(Received for publication, May 24, 1996, and in revised form, September 20, 1996)
From the Department of Biochemistry, East Carolina University School of Medicine, Greenville, North Carolina 27858
In the current study we report on the
contribution of the GLUT1 3-untranslated region (UTR) to the stability
of the GLUT1 mRNA. To facilitate these investigations, a hybrid
construct was prepared by insertion of the GLUT1 3
-UTR into a normally
stable reporter gene coding for preproinsulin. The GLUT1 3
-UTR
conferred lability to the otherwise long lived construct and
transferred an ability to be stabilized in response to treatment with
the cytokine, tumor necrosis factor-
(TNF). The destabilizing
element has been mapped to a region located between bases 2242 and 2347 of the GLUT1 3
-UTR; this same region also mediates the stabilization response to TNF. In vitro RNA-protein binding assays using
protein extracts from control and TNF-treated cells demonstrated that two proteins, one of 37 kDa and the other of 40 kDa, recognized sequence elements within the stability-determining region and were
up-regulated in response to TNF treatment. The RNA-binding activity of
these proteins coincides with the stabilization of the GLUT1 message,
suggesting that they may be involved in regulation of the turnover of
this message.
Glucose transport across the plasma membrane is the rate-limiting step for subsequent glucose metabolism and energy production within the cell (1). This process is catalyzed by a family of tissue-specific integral membrane proteins known as the glucose transporters (GLUTs)1 (reviewed by Bell et al. (2)). Transporter activity can be regulated by hormones and growth factors through the redistribution of transporter proteins from intracellular vesicles to the plasma membrane (translocation), modulation of transporter intrinsic activity, and stimulation of new protein synthesis through increased transcription and/or alteration in transcript stability (reviewed by McGowan et al. (3)).
Previously, we have demonstrated that treatment of quiescent 3T3-L1
preadipocytes with the cytokine tumor necrosis factor- (TNF)
produces a 3-4-fold stabilization of the GLUT1
(HepG2/brain/erythrocyte glucose transporter) mRNA that leads to a
parallel increase in glucose transport activity within these cells (4).
Coincident with the stabilization of the message was the up-regulation
of an RNA binding protein(s) exhibiting specificity for an in
vitro synthesized 80-base transcript containing four back to back
copies of the destabilizing motif, AUUUA. During this same period of message stabilization, proteins with molecular masses of 37 and 40 kDa,
with apparent binding specificity for the GLUT1 3
-UTR were also
up-regulated (5). While the GLUT1 3
-UTR is AU-rich, the actual
sequence to which these proteins bound was not localized, and identity
between the AU-rich binding protein and the GLUT1 3
-UTR binding
proteins could not be established. However, their binding activity
increased coordinately with the t1/2 of the message, consistent with a hypothesis of involvement in stabilization of the
GLUT1 transcript (5).
To determine if sequences within the GLUT1 3-UTR regulate decay, we
stably transfected 3T3-L1 preadipocytes with a set of chimeric reporter
constructs. These constructs contained the 5
-UTR and coding region of
preproinsulin as a reporter gene, attached to the normal and various
truncated versions of the GLUT1 3
-UTR. The preproinsulin gene was
selected as a reporter for the specific reason that, similar to the
GLUT4 gene, the resultant transcript would be translated on ribosomes
associated with the endoplasmic reticulum. We wished to preserve this
interaction, since it provides a critical structure that may influence
stability. Our results indicated that sequences within bases 2246 and
2347 mediate the rapid decay of this message in the quiescent
fibroblasts. This analysis also suggested that the same region
controlled the TNF-induced accumulation of the GLUT1 mRNA.
RNA gel mobility shift assays utilizing the full-length 3-UTR and the
various truncated forms as binding probes have detailed the occupancy
of the GLUT1 3
-UTR by RNA-binding proteins. This analysis identified
two RNA-binding proteins that bound to the stability-determining region
of the GLUT1 3
-UTR. The RNA binding activity of these proteins
increased coordinately with the stabilization of the GLUT1 message
following TNF stimulation.
Recombinant human TNF- was provided by Biogen
(Cambridge, MA). The specific activity was 9.6 × 106
units/mg of protein, based on a cytotoxicity assay using L929 cells.
