Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Glucose stabilizes the mRNA for human fatty acid synthase (FAS), an enzyme relevant to diverse human disorders, including hyperlipidemia, obesity, and malignancy. To determine the underlying mechanisms, RNA gel mobility shift assays were used to demonstrate that human Hep G2 cells contain a cytoplasmic factor that binds specifically to the 3'-terminus of the human FAS mRNA. D-Glucose increased RNA-binding activity by 2.02-fold (P = 0.0033), with activity peaking 3 h after glucose feeding. Boiling or treatment of extracts with proteinase K abolished binding. Ultraviolet cross-linking of the FAS mRNA-binding factor followed by SDS-PAGE resolved a proteinase K-sensitive band with an apparent molecular mass of 178 ± 7 kDa. The protein was purified to homogeneity using nondenaturing polyacrylamide gels as an affinity matrix. Acid phosphatase treatment of the protein prevented binding to the FAS mRNA, but binding activity was unaffected by modification of sulfhydryl groups and was not Mg2+ or Ca2+ dependent. Deletion and RNase T1 mapping localized the binding site of the protein to 37 nucleotides characterized by the repetitive motif ACCCC and found within the first 65 bases of the 3'-UTR. Hybridization of the FAS transcript with an oligonucleotide antisense to this sequence abolished binding. These findings indicate that a 178-kDa glucose-inducible phosphoprotein binds to an (ACCCC)n-containing sequence in the 3'-UTR of the FAS mRNA within the same time frame that glucose stabilizes the FAS message. This protein may participate in the posttranscriptional control of FAS gene expression.
phosphorylation; 3'-untranslated region; repetitive element; messenger ribonucleic acid stability
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INTRODUCTION |
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CHRONICALLY ELEVATED concentrations of glucose like those seen in humans with diabetes mellitus are probably toxic. Insulin is important for glucose transport in certain tissues, but the majority of glucose uptake is independent of insulin and proportional to circulating glucose concentrations (5). In insulin-resistant states such as type II diabetes, the total flux of glucose across certain tissues such as the liver is substantially increased. Glucose alone, independent of hormonal derangements, likely contributes to complications of diabetes, such as hyperlipidemia (34).
Exactly how glucose promotes hyperlipidemia is unknown. The liver is central to this process, and carbohydrates increase the expression of several hepatic genes involved in intermediary metabolism and lipogenesis, including fatty acid synthase (FAS), L-type pyruvate kinase, acetyl-CoA carboxylase, ATP-citrate lyase, and malic enzyme (9). This effect is primarily transcriptional, but glucose also stabilizes mRNAs for many feeding-responsive hepatic genes (33). Glucose promotes the stability of the message for the insulin receptor (11), another protein central to fuel flow and lipid metabolism.
Regulation of mRNA stability is an important mechanism for altering mammalian gene expression. mRNA half-life depends on at least three components as follows: 1) the enzymes that degrade mRNA; 2) cis determinants located anywhere in the mRNA but usually in the 3'-terminus of the message; and 3) trans-acting factors that interact with the message to promote or prevent decay. There are probably a small number of eukaryotic ribonucleases (30). It is likely that regulated message stability depends on the interaction of binding proteins with specific RNA sequences to either promote or prevent access to ribonucleases.
FAS is the cytosolic enzyme that synthesizes palmitate from acetyl-CoA, malonyl-CoA, and NADPH (37). It is rate limiting in the long-term control of fatty acid synthesis (36) and regulated by several different classes of nutrients (7). FAS overexpression occurs in animal models of obesity (17). Carbohydrates stimulate FAS expression and produce hypertriglyceridemia in animals and humans (15, 36).
FAS is also relevant to malignancy. Cultured carcinoma cells maintain high levels of endogenous fatty acid synthesis, even in the presence of high concentrations of exogenous fatty acids (26). FAS overexpression in breast and prostate carcinomas is a powerful predictor of a poor clinical outcome (1, 8). The enzyme is overexpressed in colorectal tumors (28). Glucose transport and catabolism are enhanced in cancer cells (29), suggesting that coordinate regulation of glucose and lipid metabolism characterizes the malignant state.
Glucose alone, independent of hormones, stabilizes the FAS message in Hep G2 cells (33). In this model system, glucose maintains message levels for several hours, although decay is rapid after glucose deprivation (32). To test the hypothesis that a glucose-regulated mRNA-binding protein is involved in this process, we studied the interaction of the 3'-terminus of the human FAS mRNA with Hep G2 cytoplasmic extracts. We have identified and purified a large glucose-inducible phosphoprotein that binds to a novel repetitive element in the 3'-UTR of the FAS mRNA.
