Characterization of the Translation-dependent Step
during Iron-regulated Decay of Transferrin Receptor mRNA*
Markus
Posch
,
Hedwig
Sutterluety
§,
Tim
Skern¶, and
Christian
Seiser
From the
Institute of Molecular Biology, Vienna
Biocenter and the ¶ Institute of Biochemistry, Medical Faculty,
University of Vienna, A-1030 Vienna, Austria
 |
ABSTRACT |
Iron regulates the stability of the mRNA
encoding the transferrin receptor (TfR). When iron is scarce, iron
regulatory proteins (IRPs) stabilize TfR mRNA by binding to the
3'-untranslated region. High levels of iron induce degradation of TfR
mRNA; the translation inhibitor cycloheximide prevents this. To
distinguish between cotranslational mRNA decay and a
trans effect of translation inhibitors, we designed a
reporter system exploiting the properties of the selectable marker gene
thymidine kinase (TK). The 3'-untranslated region of human transferrin
receptor, which contains all elements necessary for
iron-dependent regulation of mRNA stability, was fused
to the TK cDNA. In stably transfected mouse fibroblasts, the
expression of the reporter gene was perfectly regulated by iron.
Introduction of stop codons in the TK coding sequence or insertion of
stable stem-loop structures in the leader sequence did not affect on
the iron-dependent regulation of the reporter mRNA.
This implies that global translation inhibitors stabilize TfR mRNA
in trans. Cycloheximide prevented the destabilization of
TfR mRNA only in the presence of active IRPs. Inhibition of IRP
inactivation by cycloheximide or by the specific proteasome inhibitor
MG132 correlated with the stabilization of TfR mRNA. These
observations suggest that inhibition of translation by cycloheximide interferes with the rate-limiting step of iron-induced TfR mRNA decay in a trans-acting mechanism by blocking IRP inactivation.
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INTRODUCTION |
Regulated degradation of mRNA constitutes an important
mechanism for differential gene expression (reviewed in Refs. 1 and 2).
Changes in the stability of specific transcripts allow the cell to
react quickly to alterations of physiological conditions by
manipulating the pool of translatable transcripts.
Iron-dependent regulation of transferrin receptor
(TfR)1 mRNA expression
represents one of the most intensively studied eukaryotic model systems
for regulated mRNA turnover (reviewed in Refs. 3-5). Under low
iron conditions, mammalian cells induce iron uptake by increasing the
number of TfRs on the cell surface (6-9). This rise in receptor
expression is mediated by a dramatic increase in TfR mRNA stability
and is dependent on the presence of the 3'-untranslated region of the
TfR transcript (10). Five RNA motifs termed iron-responsive elements
(IREs) were identified within this region (11, 12). In iron-deprived
cells, specific RNA-binding factors, the iron regulatory proteins
(IRPs), bind to these regions and prevent the degradation of TfR
mRNA, presumably by masking a rapid turnover determinant (13, 14).
IRPs also recognize IREs located in the 5'-UTR of ferritin, erythroid
-aminolevulinic acid synthase, and succinate dehydrogenase
b mRNAs; in these cases, binding causes translational
repression (15-20).
Two distinct IRPs have been identified in cells of different mammalian
species (14, 15, 21-25). Both proteins recognize naturally occurring
IREs (23, 24), although with different affinity (26). IRP-1 binds
equally well to IREs from ferritin, TfR, m-aconitase, and
erythroid
-aminolevulinic acid synthase, whereas IRP-2 has a higher
affinity for the ferritin IRE. In addition the RNA binding activities
of IRP-1 and IRP-2 are regulated by distinct molecular mechanisms
(27-30). In iron-replete cells, IRP-1 contains an iron-sulfur cluster
and has low affinity to IREs; in iron-deprived cells, in contrast, the
protein is converted into its apo-protein form with high RNA binding
activity (31-33). IRP-2, on the other hand, is stable in iron-depleted
cells but is ubiquitinated and rapidly degraded by a
proteasome-dependent mechanism when iron is abundant (28,
29, 34). A similar type of IRP regulation was previously observed in
rabbit cells in response to modulation of cell growth (35, 36).
Finally, the IRE binding activities of IRP-1 and IRP-2 were shown to be
differentially regulated in response to non-iron environmental conditions, such as nitric oxide signaling (37-39), oxidation (40, 41), and hypoxia (42-44).
Under high iron conditions, in the absence of active IRPs, TfR mRNA
is rapidly degraded. A potential endonucleolytic cleavage site has been
identified within the 3'-UTR of the TfR transcript (45). The cutting
site was mapped within a previously characterized destabilizing domain
close to the IREs (46). The corresponding endonuclease, however, still
awaits identification.
As inhibition of transcription by high concentrations of
-amanitin
interferes with TfR mRNA decay, it was suggested that RNA
polymerase III transcripts may play a role in the degradation process
(47). In addition, inhibition of translation by cycloheximide and
puromycin was reported to block TfR mRNA decay (11, 47, 48). This
effect could either be due to the involvement of highly unstable
factors in the degradation of the transcript (trans effect) or to the fact that translation per se is necessary for the
decay of TfR mRNA (cis effect). Manipulating the
translatability by insertion of an ferritin IRE within the 5'-UTR of
TfR transcripts, Koeller et al. (48) demonstrated that
inhibition of translation in cis had no effect on the rapid
turnover of constitutively unstable TfR mutant constructs and
TfR/c-fos hybrids. These results would point toward a
trans effect of protein synthesis in the case of transferrin
receptor mRNA and also of the AU-rich elements (AREs) containing
c-fos transcript. However, the constructs used in their study did not allow one to distinguish whether inhibition of
translation affects the degradation step or the regulatory system.
