabeled RNA. Supershift assays were performed by incubating the protein in the binding buffer with 0.5 µg of mouse monoclonal antibody directed against HuR (Molecular Probes, Eugene, OR) or a nonspecific antibody at room temperature for 1 h prior to the addition of the radiolabeled transcripts. Immunoprecipitation and AChE mRNA AnalysisImmunoprecipitation of HuR from myoblasts and myotubes was performed as previously described with some modifications (28, 40). Myoblast and myotube protein extracts obtained as described above were precleared with 5 µg of normal mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and 30 µl of paramagnetic protein G Dynabeads (Dynal Biotech, Oslo, Norway) in 300 µl of immunoprecipitation buffer (10 mM Tris-HCl, pH 7.5, 1% Igepal CA-630, 1% bovine serum albumin fraction V, 2 mM EDTA, 3 mM magnesium acetate, 100 µM ZnCl2, 1 µg/ml yeast tRNA) for 1 h at room temperature with constant mixing. The precleared protein extracts were then incubated with 2 µg of a mouse monoclonal antibody directed to HuR (Molecular Probes) or normal mouse IgG (Santa Cruz Biotechnology) for 1 h at room temperature with constant mixing. The protein antibody solution was incubated with 30 µl of prewashed paramagnetic protein G Dynabeads for 1 h at room temperature with constant mixing. The protein G Dynabeads were washed with immunoprecipitation buffer three times, and the protein and mRNA were extracted with 0.5 ml of TRIzol according to the manufacturer's instructions. The mRNA was precipitated in the presence of 10 µg of RNase-free glycogen (GenHunter, Nashville, TN) and subsequently used for RT-PCR. The protein pellet was solubilized in 1x PBS with 1% SDS and used for Western blotting (see below). mRNA-binding Protein Pull-down AssayMyoblast and myotube protein extracts (100 µg) were incubated with 2 µg of biotin-labeled AChE 3'-UTR with and without the ARE or with the ARE region alone, in 1x binding buffer as used in REMSAs (20 mM Hepes, pH 7.5, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol, 0.1 mM ZnCl2, 5% glycerol, 0.2 mg/ml yeast tRNA) for 30 min at room temperature with intermittent mixing. The biotin-labeled RNA and associated mRNA-binding proteins were incubated with 50 µl of paramagnetic streptavidin Dynabeads (Dynal Biotech), prewashed in RNase-free buffers and 1x binding buffer for 1 h at room temperature with constant mixing, and washed three times with 1x binding buffer. The protein was solubilized in PBS and subjected to Western blot analysis for the detection of HuR.
Western BlotFor Western blotting, 25 µg of myoblast and
myotube protein extracts or proteins extracted from the mRNA-binding pull-down
assays were denatured in SDS loading buffer and subjected to 3% SDS-PAGE. The
separated proteins were transferred to nitrocellulose Trans-Blot transfer
medium (Bio-Rad, Hercules, CA). Following transfer the membranes were blocked
in either 10% nonfat skim milk or 10% bovine serum albumin in Tris-buffered
saline and Tween 20 for 1 h at room temperature with constant agitation. The
membranes were incubated with mouse monoclonal antibodies directed against HuR
(1:1,000) or Statistical AnalysisAn analysis of variance was performed to evaluate the effect of the AChE 3'-UTR on luciferase activity in transfected C2C12 cells. The Fisher's least squares difference test was used to determine whether the differences seen between group means were significant. The level of significance was set at p < 0.05. Data are expressed as mean ± S.E. throughout.
AChE mRNA Expression during Myogenic DifferentiationIn an initial series of experiments, we examined the levels of AChE mRNA during myogenic differentiation by RT-PCR. As shown in Fig. 1A, AChE mRNA levels are barely detectable in 50% confluent myoblasts and increase considerably as the cells become more confluent and begin to differentiate. Upon the second day of differentiation, AChE transcript levels increased by 10-fold over the levels seen in 50% confluent myoblasts. The extent of
this increase reached more than 50-fold in 4-day-old myotubes
(Fig. 1B). This
pattern of expression of AChE mRNA during myogenic differentiation is in
agreement with earlier reports
(23,
25).