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METHODS |
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Tissue culture reagents and culture conditions. Glucosefree/bicarbonate-free RPMI 1640, bicarbonate, D-glucose, L-glucose, and BSA (fraction V) were purchased from Sigma. Fetal bovine serum (FBS) was purchased from Intergen. MEM was provided by the Washington University Tissue Culture Support Center.
Hep G2 cells were grown in MEM plus 10% FBS for a large-scale preparation of cell extracts and protein purification. For experiments involving glucose regulation, cells were treated as described (32, 33). On day 3 or 4 after passage, cells were fed MEM plus 10% FBS. On day 5 or 6, culture medium was replaced with RPMI 1640 plus 10% FBS plus 4,500 mg/l D-glucose. Twenty-four hours later, medium was removed, and dishes were washed two times with phosphate-buffered saline (PBS) at 37°C. The cells were then fed RPMI 1640 plus 3% BSA plus 4,500 mg/ml D-glucose. Six hours later, the medium was removed, and dishes were washed two times with PBS at 37°C. Cells were then fed RPMI 1640 plus 3% BSA with 4,500 mg/l D-glucose or 4,500 mg/l L-glucose.
Preparation of S100 cytosolic extracts. Medium was removed, and dishes were washed two times with PBS at 4°C. The cells were scraped in lysis buffer (250 µl/100 mm dish) consisting of 10 mM Tris (pH 7.4), 40 mM KCl, 0.15 mM spermine, 2 mM EDTA, 5 mM dithiothreitol, and 100 µg/ml phenylmethylsulfonyl fluoride. Cells were homogenized using a Dounce homogenizer by 20 strokes of a tight-fitting pestle. Nuclei were removed by centrifugation at 13,000 g for 10 min. This supernatant was centrifuged at 100,000 g for 1 h in a Beckman TL-100 ultracentrifuge to yield a polysome-free extract (S100). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay (Pierce).
Preparation of RNAs by in vitro transcription. The parent plasmid used as a template for transcription of the FAS full-length 3'-untranslated region (UTR) was constructed by subcloning an Nco I-Not I restriction fragment of the human FAS cDNA (from nucleotides 1190 to 2237, as numbered in Ref. 32) into pGEM-5Z. This fragment (see Fig. 6, transcript 1) contained the coding region for the 72 COOH-terminal amino acids of the FAS protein and the entire 806 bases of the 3'-UTR. Several additional FAS plasmids (see Fig. 6) were derived from this construct by standard cloning techniques. Templates were linearized and gel purified before in vitro transcription.
Radiolabeled FAS RNA probes were prepared using T7 RNA polymerase (sense) or SP6 (antisense) and [32P]CTP and were purified using NucTrap columns (Stratagene) before use in gel mobility shift assays. Nonradioactive transcripts used for competition studies were prepared in the same way, except unlabeled CTP was used for transcription reactions. The plasmid used as a template for transcription of a portion of the coding region of the lipoprotein lipase (LPL) message has been described (31).
All of the RNAs generated by in vitro transcription were treated
identically. Probes were frozen at 70°C immediately after their preparation. The transcripts were thawed gradually at room temperature before use in gel mobility shift assays.
Gel mobility shift assays. Binding
reactions were performed with S100 cytosolic extract and 0.5-1.0 × 105 cpm of
[32P]RNA in binding
buffer [10 mM Tris (pH 7.4), 2 mM EDTA, 40 mM KCl, and 5 mM
dithiothreitol]. Binding reactions were incubated for 10 min at
4°C. Five units of RNase T1, which degrades single-stranded RNA not
bound to protein, was then added, and the incubation was continued for
10 min at room temperature. The binding reactions were then subjected
to electrophoresis through a 6% nondenaturing polyacrylamide gel. Gels
were autoradiographed at 70°C for 4-24 h.
Ultraviolet cross-linking of the RNA-protein
complex. Multiple lanes of 6% nondenaturing gels were
loaded with the products of binding reactions, some of which contained
radiolabeled RNA only. After electrophoresis, gels were ultraviolet
(UV) irradiated at 270,000 µJ/cm2. Gels were
autoradiographed, and then radiolabeled bands were excised from lanes
loaded with extracts; corresponding gel regions were also excised from
lanes run with RNA only. Samples were pooled and incubated in an equal
volume of Laemmli electrophoresis buffer at 50°C for 30 min and
then at 100°C for 1 h. Eluates from lanes containing extracts were
separated into two aliquots, one of which was treated with proteinase K
and one of which was not. Samples were subjected to SDS-PAGE in 6%
gels and then autoradiographed at 70°C.
Size exclusion chromatography. S100 cytosolic extracts from Hep G2 cells were precipitated with 25-45% ammonium sulfate. After dialysis against lysis buffer, samples were loaded onto Superose 6 columns equilibrated with 0.9 M NaCl plus 0.2 mM EDTA plus 0.2 g/l NaN3. Fractions of 0.5 ml were collected at a flow rate of 0.4 ml/min and assayed for RNA binding activity by gel mobility shift assay.