The endotoxin contamination was 0.12 ng/mg of protein, based on a
Limulus amebocyte lysate assay (Sigma). Dulbecco's modified Eagle's medium was purchased from Hazelton Research Products (Lenexa, KS). Fetal calf serum was purchased from Hyclone (Logan, UT).
All radiolabeled compounds were purchased from DuPont NEN. Hybond-N
nylon filter paper was purchased from Amersham Corp. All restriction
enzymes, DNA polymerases, and molecular biology enzymes were purchased
from Life Technologies, Inc. Actinomycin D was purchased from
Calbiochem. Protogel premixed acrylamide solution was purchased from
National Diagnostics (Atlanta, GA).
Murine 3T3-L1 fibroblasts used in these studies were the gift of the late Dr. Ora Rosen (Memorial Sloan-Kettering Cancer Center (New York, NY)). These cells were plated and grown to confluence in Dulbecco's modified Eagle's medium supplemented with 10% calf serum as described previously (4). Four days postconfluence, the medium was replaced with Dulbecco's modified Eagle's medium supplemented with 0.5% essentially fatty acid-free bovine serum albumin. Cells were characterized as quiescent 24 h after this final medium change when the aphidicolin-sensitive component of DNA synthesis was at a minimum. All experiments were initiated within 24 h after reaching quiescence. TNF was dissolved in Krebs-Ringer-Hepes buffer containing 0.1% bovine serum albumin, and a 1:100 dilution was added to the cell culture medium, producing a final concentration of 5.0 nM. The duration of all treatments was 10 h.
cDNAs and PlasmidsThe GLUT1 cDNA used in these
studies is the 3T3-L1 clone (accession number M23384[GenBank]) as reported by
Kaestner et al. (6). All position numbering is derived from
that report. The -actin probe was donated by Dr. Don W. Cleveland
(Johns Hopkins University, Baltimore, MD). The pBC12BI expression
vector (7) was purchased from Dr. Bryan Cullen (Duke University,
Durham, NC).
Transfections were performed using calcium phosphate DNA co-precipatation method as described by Sambrook et al. (8). The pBC12BI expression vector and derivatives produced for these studies do not contain a selectable marker and required co-transfection with the vector pSV2neo, which conferred neomycin resistance. All studies reported represent the results of two independent stable transfections.
Cytosolic Extract Preparation and Polysome IsolationThe
S10, S100, and polysomal protein fractionation procedure was performed
as described by Brewer and Ross (9). Cells were harvested by washing
with ice-cold phosphate-buffered saline (two times), scraping in 2 ml
of phosphate-buffered saline/plate, combining the scraped cells, and
pelleting them by centrifugation at 500 × g for 10 min
at 4 °C. The cell pellet was resuspended in 3.5 ml of buffer A (10 mM Tris-HCl, pH 7.6, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM dithiothreitol)
by gentle pipetting. Cells were lysed by applying 20 strokes with a
Teflon pestle homogenizer, and the crude lysate was spun at 10,000 × g for 10 min at 4 °C to remove nuclei. Small amounts
of the supernatant from this spin were aliquoted and designated as the
S10 fraction. The remaining supernatant was removed and layered onto a
1.5-ml cushion of buffer B (30% (w/v) sucrose solution in buffer A) in
a Beckman SW55 tube (Palo Alto, CA). These samples were centrifuged at
130,000 × g for 3 h at 4 °C. The high speed
supernatant from this spin was designated S100 and was carefully
removed from the top of the sucrose cushion and immediately frozen at
80 degrees. The remaining sucrose pad was removed by aspiration; the
pellet containing the polysomes was washed twice with buffer A and
harvested by repeated pipetting with 500 µl of buffer A. Once in
solution, the polysomes were transferred to a microcentrifuge tube and
immediately frozen. The protein yields were determined by BCA protein
analysis (Pierce), and the polysome yields were quantified by measuring
absorbances at 260 and 280 nM.