Previous studies have also shown that changes in the transcriptional
activity of the AChE gene cannot solely account for the large
increase in transcript levels seen in myotubes, therefore implicating
posttranscriptional mechanisms such as those regulating transcript stability.
Because elements located in the 3'-UTR are often implicated in the
control of mRNA turnover (41,
42), we next sought to
determine whether the AChE 3'-UTR plays a role in regulating the
relative abundance of AChE transcripts in differentiating muscle cells. To
this end, we engineered a luciferase reporter construct in which the
endogenous 3'-UTR was replaced with the mouse AChE 3'-UTR
terminating at the first polyadenylation signal (see
Fig. 2A). The short
3'-UTR was chosen over the longer 3'-UTR because previous studies
have demonstrated that the first polyadenylation signal is used preferentially
over the second signal (23,
43). As a result, there is
significantly more of the
Transfection of this construct into myoblasts resulted in a low level of expression of the luciferase reporter (Fig. 2B). In 4-day-old myotubes, however, the activity level of the luciferase reporter increased nearly 5-fold. To show the
specificity of this increase, we transfected a reporter construct containing
the 3'-UTR of utrophin. As expected on the basis of recent studies
showing a lack of posttranscriptional regulation of utrophin transcripts
during myogenic differentiation
(45,
46), no change in reporter
expression was observed between myoblasts and myotubes (data not shown). These
results highlight the specificity of the increase in reporter expression
obtained with the AChE 3'-UTR. The increased expression of the reporter
gene under these conditions supports the notion that the 3'-UTR is
involved in regulating AChE transcript levels during myogenic
differentiation. Pattern of Interactions between the AChE 3'-UTR and RNA-binding ProteinsWe next examined the intensity and binding pattern of proteins found in myoblasts and myotubes that interact with the AChE 3'-UTR using REMSA. For these experiments, we used in vitro transcribed RNA that corresponded to either the full-length AChE 3'-UTR (FL) or to ARE. AREs are well characterized cis-acting elements found in UTRs that mediate transcript stability through interactions with destabilizing trans-acting factors such as AUF1 or with stabilizing trans-acting factors including those of the Hu family (41, 47). REMSA performed with the FL probe revealed the presence of two major protein-RNA complexes (Fig. 3, indicated with black arrows) that formed in myoblasts and myotubes. Although there is no apparent change in the mobility of these bands, the binding intensity of both complexes is greatly increased in myotubes, suggesting that the abundance of mRNA-binding proteins is increased in myotubes and/or that their binding characteristics to the AChE 3'-UTR are affected. This apparent increase in binding activity in myotubes was observed in several independent experiments using different protein extracts (n = 4). REMSA performed using the ARE probe also showed some important changes in the pattern of RNA-protein interactions in myoblasts versus myotubes (Fig. 3). In particular, there was a clear increase in the band intensity using proteins extracted from 4-day-old myotubes (Fig. 3, indicated with arrows). Here, again, the increased banding intensity obtained with differentiated myotubes was consistently observed (n = 3). The specificity of the binding was demonstrated in competition experiments using a cold probe to the ARE (Fig. 3, ARE-C). Taken together, these results suggest that there are specific RNA-binding complexes that interact with the AChE 3'-UTR in which the ability to bind to the 3'-UTR increases according to the state of myogenic differentiation.
HuR Binds the AChE 3'-UTROur findings using REMSA illustrate the importance of ARE in the binding of cytoplasmic factors to the AChE 3'-UTR. Members of the ELAV-like family of mRNA-binding proteins, known to interact with ARE, have recently received an increasing amount of attention because of their ability to stabilize a variety of transcripts in cells placed under different conditions. In our studies, we focused on one family member, HuR, because it is expressed in skeletal muscle (48, 49). In an initial experiment, we performed supershift REMSA with an antibody directed to HuR and protein extracts from myoblasts and myotubes, to determine whether HuR was present in the protein complexes interacting with the AChE 3'-UTR. As shown in Fig. 4A, the addition of the HuR antibody to the RNA/protein extract mixture resulted in the supershift of an RNA-protein complex formed using myotube protein extracts. This supershift was mostly evident using the myotube extract. As a control, no supershift was apparent when a nonspecific antibody was used instead of the antibody directed against HuR.
