(Received for publication, August 3, 1995; and in revised form, September 21, 1995)
From the
To better understand the regulation of gene expression by amino
acids, we studied the effects of these macronutrients on fatty acid
synthase (FAS), an enzyme crucial for energy storage. When HepG2 cells
were fed serum-free media selectively deficient in each amino acid, the
omission of any single classic essential amino acid as well as Arg or
His (essential in some rapidly growing cells) resulted in FAS mRNA
levels that were about half of those in complete medium. Control
message levels were unaffected and omission of nonessential amino acids
did not alter FAS expression. FAS mRNA levels peaked 12-16 h
after feeding complete and Ser (nonessential)-deficient media but did
not increase in cells fed Lys (essential)-deficient medium. With Lys,
FAS mRNA increased over the physiologic concentration range of
15-150 µM, and low concentrations of lysine
decreased FAS but not apoB protein mass. Transcription inhibitors
mimicked treatment with Lys-deficient media, and nuclear run-off assays
showed that Lys-deficient media abolished FAS but not apoB
transcription. After treatment with Lys-deficient media, the
intracellular Lys pool was rapidly depleted in association with an
increase of uncharged (deacylated) tRNA from <1 to 64%
of available tRNA
. Even in the presence of the essential
amino acid His, increasing the levels of uncharged tRNA
with histidinol, a competitive inhibitor of the histidinyl-tRNA
synthetase, blocked FAS expression. Tyrosinol treatment did not alter
FAS mRNA levels. These results suggest that essential amino acids
regulate FAS expression by altering uncharged tRNA levels, a novel
mechanism for nutrient control of gene expression in mammalian cells.
Fatty acid synthase (FAS) ()is a large
multifunctional protein that synthesizes the fatty acid palmitate from
acetyl-CoA, malonyl-CoA, and NADPH(1) . This process is
essential for the conversion of dietary calories into a storage form
suitable for use during periods of fasting. Although several enzymes
are critical for the synthesis of fatty acids, it appears that FAS is
rate-limiting in the long term control of lipogenesis(2) .
Feeding increases FAS expression(3) . The intake of high carbohydrate diets following periods of fasting increases FAS mRNA levels and enzyme concentration(4) . Hormones, many of which are affected by feeding and fasting, play a role in this regulation. In diabetic rats, insulin increases hepatic FAS transcription, mRNA levels, and enzyme levels 5-15-fold(5) . Dexamethasone enhances FAS mRNA induction by insulin in primary rat hepatocyte cultures(6) . Triiodothyronine stimulates FAS gene transcription in cultured chick embryo hepatocytes (7) and mouse 3T3-L1 adipocytes(8) . Progesterone stabilizes FAS mRNA levels(9) .
Hormonal regulation of FAS expression has appropriately been the focus of many previous studies. However, individual dietary components, especially macronutrients, can also affect FAS expression. The carbohydrate glucose stabilizes FAS mRNA levels in HepG2 cells cultured in serum-free media(10, 11) . Polyunsaturated fatty acids are potent inhibitors of FAS gene transcription in rats(12) . But the role of amino acids in FAS regulation is ill-defined. Maximal induction of transcription, mRNA levels, and enzyme activity in fasted rats requires feeding of both carbohydrate and protein(5) . In cultured rat hepatocytes, a combination of essential and nonessential amino acids is necessary for maximal induction of FAS protein levels(13) , but nothing else is known about the effects of individual amino acids on FAS gene expression.
An active role for amino acids in gene regulation is not without precedence. Starvation for a single essential amino acid induces differentiation of the human promyelocytic leukemia line HL-60 into mature granulocytes(14) . Asparagine synthetase mRNA levels are increased by essential amino acid starvation in baby hamster kidney and HeLa cells(15) . In yeast, deficiencies of various amino acids will induce the respective amino acid synthetic pathway by activating the transcription factor GCN4(16) . Therefore, we used human HepG2 cells, known to functionally resemble hepatocytes(17) , to test the hypothesis that amino acids regulate FAS expression. In this paper, we present data supporting this hypothesis and show that the effect of amino acids is likely mediated by changes in the intracellular concentrations of uncharged tRNA.
