(Received for publication, July 1, 1995; and in revised form, September 14, 1995)
From the
Transferrin receptor (TfR) mRNA expression is tightly linked to
intracellular iron levels. Upon iron deprivation, the iron regulatory
protein (IRP) stabilizes TfR mRNA by binding to stem-loop structures in
its 3`-untranslated region, whereas increased iron levels result in
inactivation of the mRNA-binding protein and rapid degradation of TfR
mRNA. Although IRP and the regulation of its RNA binding activity have
been studied intensively, little is known about the mechanism of TfR
mRNA degradation. In order to get more information about factors
involved in this process we investigated the in vivo IRP-RNA
interaction and the effect of transcription inhibitors on the
iron-dependent decay of TfR mRNA. Here we demonstrate that part of the
active IRP co-localizes with TfR mRNA to the rough endoplasmic
reticulum. High intracellular iron levels led to a drastic reduction of
this active RNA-bound IRP in vivo, indicating that IRP
dissociates prior to TfR mRNA decay. Furthermore, the transcription
inhibitor actinomycin D and translation inhibitor cycloheximide
suppressed TfR mRNA degradation but did not interfere with the IRP
dissociation step. Other inhibitors of RNA polymerase II had no effect
on iron-dependent degradation of TfR mRNA. However, high concentrations
of -amanitin known to block transcription by RNA polymerase III
interfered with mRNA decay suggesting the involvement of polymerase III
transcripts in the degradation pathway.
Cellular iron acquisition is a prerequisite for cell
proliferation. In most vertebrate cells, iron uptake is mediated by
transferrin receptor (TfR), ()whose mRNA expression is
modulated inversely proportional to iron availability. This regulation
occurs mainly at the post-transcriptional level through the control of
mRNA stability. A change in mRNA turnover is mediated by sequences
within the 3`-untranslated region (UTR) of the TfR
transcript(1) . This 3`-UTR contains both rapid turnover
determinant(s) and five hairpin elements referred to as iron responsive
elements (IREs)(2, 3) . Deletion of the entire 3`-UTR
or the rapid turnover determinant yields a constitutively high
expression of TfR(3) , whereas mutagenesis of all IREs results
in rapid unregulated degradation of TfR mRNA(4) . IREs are
recognized by a cytosolic RNA-binding protein named iron regulatory
protein (IRP), previously also referred to as IRE-binding
protein(5, 6) , iron regulatory factor (7) ,
ferritin repressor protein (8) or p90(9) . The RNA
binding activity of IRP is reversibly regulated by intracellular iron
levels and gets induced under conditions of iron deprivation. IRP binds
also to single copy IREs in other mRNAs, notably in the 5`-UTR of mRNAs
coding for H- and L-ferritins (2, 10) as well as the
erythroid form of 5-aminolevulinate synthase(11, 12) .
Here, IRP binding inhibits initiation of translation. In the case of
TfR mRNA at least four of the five IREs are simultaneously bound by
IRPs(7, 13) , whereas only three of these elements are
required for the iron-dependent control of RNA
degradation(3, 4) . Recently, a potential
endonucleolytic cleavage site has been mapped near to one of these
indispensable IREs(14) . These results, together with the fact
that the IRP activity in vivo parallels TfR mRNA levels,
suggest that binding of IRP to IREs masks the recognition site for a
putative nuclease. However, the pathway of TfR decay and the components
involved in this process remain to be characterized. One of the factors
essential for the degradation seems to be a short-lived polypeptide
because inhibition of protein synthesis by cycloheximide blocks the
iron-dependent decay of TfR mRNA(3) . This hypothesis is
supported by results of Koeller et al.(15) , who found
that translation inhibition through an IRE in the 5`-UTR had no
influence on the decay of a constitutively unstable TfR mRNA.
Furthermore, inhibition of transcription by actinomycin D has also been
reported to interfere with the iron-dependent degradation of TfR
mRNA(3) .
In this report we investigated whether the
previously observed effects of transcription and translation inhibitors
can be attributed to impaired IRP function and/or whether transcription
by a particular RNA polymerase is required for the TfR mRNA degradation
pathway. We show by cell fractionation that part of the IRP
co-localizes with TfR mRNA in the rough endoplasmic reticulum and gets
iron-dependently released. However, dissociation of IRP from this
fraction was neither impaired by actinomycin D nor cycloheximide,
although TfR mRNA degradation was blocked. Specific inhibition of RNA
polymerase II-dependent mRNA synthesis failed to interfere with the
decay process. However, treatment of mouse L cells with high
concentrations of -amanitin significantly inhibited TfR mRNA
degradation. These results suggest that polymerase III transcripts may
play a crucial role in the pathway of TfR mRNA decay.