We designed a novel reporter system that reflects all aspects of the
iron-dependent regulation of TfR transcripts. To this end,
the 3'-UTR of human transferrin receptor gene containing all five IREs
and the rapid turnover determinant (11, 46) was linked to the coding
part of mouse thymidine kinase (TK) cDNA. Efficient inhibition of
TK protein synthesis could be easily assayed by enzyme activity assays,
immunoblots, and, on a cellular level, by selection for HAT-sensitive
transfectants. Using this reporter system, we set out to characterize
the translation-dependent step during the
iron-dependent decay of TfR mRNA.
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MATERIALS AND METHODS |
Plasmid Constructions--
All expression vectors are based on
the mammalian expression vector pSVL (Amersham Pharmacia Biotech). The
2.3-kb BamHI/BglII fragment of the plasmid
pcD-TR1 (49), representing the 3'-UTR of the human transferrin receptor
cDNA with the complete regulatory region, was ligated in the
correct orientation into the BamHI site of the construct
pSVLTK
30 described previously in Ref. 50. This parental reporter
plasmid was termed pTK-hTfR.
The frameshift constructs pSTOP1 and pSTOP2 were obtained by
linearization with ApaI and ClaI, respectively,
of the plasmid pTK-hTfR, followed by a Klenow fill-in reaction and
religation. The changes in the TK open reading frame were confirmed by
sequencing and expression of the encoded polypeptides by in
vitro transcription/translation in reticulocyte lysates (Promega).
To create the construct pSL, the original NcoI site at
position 333 of pSVL was converted to a StuI site by
standard PCR reaction. 1130 base pairs of the pSVL vector containing
the SV40 intron were deleted by ligation to the StuI site at
position 1463. The resulting plasmid pSL lacks the SV40 intron and a
stretch of untranslated nucleotides of pSVL; it encodes a transcript
with a 5' leader sequence shortened from 265 to 50 bases.
For the plasmids pSTEM1 and pSTEM2, encoding for mRNAs with
stem-loop structures in their 5'-UTR, synthetic oligonucleotides were
phosphorylated with T4 polynucleotide kinase (Promega), annealed, and
ligated into the XhoI site of pTK-hTfR. Oligonucleotides 1s (5'-TCG ACG GCG GCG GCC GGC CGC GCA AAC AAA GCG CGG CCG GCC GGC CGG
G-3') and 1a (5'-TCG ACC CCG GCC GGC CGG CCG CGC TTT GTT TGC GCG GCC
GGC CGG CCG CCG-3') were annealed for stem-loop STEM1, which
corresponds to STEMB in Ref. 51. Oligonucleotide 2 (5'-TCG AGC AGC GGA
CGC CCG CCA AAT TTG GCG GGC GTC CGC TGC-3') of stem-loop STEM2 is
similar to STEMN in Ref. 51. The presence of inserted oligonucleotides
was confirmed by sequencing.
Cell Culture and DNA Transfection--
Mouse LTK
fibroblasts (ATCC CCL 1.3) were grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum and antibiotics (30 µg/liter penicillin, 50 µg/ml streptomycin-sulfate). Transfection
and selection for TK expressing transformants was done as described
(50). Cell lines expressing TK frameshift mutants and stem-loop
constructs were established by co-transfection with 20 ng of a plasmid
conferring hygromycin resistance. Stable transformants were selected in
medium containing 250 µg/ml hygromycin B (Roche Molecular
Biochemicals). Pools of 50-100 clones and single colonies were
propagated for analysis in further experiments. The regulation of
reporter gene expression was essentially the same in mixed cell
populations and single clones. Iron chelation was performed by the
addition of 50 µM desferrioxamine (Desferal; gift from
Ciba-Geigy), whereas iron was added as ferric ammonium citrate at 20 µg/ml. Where indicated, cells were treated with the transcription
inhibitor 5,6-dichloro-1-
-ribofuranosyl-benzimidazole (DRB, Sigma)
at 30 µg/ml. To inhibit protein synthesis, cycloheximide was added to
the medium at 10 µg/ml. For proteasome inhibition studies, cells were
treated with 50 µM MG132 (Peptides International, Louisville, KY).
RNA Isolation and Northern Blot Analysis--
Isolation and
analysis of cytoplasmic RNA was performed as described previously (52).
The 1.2-kb fragment of the murine TK cDNA (53), the 2.25-kb
cDNA of the murine transferrin receptor cDNA clone TR2 (54),
and the 1.4-kb fragment of glyceraldehyde-3-phosphate dehydrogenase
(55) were used as probes. DNA fragments were labeled with
[
-32P]dCTP (Rotem Industries, Israel) using the
random-primed DNA labeling kit (Roche Molecular Biochemicals).
Polysome Profile Analysis--
Iron-deprived cells were
harvested by trypsinization and washed with 1× phosphate-buffered
saline. To prepare cytoplasmic lysates, cells were thoroughly
resuspended in 1 ml of ice-cold lysis buffer (1 mM
potassium acetate, 2 mM magnesium acetate, 2 mM
dithiothreitol, 10 mM Tris acetate, pH 7.5, 150 µg/ml
cycloheximide) by pipetting 10 times. Cells were kept on ice for 10 min; then the nuclei were pelleted by centrifugation at 1,500 × g for 10 min at 4 °C. The supernatant was layered on a
continuous sucrose gradient (15-40% sucrose in 10 mM Tris
acetate, pH 7.5, 140 mM NaCl, 1.5 mM
MgCl2, 10 mM dithiothreitol, 100 µg/ml
cycloheximide) and separated by centrifugation at 38,000 rpm for 2 h at 4 °C in a SW41Ti rotor (Beckman). 22 fractions (500 µl) were
collected, and each fraction was vigorously mixed with 200 µl of a
buffer containing 7 M urea, 1% SDS, 0.35 M
NaCl, 10 mM EDTA, 10 mM Tris-HCl, pH 7.5, and
400 µl of phenol-chloroform. After centrifugation at 14,000 × g for 30 min at 4 °C, 600 µl of the aqueous phase were
precipitated with 1 ml of ethanol containing 20 µg of yeast tRNA
carrier. The RNA pellet was dissolved in 20 µl of water and loaded on
1.2% denaturing gels. RNA of all fractions was separated by
electrophoresis, transferred to GeneScreen nylon membranes (NEN Life
Science Products), and submitted to Northern blot analysis. A similar
protocol was used for the analysis of transcripts after EDTA-induced
ribosome release. Cycloheximide was substituted by 10 mM
EDTA in both the lysis buffer and the 15-40% sucrose gradients in
these experiments. Similarly, high salt gradients contained 500 mM NaCl and 10 mM EDTA.