Ion exchange chromatography. After ammonium sulfate fractionation, samples were applied to prepacked anion (Mono Q) or cation exchange columns (Mono S) using a Pharmacia fast-performance liquid chromatography apparatus. For the Mono S column, both 50 mM malonate (pH 5.0) and 50 mM MES (pH 6.0) were used for column equilibration in separate experiments. For the Mono Q column, equilibration was performed using 20 mM Tris (pH 7.5); the elution buffer was 20 mM Tris (pH 7.5) plus 0-1.0 M KCl. For both ion exchange columns, the flow rate was 0.5 ml/min, and collected fractions were assayed for RNA binding activity. The binding protein did not bind to a Mono S column under a variety of conditions. The protein did bind to a Mono Q column and was eluted with 0.3 M KCl at pH 7.5.
RNA binding protein purification. In a
typical purification, S100 cytosolic extracts were prepared from
~109 Hep G2 cells. After
fractionation with 25-45% ammonium sulfate and dialysis, samples
were subjected to anion exchange chromatography as described above.
Fractions containing the RNA-binding protein were pooled, concentrated
with Centricon-30 filters at 5,000 g, and then subjected to a large-scale gel shift procedure using 40-45 nondenaturing polyacrylamide gels. Bands were cut out and placed into an electroelution apparatus (model 422; Bio-Rad). Elution
was performed at 10 mA/glass tube for 5 h in 1×
Tris-borate-EDTA. Samples were desalted using Centricon
filters and then subjected to SDS-PAGE using 2-15% gradient gels.
Gels were stained with amido black B followed by autoradiography at
70°C. Protein recovery was followed using the BCA protein
assay (Pierce) except at the final step when values were determined by
comparison of protein-stained bands with standards.
Dephosphorylation of the RNA-binding protein. S100 extracts were subjected to ammonium sulfate fractionation and anion exchange chromatography. Partially purified protein was incubated with potato acid phosphatase (Boehringer Mannheim) in 3-(N-morpholino)propanesulfonic acid (pH 6.5), 1 mM MgCl2, 100 mM KCl, and 0.2 mM leupeptin for 30 min at 30°C, as described by Kwon and Hecht (18). The phosphatase inhibitor okadaic acid (10 µM) was added to some samples before the incubation.
RNase T1 mapping. Gel mobility shift assays were performed using the 25-45% ammonium sulfate fraction from Hep G2 cells. Bands were cut from the gel and electroeluted as described above. Samples were extracted with phenol/chloroform and then ethanol precipitated. Radioactive RNA (~10,000 cpm) was separated into two aliquots, one of which was digested with 20 units of RNase T1 at room temperature for 30 min. The second aliquot was sham digested. Samples were then separated on a 15% denaturing polyacrylamide gel with end-labeled size markers and autoradiographed.
Oligonucleotide competition. Radiolabeled RNA was heated to 75°C for 10 min, a competitor oligonucleotide at various concentrations was added, and the mixture was gradually cooled to room temperature over 30-60 min to allow hybridization of the oligonucleotide with the RNA transcript. The RNA/DNA hybrid was then incubated with Hep G2 extracts and assayed for gel shift activity. The sequences of the competitor oligos were as follows: antisense oligo, 5'-GGGGGTGGGG TGGGGTGGGG TGGGGATGGT GGAGTGA-3'; control oligo "A," 5'-GATCTAGCTT CCCGCTGATG AGTCCGTGAG GACGAAACAT GCCGGCA-3'; control oligo "B," 5'-GATCTGCCGG CATGTTTCGT CCTCACGGAC TCATCAGCGG GAAGCTA-3'.
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RESULTS |
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When Hep G2 extracts were incubated with a transcript corresponding to the FAS 3'-terminus, a discrete band was seen in RNA gel shift assays (Fig. 1, lane 2), which did not appear with RNA alone (Fig. 1, lane 1). The addition of a 10-fold molar excess of nonradioactive FAS sense transcript essentially eliminated the gel shift signal (Fig. 1, lane 5), whereas nonradioactive FAS antisense (Fig. 1, lane 10) and LPL sense (Fig. 1, lane 12) transcripts had no effect. Identical results were seen in five independent experiments. In other experiments, the addition of up to a 100-fold molar excess of the FAS antisense and LPL sense transcripts had no effect on binding. No complexes were seen when Hep G2 extracts were incubated with radiolabeled LPL RNA (not shown).
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The intensity of the gel-shifted band was dependent on the input protein in the assay. In five independent experiments, band intensity increased linearly with protein concentrations from 5 to 20 µg.