An 876-base
SacI-EcoRI fragment (bases 1668-2544 relative to
the transcription initiation site) of the GLUT1 cDNA was ligated into identical sites within the vector pBluescript (Stratagene) to
generate the plasmid pGT1UTR. Site-directed mutagenesis was performed
on this plasmid, according to the method of Kunkel (10), to introduce a
SpeI restriction enzyme site at base 1902. This new plasmid
was named pGT1:SDM1902 and was used as the starting material for
producing truncation deletion mutants using exonuclease III digestion
as described below. The plasmids that resulted from the exonuclease III
digestion procedure were assigned names according to the portion of the
GLUT1 3-UTR that remained (i.e. TD2088 contained the
segment between base 2088 and the 3
terminus). These plasmids were
named pTD1902, pTD2088, pTD2242, and pTD2347. These plasmids as well as
the pGT1UTR construct were still contained within the original
pBluescript vector. This allowed for the production of radiolabeled
transcripts derived from these constructs that were used in the RNA
binding studies.
The expression vectors used in the analysis of chimeric transcript
half-lives were derived from the vector pBC12BI. This vector contains
the gene for preproinsulin under the control of a Rous sarcoma virus
long terminal repeat promoter. The plasmids used for this study were
constructed through ligation into the pBC12BI using a SmaI
site at base 1207 and a BstEII site at base 1215. These
sites are located 50 bases after the translation stop sequence such
that ligation into this position will replace the 3-UTR of the
preproinsulin gene, provided that the inserted DNA contains a
polyadenylation sequence. Polymerase chain reaction with the primers
(SmaI, 5
-ACAACCCGGGACTCCCAAGT-3
; BstEII,
5
-GTGGTCACCCTCCAGCCATTTA-3
) was used to incorporate SmaI
and BstEII sites into the 5
and 3
termini, respectively,
of products amplified from the vectors pGT1UTR, pTD1902, pTD2088,
pTD2242, and pTD2347. The polymerase chain reaction products were
purified, digested with SmaI and BstEII, and
ligated into the pBC12BI vector to generate the plasmids pBC:GT1UTR,
pBC:TD1902, pBC:TD2088, pBC:TD2242, and pBC:TD2347. These vectors
produced transcripts that contained the 5
-UTR and coding region of the
preproinsulin gene with either the full-length or truncated forms of
the GLUT1 3
-UTR.
The plasmids pTD1902,
pTD2088, pTD2242, and pTD2347 were constructed through the
unidirectional digestion of the plasmid pGT1:SDM1902. This procedure
was modified from the method previously described by Henikoff (11). The
plasmid pGT1:SMD1902 was digested with SacI and
SpeI, purified, and resuspended in exonuclease III digestion buffer (60 mM Tris-HCl, pH 8.0, 0.6 mM
MgCl2). Unidirectional digestion occurred in a 5
3
direction from the SpeI site but did not proceed in the 3
5
direction past the SacI site of the GLUT1 3
-UTR.
Exonuclease III digestion was performed with 500 units of exonuclease
III at 22 °C. To stop the reaction, aliquots were removed at 2-min
intervals, placed into S1 nuclease buffer (40 mM potassium
acetate, 300 mM NaCl, 1.5 mM ZnSO4,
5% (v/v) glycerol), and digested with 2 units of S1 nuclease/aliquot
for 30 min at room temperature. The extent of truncation was assessed by agarose gel electrophoresis of a portion of each aliqout. Those samples that visually represented significant deletions (~100-200 bases) were ligated and used to transform competent bacteria. The
extent of truncation was determined through dideoxy sequencing of
miniprep DNA.
The
GLUT1 3UTR contained in the PBC:GLUT1 construct was cleaved at
position 2246 with MmeI and at 2347 with BbvII;
the ends were first blunted with T4 DNA polymerase and then ligated
with T7 DNA ligase. The resulting deletion was confirmed by restriction mapping and dideoxysequencing of the miniprep DNA.
The region was
isolated using polymerase chain reaction. The forward primer for the
reaction was
5-GCCTAATACGACTCACTATAGGGGACCAGTTGGAAGCACTGG-3
, while the 3
-primer was 3
-AGTTAAGTTCTCAGCCTC-5
. Bases 4-21 of the forward primer comprise the T7 RNA polymerase promoter, which was
contained in the amplified fragments, permitting production of sense
RNA transcripts following in vitro transcription. Bases 23-41 (boldface type) correspond to positions 2242-2257 of the 3
-UTR. The 3
-primer corresponds to positions 2352-2370 of the 3
-UTR.