Protein Extraction and Immunological Methods--
Cytoplasmic
proteins were prepared according to the procedure described by Sherley
and Kelly (56). Cells were trypsinized, pelleted at 200 × g for 4 min, washed, and lysed in digitonin buffer (0.8 mg/ml digitonin, 250 mM sucrose, 1.5 mM
MgCl2, 160 mM KCl, 3 mM
2-mercaptoethanol, 50 mM
-amino-n-caproic
acid, 10 mM Tris-HCl, pH 7.5) by incubating the cells for
15 min on ice. Cell fragments were removed by centrifugation at
1.000 × g for 10 min at 4 °C, and the supernatant
was used for further analysis. Protein concentrations were determined
by the Coomassie Brilliant Blue protein assay (Bio-Rad). Thymidine
kinase enzyme assays, immunoprecipitation, and Western blot analysis of
the epitope-tagged mouse thymidine kinase protein have been described (50).
Preparation of RNA Transcripts--
32P-Labeled RNA
was transcribed from the linearized plasmid pSPT-fer containing the IRE
from the 5'-UTR of human ferritin H chain mRNA as described by
Mullner et al. (14). Transcription was carried out in the
presence of 1.5 mM ATP, GTP, and UTP, 60 µCi of
[
-32P]CTP (800 Ci/mmol, NEN Life Science Products),
and T7 RNA polymerase (Promega).
RNA-Protein Band Shift Assays--
The IRP-IRE interaction was
analyzed as described previously (14). Briefly, protein extract (1-2
µg) was incubated for 10 min at room temperature with an excess (0.2 ng) of 32P-labeled IRE in vitro transcript in a
total volume of 20 µl. After addition of heparin for another 10 min,
RNA-protein complexes were resolved on a 6% nondenaturing
polyacrylamide gel and processed for autoradiography.
 |
RESULTS |
TK-hTfR Reporter mRNA Stability Is Regulated by Iron--
To
analyze the effect of translation on the iron-dependent
decay of RNA molecules containing TfR control elements, we established a selectable reporter system. The coding region of the mouse thymidine kinase cDNA was fused to the entire 3'-UTR of human TfR cDNA to give the construct TK-hTfR (Fig.
1A). This mRNA, which is
expressed from the constitutive SV40 promoter, contains all elements
that have been demonstrated to be essential for
iron-dependent regulation of mRNA stability (11, 12).
The open reading frame encodes a highly stable COOH-terminally
truncated murine thymidine kinase protein TK
30 which contains a
c-myc epitope at its amino terminus (50). The expression of
this protein has been shown to be independent of growth conditions
(50). Cellular levels of the epitope-tagged TK polypeptide can be
easily monitored by immunological techniques.

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Fig. 1.
Iron-dependent regulation of
TK-hTfR reporter mRNA expression. Northern blot analysis of
stably transfected LTK-hTfR cells (A) and LTK (B)
cells. Log-phase cells were exposed to 50 µM
desferrioxamine for the times indicated (hours). Expression of the
reporter constructs and endogenous TfR mRNA was determined by
sequential hybridization with radiolabeled probes for TK, murine TfR,
and GAPDH. C, half-life of the TK-hTfR reporter mRNA
under low (DES) and high (FE) iron conditions.
LTK-hTfR cells were treated for 18 h with desferrioxamine
(DES) or 20 µg/ml ferric ammonium citrate for 12 h
(FE). Cytoplasmic RNA was prepared 0, 1, 2, 4, and 6 h
after addition of 30 µg/ml DRB. TK-hTfR mRNA levels were
determined by Northern blot analysis with a radiolabeled TK probe. The
Northern blot of iron-treated cells (FE) was exposed about
10 times longer than that of Desferal-treated cells. To confirm equal
loading of RNA, the blot was rehybridized with a probe for GAPDH.
Essentially identical results were obtained on repeating the
experiments.
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Mouse LTK
cells were stably transfected with the
construct TK-hTfR to give the corresponding cell line LTK-hTfR. To test
whether levels of TK-hTfR mRNA were regulated by iron availability,
logarithmically growing cells were depleted of iron by the addition of
50 µM desferrioxamine to the medium. After different
periods of time, cytoplasmic RNA was prepared and analyzed by Northern
hybridization. LTK
cells stably transfected with the
parental construct pSVLTK
30 giving cell line LTK served as a control
to rule out an effect of the iron status on the coding region of the
reporter transcript. TK-hTfR mRNA was about 12-fold induced upon
iron deprivation (Fig. 1A), whereas the expression of TK
transcripts in the control cells was not increased by desferrioxamine
(Fig. 1B). As shown in the lower
panel, rehybridization with a probe specific for the coding part of murine TfR mRNA revealed a similar induction pattern for the endogenous mTfR transcript. TK-hTfR mRNA expression is
therefore clearly dependent on intracellular iron levels. To confirm
that the iron-dependent regulation of TK-hTfR reporter
mRNA is due to a change in the mRNA stability, we determined
the mRNA half-life both under conditions of iron deprivation and
abundance. TK-hTfR cells were iron-starved by addition of the iron
chelator desferrioxamine for 18 h or iron-loaded by addition of
ferric ammonium citrate to the medium for 12 h. Transcription was
inhibited by the addition of DRB, and TK-hTfR mRNA levels were
determined by Northern blot analysis after different periods of time.