Figure 1:
Scheme of
cell fractionation. Ltk cells were gently lysed in
0.2% Nonidet P-40 to preserve the ER (for details of the procedure see
``Materials and Methods''). Under these conditions, a
substantial part of ER membranes remained associated with nuclei and
could be pelleted by low speed centrifugation at 1500
g. Some membranes, however, formed smaller ER vesicles, which
were separated from cytosol by high speed centrifugation through a 30%
sucrose cushion. The low speed pellet was washed, and
membrane-associated proteins, including ribosomes, were extracted with
0.2% deoxycholate (DOC). Remaining nuclei were repelleted. The high
speed supernatant and corresponding polysome pellet (P), as
well as the low speed wash (W), DOC-extract (D), and
nuclei (N) were harvested and further
analyzed.
The pellet
from the low speed centrifugation contained the nuclei as well as a
substantial fraction of ER membranes. After a wash with 0.5 ml lysis
buffer, the membranes were extracted from the pellet by a wash with 0.5
ml of lysis buffer supplemented with 0.2% deoxycholate (DOC). The
remaining nuclei were pelleted by another low speed centrifugation
(1500 g for 8 min) at 4 °C and resuspended in 0.5
ml of lysis buffer. RNA was prepared from 0.4 ml of the wash fraction,
the DOC extract, and the nuclear fraction. 50 µl of the resuspended
nuclear pellet were adjusted to 0.4 M KCl, and IRP was
extracted by shaking for 30 min at 4 °C. For Western blot analysis,
50 µl of the nuclear fraction were extracted with lysis buffer
containing 1% DOC.
In a typical experiment we tested whether TfR mRNA and IRP changed their cellular localization in response to iron availability (Fig. 2). Cells were treated overnight with the iron chelator desferrioxamine to induce the RNA binding activity of IRP and TfR mRNA or incubated for additional 3 h with ferric ammonium citrate to revert this induction. Cells were then lysed, and fractions were isolated (Fig. 1). Each fraction was analyzed on Western blots with a polyclonal serum raised against the rough ER marker protein ribophorin I(19) . Ribophorin was detected mainly in the low speed pellet and DOC extract, as well as to some extent in the pellet of the high speed centrifugation (Fig. 2B). The distribution of TfR mRNA in iron chelator-treated cells, as measured by Northern blot analysis, correlated perfectly with that of ribophorin (Fig. 2A). More than 95% of the total TfR mRNA was found associated with ER membrane fractions. Some TfR mRNA that remained associated with nuclei after extraction with 0.2% DOC could be removed entirely with 1% DOC (not shown). As expected, administration of iron salt to chelator-treated cells strongly diminished the total amount of TfR mRNA. But there was no evidence for TfR mRNA dissociation from the ER containing fractions.
Figure 2:
Intracellular distribution of IRP and TfR
mRNA. Murine Ltk fibroblasts were treated first with
desferrioxamine for 20 h (DES) and then for 3 h with ferric
ammonium citrate (FE). RNA and protein were fractionated
according to the scheme described in Fig. 1. A,
Northern blot analysis of TfR mRNA. RNA was prepared from each cellular
fraction and dissolved in 20 µl of water. Identical volumes of RNA
(10 µl) were loaded on a 1.2% denaturing gel, transferred to nylon
membrane, and hybridized with a radiolabeled fragment of the murine TfR
cDNA. B, Western blot analysis of the rough ER marker protein
ribophorin I in different cellular fractions. 50 µl of each
fraction were separated on a 10% polyacrylamide gel in SDS, and
ribophorin I was detected with a polyclonal serum by the ECL method. C, RNA-protein band shift analysis of intracellular IRP. 2
µl of each fraction was analyzed for presence of active IRP. RNA
binding activity was determined by a gel retardation assay using
radiolabeled ferritin H chain IRE as a probe. Where indicated, samples
were preincubated in 2%
-me.