For most experiments, cells were split 1:25 from 90% confluent T-75 flasks to 100-mm tissue culture dishes on day 1. On day 3 or 4, cells were fed modified RPMI 1640 with 10% fetal bovine serum. After 2 or 3 days when the cells were 50-75% confluent, the media were removed, the dishes were washed with phosphate-buffered saline (PBS), and the cells were fed modified RPMI 1640 with 3% bovine serum albumin.
Gel-purified
FAS and apoB cDNA inserts as well as linearized pBluescript were
denatured and then neutralized(10) , and 10 µg of DNA per
slot was applied to nitrocellulose filters using a slot blot apparatus.
After UV irradiation and prehybridization at 42 °C for 12-18
h, filters were hybridized with equal amounts of radiolabeled
transcripts (10-35 10
cpm) per condition for
48-72 h at 42 °C, washed, and exposed to film with
intensifying screens for 7 days. Data were quantitated by image
processing analysis using ImagePro Plus software.
For each experiment, cells were fed complete, serine-deficient, or lysine-deficient media (twenty 100-mm diameter dishes/condition). 2 h later, total RNA was isolated from these cells under mildly acidic conditions and divided into two aliquots for each condition. One aliquot was oxidized with 2 mM sodium periodate at 25 °C for 15 min in the dark, and the other was left unoxidized. All samples were ethanol precipitated, washed with 70% ethanol, dissolved in 0.17 M Tris (pH 8.8), incubated for 3 h at 37 °C to strip tRNA of bound amino acids, and then again ethanol precipitated.
A
partially purified aminoacyl-tRNA synthetase preparation suitable for
the charging assay was obtained in a procedure modified from that in (23) . Cells from ten 100-mm diameter dishes were washed with
cold PBS, scraped into 15 ml of 0.01 M NaCl, 0.0015 M MgCl, 0.01 M Tris (pH 7.5), briefly sonicated
with a Branson Sonifier 185, and then centrifuged at 300
g for 5 min at 4 °C. The supernatant was recovered and
centrifuged at 12,000
g for 15 min at 4 °C. This
supernatant was recovered and applied by batch to DEAE-52 (Whatman)
equilibrated with 20 mM Tris (pH 7.5), 2 mM
MgCl
, 1 mM dithiothreitol, 0.1 EDTA. The extract
was rotated for 1 h at 4 °C, centrifuged at 900
g for 5 min; then DEAE-52 was washed with equilibration buffer, and
a crude aminoacyl-tRNA synthetase fraction was eluted with
equilibration buffer raised to 0.3 M NaCl(23) . The
eluted protein was quantitated with the BCA Protein Assay (Pierce).
The actual charging assay was performed as follows after preliminary
experiments showed these conditions yielded results within the linear
response range of the assay. The assay mixture for each sample
contained 500 µg of total RNA, 50 µg of partially purified
aminoacyl-tRNA synthetases, 2.5 mM ATP, 0.25 mM CTP,
50 mM Tris (pH 7.5), 150 mM NaCl, 0.1 mM
EDTA, 2 mM MgCl, 2.5 µCi of
C-lysine (or
C-serine), 100 mM
unlabeled lysine (or serine) in a total volume of 500 µl. The
samples were incubated at 37 °C for 30 min, precipitated with 10%
trichloroacetic acid solution containing 1 mg/ml unlabeled lysine (or
serine), applied to 2.4-cm filters (Whatman 540), washed twice with 5%
trichloroacetic acid and once with 95% ethanol, and then dried.
Radioactivity was quantitated with a Beckman LS 2800 scintillation
counter. Each assay included blanks consisting of assay mixtures
without RNA. The degree of in vivo charged tRNA was calculated
by dividing the charging capacity of periodate-oxidized RNA by the
charging capacity of unoxidized RNA. The data are expressed in Table 3as the percentage of uncharged tRNA (determined
by subtracting the percentage of charged tRNA from 100).