In iron-depleted cells, we observed a half-life of TK-hTfR reporter
mRNA of about 6 h (Fig. 1C, left
panel). The presence of ferric ammonium citrate in the
culture medium dramatically reduced the half-life to about 50 min (Fig.
1C, right panel). Similar data have
previously been shown for murine TfR mRNA (47) and were confirmed
by rehybridization with a probe specific for the endogenous murine TfR
transcripts (data not shown). These results clearly indicate that the
reporter mRNA and the endogenous TfR mRNA both are regulated by
the same mechanism.
Iron-dependent Degradation of TK-hTfR Reporter mRNA
Is Uncoupled from Ongoing Translation--
A number of rapidly
degraded transcripts are markedly stabilized upon the addition of
global translation inhibitors. A similar effect of cycloheximide on
iron-mediated decay of TfR mRNA has been reported previously (11).
Similarly, we observed inhibition of TK-hTfR mRNA degradation by
cycloheximide and puromycin in the course of iron repletion in our
reporter system (data not shown). Two mechanisms have been proposed to
account for the ability of inhibitors of protein synthesis to stabilize
labile mRNAs; either highly unstable proteins are involved in the
degradation of these mRNAs (trans effect), or
translation of the labile mRNAs itself is required for decay
(cis effect).
In order to distinguish between these alternatives, two approaches to
influence protein synthesis from our reporter mRNA were chosen.
First, we investigated whether complete translation of the coding
region is a prerequisite for TK-hTfR mRNA degradation by
introducing stop codons into the open reading frame of pTK-hTfR. Plasmids pSTOP1 and pSTOP2 encode truncated TK proteins of 51 and 128 amino acids, respectively. In vitro expression in a
reticulocyte lysate transcription/translation system yielded
polypeptides of the calculated sizes of 5.6 and 14 kDa, respectively
(data not shown). The corresponding cell lines LSTOP1 and LSTOP2
expressed no full-length TK protein, as judged by Western blot analysis (Fig. 2A) and enzyme activity
assays (data not shown), and were not viable in HAT medium, which
selects for transfectants expressing functional TK protein. Although
truncated TK protein could be immunoprecipitated from in
vitro translation reactions, we were unable to do so in cellular
extracts of LSTOP1 and LSTOP2 cells, indicating that the polypeptides
might be unstable in vivo. Nevertheless, although the
reporter mRNAs of LSTOP1 and LSTOP2 cells were not fully
translated, their expression remained strictly iron-regulated (Fig.
2B). We conclude that translation of the complete coding region is not required for the enhanced turnover of TK-hTfR mRNA in
response to iron.

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Fig. 2.
Expression of reporter constructs with
premature stop codons. A, TK protein expression in the
different reporter cell lines. Cytosolic proteins were isolated from
LTK-hTfR, LSTOP1, and LSTOP2 cells after treatment with Desferal for
18 h. TK protein was immunoprecipitated from 60 µg of total
protein from LTK-hTfR cells or 1200 µg of total protein from the
other cell lines and analyzed on a Western blot. B,
expression of reporter transcripts with nonsense codons under low and
high iron levels. Logarithmically growing LSTOP1 and LSTOP2 cells were
iron-starved for 18 h (DES) or iron was added for
4 h (FE) and mRNA levels determined by Northern
blot analysis. The blots were sequentially hybridized with radiolabeled
probes for TK and GAPDH as loading control. A schematic drawing of the
reporter mRNAs STOP1 and STOP2 with a translation stop at codons 51 and 128, respectively, is shown on the left. C,
polysome profiles of LTK, LTK-hTfR, and LSTOP1 cells. Extracts from
iron-deprived cells were separated on 15-40% sucrose gradients as
described under "Materials and Methods." RNA was prepared from all
22 fractions and separated on denaturing 1,2% agarose gels.
Distribution of ribosomal complexes is represented by the ethidium
bromide stain of one typical gel. mRNA distribution was visualized
by Northern hybridization with a radiolabeled TK probe. D,
sucrose gradients of LTK and LTK-hTfR cells after EDTA-induced mRNA
release from ribosomes. Cellular lysates of iron-starved cells were
separated on 15-40% gradients containing 10 mM EDTA.
Top panel shows representative distribution of
ribosomal RNA from a typical experiment. Northern analysis in the lower
three panels was performed with radiolabeled fragments of TK cDNA
and mouse TfR cDNA. E, polysome profiles of LTK and
LTK-hTfR cells under high salt conditions. Cellular lysates from
iron-deprived cells were separated on 15-40% gradients containing 500 mM NaCl and 10 mM EDTA. Representative
distribution of ribosomal RNA is shown on top
panel. Distribution of TK, TK-hTfR, and mouse TfR RNA was
visualized by Northern analysis (lower three
panels); Northern blots were hybridized with radiolabeled TK
cDNA and mouse TfR cDNA probes. Each experiment was performed
twice with the same results.