In parallel, IRP was determined
both in cytosolic and membrane fractions. Soluble IRP was directly
tested for IRE binding in the six sucrose fractions, whereas membrane-
and polysome-associated IRP was first dissociated by high salt
extraction with 0.4 M KCl and separated from remaining
insoluble material by centrifugation. As shown in Fig. 2C (upper part), most IRP was present in the sucrose
fractions and activated due to iron chelation. This soluble IRP was
clearly inactivated in Ltk cells grown with iron
salt. A significant amount (about 14%) of total IRP was also detected
in membrane-derived fractions of iron chelator-treated cells (Fig. 2C, lanes P, D, and N). This IRP bound to a
ferritin IRE with high affinity, even in the absence of in vitro preincubation with
-mercaptoethanol (
-me), indicating
its active state(20, 21) . Membrane-associated IRP
also declined in high iron medium but unlike soluble IRP could not be
reactivated by in vitro treatment with
-me (Fig. 2C). We conclude that most likely bound IRP had
detached from ER membranes in response to high cellular iron supply.
Alternatively, membrane-associated IRP might have been degraded in
response to iron, but this is rather unlikely, because total IRP is
usually unchanged when iron is added to cells(22) . Control
experiments also showed that activation of IRP by
-me was not
inhibited by DOC at concentrations used for IRP isolation (data not
shown).
Recently, a second protein with high affinity for IREs was
characterized in cells from mice(23) , humans(24) , and
rats(25) . This factor is called IRP-2 and exhibits many of the
characteristics of IRP but has also some distinct features. In
RNA-protein gel shifts, mouse IRP-2 forms a faster migrating complex
than mouse IRP(23) . It is interesting to notice that IRP-2 was
clearly present in both the soluble and membrane-associated fractions
of Ltk cells (Fig. 2C). Thus, the
intracellular distribution of this protein is similar to the one of
IRP. Because IRP and IRP-2 have similar affinities for
IREs(23) , it is likely that they are both bound to mRNAs on
membrane-associated polysomes.
A first set of
experiments was performed without the inhibitors. TfR mRNA was more
than 20-fold induced after desferrioxamine treatment (Fig. 3A), whereas subsequent addition of ferric
ammonium citrate resulted in a rapid disappearance of TfR mRNA within 2
h. In control hybridizations, murine plasminogen activator inhibitor 1
(PAI-1) mRNA, unlike TfR mRNA, was markedly induced by iron (Fig. 3A). A similar effect was also observed after the
addition of new medium with desferrioxamine and is presumably due to
fresh serum. ()Analysis of free cytosolic IRP revealed that
IRE binding activity was modulated as a function of iron levels, in
parallel to changes in TfR mRNA (Fig. 3B). Inactivated
IRP in cytosolic extracts could always be reactivated by in vitro incubation with 2%
-me to yield a constant total IRE binding
activity. Membrane-associated IRP was also perfectly regulated by
modulations of intracellular iron (Fig. 3C). However,
unlike inactivated IRP of cytosol, it responded only slightly to 2%
-me (Fig. 3C, lower panel). Again we
conclude that the disappearance of this active IRP from membranes under
high iron conditions can only be due to its physical dissociation from
this fraction (a small response to 2%
-me was always observed and
is likely due to prior limited oxidation of IRP during extraction).
Hence, inactivation and disappearance of membrane-associated IRP and
the decline of TfR mRNA correlated in time, compatible with the
hypothesis of a causal relationship between the IRE-IRP interaction and
TfR mRNA stability(7) .
Figure 3:
Iron-dependent regulation of
membrane-associated TfR mRNA and IRP. Exponentially growing
Ltk cells (EXP) were treated with
desferrioxamine for 20 h (DES) and then transferred into
medium containing 20 µg/ml ferric ammonium citrate (FE).
After different periods of time, cells were harvested and lysed, and
membrane fractions were isolated by a 0.5% DOC extraction of a rough
nuclear pellet as described under ``Materials and Methods.'' A, Northern blot analysis of membrane-associated RNA (20
µg/lane) was performed by sequential hybridization with labeled
cDNA fragments for mouse TfR and PAI-1. B, IRE binding
activity of IRP in the cytosolic fractions was determined by a gel
retardation assay using a human ferritin H chain IRE probe as described
under ``Materials and Methods.'' Where indicated, samples
were preincubated in 2%
-me. C, IRP bound to
membrane-associated RNA was recovered by high salt extraction (0.4 M KCl) of the rough nuclear pellet. The extract was diluted to
80 mM KCl, and the IRE binding activity determined as above.
Where indicated, samples were preincubated in 2%
-me. The
experiment was performed twice with the same
result.