When HepG2 cells were incubated for 12 h in serum-free experimental media resembling the amino acid composition of human serum (Table 1), the omission of a single essential amino acid, lysine, resulted in lower FAS message levels compared with complete medium as shown by the RNase protection gel depicted in Fig. 1. Omission of histidine, essential in infancy in humans (24) , had the same effect, but the omission of serine, a nonessential amino acid, did not affect FAS mRNA levels.
Figure 1:
Deficiency of His and Lys but not Ser
inhibits FAS mRNA expression. HepG2 cells cultured as described under
``Materials and Methods'' were washed with PBS and fed
serum-free complete modified RPMI 1640 (Table 1) with 3% bovine
serum albumin (Complete Medium), or the same medium minus Ser,
His, or Lys. 12 h later, total RNA was isolated, and FAS mRNA was
assayed by RNase protection using a human FAS riboprobe. Protected
fragments were separated by denaturing SDS-polyacrylamide gel
electrophoresis and then exposed to film for 24 h. The arrowhead denotes the predicted position (450 nucleotides) of protected
fragments as determined by comparison with the migration of end-labeled
X174/HaeIII fragments run on the same gel. The first
four lanes were generated using 20 µg of HepG2 total RNA. The lane
marked tRNA represents a negative control obtained by
incubating 20 µg of yeast transfer RNA with the FAS riboprobe.
There were no differences in the intensities of apoB or
-actin
bands using the same HepG2 RNA (data not
shown).
These analyses were extended to all 20
amino acids (Fig. 2). HepG2 cells were cultured for 12 h in
complete medium and in 20 separate media, each selectively deficient in
a single amino acid. Fig. 2shows the relative levels of FAS (Fig. 2A) and apolipoprotein B (apoB) (Fig. 2B) mRNA expression after incubation in
selectively deficient media with data expressed relative to complete
medium containing all 20 amino acids. Each one-letter amino acid
abbreviation on the horizontal axis of Fig. 2denotes the amino
acid absent from the medium. FAS mRNA levels were unchanged
relative to complete medium after 12 h of deficiency in any one of the
10 amino acids on the left half of the panel: alanine,
asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,
proline, serine, or tyrosine. All are nonessential; they can be
synthesized from precursors. However, deficiency in any one of the 10
amino acids on the right half of the panel resulted in a
50% decrease in FAS message level. This group includes the 8
classic essential amino acids: isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, tryptophan, and valine(25) .
Deficiency of arginine or histidine also resulted in lower FAS mRNA.
These two amino acids have been shown to be functionally essential in
rapidly growing cell lines(26) . The effect appeared to be
relatively specific for FAS, because message levels for apoB (B) and
-actin (data not shown) were unchanged by amino
acid deficiency.
Figure 2: Deprivation of any single essential amino acid is associated with decreased FAS mRNA levels. HepG2 cells were treated as described in the legend to Fig. 1. Separate dishes received 21 different media, complete medium as well as 20 media each deficient in a single amino acid. The one-letter code on the horizontal axis denotes the amino acid deficient from the medium in which the cells were cultured. FAS (A) and apoB (B) mRNA levels were assayed by RNase protection as in Fig. 1except protected fragments were collected on filters and counted as described under ``Materials and Methods.'' Results represent mRNA levels after 12 h in each selectively-deficient medium and are expressed relative to message detected in complete medium (containing all amino acids). Data shown represent the mean of two independent experiments.
Omission of a single essential amino acid blocked the time-dependent induction of FAS expression after feeding (Fig. 3). 2-4 hours after feeding complete or serine (nonessential)-deficient media, FAS mRNA levels (Fig. 3A) began to increase and peaked 12-16 h post-feeding. No FAS induction occurred when cells were fed lysine (essential)-deficient medium (Fig. 3A, open circles). No differences in apoB expression were observed after feeding the experimental media (Fig. 3B).