RNA isolation,
Northern analysis, and mRNA half-life determinations were performed
as described previously (12) using either radiolabeled cDNA probes
or cRNA probes (described below). All cDNA probes were labeled with
[-32P]-dATP (3000 Ci/mmol) by the random priming
method described by Feinberg and Vogelstein (13) and used at an
activity of 1 × 107 cpm/ml. cRNA probes were prepared
as described under "In Vitro Transcription Reactions"
(see below). Hybridization with cRNA probes was performed at 65 °C,
and the probes were used at a concentration of 1 × 106 cpm/ml of hybridization solution. The hybridization
solutions and washes were the same as described previously (14).
The half-life data presented in the text represent the mean ± S.E. from a total of four independent experiments performed on each of two cell lines derived from two independent transfections. Quantitation was performed by densitometric analysis using ImageQuant software on a Molecular Dynamics PhosphorImager. Half-lives were determined through linear regression analysis for the best fit line on logarithmic plots of control and TNF-treated data points, as described previously (5).
In Vitro Transcription ReactionsIn vitro
transcription was performed according to the manufacturer's procedures
for the MAXI-script transcription kit (Ambion Inc., Houston, TX). These
probes are radiolabeled through the inclusion of
[-32P]UTP (800 Ci/mmol) in the reaction. The resulting
transcripts had a specific activity of approximately 1 × 108 cpm/µg as determined by trichloroacetic acid
precipitation.
RNA band shift analysis was performed as described by Nagy and Rigby (15). Radiolabeled RNA (105 cpm, ~1.0 ng) was prepared as described above and incubated with 30 µg of S10, S100, or polysomal protein fractions. The incubations were performed at 37 °C for 30 min in a final buffer concentration of 10 mM HEPES, pH 7.9; 15 mM KCl; 3 mM MgCl2; 0.2 mM dithiothreitol; 1 µg/ml yeast tRNA; 25 µg/ml poly(I) and 10% glycerol. The reactions were then placed on ice and cross-linked using a Stratalinker 1800 (Stratagene) for 5 min at 3000 microwatts/cm2. This was followed by RNase digestion (10 units of RNase T1, 10 µg of RNase A) for 30 min at room temperature. The cross-linked complexes were analyzed by 12% SDS-polyacrylamide gel electrophoresis, dried, and subjected to autoradiography.
3T3-L1 preadipocytes were stably transfected with an
expression vector containing the preproinsulin gene or a construct
where the 3-UTR of the preproinsulin gene has been replaced with the GLUT1 3
-UTR. All transfectants required cotransfection with the vector
pSV2-neo that contained a neomycin resistance gene to allow for antibiotic selection. Following transfection, G418-resistant colonies were pooled to ensure a heterogeneity of integration sites,
and permanent transfected cell lines were established. These
transfectants along with a transfection control, which contained only
the pSV2 neomycin resistance vector, were analyzed for
GLUT1 mRNA turnover under control and TNF-treated (5 nM
for 10 h) conditions; a typical analysis is shown in Fig.
1. The following summary half-life data represent the
mean ± S.E. from a total of four independent experiments
performed on each of two cell lines derived from two independent
transfections. The results of the study demonstrate that the cells
transfected with parental preproinsulin vector produce preproinsulin
mRNA that is extremely stable under control conditions and does not
respond to TNF (t1/2 values: control, 675 ± 20 min; TNF-treated, 682 ± 20 min). Replacing the preproinsulin 3
-UTR with the GLUT1 3
-UTR results in a transcript with a much shorter half-life (control t1/2 = 51 ± 9 min)
that is stabilized in response to TNF treatment (TNF
t1/2 = 181 ± 18 min). The turnover rate for
this chimeric transcript resembled those of the GLUT1 mRNA,
suggesting that the GLUT1 3
-UTR conferred mRNA decay
characteristics consistent with the GLUT1 message to the normally
stable preproinsulin reporter gene. Control transfectants that
contained only the pSV2-neo vector were also analyzed for
GLUT1 mRNA turnover. The half-life of GLUT1 within these cells was
calculated to be 39 ± 8 min under control conditions and 168 ± 20 min following TNF treatment, indicating that the transfection
procedure does not alter the behavior of the GLUT1 transcript.