Next, we analyzed the effect of
actinomycin D. Exponentially growing Ltk cells were
cultured in medium with desferrioxamine for 20 h, and then actinomycin
D was added for 1 h prior to the addition of iron salt. Fresh medium
containing the inhibitor and ferric ammonium citrate was then added to
reverse the iron chelation conditions, and the cells processed as above
at different time points. The chelation-induced level of TfR mRNA
remained high in the presence of actinomycin D (Fig. 4A). However, when added simultaneously with iron
salt, the effect of the drug was less pronounced (data not shown).
Actinomycin D inhibited also the serum-dependent induction of PAI-1
mRNA suggesting the necessity of on-going transcription for this rise.
Inactivation of cytosolic IRP, however, was not influenced by the
inhibitor (Fig. 4B). More importantly,
membrane-associated IRP declined with the same kinetics as in the
absence of actinomycin D (compare Fig. 4C with Fig. 3C). Thus, actinomycin D, a compound known to
inhibit transcription by intercalation into DNA, did not affect the
dissociation of IRP from membrane-bound transcripts.
Figure 4:
Actinomycin D inhibits the iron-dependent
decay of TfR mRNA but not the dissociation of the IRP-RNA complex.
Exponentially growing Ltk cells (EXP) were
treated for 20 h with desferrioxamine (DES), and the
transcription inhibitor actinomycin D (6 µg/ml) was then added for
1 h to the medium (D/A). Thereafter, fresh medium containing
the inhibitor and 20 µg/ml ferric ammonium citrate was added for
different periods of time, and cells were processed by the simplified
cell fractionation (see ``Materials and Methods''). A, membrane-associated RNA was analyzed on a Northern blot by
hybridization with labeled cDNA fragments for mouse TfR and PAI-1. B, cytosolic proteins were analyzed for IRE binding activity
in the presence or absence of 2%
-me. C, RNA band shift
assays were performed using equal amounts of high salt extracted
protein (1 µg) from the low speed pellet as described under
``Materials and Methods.'' Where indicated, samples were
preincubated in 2%
-me. The figure shows typical results from two
experiments.
Similar results were obtained with the translation inhibitor cycloheximide. It inhibited completely the iron-dependent decay of TfR mRNA (Fig. 5A). In contrast, inactivation of cytosolic IRP as well as the dissociation of membrane-associated IRP were unaffected (Fig. 5, A and B). Treatment of cells with cycloheximide for 1 h still in the presence of desferrioxamine led to a slight reduction of the total cytosolic IRP activity. The induction of PAI-1 mRNA was not influenced by cycloheximide.
Figure 5:
Effect of cycloheximide on the
iron-dependent degradation of TfR mRNA and the IRP-IRE interaction.
Exponentially growing Ltk cells (EXP) were
treated with desferrioxamine for 20 h (DES). 1 h after the
addition of 10 µg/ml cycloheximide to the medium (D/C),
new medium with 20 µg/ml ferric ammonium citrate and cycloheximide (CHX + FE) was added for different periods of time.
Membrane-associated RNA and IRP bound to this RNA were prepared as
described under ``Materials and Methods.'' A,
membrane-associated RNA was analyzed on a Northern blot using
radiolabeled cDNA fragments for mouse TfR and PAI-1 as probes. B, a gel retardation assay with cytosolic extracts was
performed. Where indicated, samples were preincubated in 2%
-me. C, IRP bound to membrane-associated RNA was recovered by high
salt extraction and detected by band shift analysis. Where indicated,
samples were preincubated in 2%
-me. The experiment was done twice
with similar results.
Thus, inhibition of neither transcription nor translation interfered with the iron-induced dissociation of IRP from ER membranes. It seems therefore that the degradation of TfR mRNA, rather than IRP inactivation, depends on RNA and protein synthesis. Consequently, the simplest explanation for the action of these inhibitors would be to postulate a labile trans-acting factor encoded by a very unstable mRNA. Similar conclusions were reached by Koeller et al.(15) , who found that the degradation rate of a constitutively unstable TfR transcript was unaffected by a cis-acting translation block through an IRE in the 5`UTR. Both results suggest the presence of a labile protein essential for TfR mRNA degradation.
TfR mRNA in Ltk fibroblasts was first induced by desferrioxamine. Prior to
changing iron levels, transcription inhibitors were added for 1 h and
then maintained for 3 h further after the addition of iron salt. In
contrast to actinomycin D, neither low concentrations of
-amanitin
nor DRB had an effect on the iron-induced decay (Fig. 6).