Figure 3:
Omission of an essential amino acid blocks
the time-dependent induction of FAS expression after feeding. HepG2
cells were washed with PBS and then fed either control medium ()
containing all 20 amino acids, serine-deficient medium (
), or
lysine-deficient medium (
). Total RNA was isolated at different
time points as indicated on the horizontal axis. FAS (A) and
apoB (B) mRNA levels determined by RNase protection are
expressed relative to message levels in control cells harvested at time
0. Data shown are representative of three independent
experiments.
Two lines of evidence suggested that decreased cell viability did not explain the effect of lysine-deficiency on FAS expression. First, when cells were cultured for 12 h in lysine-deficient, serine-deficient, or complete media followed by addition of MTT (cleaved by mitochondrial dehydrogenases of living cells), MTT cleavage products (quantitated as described under ``Materials and Methods'') in each of the three media were virtually identical (data not shown). Second, the inhibition of FAS mRNA induction by essential amino acid deficiency was reversible (Fig. 4). Cells incubated in lysine-deficient medium from 0-12 h showed no increase in FAS mRNA, but replacing lysine-deficient with complete medium at 12 h resulted in the expected increase in FAS message at 24 h (Fig. 4A, inverted triangles). ApoB expression was again unchanged by incubation in these experimental media (Fig. 4B).
Figure 4:
Inhibition of FAS mRNA induction by
essential amino acid deficiency is reversible. HepG2 cells were washed
with PBS and then fed either complete () or lysine-deficient
medium (
). 12 h later, some cells were harvested, and FAS (A) and apoB (B) mRNA were assayed by RNase
protection. Half of the remaining dishes were washed and fed complete
medium, while the remainder were left in their original medium. After
an additional 12 h (hours 12-24 as indicated on the horizontal
axis), RNA was isolated from the remaining dishes and assayed for FAS
and apoB message. Cells receiving lysine-deficient medium for hours
0-12 and then complete medium for hours 12-24 are indicated
(
). Data represent the means of two independent
experiments.
The effect of the essential amino acid lysine on FAS expression was concentration-dependent (Fig. 5). HepG2 cells were incubated for 12 h in different concentrations of lysine from 0-300 µM(18) . FAS expression increased linearly over this physiologic range, reaching a peak at approximately 150 µM lysine. ApoB mRNA levels were unaffected by lysine concentration.
Figure 5:
Inhibition of FAS mRNA induction by
essential amino acid deficiency is concentration-dependent. Dishes in
triplicate were washed and then fed modified RPMI 1640 media containing
0, 1.5, 15, 75, 150, or 300 µM lysine. 12 h later, RNA was
isolated. FAS () and apoB (
) message levels are expressed
relative to message in cells incubated in lysine-deficient (0
µM) medium. Data are expressed as means ± S.E.; for
most points the S.E. is smaller than the size of the data
symbol.
To determine if the regulation of FAS mRNA by essential amino acids is reflected at the protein level, FAS mass was assayed by Western blotting (Fig. 6). Obviously, cells deprived of an essential amino acid will be unable to synthesize proteins containing that amino acid. So instead of lysine-deficient medium, cells were cultured in medium containing 1.5 µM lysine, a low concentration empirically found to be sufficient for protein synthesis. Fig. 6shows Western blots of FAS and apoB protein after incubation in serine-deficient and 1.5 µM lysine medium for 12 h, longer than the half-life of the FAS protein in HepG2 cells(10) . ApoB is a suitable control protein for this experiment because it is extremely short-lived(27) . FAS protein mass was strikingly lower in the 1.5 µM lysine medium whereas apoB protein was not, confirming that the cells had sufficient lysine for protein synthesis. In fact, apoB protein levels were higher in the low lysine medium, consistent with previous studies showing that incubation of HepG2 cells in low concentrations of amino acids increases apoB synthesis(28) . For both FAS and apoB protein, mass was the same in complete and serine-deficient media (not shown). These results suggest that changes in FAS message levels induced by changes in essential amino acid concentrations can be reflected at the level of the FAS protein.