Truncation Analysis of the GLUT1 3
The initial findings
suggested that the GLUT1 3-UTR contained elements that could regulate
the turnover of the normally stable preproinsulin transcript.
Exonuclease III digestion was used to produce a series of constructs
that were truncated from the 5
-end of the 3
-UTR (Fig.
2A). These constructs were transfected into 3T3-L1 preadipocytes, selected for resistance to G418, and pooled as
described above. The expression of these constructs following transfection was verified by Northern blot analysis using a cRNA (riboprobe) generated against the terminal 250 bases of the GLUT1 message (Fig. 2B).
To investigate whether the removal of any portion of the GLUT1 3-UTR
altered the behavior of the pBC:GLUT1 chimera, the transfectants containing the truncation deletions were analyzed for the decay of the
chimeric transcripts under control and TNF-treated conditions (Fig.
3). Under control conditions, transcripts encoded by
PBC:TD1902, PBC:TD2088, and PBC:TD2242 possessed decay rates of 56 ± 11, 51 ± 10; and 52 ± 8 min, respectively. Treatment of
the cells with TNF resulted in a significant (p < 0.05, Student's t test) stabilization of the chimeric
mRNA with values determined to be 176 ± 20 min for
PBC:TD1902; 184 ± 9 min for PBC:TD2088; and 180 ± 9 min for PBC:TD2242. These values were comparable with that observed for the
half-life for endogenous GLUT1 in control and TNF-treated cells.
However, the construct PBC:TD2347 was remarkably stable in untreated
quiescent cells with a half-life of 477 ± 30 min, a value that
approximated the t1/2 of the preproinsulin
mRNA (675 ± 20 min) encoded by the parental PBC12BI vector
more than the PBC:GLUT1 chimera (51 ± 8 min). TNF treatment
yielded a half-life of 497 ± 35 min for the PBC:TD2347 chimera, a
value that was not statistically different from the value obtained
under control conditions (p > 0.05, Student's t test). These data indicate that the ability to
destabilize the PBC:GLUT1 transcript resides within the region defined
by bases 2242 and 2347 of the GLUT1 3
-UTR. Transcripts that contained
this segment of the 3
-UTR remained unstable, while those that do not
have this segment are inherently stable. The dramatic increase in
stability produced by the removal of this region (2242-2347)
complicated the identification of a TNF-responsive element. Prior to
the removal of this portion of the GLUT1 3
-UTR, all of the chimeras
exhibited significant stabilization in response to TNF (Fig. 3 and see
above). This suggests that the TNF-responsive element resides somewhere
between base 2242 and the 3
terminus.
Stabilization of the Message and Loss of TNF Response upon Deletion of the Region between 2246 and 2347 of the PBC:GLUT1 Chimera
The
findings described above were consistent with the hypothesis that the
region between 2242 and 2347 contained an element essential for the
determination of the stability of this message. In addition, the region
potentially contained a TNF-responsive element. Restriction sites at
positions 2246 and 2347 of the construct permitted removal of the
region to examine these possibilities (detailed under "Experimental
Procedures"). The deletion mutant PBC:GLUT1 2246-2347 was
prepared (Scheme I) and stably transfected into the 3T3-L1 cells as
described above.
Scheme I.
Expression and TNF regulation of the mutant chimeric RNA were then
examined. As shown in Table I, deletion of this region of the GLUT1 3-UTR resulted in a significant stabilization of the
message in control cells (2.4-fold over the PBC:GLUT1 mRNA, p < 0.05). These data were consistent with the deleted
region containing an element essential for the rapid turnover of the mRNA. Moreover, the half-life of the message did not change when the cells were exposed to TNF, supporting a role for the region in
mediating the stabilizing effects of this cytokine.
|
The occupancy of the GLUT1 3-UTR by
RNA-binding proteins was assessed through gel mobility shift analysis.