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In order to investigate whether the polysome distribution of the
TK-hTfR reporter RNAs was affected by premature termination of
translation, we analyzed the association of these transcripts with
ribosomes in linear 15-40% sucrose gradients. The resulting RNA
distribution, analyzed on ethidium bromide-stained gels, clearly revealed fractions containing 40, 60, and 80 S ribosomal particles as
well as polysomes (Fig. 2C). The localization of the STOP1 mRNA was essentially identical to that of the fully translated TK-hTfR transcript, with both RNAs being predominantly detectable in
the polysome fractions 11-15. The same distribution also was found for
the STOP2 transcripts (data not shown). Thus, premature termination of
translation was without significant consequence for the polysome
distribution on the mutant mRNAs.
Interestingly, TK-hTfR reporter mRNA sedimented faster than
parental TK mRNA, which was primarily found in fractions 10-13 (Fig. 2C). The relative shift in apparent density was
independent of the presence of divalent cations, as removal of
ribosomes and other divalent cation-dependent binding
proteins by EDTA failed to abrogate the difference in sedimentation. As
shown in Fig. 2D, the TK transcript sedimented slowly in an
EDTA-containing sucrose gradient (fractions 5-8), whereas TK-hTfR
transcripts and endogenous mouse TfR mRNA showed higher
sedimentation rates (fractions 7-10). Thus, transcripts containing the
3'-UTR of the TfR mRNA seem to form larger RNA-protein complexes.
It is unlikely that this is due to the binding of IRPs to IREs, because
a similar polysome profile was found for the low abundant TfR mRNA
(and the TK-hTfR transcripts) under high iron conditions, at which IRPs
are dissociated from IREs ( (14) and data not shown). Even in a
gradient with more stringent conditions (500 mM NaCl, 10 mM EDTA) TK mRNA sedimented about two fractions slower
than TK-hTfR and mTfR transcripts (Fig. 2E). Presumably,
other EDTA-insensitive and high salt-resistant factors are bound to the
TfR-untranslated region, increasing sedimentation of the respective mRNAs.
Next, we addressed the question whether targeted inhibition of
translation initiation might interfere with the iron-induced destabilization of TK-hTfR reporter mRNA. To prevent ribosome association on TK-hTfR mRNA, two hairpin structures (STEMB and STEMN in Ref. 51), which had been previously shown to efficiently block
initiation of protein synthesis in eukaryotic cells, were introduced
into the 5'-UTR of the reporter. As such secondary structures are only
effective if they are located close to the transcription start site, we
first drastically shortened the 265-nucleotide leader sequence of
TK-hTfR mRNA. The resulting SL mRNA differs from TK-hTfR
mRNA only in possessing a shorter 5'-UTR. As expected, expression
of the fully translated SL mRNA was regulated by the iron level
(Fig. 3A, upper
panel). To block translation initiation of SL mRNA, two
different hairpin cDNAs were introduced into the unique
XhoI cloning site 50 base pairs downstream of the
transcription initiation site resulting in plasmids pSTEM1 and pSTEM2.
By stably transfecting LTK
cells with these constructs,
we established the cell lines LSTEM1 and LSTEM2. Both cell lines were
HAT-sensitive, indicating that TK protein synthesis was efficiently
inhibited. TK protein was undetectable in cytosolic extracts of these
cells, as judged by immunoprecipitations and enzyme activity assays
(Fig. 3B and data not shown). Translational inhibition of
the stem-loop structures seems to be very efficient, because owing to a
half-life of more than 18 h (50), TK protein would accumulate even
at low translation rates. To directly demonstrate that the hairpin
insertion affected translation initiation of STEM1 mRNA, we
evaluated the association with ribosomes in polysome gradients (Fig.
3C). Although the control SL mRNA was predominantly
found in polysome-bound fractions (fractions 12-17), insertion of the
hairpin (as shown for STEM1) resulted in a shift of the transcripts
toward polysome-free fractions (fractions 8-12). The fact that STEM1
transcripts do not shift beyond the 40 S complexes is most probably due
to the formation of high molecular weight RNA-protein complexes. This
idea is further corroborated by only a minor shift in the polysome
profile of STEM1 mRNA after EDTA induced ribosome release (Fig.
3D, compare also with Fig. 2D). Similar results
were obtained with cell line LSTEM2 (data not shown). Despite this
efficient block of TK translation by insertion of stem-loops, both
STEM1 and STEM 2 transcripts are still regulated by changes in iron
levels as shown by Northern blot analysis (Fig. 3A).
Therefore, we concluded that introduction of stable secondary
structures into the 5'-UTR of TK-hTfR transcripts results in a
considerable change in the polysome profiles of the mRNAs but has
no effect on their destabilization by iron. These data are in good
agreement with the results of Koeller et al. (48) and
indicate a trans effect of translation inhibitors.

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Fig. 3.
Effect of stem-loop structures on the
expression and polysome profiles of TK-hTfR reporter mRNA.
A, Northern blot analysis of SL, STEM1, and STEM2 mRNA
expression in response to changes of iron levels. RNA was prepared from
logarithmically growing cells treated with Desferal (DES)
for 18 h or ferric ammonium citrate (FE) for 4 h.
Hybridization was performed with radiolabeled TK and GAPDH probes.
B, TK protein expression in cell lines LSL, LSTEM1, and
LSTEM2 after 18 h of Desferal treatment. Immunoprecipitates from
60 µg of total protein from LSL cells or 1200 µg of total protein
from the other cell lines were analyzed for TK protein on a Western
blot. C, polysome profiles of SL and STEM1 mRNA from
iron-starved LSL and LSTEM1 cells. Cytoplasmic extracts were separated
on sucrose gradients; all fractions were subjected to Northern
analysis. rRNA distribution is shown in one representative ethidium
bromide stain; Northern membranes were hybridized with a radiolabeled
TK probe. D, sucrose gradient of STEM1 cells after
EDTA-induced mRNA release from ribosomes. Cells were lysed and
fractionated in the presence of 10 mM EDTA. RNA from each
fraction was analyzed on denaturing gels (ethidium bromide stain,
top panel) and by Northern hybridization
(lower panel) with a radiolabeled fragment of TK
cDNA. Each experiment was performed twice with the same
results.