Figure 6:
Essential amino acid effects are reflected
at the protein level in HepG2 cells. Cells were incubated in
serine-deficient (Minus Serine) or low lysine (1.5
µM Lysine) media for 12 h followed by preparation of
postnuclear cell extracts. 20 µg of protein was loaded in each
lane, subjected to SDS-PAGE, and then Western blotted using anti-FAS
and anti-apoB antibodies. The arrowheads denote the positions
of the FAS (M =
260,000) and apoB (M
=
550,000)
proteins.
To address the possibility of transcriptional regulation of FAS expression by essential amino acids, cells were cultured for 4 h in complete, serine-deficient, or lysine-deficient medium in the presence of the transcriptional inhibitors actinomycin D, cordycepin, and dichlororibofuranosylbenzimidazole. Although these compounds inhibit transcription by different mechanisms, each mimicked the effect of essential amino acid deficiency on FAS message levels (Fig. 7). In the same cells, there were no differences in apoB mRNA levels between cells cultured in complete, serine-deficient, or lysine-deficient medium in the absence of transcriptional inhibitors, and there were no differences in apoB mRNA levels between cells cultured in the different media in the presence of transcriptional inhibitors (data not shown). These results suggest that essential amino acid regulation of FAS is transcriptional.
Figure 7: Treatment with different transcriptional inhibitors mimics the effect of essential amino acid deficiency on FAS mRNA levels. HepG2 cells were washed and fed either complete, serine-deficient, or lysine-deficient media in the presence of carrier (Controls), 4 µM actinomycin D, 80 µM cordycepin, or 65 µM dichlororibofuranosylbenzimidazole (DRB). 4 h later, RNA was isolated from triplicate dishes for each condition, and FAS mRNA was determined by RNase protection. FAS mRNA levels are expressed relative to message in cells fed lysine-deficient control medium. Data are expressed as means ± S.E. The same results were obtained in two independent experiments.
Nuclear run-off assays provided more direct evidence of transcriptional regulation. As shown in Table 2, there was no detectable initiation of FAS transcription after 4 h of incubation in lysine-deficient medium. ApoB transcription also decreased under these conditions but to a lesser extent than FAS. Unlike FAS, the decrease in apoB transcription was not associated with changes in apoB message levels (see Fig. 2and Fig. 3).
To explore how deficiency of a single essential amino acid might affect FAS transcription, the role of uncharged or deacylated transfer RNA (tRNA not bound to its cognate amino acid) was studied. Uncharged tRNA is known to regulate transcription in bacteria(29) . One way to alter uncharged tRNA levels is to change intracellular amino acid concentrations(30) . If amino acids alter uncharged tRNA levels to regulate FAS expression, the effect must be rapid. Message levels increase within 2-4 h of feeding (Fig. 3), and substantial changes in amino acid pool sizes should precede this increase. As shown in Fig. 8, feeding lysine-deficient medium depleted intracellular lysine pools within 1 h (Fig. 3A). In contrast, there was no decrease in intracellular serine pools (Fig. 3B) after incubation in serine-deficient medium, an expected result because cells readily synthesize this amino acid.
Figure 8:
Intracellular lysine is rapidly depleted
after incubation in lysine-deficient medium. HepG2 cells were washed
and then fed either complete (), serine-deficient (
), or
lysine-deficient media (
). At various time
points, cells were collected, and intracellular amino acid pools were
determined by high pressure liquid chromatography as described under
``Materials and Methods.'' Intracellular lysine (A)
and serine (B) are expressed relative to the respective amino
acid pool size determined in time 0 controls. The data shown represent
the means of two independent experiments.
To determine if the decrease in
intracellular lysine affects uncharged tRNA levels in HepG2 cells, the
amount of uncharged tRNA was assayed by a periodate oxidation method
described under ``Materials and Methods.'' 2 h after feeding
lysine-deficient medium, 64% of available tRNA was
uncharged as shown in Table 3. At the same time point, the
uncharged fraction accounted for less than 1% of available
tRNA
in cells fed complete or serine-deficient medium. As
expected, the percentage of uncharged tRNA
did not
increase in serine-deficient medium. Uncharged tRNA
levels in complete medium were substantially higher than those of
uncharged tRNA
but similar to uncharged tRNA
levels reported in Escherichia coli(31) .