However, the RNA polymerase II-specific inhibitors abolished
transcription of PAI-1 mRNA. This excludes the possibility of
insufficient cellular uptake of these drugs. Expression of
glyceraldehyde-3-phosphate dehydrogenase mRNA, a highly stable
transcript, was not affected by the inhibitors. Similarly, poly(A)-tail
shortening, a prerequisite for the degradation of c-myc mRNA (28) did not seem to affect the TfR mRNA half-life; cordycepin
had no effect on iron-dependent decay or steady state levels of TfR
mRNA, even in a more extended kinetic study, whereas c-myc transcript levels were strongly reduced (data not shown). These
results are consistent with the recently published observation that
shortening of the poly(A)-tail length is not necessary to create a 3`
degradation intermediate of TfR mRNA(14) . By using DRB rather
than actinomycin D, we have also been able to measure the rate of TfR
mRNA decay with accuracy (data not shown). The half-life of TfR mRNA
was about 3.5 h in exponentially growing Ltk
cells in
normal medium. This value increased to more than 6 h in cultures
treated with 50 µM desferrioxamine. In contrast, high
intracellular iron levels reduced the half-life of TfR mRNA to less
than 60 min.
Figure 6:
Effect of different RNA synthesis
inhibitors on TfR mRNA decay. Murine Ltk fibroblasts
were treated with desferrioxamine for 20 h (DES) and then for
3 h with ferric ammonium citrate (FE). Where indicated
actinomycin D (6 µg/ml), cordycepin (25 µg/ml),
-amanitin
(10 µg/ml), or DRB (30 µg/ml) was added together with the iron
salt (FE+) after a 1-h preincubation with the inhibitor
in the presence of desferrioxamine. The Northern blot was hybridized
with probes for murine TfR, PAI-1, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). The results are representative for two
independent experiments.
Taken together, the data indicate that mRNA synthesis
by RNA polymerase II is not essential for the iron-dependent regulation
of TfR mRNA expression. Because in contrast to the other inhibitors,
actinomycin D affects all three RNA polymerases, we tested the
possibility that RNA polymerase III might be necessary for TfR mRNA
degradation. This enzyme is responsible for the transcription of tRNAs,
5 S ribosomal RNA, and some other small RNA molecules. High
concentrations of -amanitin are known to block polymerase III by
direct interaction with the protein(29, 30) . We
therefore tested the effect of a high dose of
-amanitin (200
µg/ml) on the regulation of TfR mRNA stability. Pretreatment with
the inhibitor for 1 h resulted in nearly complete inhibition of
iron-induced degradation during the first 2 h (Fig. 7). After 4
h, 20% of the TfR mRNA were still left, whereas TfR transcript levels
in cells treated with iron alone were undetectable. Simultaneous
addition of 200 µg/ml
-amanitin and iron salt, however, had no
inhibitory effect on the TfR mRNA decay. As expected, induction of the
control PAI-1 mRNA was completely abolished by
-amanitin. These
results suggest that the presence of labile polymerase III transcripts
is essential for the degradation of TfR mRNA. The stabilization of TfR
mRNA in cells treated with
-amanitin was not complete, but the
decay is clearly delayed by the drug. The slight difference in the
effect of actinomycin D and
-amanitin is possibly due to a slower
uptake of the latter by the cell. This could also explain why
-amanitin has no effect when added simultaneously with iron salt.
Figure 7:
High concentrations of -amanitin
interfere with the decay of TfR mRNA. Mouse Ltk
cells
were incubated for 20 h with desferrioxamine (DES) and then
treated for 2 h or 4 h with iron salts. Ferric ammonium citrate was
added without inhibitor (FE), simultaneously with 200
µg/ml
-amanitin (FE/
), or after a 1-h
preincubation with the inhibitor (
/FE). One sample was
incubated with
-amanitin without changing the iron level (
/DES). Cytoplasmic RNA was isolated, and Northern blot
analysis was performed using radiolabeled cDNA probes for TfR, PAI-1,
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The
experiment was done twice with similar
results.
The iron-dependent regulation of TfR mRNA is one of the best
studied examples for a post-transcriptional control of mRNA stability.