The interaction between HuR and the AChE 3'-UTR was further confirmed using an mRNA-binding pull-down assay. In this assay, excess in vitro transcribed biotin-labeled AChE 3'-UTR probes (both FL and ARE) were incubated with protein extracts from myoblasts or myotubes and subsequently pulled out of solution along with any proteins that might bind to the probes with streptavidin-coated paramagnetic beads. The pulled-down proteins were then resolved by SDS-PAGE and used for Western blotting. A Western blot to detect the presence of HuR (Fig. 4B) showed that indeed HuR could be pulled down with both the AChE full-length 3'-UTR and the ARE in experiments using myotube extracts. As observed with the supershift REMSAs, the FL probe was not able to bind HuR proteins from myoblast extracts, although a small amount of HuR could be detected in pull-down assays using the ARE probe. To further examine the binding of HuR to the ARE, we performed an additional set of pull-down experiments using AChE 3'-UTR probes that included (Fig. 4C, +ARE) or not (ARE) the ARE and myotube extracts. As shown in Fig. 4C, the ARE is essential for the binding of HuR to the AChE 3'-UTR because we observed a significant decrease in the binding of HuR to the mutant AChE 3'-UTR probe (ARE). In these assays, we confirmed that HuR was still present in the protein extract following incubation of the extract with the mutant AChE 3'-UTR probe that did not contain the ARE (data not shown). Together, the results of the supershift and pull-down assays clearly indicate that HuR expressed in myotubes can interact with the AChE 3'-UTR via the ARE.
In complementary experiments, we determined whether HuR could bind to endogenous AChE transcripts in living cells. For these assays, we immunoprecipitated HuR from myoblasts and myotubes, extracted total RNA, and performed RT-PCR to amplify AChE. As shown in Fig. 4D, AChE mRNAs were present in immunoprecipitates obtained from myotubes but not in those from myoblast extracts. As a control, AChE could not be amplified from RNA isolated following immunoprecipitation with normal mouse IgG (Fig. 4D) or protein G paramagnetic beads alone (data not shown).
HuR Increases the Expression of AChE mRNATo further
demonstrate that HuR has a direct effect on the level of AChE mRNA, we created
stable C2C12 cells that overexpressed HuR (pHuR). As shown in
Fig. 5A, endogenous
HuR protein levels are increased by severalfold in HuR-overexpressing muscle
cells. Stable transfections with the empty vector (pCI-neo), which was used as
a control in these experiments, showed no effect on endogenous HuR levels. We
next measured endogenous levels of AChE mRNA in these cells by RT-PCR. In
undifferentiated myoblasts, we did not observe any significant changes in AChE
mRNA levels between control and pHuR cells (data not shown, see
"Discussion"). In 2-day differentiated myotubes, however, we
observed an
We next performed in vitro stability assays using protein extracts from control and pHuR cells and used poly(A)+ RNA from mouse brain to determine whether overexpression of HuR affected AChE mRNA stability. As shown in Fig. 6, A and B, the rate of AChE transcript decay was slower in the presence of protein extracts from the pHuR cells, thereby confirming the importance of posttranscriptional regulation in mediating the changes seen in steady-state AChE mRNA levels in these cells. Using these stably transfected cells, we also determined whether HuR, through its interaction with the AChE 3'-UTR, could increase the expression of the luciferase-AChE 3'-UTR reporter construct. For these experiments, the luciferase-AChE 3'-UTR reporter construct was transfected into control and pHuR stable C2C12 cells. As shown in Fig. 6C, expression of the reporter construct was increased by 23-fold in C2C12 cells that stably overexpressed HuR. In
additional control experiments, we verified that the ARE plays an important
role in this process because transfection of a reporter construct containing
the mutant AChE 3'-UTR fragment without the ARE decreased significantly
expression of the reporter. These results indicate that through its
interaction with the ARE located in the 3'-UTR, HuR increases the
expression of AChE transcripts in differentiating skeletal muscle cells.