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Cycloheximide Fails to Stabilize TfR mRNA when Iron Is
Abundant--
Subsequently, we focused our attention on how global
translation inhibitors stabilize TfR mRNA by a
trans-acting mechanism. Koeller et al. had
suggested that a short-lived component of the degradation apparatus
might be affected by global translation inhibitors (48). In their
system, translation inhibitors markedly increased levels of a TfR
construct, which was intrinsically and constitutively unstable owing to
mutation within its regulatory region. To test the effect of
cycloheximide on the levels of destabilized mouse TfR mRNA, the
translation inhibitor and iron salt were added to iron-depleted L
fibroblasts at different time points (Fig. 4A). mTfR mRNA was
stabilized when iron-starved cells were treated with cycloheximide and
eventually incubated in in new medium containing iron salt and
cycloheximide (Fig. 4A, lane b).
Similarly mTfR mRNA could be stabilized when iron salt and the
translation inhibitor were applied simultaneously (lane
c). In contrast, cycloheximide did not affect the low
expression of TfR mRNA in iron-loaded cells when added after 4 h of iron treatment (Fig. 4A, lane d).
The fact that the translation inhibitor is not sufficient to stabilize TfR transcripts in iron-loaded cells strongly argues against the idea
that a labile factor of the degradation machinery is a target for
cycloheximide.

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Fig. 4.
Effect of cycloheximide on the expression of
TfR mRNA in iron-loaded cells. A, Northern blot
analysis of mTfR mRNA levels under high iron conditions with
cycloheximide added at different time points. LTK-hTfR cells were
iron-starved for 18 h with 50 µg/ml Desferal (DES);
in lane a, RNA was extracted at this time point.
In lane b, 10 µg/ml cycloheximide
(CHX) was added during the last hour of iron depletion
before cells were incubated in new medium containing CHX and 20 µg/ml
ferric ammonium citrate (FE) for 4 h. Iron-starved
cells were incubated in new medium containing FE and CHX for 4 h
in lane c. In lane d
iron-depleted cells were incubated in new medium containing iron salt
for 4 h; then CHX was added for another 4 h. In
lane e, iron-starved cells were incubated in new
medium supplemented with ferric ammonium citrate for 4 h.
Cytoplasmic RNA from all cells was analyzed by Northern blotting. The
membrane was probed with radiolabeled cDNA fragments of mouse TfR
and GAPDH. B, half-life of TK-hTfR mRNA and endogenous
TfR mRNA after CHX addition in iron-abundant LTK-hTfR cells.
LTK-hTfR cells were incubated with 20 µg/ml ferric ammonium citrate
for 4 h, and 10 µg/ml cycloheximide was then added to the
culture medium. After 2 h, DRB was added to 30 µg/ml and
cytoplasmic RNA was prepared 0, 1, 2, 4, and 6 h after addition of
the transcription inhibitor. mRNA levels were determined by
Northern analysis with radiolabeled cDNA fragments of TK and mouse
TfR; blots were exposed about 10 times longer than in A.
Equal loading was confirmed by rehybridization with a GAPDH probe.
Experiments were performed twice with similar results.
|
|
In order to exclude the possibility that the translation inhibitor
influences transcription of mouse TfR mRNA, we uncoupled RNA
synthesis from the degradation process with the transcription inhibitor
DRB. The half-lives of both endogenous TfR and TK-hTfR reporter
mRNA were measured in iron-loaded cells as in Fig. 1C, except they were treated with cycloheximide for 2 h prior to DRB addition. As depicted in Fig. 4B, cycloheximide was not
sufficient to impede the rapid decay of the transcripts destabilized by
iron treatment. In fact the half-life of TK-hTfR mRNA in
iron-replete cells was, as analyzed by densitometric scanning of
autoradiograms, very similar (about 50 min) in the presence or absence
of cycloheximide (compare Fig. 4B with Fig. 1C,
right panel). Therefore, the degradation of TfR
mRNA under high iron conditions is equally efficient in the absence
of de novo synthesis of proteins; consequently,
cycloheximide failed to induce TfR mRNA levels when added to
iron-loaded L fibroblasts. Previous studies (11, 47) have shown that
cycloheximide prevents TfR mRNA degradation when added
simultaneously with iron salts; in contrast, we observed that the
translation inhibitor was not sufficient to interfere with TfR
transcript decay in iron-replete cells (Fig. 4, A and
B). This implies that the stabilizing effect of translation
inhibitors is dependent on the iron level at the time point of
cycloheximide addition.
Inhibition of IRP Inactivation Mediates Stabilization of TfR
mRNA during Iron Repletion--
Our data suggest that, in mouse L
fibroblasts, the mechanism by which inhibitors of protein synthesis
stabilize TfR mRNA involves rather the system that regulates TfR
mRNA stability (i.e. the IRPs) than factors degrading
TfR mRNA. Therefore, we set out to investigate whether the presence
of active IRPs in iron-loaded cells is sufficient for TfR mRNA stabilization.
Recently, it was demonstrated that cycloheximide prevented the
iron-induced proteolysis of IRP-2 (27, 29). Thus, it was suggested that
a labile, yet unidentified factor had to be synthesized in order to
inactivate this iron regulatory protein. The same component might also
be the key protein whose function is necessary for the
iron-dependent degradation of TfR mRNA. To address this question, we first verified the effect of cycloheximide on IRP inactivation in our model system. Desferal-treated LTK-hTfR cells were
incubated with iron salts in the absence (Fig.