Glucose regulates the expression of the FAS mRNA-binding factor (Fig. 2). When cells were cultured in serum-free medium, D-glucose (Fig. 2A, lane 3) but not L-glucose (Fig. 2A, lane 4) increased binding activity in gel shift assays. At the 3-h time point in three independent experiments using protein concentrations within the linear response range of the assay, binding intensity (means ± SE) as shown in Fig. 2B was 14.95 ± 1.20 with D-glucose and 7.40 ± 0.18 for L-glucose (P = 0.0033 by unpaired, 2-tailed t-test). The time course for the glucose-induced increase in binding is shown in the representative experiment of Fig. 2C. For this experiment, the band indicated by the arrowhead in Fig. 2A was scanned. After feeding 4,500 mg/l D-glucose, binding intensity increased by 1 h and peaked at 3 h. The return to baseline was variable, occurring between 6 and 11 h after feeding. L-Glucose had no effect. Results were confirmed in three independent experiments.
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In Fig. 2A, there is a faint upper band (above the predominant band indicated by the arrowhead) that is also induced by glucose. It is possible that this band has the same function as the band at the arrowhead. Because gel shift assays are performed under nondenaturing conditions, it is also possible that this upper band represents aggregates of the RNA-binding protein-RNA complex.
Treatment of Hep G2 extracts with proteinase K or boiling abolished the binding activity (Fig. 3A). The same results were seen in three independent experiments. Figure 3A, lane 2, shows a dominant upper band (denoted by the large arrowhead) and a lower band with faster mobility (denoted by the small arrowhead). The latter probably represents a degradation product. It was faint in fresh extracts and increased in intensity over time. The faint upper band seen above the band indicated by the arrowheads in Figs. 1 and 2 is not seen in Fig. 3 but was present in longer exposures of this blot. The intensity of this faint upper band did not decrease with extract storage over time, making it unlikely that the dominant band at the large arrowhead is a proteolysis product of the upper band visible in Figs. 1 and 2.
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The band at the large arrowhead in Fig. 3A was resolved by SDS-PAGE after UV cross-linking, as described in METHODS. Bands were cut from multiple lanes, eluted, pooled, and electrophoresed in SDS gels (Fig. 3B). The FAS mRNA-binding protein (Fig. 3B, lane 1, arrowhead) analyzed under these conditions had an apparent molecular mass of 178 ± 7 kDa (mean ± SE of 4 independent experiments). In 2 of the 4 experiments, despite the fact that only the upper, dominant band was cut from native gels, SDS-PAGE showed a lower molecular weight protein just below the 178-kDa band, consistent with the hypothesis that the pattern sometimes seen in gel shift assays is due to proteolysis. The 178-kDa signal did not appear when eluted bands were treated with proteinase K before SDS-PAGE (Fig. 3B, lane 2) and was absent from lanes without extracts (Fig. 3B, lane 3).
Because the apparent size as determined by SDS-PAGE included cross-linked ribonucleotides, the molecular weight of the FAS RNA-binding protein was also determined by gel filtration chromatography, as described in METHODS. Fractions with RNA-binding activity eluted at an apparent molecular weight of 160-170 kDa.
The protein was purified as described in METHODS from Hep G2 S100 extracts by a combination of ammonium sulfate precipitation, anion exchange chromatography, and an affinity step. Instead of making an RNA affinity column, we used nondenaturing polyacrylamide gels as the chromatography matrix. Preparative scale gels were UV cross-linked and autoradiographed, and then bands representing the RNA-binding protein were excised and electroeluted. Results of a typical purification are shown in Fig. 4. Figure 4, lane 6, is an autoradiograph of the purified protein from lane 5. The protein is visible after autoradiography because radiolabeled FAS RNA was UV cross-linked to the protein during purification. The same results were seen in four independent purifications.
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Protein recovery data from a typical purification were as follows: S100 cytoplasmic extract = 280 mg (100%), ammonium sulfate fractionation = 42 mg (15%), Mono Q column = 4 mg (1.4%), electroelution = 0.58 mg (0.21%), SDS-PAGE = 0.01-0.02 mg (0.004-0.007%).
Purification of the 178-kDa protein allowed us to define its properties. Phosphorylation is required for the activity of some RNA-binding proteins (18, 21). Acid phosphatase decreased binding activity of Mono Q-purified protein in a dose-dependent fashion, an effect that was reversed with the phosphatase inhibitor okadaic acid (Fig. 5). Results in Fig. 5 represent the means ± SE of three independent experiments.