If
uncharged tRNAs for essential amino acids regulate FAS expression, then
inhibiting the charging reaction for an essential amino acid, even in
the presence of normal concentrations of that nutrient, should mimic
the effect of essential amino acid deficiency. Amino acid alcohols are
competitive inhibitors of the specific aminoacyl-tRNA synthetases that
facilitate the binding of amino acids to their cognate
tRNAs(23, 32) . Compounds such as histidinol (the
alcohol of the essential amino acid histidine) and tyrosinol (the
alcohol of the nonessential amino acid tyrosine) increase uncharged
tRNA and tRNA
, respectively, even at normal
amino acid concentrations. As shown in Fig. 9A,
culturing HepG2 cells for 12 h in complete medium with 2 mM histidinol had the same effect on FAS expression as
histidine-deficient medium; FAS mRNA levels were unaffected by
incubation in complete medium with 2 mM tyrosinol and
tyrosine-deficient medium. The effects of histidinol on FAS expression
were dose-dependent (data not shown) and these amino acid alcohols did
not affect HepG2 viability within the time frame of these experiments.
These data support the hypothesis that essential amino acids regulate
FAS expression by altering levels of uncharged tRNA.
Figure 9: Inhibition of tRNA charging mimics the effect of essential amino acid deficiency on FAS mRNA levels even in the presence of essential amino acids. HepG2 cells were washed and then fed one of the following media: complete, tyrosine-deficient (Minus Tyrosine), complete with 2 mM tyrosinol (Tyrosinol), histidine-deficient (Minus Histidine), or complete with 2 mM histidinol (Histidinol). 12 h later, FAS and apoB mRNA were assayed by RNase protection. FAS (A) and apoB (B) message levels are expressed relative to message in cells incubated in histidine-deficient medium. Data are expressed as means ± S.E. and are representative of three independent experiments. The asterisks indicate p < 0.001 versus complete medium (Bonferoni multiple comparisons test).
In this paper, we present evidence that essential amino acids in HepG2 cells regulate the expression of a critical enzyme in lipogenesis, fatty acid synthase. Deficiency of a single essential amino acid in serum-free media reversibly blocks the induction of FAS normally associated with feeding, and essential amino acid effects are concentration-dependent. Essential amino acids appear to regulate FAS expression via a transcriptional mechanism mediated through uncharged tRNA.
There is precedence for the involvement of amino acids in gene regulation. Global deprivation of amino acids predictably produces a myriad of effects. Removal of all amino acids from the culture medium up-regulates several types of amino acid transporters in the bovine renal epithelial cell line NBL-1(33) . In primary rat hepatocyte cultures, such amino acid deprivation decreases gene transcription of IGF-I while simultaneously increasing mRNA abundance for one of its binding proteins(34, 35) .
Deficiencies of single amino acids also evoke responses. In bacteria, starvation for an individual amino acid typically increases the level of its cognate aminoacyl-tRNA synthetase(29, 36) . Asparagine deprivation increases expression of asparaginyl-tRNA synthetase in baby hamster kidney and HeLa cells(15) , mammalian cell lines. Amino acid synthetic pathways are induced in yeast by deficiency of the respective amino acid(16) . Deficiency of a single essential amino acid induces differentiation of the human promyelocytic leukemia line HL-60 into mature granulocytes(14) . This effect is mimicked by treatment with the aminoacyl-tRNA synthetase inhibitor histidinol (also used in the current study, see Fig. 9), suggesting an important role for tRNA during HL-60 differentiation.