According to the prevalent model, iron-deprived IRP, the
[Fe-S]-free apoprotein form of cytoplasmic aconitase (21, 31, 32, 33) acts as a
RNA-binding protein with high affinity for IREs and thereby protects
the TfR mRNA 3`UTR from endonucleolytic attack(4, 7) .
This hypothesis is based on functional assays with numerous 3`-UTR
mutants (3, 4) and has recently gained support from
the identification of a putative endonuclease cleavage site next to the
IREs(14) . The model implies that iron favors dissociation of
IRP from TfR mRNA, which then becomes accessible to a nuclease.
However, neither the in vivo IRE-IRP dissociation nor the
enzymes involved in RNA degradation have been characterized. Most
intriguingly, we have observed that cycloheximide and actinomycin D are
inhibitory to rapid degradation of TfR mRNA(3) . As the decay
of several other short-lived mRNAs was reported to be blocked by
translation and/or transcription inhibitors too, it seemed of interest
to elucidate whether the inhibitors stabilize TfR mRNA by effects on
IRP or at a subsequent step. To approach this question we developed a
cell fractionation procedure that permits the simultaneous analysis of
rough ER-associated IRP and TfR mRNA. The method takes advantage of a
mild detergent lysis that leaves the rough ER mostly intact and
attached to nuclei, such that it is readily separated from the
cytoplasm by low speed centrifugation. mRNA was then extracted with
DOC, and IRP was dissociated in high salt. Using this procedure we find
an iron-dependent association of IRP with the ER, which comprises about
14% of the active cellular IRP under iron-chelating conditions, and
virtually none under conditions of high iron supply. This relative
amount as compared with total IRP may be slightly overestimated,
because we did not measure all IRP in the cytosol where some is also
bound to mRNAs(34) . As IRP interacts strongly with IREs in a
1:1 stoichiometry but was never reported to bind to other cellular
components, we conclude that rough ER-associated IRP consisted mainly
of active IRP that was bound in vivo to transcripts with an
IRE. This conclusion is supported by the properties of the extracted
IRP, as well as some IRP-2, that showed a high IRE binding affinity and
no further in vitro activation by 2% -me.
Very importantly, IRP in ER fractions coincided with the presence of TfR mRNA, suggesting strongly its interaction with this mRNA, but possibly also other, hitherto unidentified, IRE-containing transcripts. Whereas high iron, either in the absence or presence of cycloheximide and actinomycin D, dislodged IRP from the rough ER, TfR mRNA decayed only in the absence of inhibitors but remained ER-bound and stable in their presence. This demonstrates for the first time iron-dependent in vivo dissociation of IRP from IRE-containing mRNAs without an obligatory degradation of these mRNAs or their release from the ER. The results are in agreement with the prevalent model, according to which the initially active RNA-protecting IRP gets inactivated by iron and dissociates from TfR mRNA, thereby unmasking a ribonuclease cleavage site. Thus, actinomycin D and cycloheximide interfere with this latter step by stabilizing the unprotected TfR transcript but have no effect on IRP dissociation.
A link between translation and RNA degradation has been shown in numerous instances, notably through the inhibition of mRNA decay by cycloheximide(35, 36, 37) . In the case of early immediate transcripts with AU-rich elements, like c-myc and c-fos mRNA, degradation is preceded by poly(A)-tail shortening(38) . For c-myc mRNA this step was reported to be blocked by translation inhibition(28) . In the yeast Saccharomyces cerevisiae, a similar deadenylation step is presumably followed by decapping and 5` to 3` exonuclease degradation (reviewed in (39) ), and cycloheximide seems to inhibit the decapping reaction rather than translation elongation(40) . For vertebrate mRNAs the role of decapping is not yet clarified, and certain data speak in favor of a trans- rather than a cis-acting effect of cycloheximide on c-fos and TfR mRNA degradation(15) . When translation rates of chimeric mRNAs with 3` instability elements of c-fos or TfR mRNA were modulated 20-fold through a 5` IRE, no change in RNA half-life was observed(15) . Moreover, in contrast to most other degradation pathways, decay of TfR mRNA is not preceded by the shortening of its poly(A)-tail(14) . The trans-acting factor that is sensitive to translation inhibition remains to be characterized. Yet, our study eliminates IRP as a possible target of cycloheximide.