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The iron-responsive protein (13), the AU-binding factor (21), and a protein that binds to the c-myc message (27) represent examples of RNA-binding proteins that require intact sulfhydryl (SH) groups for binding activity. To test the role of sulfhydryl groups in the activity of the human FAS mRNA-binding protein, Mono Q-purified protein was treated with the SH-oxidizing agent diamide (0-10 mM) or the SH-alkylating agent N-ethylmaleimide (0-2 mM) in the presence or absence of dithiothreitol. In three independent experiments, modification of sulfhydryl groups had virtually no effect on RNA-binding activity (not shown).
Divalent cations are required by some RNA-binding proteins (21). In four independent experiments using Mono Q-purified protein, magnesium concentrations between 0 and 4 mM and calcium concentrations between 0 and 2 mM had no effect on gel shift activity (not shown).
Another important property of the 178-kDa protein is its binding element. A series of deletion plasmids was constructed and used to generate transcripts for gel shift assays (Fig. 6). For these experiments, we measured the intensity of the band indicated by the arrowhead in Figs. 1-3 (large arrowhead) and 8 (see below). Binding activity was normalized to the signal for transcript 3 in Fig. 6. Transcript 2, representing the distal 3'-UTR, had no binding activity. Transcripts antisense to transcripts 1, 3, 4, and 5 also had no binding activity. Activity for transcript 7 was 6% of the activity of transcript 3 (6 ± 1 vs. 100 ± 2, P < 0.0001). Full binding activity was retained by transcript 8, a 126 nucleotide transcript containing the proximal 3'-UTR.
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The specific sequence mediating high-level binding activity was identified within the first 65 bases of the FAS 3'-UTR. RNase T1 mapping using transcripts 4 and 5 from Fig. 6 identified a 37 nucleotide fragment (Fig. 7). The fragment did not contain internal Gs because RNase T1 digestion of RNA isolated from bound protein had no effect on the migration of the isolated RNA (Fig. 7, lanes 2 and 4).
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There is one G-free region of exactly 37 nucleotides within the FAS 3'-UTR (5'-UC-ACUCC-ACC-AUCCCC-ACCCC-ACCCC-ACCCC-ACCCCC-3'). When an oligonucleotide antisense to this sequence was hybridized to the FAS transcript, RNA-binding activity was abolished (Fig. 8). Control oligo A at 10- to 80-fold molar excess had no effect on binding activity (Fig. 8, lanes 1 and 2). The same results were seen with control oligo B (not shown). In this representative experiment, assays were performed using transcript 4 from Fig. 6. Identical results, i.e., the disruption of binding by antisense oligo and no effect of control oligos, were seen using transcripts 1, 3, 5, and 6 from Fig. 6 (not shown).
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DISCUSSION |
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We have purified a large cytosolic protein from Hep G2 cells which binds specifically to a repetitive ACCCC motif in the proximal 3'-UTR of the human FAS mRNA. Binding is induced by glucose and requires phosphorylation.
Glucose deprivation leads to the rapid decay of FAS mRNA, which is prevented by D-glucose feeding (32). Either glucose deprivation is associated with a factor promoting FAS mRNA decay, or glucose feeding is associated with a factor protecting the message from decay. In the current study, D-glucose increases 178-kDa binding activity precisely within the time frame that D-glucose protects FAS mRNA from decay, suggesting that this protein is a protective trans-acting mRNA-binding protein. Although consistent with such a mechanism, these results do not prove that the 178-kDa protein mediates glucose regulation of FAS expression. Directly addressing this issue will await overexpression and inactivation of the 178-kDa protein once the complete cDNA for this protein is characterized.
Hep G2 cells are of human hepatic origin. Liver is a major lipogenic organ, but FAS is also expressed in many other tissues (32). If the 178-kDa protein plays an important role in mediating FAS expression, one might predict that its expression is not limited to liver. We have recently used S100 extracts of mouse tissues to show that 178-kDa binding activity is present in multiple tissues but highest in liver, adipose tissue (a major site of lipogenesis in rodents; see Ref. 19), and brain (Li and Semenkovich, unpublished observation).
If our hypothesis is correct that the glucose-inducible 178-kDa protein is a protective mRNA-binding protein, this protein might also interact with messages for other lipogenic enzymes. Carbohydrates regulate mRNA stability for glucose-6-phosphate dehydrogenase, malic enzyme, and L-pyruvate kinase (14), raising the possibility that the 178-kDa protein is involved in message stability for lipogenic enzymes other than FAS. This does not imply that message stability is the dominant form of regulation for lipogenesis. In both cultured hepatocytes and adipose tissue explants, glucose has been shown to stimulate FAS transcription (14). However, carbohydrate feeding to rodents increases hepatic FAS mRNA by stimulating FAS transcription and stabilizing the message (16). In addition, mRNA stabilization has been shown to be the predominant form of regulation of FAS gene expression in 3T3-L1 adipocytes during differentiation (24), human breast cancer cell lines (2), and in fetal rat lung (38). Taken together, these findings suggest that characterizing a protective RNA-binding protein that regulates FAS mRNA stability will have physiological relevance.