The tRNA molecule carries out the
adapter function necessary for translation of the genetic code from
nucleic acid to protein. Aminoacyl-tRNA synthetases (37, 38, 39) catalyze the aminoacylation of
tRNAs with the correct amino acids. tRNA is not simply a passive
conduit in this reaction. Each tRNA contains sequence, independent of
the anticodon trinucleotide, and structure information targeting it to
the appropriate aminoacyl-tRNA synthetase(40) . The tRNA itself
can also signal amino acid availability through its covalent
modification by amino acids(41) . Uncharged tRNA (tRNA not
bound to its cognate amino acid) increases when the intracellular pool
of that amino acid falls(30) . This species can regulate gene
expression in some systems. In addition to the induction of HL-60
differentiation discussed above (14) , uncharged tRNA is
involved in transcriptional regulation of aminoacyl-tRNA synthetase
genes in Bacillus subtilis(29) . In this system,
uncharged tRNA likely has a conformation that stabilizes
an antiterminator structure allowing transcription of the tyrosyl-tRNA
synthetase gene. Thus when Tyr levels are low in this bacteria,
uncharged tRNA
signals the increased expression of the
enzyme that attaches Tyr to its tRNA resulting in more efficient
utilization of the remaining low levels of Tyr.
Our data suggest
that uncharged tRNA is also involved in the regulation of FAS
expression. An amino acid itself or a related factor could be
responsible, but the aminoacyl-tRNA synthetase inhibition studies (Fig. 9) argue otherwise. Even in the presence of histidine,
increasing uncharged tRNA levels by inhibiting the histidinyl-tRNA
synthetase mimicked the effect of essential amino acid deficiency. Our
data further suggest that there are fundamental differences between the
tRNAs for essential and nonessential amino acids because increased
uncharged tRNAs for nonessential amino acids have no effect on FAS
expression. About half of tRNA molecules
were always uncharged (similar to results reported in bacteria) (31) without affecting FAS expression; a similar level of
uncharged tRNA
was associated with inhibition
of FAS (Table 3). Increasing uncharged tRNA
levels with tyrosinol (Fig. 9) did not
affect FAS message levels.
How might uncharged tRNAs regulate FAS expression? Similar to the antitermination mechanism discussed above, uncharged tRNA may regulate FAS by interacting with the FAS transcriptional apparatus. However, unlike prokaryotes, components of transcription and translation are separated by the nuclear membrane in eukaryotes, and it is unknown if cytoplasmic uncharged tRNAs have access to the nucleus.
A second potential mechanism involves tRNA genes as transcriptional repressor elements. Scattered throughout eukaryotic genomes are moderately to highly repeated RNA polymerase III promoters(42) , sequences that include tRNA genes. These may influence RNA polymerase II promoters (43, 44) to affect mRNA levels. At least 1300 tRNA genes and pseudogenes are scattered throughout the human genome(45) , but their locations are largely unknown. If a tRNA gene or pseudogene exists near the human FAS gene, activation of this locus in response to decreased levels of essential amino acids could influence transcription of the nearby FAS gene. Consistent with this mechanism, tRNA genes have been recently shown to act as repressor elements for RNA polymerase II transcription in yeast(46) .
A third and perhaps the most likely mechanism underlying uncharged tRNA regulation of FAS expression would involve a transcription factor essential for FAS expression. This transcription factor would be under predominantly translational control; elevated levels of uncharged tRNAs for essential amino acids would inhibit translation of this factor, thereby blocking FAS expression. A similar mechanism is responsible for the activation of the transcription factor GCN4 in yeast after amino acid starvation(16) . Uncharged tRNA binds and activates a kinase, GCN2, which phosphorylates eukaryotic initiation factor 2, which stimulates translation of GCN4(47, 48, 49) . We speculate that uncharged tRNAs for essential amino acids interact with translational components in the cytoplasm to block translation of a critical FAS transcription factor and prevent FAS induction with feeding.
In summary, depriving HepG2 cells of any essential amino acid prevents the induction of FAS expression normally associated with feeding. The likely role of tRNA in this process represents a novel form of gene regulation in mammalian cells. Obvious next steps include the overexpression of authentic uncharged transfer RNAs for essential amino acids in hepatocytes, and an appropriate search for tRNA response elements in the FAS gene.