In recent years, there has been considerable interest in characterizing the transcriptional and posttranscriptional mechanisms that regulate AChE expression during myogenic differentiation (23, 2527, 51, 52). In contrast to the recent progress made in the elucidation of some of the transcriptional events (see the Introduction), there is essentially no information available concerning the nature of the specific trans- and cis-acting factors that participate in the posttranscriptional regulation of AChE in differentiating skeletal muscle cells. In the present study, we have therefore sought to begin the characterization of these posttranscriptional events that act to stimulate the production and maintenance of high levels of AChE transcripts during myogenic differentiation. Transfection experiments performed using a luciferase reporter construct in C2C12 cells showed the important contribution of the 3'-UTR in controlling AChE transcript levels. Furthermore, we found that the AChE 3'-UTR interacts with several distinct complexes using muscle protein extracts and that the intensity of the binding clearly increases during differentiation. Using a combination of different assays, we established that the stabilizing mRNA-binding protein HuR binds to the AChE 3'-UTR. Finally, we have shown a functional role for HuR in increasing AChE mRNA levels in muscle cells via stabilization through its 3'-UTR.
The Hu Family of Proteins Regulates AChE Expression at the Posttranscriptional LevelThe role of posttranscriptional regulatory mechanisms has recently been implicated as an important component of the molecular events regulating AChE transcript levels (23, 25). In recent work, we observed for example a discrepancy between the increase in transcription and the extent of AChE mRNA induction during muscle differentiation (25). Here, we show that the presence of the AChE 3'-UTR resulted in an increase in the expression of a reporter construct in differentiating muscle cells, thereby demonstrating that posttranscriptional mechanisms indeed regulate the expression of AChE during myogenic differentiation through the 3'-UTR. It is well established that transcript stability is governed by the presence of the 5'-cap, the 3'-poly(A)+ tail, and mRNA-binding proteins that bind to specific sequences or structures in the 3'-UTR. In skeletal muscle, recent studies have found that in response to different stimuli, the stability of certain transcripts, including vascular endothelial growth factor and cytochrome c oxidase (COX), is altered (49, 53). During myogenic differentiation, transcripts encoding the myogenic factors myoD and myogenin display increased stability that results, at least in part, from the specific binding of the mRNA-binding protein HuR (50, 54). The increase in the stability of these transcripts is important in dictating, in turn, the overall levels of proteins encoded by these mRNAs during myogenic differentiation. HuR is a member of the ELAV (embryonic lethal abnormal vision)-like family of mRNA-binding proteins that includes the neuronally expressed HuB (HelN1), HuC, and HuD proteins (48). These proteins contain three RNA recognition motifs that are known to bind AREs: the AUUUA general sequence, U-rich sequences, and CU-rich motifs (55, 56). The function of this family of mRNA-binding proteins is to stabilize transcripts by inhibiting the de-adenylation and targeting of the transcripts to the exosome or by blocking the activity of specific endonucleases that recognize the AREs (5759). Using several complementary approaches, we have shown here that HuR can directly interact with AChE transcripts via their 3'-UTR. Additionally, overexpression of HuR in stably transfected C2C12 cells, as well as down-regulation of HuR via siRNA, clearly affected the endogenous levels of AChE mRNA through the 3'-UTR. In parallel experiments, we have also found that HuD, although not normally expressed in muscle, can increase AChE transcript levels in differentiated cells. This indicates that the Hu proteins share similar target preferences. Thus, AChE joins a growing family of transcripts that contain varying types of AREs that are stabilized by the Hu family of proteins in several distinct tissues (40, 41, 49, 60, 61). Transcriptional and Posttranscriptional Regulation of AChEMyogenic differentiation is a complex process involving the expression of several genes including those encoding proteins found at the neuromuscular junction (for review see Refs. 6265). Previous studies have established that the expression of genes encoding synaptic proteins in muscle is most often induced by myogenic regulatory factors such as myoD and myogenin (63). As mentioned above, recent studies have demonstrated that HuR binds to elements in the 3'-UTR of myoD and myogenin during myogenic differentiation, resulting in an increase in the stability of these transcripts (50, 54). Thus, transcription factors that are key to the process of myogenic differentiation are themselves regulated posttranscriptionally by HuR (50, 54). In the present study, we found that AChE, for which transcriptional regulation during myogenic differentiation is known to involve myogenin and an E-box (25, 26), is also a direct posttranscriptional target for the stabilizing effects of HuR. Together, the results of these and other studies show that AChE expression is regulated by increases in transcription as well as by increases in transcript stability during myogenic differentiation (28). In this context, our preliminary studies performed with pHuR stable C2 cells have shown that overexpression of HuR does increase the expression of an AChE promoter-reporter construct.3 Accordingly, combined with earlier studies that focused on the role of HuR in myogenic differentiation, our findings further indicate therefore that HuR assumes a key regulatory function: (i) by controlling the stability of mRNAs encoding transcription factors known to modulate the transcriptional activity of the AChE gene during myogenic differentiation; and (ii) by also directly controlling the stability of AChE transcripts. Such dual roles for HuR in differentiating myogenic cells would confer to this RNA-binding protein a master regulatory function necessary to optimize gene expression via both transcriptional and posttranscriptional events (see further discussion in Refs. 60 and 6668).