Several protective mRNA-binding proteins are induced in response to
nutritional or environmental signals. Iron deprivation induces the
iron-responsive protein to bind to the transferrin receptor mRNA and
protect it from endonucleolytic attack (13). Estrogens induce protein
binding to the vitellogenin 3'-UTR in association with message
stabilization (6). Hypoxia stabilizes the tyrosine hydroxylase mRNA and
increases protein binding to its 3'-UTR (4). Tumor necrosis
factor- stabilizes glucose transporter-1 (GLUT-1) mRNA and induces
protein binding to a GC-rich region of the GLUT-1
3'-UTR (22). Repeated copies of the sequence AUUUA confer
instability to mRNA; proteins bind the AUUUA motif in association with
message stabilization (39).
The 178-kDa binding element, (ACCCC)n, appears to be unique. It resembles the AUUUA motif with cytidines substituted for uridines. Recent data suggest that UUAUUUAUU may be the minimal sequence of the repeated AUUUA motif that confers instability (40). If our element is analogous, the functional motif may be CCACCCCACC; there are three overlapping copies of this sequence in the 178-kDa binding element. Mutagenesis of the (ACCCC)n motif and determining whether this sequence can confer glucose-induced stability to unrelated messages will be critical for characterizing its function.
Exactly how a protective 178-kDa RNA-binding protein might work is unknown. Binding may block the action of constitutive endonucleases at critical sites in the message, such as repeated ACCCC motifs. Another possibility is that the 178-kDa protein sequesters FAS mRNA so that it cannot access cytoplasmic decay complexes. In our system, polyribosomes protect FAS mRNA from decay after glucose deprivation (32), raising the possibility that the 178-kDa protein promotes the association of the FAS message with the translational apparatus.
Although the binding activity that we describe is likely to be novel (we are unaware of similar size mRNA-binding proteins), it is possible that the 178-kDa protein is not. More than a dozen classic metabolic enzymes have been shown to bind nucleic acids. Dinucleotide binding domains common to these enzymes may be involved (12). The iron-responsive protein is also an aconitase, catalyzing the conversion of citrate to isocitrate (10). Glyceraldehyde-3-phosphate dehydrogenase binds tRNA (35) and AUUUA motifs (25). Catalase binds and stabilizes its own mRNA (3).
How does glucose increase the binding of the 178-kDa factor to the FAS mRNA? Phosphorylation is required for optimal binding (Fig. 5). A reasonable hypothesis is that glucose metabolism induces phosphorylation of the 178-kDa protein to promote binding to the FAS mRNA. There is precedence for glucose regulation of gene expression through phosphorylation. In yeast, glucose initiates a complex phosphorylation cascade. Malate dehydrogenase (23) and the transcriptional activator SIP4 (20) are examples of proteins differentially phosphorylated in response to glucose availability.
In summary, glucose transiently increases binding of a large phosphoprotein to an ACCCC repetitive element in the 3'-UTR of the human FAS mRNA within the same time frame that glucose prevents FAS mRNA decay. Because aberrant glucose and lipid metabolism are commonly associated, characterizing this protein could link glycolysis and lipogenesis in such a way that mechanisms underlying such diverse disorders as diabetic dyslipidemia and malignancy are better understood.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the American Heart Association, the American Diabetes Association, the Washington University/Monsanto/Searle Biomedical Research Agreement, and the Washington University Diabetes Research and Training Center (DK-20579). Q. Li was the recipient of a postdoctoral fellowship from the Missouri Affiliate of the American Heart Association. M. S. Chua was supported in part by a summer fellowship from the Howard Hughes Medical Institute. This work was done during the tenure of an Established Investigatorship from the American Heart Association.
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FOOTNOTES |
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Address for reprint requests: C. F. Semenkovich, Div. of Atherosclerosis, Nutrition, and Lipid Research, Washington Univ. School of Medicine, 660 S. Euclid Ave., Box 8046, St. Louis, MO 63110.
Received 1 August 1997; accepted in final form 16 December 1997.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alo, P. L.,
P. Visca,
A. Marci,
A. Mangoni,
C. Botti,
and
U. Di Tondo.
Expression of fatty acid synthase (FAS) as a predictor of recurrence in stage I breast carcinoma patients.
Cancer
77:
474-482,
1996[Medline].
2.
Chalbos, D.,
F. Galtier,
S. Emiliani,
and
H. Rochefort.
The anti-progestin RU486 stabilizes the progestin-induced fatty acid synthetase mRNA but does not stimulate its transcription.
J. Biol. Chem.
266:
8220-8224,
1991
3.