Postnuclear (S10), cytosolic (S100), and polysomal protein extracts
were prepared from control and TNF-treated quiescent 3T3-L1 cells.
These proteins were incubated with a radiolabeled transcript
corresponding to the full-length GLUT1 3
-UTR. A total of five
RNA-protein complexes from the S10, S100, and polysomal protein
extracts with molecular masses of approximately 67, 45, 40, 37, and 26 kDa were detected by the label transfer (Fig. 4). The
postnuclear S10 fraction displayed all five bands, and the RNA binding
activities at 67, 40, and 37 kDa appeared to increase with TNF
treatment. The 67-, 46-, and 37-kDa proteins partitioned into the
postmitochondrial S100 fraction, but no differences in their RNA
binding activities between control and TNF-treated extracts were
observed. The TNF-treated polysomal protein fraction contained all five
RNA binding activities including the 40- and 37-kDa bands that were not
observed within the polysomal preparations from control cells.
Comparison of Gel Mobility Shifts with the Full-length 3
A comparison study was carried out
with the S10, S100, and polysomal fractions using radiolabeled
transcripts prepared from the full-length GLUT1 3-UTR and the various
truncation deletions. The primary goal of this study was to determine
if any of the RNA-protein interactions observed in Fig. 4 were specific
for the destabilizing region located between bases 2242 and 2347 of the
GLUT1 transcript. The sequential removal of regions within the GLUT1
3
-UTR did not alter the binding pattern within any of the control
extracts (Fig. 5, A and C).
However, significant alterations in the RNA binding activity were found
within the postnuclear (S10) and polysomal protein fractions obtained
from TNF-treated cells. Removing the portion of the GLUT1 3
-UTR
located between bases 2242 and 2347, the stability-determining element, reduced the label transfer to both the 37- and 40-kDa binding activities found within these extracts (Fig. 5, A and
C). The TNF-treated S100 fraction demonstrated no alteration
in binding activity when either transcript was used in the assay.
Mapping of the RNA binding activity to the
stability-determining element. RNA gel mobility shift analysis was
performed as described in the legend to Fig. 4 with radiolabeled
ligands corresponding to the various truncated forms of the GLUT1
3-UTR described in the legend to Fig. 2A. Analysis was
performed with S10 (A), S100 (B), and polysomal
protein extracts (C) from control and TNF-treated (5 nM for 10 h) 3T3-L1 cells. The molecular weights for
the various binding activities are indicated and were determined as
described in the legend to Fig. 4. The data are representative of an
experiment performed three times with similar results.
Protein Binding to the Region Located between Bases 2242 and 2347 of the GLUT1 3
As described above, deletion of the region
between 2246 and 2347 of the GLUT1 3-UTR resulted in stabilization of
the message and loss of the TNF response. Moreover, it has been
identified by deletion analysis (Figs. 4 and 5) as the site of
TNF-inducible protein binding. Thus, it was of interest to determine if
this region upon isolation remained a ligand for the TNF-inducible protein. A riboprobe corresponding to the region was prepared as
described under "Experimental Procedures," and RNA gel mobility shift assays were performed. The data displayed in Fig.
6 indicate that TNF treatment resulted in the
up-regulation of binding activity in the S10/polysome-containing
fraction of proteins of 37 and 40 kDa as well as a doublet at 67 kDa.
These data, similar to those obtained with the 3
-UTR truncations
described in Figs. 4 and 5A, support the identification of
the region between 2246 and 2347 as the site for TNF-inducible protein
binding. Interestingly, use of this "binding site-specific
riboprobe" demonstrated decreased binding in both the 67- and 37-kDa
regions of the gel when the TNF-treated S100 fraction was used as a
source of ligand. This was not evident in the data presented in Figs. 4
and 5, where larger portions of the 3
-UTR were used as probes and may
reflect an alteration in affinity of the proteins for the smaller probe after TNF treatment or the ability to detect the partitioning of the
binding proteins from soluble to particulate fractions of the cell.
These possibilities are currently under investigation. However, the
ability to use the specific binding site probe to demonstrate
TNF-inducible binding provides a convenient assay for the purification
and characterization of the proteins involved.