5A, lanes 1 and 2) or presence (Fig. 5A,
lanes 3 and 4) of the translation inhibitor. It can be seen that cycloheximide had only a slight effect
on the deactivation of IRP-1, but completely inhibited the
disappearance of IRP-2 during iron repletion as shown previously (27,
29). Concurrently, mTfR mRNA was stabilized by the translation inhibitor (Fig. 5B, compare lanes 2 and 4). These results fit well into a model according to
which a block in IRP-2 degradation by cycloheximide leads to
stabilization of TfR mRNA. One prediction would be that
stabilization of active IRPs would lead to stable TfR mRNA in
iron-replete cells.

View larger version (53K):
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|
Fig. 5.
Effect of cycloheximide and MG132 on IRP
activity and TfR mRNA expression. A, gel
retardation assays of cellular extracts with a radiolabeled transcript
of a human ferritin IRE. Extracts were prepared from LTK-hTfR cells
iron-deprived for 18 h (DES, lanes
1 and 5). Where indicated, 10 µg/ml
cycloheximide was added during the last hour of iron chelation
(DES-CHX, lanes 3 and 7).
Desferal-treated cells were eventually iron-repleted for 3 h in
new medium containing 20 µg/ml ferric ammonium citrate
(FE, lanes 2 and 6) or both
iron salt and the translation inhibitor (FE-CHX,
lanes 4 and 8); cells in
lane FE-CHX received cycloheximide treatment
throughout the last 4 h before harvesting. In vitro
reduction with 2% 2-mercaptoethanol to restore IRP activity is
indicated by 2-ME (lanes 5-8).
RNA-protein complexes containing IRP-1 or IRP-2 are indicated by
arrows. B, Northern blot analysis of cytoplasmic
RNA from cells treated with Desferal (lanes 1,
3, and 5) or iron-repleted with ferric ammonium
citrate (lanes 2, 4, and 6)
in the absence (lanes 1 and 2) or
presence (lanes 3 and 4) of
cycloheximide as described under A. Where indicated
(lane 5), iron-starved cells were incubated with
50 µM proteasome inhibitor MG132 for 1 h. In
lane FE-MG132 (lane 6),
iron-starved cells were treated with MG132 for 1 h; subsequently
they were incubated in new medium containing iron salt and the
inhibitor for another 3 h. Northern blots were probed with
radiolabeled cDNA probes for murine TfR and GAPDH. C,
RNA band shift assay with MG132-treated cells. Cells were iron-starved
or repleted with iron salt as described in A. Where
indicated, cells were incubated with 50 µM MG132 during
the last hour of iron starvation. Cells in lane FE-MG132 received MG132
treatment throughout the last 4 h before harvesting. Experiments
were performed three times with similar results; the variation, as
determined by densitometric scanning, was less than 7%.
|
|
To test this hypothesis, we utilized the specific proteasome inhibitor
MG132 that had been shown previously to protect IRP-2 against
iron-mediated proteolysis (57, 58). RNA-protein band shift analysis
confirmed that, in LTK-hTfR cells, the effect of MG132 on the RNA
binding activity of IRP-2 was similar to that of cycloheximide. The
drug efficiently blocked the inactivation of IRP-2 (Fig.
5C). In addition, a considerable fraction of IRP1 (about
25%) remained active in the presence of the proteasome inhibitor (Fig.
5C). This result was reproducible in three independent experiments with less than 7% variation. Similar results were obtained
with another proteasome inhibitor, MG115 (data not shown). Importantly,
MG132 was equally potent in inhibiting iron-induced TfR mRNA decay
as cycloheximide (Fig. 5B, lanes 4 and
6). These data strongly support the idea that cycloheximide
stabilizes TfR mRNA in mouse fibroblasts by impairing the function
of a labile protein involved in IRP inactivation.
 |
DISCUSSION |
In this report, we have analyzed the
translation-dependent step during the iron-mediated
degradation of TfR mRNA. Interrelationships between protein
synthesis and mRNA degradation have been observed for a number of
different eukaryotic transcripts in specific cellular systems (recently
reviewed in Ref. 59). For example, inhibition of translation in
cis was observed to have completely different effects on the
stability of different transcripts. On one hand, insertion of a stable
secondary structure in the otherwise stable yeast phosphoglycerate
kinase 1 mRNA resulted in about 7-fold reduction of its half-life
(60). On the other hand, stem-loop insertion into the 5'-UTR stabilized
transcripts containing the AU-rich element of granulocyte-macrophage
colony-stimulating factor (GM-CSF) mRNA (61), but was without
consequence for the stability of chloramphenicol acetyltransferase
transcripts in yeast (62).
The best understood mammalian example for a direct link between
translation and mRNA decay is the autoregulation of
-tubulin expression (63, 64). Polysome-associated tubulin transcripts have been
shown to be targeted as a substrate for degradation through recognition
of the amino-terminal tetrapeptide sequence of the nascent
-tubulin protein after its emergence from the ribosome.
Cotranslational mRNA degradation was also reported for the cell
cycle-dependent regulation of histone mRNA expression (65). In both cases the presence of a polysome-associated nuclease was
suggested (66, 67).