Clerch, L. B.,
J. Iqbal,
and
D. Massaro.
Perinatal rat lung catalase gene expression: influence of corticosteroid and hyperoxia.
Am. J. Physiol.
260 (Lung Cell. Mol. Physiol. 4):
L428-L433,
1991
4.
Czyzyk-Krzeska, M. F.,
Z. Dominski,
R. Kole,
and
D. E. Millhorn.
Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA.
J. Biol. Chem.
269:
9940-9945,
1994
5.
Dinneen, S.,
J. Gerich,
and
R. Rizza.
Carbohydrate metabolism in non-insulin-dependent diabetes mellitus.
N. Engl. J. Med.
327:
707-713,
1992[Medline].
6.
Dodson, R. E.,
and
D. J. Shapiro.
An estrogen-inducible protein binds specifically to a sequence in the 3' untranslated region of estrogen-stabilized vitellogenin mRNA.
Mol. Cell. Biol.
14:
3130-3138,
1994[Abstract].
7.
Dudek, S. M.,
and
C. F. Semenkovich.
Essential amino acids regulate fatty acid synthase expression through an uncharged transfer RNA-dependent mechanism.
J. Biol. Chem.
270:
29323-29329,
1995
8.
Epstein, J. I.,
M. Carmichael,
and
A. W. Partin.
OA-519 (fatty acid synthase) as an independent predictor of pathologic state in adenocarcinoma of the prostate.
Urology
45:
81-86,
1995[Medline].
9.
Goodridge, A. G.
Dietary regulation of gene expression: enzymes involved in carbohydrate and lipid metabolism.
Annu. Rev. Nutr.
7:
157-185,
1987[Medline].
10.
Gray, N. K.,
S. Quick,
B. Goosen,
A. Constable,
H. Hirling,
L. Kuhn,
and
M. Hentze.
Recombinant iron-regulatory factor functions as an iron-responsive-element-binding-protein, a translational repressor and an aconitase.
Eur. J. Biochem.
218:
657-667,
1993[Abstract].
11.
Hauguel-de-Mouzon, S.,
C. Mrejen,
F. Alengrin,
and
E. Van Obberghen.
Glucose-induced stimulation of human insulin-receptor mRNA and tyrosine kinase activity in cultured cells.
Biochem. J.
305:
119-124,
1995[Medline].
12.
Hentze, M. W.
Enzymes as RNA-binding proteins: a role for (di)nucleotide-binding domains?
Trends Biochem. Sci.
19:
101-103,
1994[Medline].
13.
Hentze, M. W.,
T. Rouault,
J. B. Harford,
and
R. D. Klausner.
Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction.
Science
244:
357-359,
1989[Medline].
14.
Hillgartner, F. B.,
L. M. Salati,
and
A. G. Goodridge.
Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis.
Physiol. Rev.
75:
147-176,
1995.
15.
Hudgins, L. C.,
M. Hellerstein,
C. Seidman,
R. Neese,
J. Diakun,
and
J. Hirsch.
Human fatty acid synthesis is stimulated by a eucaloric low fat, high carbohydrate diet.
J. Clin. Invest.
97:
2081-2091,
1996
16.
Iritani, N.
Nutritional and hormonal regulation of lipogenic-enzyme expression in rat liver.
Eur. J. Biochem.
205:
433-442,
1992[Medline].
17.
Jones, B. H.,
J. H. Kim,
M. B. Zemel,
R. P. Woychik,
E. J. Michaud,
W. O. Wilkison,
and
N. Moustaid.
Upregulation of adipocyte metabolism by agouti protein: possible paracrine actions in yellow mouse obesity.
Am. J. Physiol.
270 (Endocrinol. Metab. 33):
E192-E196,
1996
18.
Kwon, Y. K.,
and
N. B. Hecht.
Binding of a phosphoprotein to the 3' untranslated region of the mouse protamine 2 mRNA temporally represses its translation.
Mol. Cell. Biol.
13:
6547-6557,
1993[Abstract].
19.
Laybutt, D. R.,
D. J. Chisholm,
and
E. W. Kraegen.
Specific adaptations in muscle and adipose tissue in response to chronic systemic glucose oversupply in rats.
Am. J. Physiol.
273 (Endocrinol. Metab. 36):
E1-E9,
1997
20.
Lesage, P.,
X. Yang,
and
M. Carlson.
Yeast SNF1 protein kinase interacts with SIP4, a C6 zinc cluster transcriptional activator: a new role for SNF1 in the glucose response.
Mol. Cell. Biol.
16:
1921-1928,
1996[Abstract].
21.
Malter, J. S.,
and
Y. Hong.
A redox switch and phosphorylation are involved in the post-translational up-regulation of the adenosine-uridine binding factor by phorbol ester and ionophore.