Regulation of transcript stability plays a major role in controlling the expression of the HepG2/erythrocyte/brain glucose transporter, GLUT1 (3). GLUT1 is the predominant glucose transporter within several biologically important tissues including blood-tissue barriers such as placenta and the blood-brain barrier (16, 17). Several laboratories have reported that conditions such as glucose deprivation (18, 19), experimental diabetes (20), and inhibition of oxidative phosphorylation (21) as well as cytokine and hormone stimulation (4, 22, 23, 24) alter the stability of the GLUT1 transcript. The mechanisms that produce this effect, including the role of cis-acting sequence elements and trans-acting factors, have remained unclear.
The current study provides evidence that the GLUT1 3-UTR regulates the
decay of the GLUT1 message. Insertion of the GLUT1 3
-UTR into the
otherwise long lived preproinsulin transcript is sufficient to
destabilize the resulting mRNA. Additionally, this chimeric
transcript can also be stabilized by treatment with TNF, a
characteristic of the endogenous GLUT1 mRNA. The 3
-UTR of a number
of short-lived mRNAs such as oncogenes and growth factors has also
been shown to promote the degradation of heterologous RNAs (25, 26).
The 3
-UTRs of these early response genes contain a destabilizing motif
known as an adenylate-uridylate-rich element that mediates their rapid
decay. The destabilizing element within the GLUT1 3
-UTR was mapped to
a portion located between bases 2242 and 2347. Importantly, when this
region was deleted, the message was markedly stabilized (Table I). The
-fold stabilization was not as large as we would have predicted based
on the truncation mutations. As opposed to those experiments, the
effect was observed in the context of the nearly intact 3
-UTR (Scheme
I), suggesting that other regions may facilitate the stabilization of
the message on their own or interact with the 2242-2347 region to
affect stability. This region is predominantly GC-rich (~60%) and
was not homologous to either a GC-rich stability element reported for
the transforming growth factor-
1 gene (27) or to a TNF-responsive
stability element identified within the surfactant-B protein mRNA
(28).
A comparison of this 105-base sequence against sequences within
GenBankTM revealed conservation of this element among GLUT1
isoforms across species lines (human, mouse, and chicken) conferring a
degree of evolutionary significance to the reported observations. In addition to the homology found within 3-UTRs, significant homology was
also found to a portion of the GLUT1 coding region for both mice and
humans beginning at base 290. The redundant expression of destabilizing
sequences has been observed within the c-myc and
c-fos protooncogene mRNAs. These mRNAs have been
demonstrated to contain destabilizing sequences within their open
reading frames in addition to elements within their 3
-UTRs (29, 30, 31). These observations have led to the suggestion that multiple
destabilizing elements may be employed in order to prevent the
overexpression of a protein within normal cells. It is also possible
that this sequence may affect other aspects of the post-translational
processing of the GLUT1 message such as translational initiation (32). This said, we did not observe any TNF-inducible binding to probes prepared from this coding region element2
and have not as yet investigated whether this element actually functions as a stability-determining sequence.
Our chimeric transcript analysis did not directly identify sequences that mediate the stabilizing effect of TNF that was observed with several of the chimeric transcripts as well as the endogenous GLUT1 message. However, when the region from 2246 to 2347 was deleted, the ability of TNF to stabilize the message was lost. These data suggest that the same region responsible for the inherent instability of the GLUT1 transcript also functions to stabilize this message in response to TNF.
The production of hybrid transcripts demonstrated that specific
sequences within the GLUT1 3-UTR regulate the decay of this message.
To gain some insight into how these sequences may function to control
GLUT1 mRNA turnover, we searched for trans-acting
factors, such as RNA-binding proteins, using gel mobility shift
analysis. We observed several binding activities within extracts
prepared from control and TNF-treated cells (Fig. 4). Two of these
proteins, with apparent molecular masses of 37 and 40 kDa, demonstrated enhanced RNA-binding activity following TNF treatment. These proteins were contained within the polysomal protein fraction and were also
detected within the postnuclear S10 fraction (Figs. 4 and 5). The
involvement of these RNA-binding activities in regulating the turnover
of the hybrid constructs was investigated by comparing the RNA binding
patterns obtained with the full-length GLUT1 3
-UTR with the various
truncated versions used for the in vivo analysis (Fig.