Another group of highly unstable mRNAs encoding proto-oncogenes,
cytokines, and transcription factors contains AU-rich elements (AREs)
within their 3'-UTRs (reviewed in Ref. 68). AU-rich sequences of the
GM-CSF and c-fos 3'-UTRs have been shown to destabilize the
otherwise stable
-globin reporter transcripts (69, 70). Numerous
studies have addressed the mechanism of the decay of ARE-containing
transcripts (70-73). The common degradation pathway involves two
steps: shortening of the poly(A) tail and subsequent degradation of the
mRNA body (71, 74-77). In the case of c-myc mRNA,
the deadenylation step was identified as the
translation-dependent step during the decay (78). In
contrast, deadenylation does not seem to be a prerequisite for TfR
mRNA degradation as it was shown that the average length of poly(A)
tails of both degradation intermediates and the full-length TfR
transcripts are very similar (45).
Studies concerning a cis effect of translation on the decay
of AU-rich transcripts yielded divergent results. Although the GM-CSF
ARE-directed decay was shown to be translation-dependent (61, 79), degradation of transcripts containing the c-fos ARE was found to be uncoupled from protein synthesis in cis
(48). Direct comparison of the two destabilizing elements by different methods in different systems again gave contradictory results (57,
80).
We have analyzed the effect of translation on the decay of TfR mRNA
by using a TK-hTfR reporter that contains all five stem-loops and the
rapid turnover determinant of the human wild-type TfR mRNA. In
their studies, Koeller et al. had used a synthetic minimal construct (TRS-1) containing three stem-loops and the rapid turnover determinant. TRS-1 was recently shown to contain a mutation in stem-loop C at a key residue for IRP binding (81). Although iron-dependent regulation of both TK-hTfR and TRS-1
transcripts is similar to the one of endogenous TfR mRNA, the two
reporter constructs showed clear differences in the response to
cycloheximide. The steady state levels of TRS-1 and a constitutively
unstable derivative were induced by the translation inhibitor in
iron-loaded cells (48), suggesting that cycloheximide affects the
function of a short-lived participant in mRNA turnover. In
contrast, we find that TK-hTfR transcripts and endogenous mouse TfR
mRNA both are rapidly degraded in iron-loaded L fibroblasts in the
presence of cycloheximide.
However, cycloheximide interferes with iron-induced decay of TfR
mRNA when added simultaneously with iron salts to L cells (11, 47).
These results strongly argue against the idea that translation
inhibitors act via a labile protein directly mediating the
iron-dependent endonucleolytic cleavage of TfR transcripts. The simplest hypothesis to explain the iron-dependent
effect of cycloheximide is that the regulatory system of TfR stability
is a target for the translation inhibitor.
The regulatory proteins that determine the fate of TfR mRNA display
unique features. IRP-1 is mutually active as cytosolic aconitase or
mRNA-binding protein (41, 57, 58, 82, 83). The enzymatically active
form contains a cubane 4Fe-4S cluster and has no RNA binding activity.
Disassembly of the Fe-S structure in iron-depleted cells results in a
gain of RNA binding activity but a loss of enzymatic activity. IRP-2,
on the other hand, has no aconitase activity, but contains a unique
domain of 73 amino acids that is required for its
iron-dependent decay (24, 28, 29).
Stabilization of IRP-2 by cycloheximide and MG132 prevents not only
proteolysis but also the inactivation of this regulatory protein
(27-29). Here, we demonstrate that this block in IRP inactivation correlates with the stabilization of TfR transcripts. Surprisingly, MG132 also affected the iron-dependent decrease in IRP-1
binding activity. This result is divergent from data obtained with
hamster FTO2B cells (29), but was also found in independent studies with mouse L cells (84), indicating cell type-specific differences in
the regulation of IRP activity. A possible explanation for the effect
of MG132 on IRP-1 inactivation might be the involvement of another
labile factor that is a target of the proteasome. Due to the effect of
MG132 on both IRPs, it is difficult to distinguish which of the two
proteins mediates the effect of the proteasome inhibitor on TfR
mRNA decay. Most likely both RNA-binding proteins, IRP-1 and IRP-2,
stabilize TfR mRNA by binding to its IREs. This idea is supported
by the findings that both proteins bind TfR and ferritin IREs in
vitro (23, 24), that IRP-1 and IRP-2 were found to be bound to
membrane-associated mRNA (47), and that IRP-2 is able to regulate
TfR mRNA expression in a murine pro-B lymphocyte cell line in the
absence of detectable levels of IRP-1 (85).
This report documents an interesting example of the relationship
between translation and mRNA degradation. Future studies will
reveal the function of the cycloheximide target factor(s) and allow a
better understanding of the remarkable link between specific protein
degradation and regulated mRNA decay.
 |
ACKNOWLEDGEMENTS |
We thank F. Yeong, L. Kühn, E. Müllner, A. von Gabain, and B. Henderson for critically reviewing
this manuscript.
 |
FOOTNOTES |
*
This work was supported by Grant 70.003/2-Pr/4/95 from the
Austrian Ministry of Science, and by the Josefine Hirtl Foundation, the
Herzfelder Foundation, and the Anniversary Fund of the Austrian National Bank.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Friedrich Miescher Institute, CH-4002 Basel, Switzerland.
To whom all correspondence should be addressed: Inst. of
Molecular Biology, University of Vienna, Vienna Biocenter, Dr.
Bohr-Gasse 9, A-1030 Vienna. Tel.: 431-4277-61770; Fax: 431-4277-9617;
E-mail: cs{at}mol.univie.ac.at.
 |
ABBREVIATIONS |
The abbreviations used are:
TfR, transferrin
receptor;
kb, kilobase pair(s);
UTR, untranslated region;
TK, thymidine
kinase;
SL, stem-loop;
GM-CSF, granulocyte-macrophage
colony-stimulating factor;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
IRE, iron-responsive element;
IRP, iron regulatory
protein;
ARE, AU-rich element;
DRB, 5,6-dichloro-1-
-ribofuranosyl-benzimidazole;
HAT, hypoxanthineaminopterine-thymidine.
 |
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