Additional Factors Regulate AChE Expression
PosttranscriptionallyOn the basis of our studies, however, it
appears that HuR is not the sole factor important for modulating the abundance
of AChE transcripts in differentiating myotubes at the posttranscriptional
level, as suggested by the 50100-fold increase in AChE mRNA
versus the In addition to the myriad of trans-acting factors that are expressed in cells under specific conditions, there are other elements that also need to be considered because they may regulate the binding ability and specificity of these RNA-binding proteins. In this context, one intriguing finding in our study was the inability of HuR from myoblasts to bind exogenous AChE 3'-UTR, despite the presence of HuR in these cells (data not shown). In fact, RT-PCR assays and Western blotting experiments revealed that HuR is equally abundant in myoblasts and myotubes (data not shown) as was also observed by others (see also Ref. 50). This indicates therefore that additional regulatory events favor HuR-RNA interactions in myotubes. One possibility involves the cellular localization of HuR in myoblasts versus myotubes. Previous studies have demonstrated that HuR is predominantly localized in nuclei in myoblasts and that it shuttles out to the cytoplasm during myogenesis where it can interact with and stabilize specific transcripts (50, 54). Because in our study we used total protein extracts containing equivalent amounts of HuR protein and found that HuR from myoblast extracts was unable to bind the AChE 3'-UTR, we speculate that there must be some modifications or other factors that affect the ability of HuR to bind to exogenous, in vitro transcribed, AChE transcripts or to those already present in the cytoplasm. The regulation of HuR binding could indeed be mediated by posttranslational modifications, such as phosphorylation or methylation. In this context, metabolic stress was recently shown to regulate the cytoplasmic localization of HuR and its binding through activation of the AMP-activated kinase (75). In addition, HuR has been shown recently to be methylated by the arginine methyltransferase CARM1 (co-activator-associated arginine methyltransferase) in the hinge region adjacent to the novel nuclear localization signal, resulting in a nuclear-cytoplasmic shuttling ability and/or enhanced interactions with other proteins or transcripts (76, 77). Therefore, although our study clearly highlights the important role of HuR in the regulation of AChE mRNA in differentiating myogenic cells, there are several aspects relevant for our complete understanding of the nature of these posttranscriptional regulatory events that are obviously complex and that will hence necessitate further experimentation.
* This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR) (to B. J. J. and R. J. P.) and by National Institutes of Health Grant NS-30255 (to N. P.-B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a fellowship from the Association Française contre les
Myopathies. ¶¶ A CIHR investigator. To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada. Tel.: 613-562-5800 (ext. 8383); Fax: 613-562-5636; E-mail: jasmin{at}uottawa.ca.
1 The abbreviations used are: AChE, acetylcholinesterase; ARE, AU-rich
element; FL, full-length AChE 3'-UTR; PBS, phosphate-buffered saline;
REMSA, RNA electrophoretic mobility shift assay; UTR, untranslated region; RT,
reverse transcription; siRNA, small interfering RNA; S12, S12 ribosomal
protein; MOPS, 4-morpholinepropanesulfonic acid.
2 R. J. Parks, unpublished observations.
3 G. Bélanger and B. J. Jasmin, unpublished observation..
We thank Lauren Smith and Kathy Sargeant for technical assistance with specific portions of this project.
This article has been cited by other articles:
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
Advertisement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||