2A). Two RNA binding proteins, one of 37 kDa and the other of 40 kDa, previously detected within the TNF-treated extracts using
the full-length GLUT1 3
-UTR, could not be detected when the TD2347
probe was used (Fig. 5). We demonstrated that these two proteins bound
all forms of the GLUT1 3
-UTR except the one in which the
stability-determining region had been removed (TD2347). This suggests
that the RNA binding activity of these proteins is specific for the
stability-determining region (2242-2347) and can be activated by TNF.
This finding further supports the hypothesis that the destabilizing
element located between bases 2242 and 2347 functions to stabilize the
GLUT1 message through TNF-controlled RNA-protein interactions.
Interestingly, the data displayed in Fig. 5B demonstrate
that a 37-kDa protein present in the S100 (cytosolic fraction) bound
all forms of the 3
-UTR and did not appear to be regulated by TNF. One
possible explanation of this observation is that the regulated binding
activities measured in the S10 and polysomal fractions are not derived
from the same protein found in the S100. The S100 37-kDa protein that
binds to the 2347 mutant may be a nonspecific RNA binding activity, many of which coincidentally are in this molecular mass range. This
hypothesis is reinforced by our recent study of dehydrogenase binding
to the GLUT1 3
-UTR, which indicated that both lactate and
glyceraldehyde 3-phosphate dehydrogenases bind to this region of the
UTR (33). Additionally, both enzymes have subunit molecular sizes in
the appropriate range and are particularly abundant in the cytosolic
fraction of these cells. A second explanation addresses the issue that
only the S10 and polysomal fractions contain the translational
machinery and that specific interactions mediated by the presence of
these components may be necessary to observe the regulation. These
issues are currently under investigation.
Our work has documented that in the GLUT1 3-UTR, a GC-rich region
(bases 2242-2347) contributes significantly to the
post-transcriptional regulation of this gene. The interaction between
this region and two RNA-binding proteins following TNF treatment
resembles a similar mechanism through which the expression of the
transferrin receptor mRNA is regulated post-transcriptionally (34).
A destabilizing element within the transferrin receptor 3
-UTR serves
as a nuclease recognition site that normally allows for the rapid decay
of this message (35). In response to changes in cellular iron
concentration, the iron-responsive binding protein binds to this
destabilizing sequence, blocks the activity of the nuclease, and
prevents the decay of the transferrin receptor mRNA (36). The
similarities between the two systems have led us to propose the model
shown in Fig. 7, where in control cells, the
destabilizing region is unprotected and the message exhibits a rapid
turnover. Treatment of the cells with TNF leads to occupation of the
region by a complex composed of at least two proteins (37 and 40 kDa),
protection against nucleolytic attack, and stabilization of the
mRNA. The model presented in Fig. 7 represents the simplest of all
models and a good place to start. However, the data presented in this study do not rule out more complex models where the proteins may bind
at the defined site but interact with accessory proteins and/or other
regions of the message. Further definition of the system requires the
purification and cloning of these binding activities, a task that is
currently under way. However, the results presented in the current
study represent a unique contribution to the understanding of glucose
transporter gene expression. Additionally, these findings support
previous studies suggesting that TNF works through inducible
RNA-binding proteins to influence gene expression at the
post-transcriptional level (5, 32), a result that has important
implications for the understanding of TNF involvement in numerous
biological systems.
We are grateful for the expert technical assistance of Amy Heffner and Donna Hardee in the performance of these studies. In addition, we thank Drs. Sheree Long, James McCubrey, and Mike McIntosh for critically reading the manuscript; Drs. Richard Hanson, Ashok Aiyar, and Parvin Hakimi (Case Western Reserve University, Cleveland, OH) and Dr. Tom Kunkel (NIEHS, National Institutes of Health, Raleigh, NC) for assistance with site-directed mutagenesis; and Drs. Gary Brewer and Belinda Wagner (Wake Forest University, Winston-Salem, NC) for useful discussions. Finally, we thank Renee McGowan for the artwork presented in Fig. 7.