J. Biol. Chem.
266:
3167-3171,
1991
22.
McGowan, K. M.,
S. Police,
J. B. Winslow,
and
P. H. Pekala.
Tumor necrosis factor- regulation of glucose transporter (GLUT1) mRNA turnover.
J. Biol. Chem.
272:
1331-1337,
1997
23.
Minard, K. I.,
and
L. McAlister-Henn.
Glucose-induced phosphorylation of the MDH2 isozyme of malate dehydrogenase in Saccharomyces cerevisiae.
Arch. Biochem. Biophys.
315:
302-309,
1994[Medline].
24.
Moustaid, N.,
and
H. S. Sul.
Regulation of expression of fatty acid synthase gene in 3T3-L1 cells by differentiation and triiodothyronine.
J. Biol. Chem.
266:
18550-18554,
1991
25.
Nagy, E.,
and
W. F. Rigby.
Glyceraldehyde-3-phosphate dehydrogenase selectively binds AU-rich RNA in the NAD(+)-binding region (Rossman fold).
J. Biol. Chem.
270:
2755-2763,
1995
26.
Pizer, E. S.,
F. D. Wood,
G. R. Pasterack,
and
F. P Kuhajda.
Fatty acid synthase (FAS): a target for cytotoxic antimetabolites in HL60 promyelocytic leukemia cells.
Cancer Res.
56:
745-751,
1996[Abstract].
27.
Prokipcak, R. D.,
D. J. Herrick,
and
J. Ross.
Purification and properties of a protein that binds to the C-terminal coding region of human c-myc mRNA.
J. Biol. Chem.
269:
9261-9269,
1994
28.
Rashid, A.,
E. S. Pizer,
M. Moga,
L. Z. Milgraum,
M. Zahurak,
G. R. Pasternak,
F. P. Kuhajda,
and
S. R. Hamilton.
Elevated expression of fatty acid synthase and fatty acid synthetic activity in colorectal neoplasia.
Am. J. Pathol.
150:
201-208,
1997[Abstract].
29.
Rempel, A.,
S. P. Methupala,
and
P. L. Pederson.
Glucose catabolism in cancer cells: regulation of the type II hexokinase promoter by glucose and cyclic AMP.
FEBS Lett.
385:
233-237,
1996[Medline].
30.
Ross, J.
Control of messenger RNA stability in higher eukaryotes.
Trends Genet.
12:
171-175,
1996[Medline].
31.
Seip, R. L.,
T. J. Angelopoulos,
and
C. F. Semenkovich.
Exercise induces human lipoprotein lipase gene expression in skeletal muscle but not adipose tissue.
Am. J. Physiol.
268 (Endocrinol. Metab. 31):
E229-E236,
1995
32.
Semenkovich, C. F.,
T. Coleman,
and
F. T. Fiedorek, Jr.
Human fatty acid synthase mRNA: tissue distribution, genetic mapping, and kinetics of decay after glucose deprivation.
J. Lipid Res.
36:
1507-1521,
1995[Abstract].
33.
Semenkovich, C. F.,
T. Coleman,
and
R. Goforth.
Physiologic concentrations of glucose regulate fatty acid synthase activity in HepG2 cells by mediating fatty acid synthase mRNA stability.
J. Biol. Chem.
268:
6961-6970,
1993
34.
Semenkovich, C. F.,
and
J. W. Heinecke.
The mystery of diabetes and atherosclerosis: time for a new plot.
Diabetes
46:
327-334,
1997[Abstract].
35.
Singh, R.,
and
M. R. Green.
Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogenase.
Science
259:
365-368,
1993[Medline].
36.
Volpe, J. J.,
and
P. R. Vagelos.
Mechanisms and regulation of biosynthesis of saturated fatty acids.
Physiol. Rev.
56:
339-417,
1976
37.
Wakil, S. J.,
J. K. Stoops,
and
V. C. Joshi.
Fatty acid synthesis and its regulation.
Annu. Rev. Biochem.
52:
537-579,
1983[Medline].
38.
Xu, Z. X.,
W. Stenzel,
S. M. Sasic,
D. A. Smart,
and
S. A. Rooney.
Glucocorticoid regulation of fatty acid synthase gene expression in fetal rat lung.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L140-L147,
1993
39.
Zhang, W.,
B. J. Wagner,
K. Ehrenman,
A. W. Schaefer,
C. T. DeMaria,
D. Crater,
K. DeHaven,
L. Long,
and
G. Brewer.
Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1.
Mol. Cell. Biol.
13:
7652-7665,
1993[Abstract].
40.
Zubiaga, A. M.,
J. G. Belasco,
and
M. E. Greenberg.
The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.
Mol. Cell. Biol.
15:
2219-2230,
1995[Abstract].