The conclusion concerning actinomycin D is similar to that for cycloheximide: actinomycin D does not block RNA turnover by interference with IRP inactivation. Thus, its effect on the iron-dependent degradation of TfR mRNA must be at a subsequent step. This block was strongest when the inhibitor was added to iron-deprived cells simultaneously with or shortly before the iron salt (this report and (3) ) and was present during the time period when most of the TfR mRNA gets degraded. However, in a quite different physiological situation, a similar effect has been observed; in the mouse T cell line B6.1, a decrease in TfR mRNA expression after IL-2 withdrawal was also blocked by actinomycin D(41) . These findings seem somewhat different from a study where chimeric TfR mRNA constructs were analyzed in murine B6 fibroblasts and where actinomycin D was successfully used as a transcriptional inhibitor to determine mRNA stability in different iron conditions(15) . In these experiments, however, the iron source hemin was added many hours before the transcription inhibitor. It seems possible, therefore, that actinomycin D affects an iron-dependent step in mRNA decay other than IRP inactivation and fails to block TfR degradation when added long after iron. Alternatively, different cell lines might respond to actinomycin D in different ways. Independent of the possible cause for these differences, it was rewarding to find that DRB, a ribonucleotide analog that interacts with the RNA polymerase II transcription complex(42, 43) , apparently did not interfere with RNA decay and provided an excellent tool to measure the stability of TfR mRNA. Consequently, it became possible for the first time to measure accurately the half-life of wild-type TfR mRNA in conditions of high intracellular iron levels. A half-life of less than 1 h was found, which is in good agreement with the results obtained with TfR mRNA constructs containing the minimal regulatory region (15) . As expected, the stability of TfR mRNA increased dramatically to a half-life of more than 6 h after treatment of cells with desferrioxamine.
Actinomycin D-induced stabilization
was observed for several other mRNAs besides TfR mRNA. The transcript
for the RII subunit of protein kinase A is transiently induced by
cAMP and thought to be stabilized in the nucleus by polymerase II
transcription inhibitors (44) . Likewise, there is a
transcription-dependent degradation of neurofilament mRNA in axotomized
sensory neurons (45) . During differentiation of rat myoblast
cells in culture, creatine phosphokinase is strongly inducible by
insulin but disappears rapidly after hormone removal. This apparently
cytoplasmic degradation of mRNA is inhibited by actinomycin
D(46) . A dexamethasone-induced modulation of the mRNA turnover
has been observed for several AU-rich mRNAs and is, at least in the
case of the urokinase type plasminogen activator mRNA, inhibited by
actinomycin D(47) . In some cases DRB was shown to exhibit an
effect similar to that of actinomycin D(44, 45) .
These results were usually interpreted by a model that predicts the
involvement in mRNA turnover of a labile protein encoded by an unstable
transcript. In the case of TfR mRNA decay, however, the effect of
actinomycin D seems to be distinct because specific inhibitors of mRNA
synthesis like DRB or
-amanitin (at 10 µg/ml) fail to
interfere with the iron-dependent decay of TfR mRNA (Fig. 6).
Thus, it remains an important question how actinomycin D acts.
Besides its effect on transcription by intercalating into DNA,
actinomycin D might interfere with mRNA degradation by intercalating
into double-stranded RNA regions. However, this would not provide a
satisfactory explanation for why the high concentrations of
-amanitin, a transcription inhibitor which directly interacts with
RNA polymerases, can also affect TfR mRNA degradation. In fact it is
known that actinomycin D and high concentrations of
-amanitin are
able to inhibit RNA polymerase III, an enzyme responsible for the
synthesis of small transcripts like 5 S ribosomal RNA and transfer RNA (30) . Because we demonstrate that both inhibitors are able to
block TfR mRNA decay, whereas other conditions of transcriptional
inhibition do not, we propose that a RNA polymerase III product might
be important as a component of the TfR mRNA decay machinery. Small RNAs
have been shown to play a role in RNA splicing (48) and
processing of histone pre-mRNAs (49, 50, 51) and are suspected as potential
components for mRNA turnover and translation. RNase P and RNase MRP are
related ribonucleoproteins involved in cleaving of tRNA precursors and
processing of mitochondrial primer RNAs(52, 53) . Both
functions require the presence of RNA components. A small RNA
polymerase III transcript was also reported to be responsible for the
regulation of translation of the myosin heavy chain
mRNA(54, 55) . Furthermore, a potential role of
polymerase III transcripts from B2 repeats was discussed in the
regulation of mRNAs containing AU-rich elements (56) , and
Brewer and Ross (57) purified a labile destabilizer that
accelerates the degradation of c-myc mRNA in an in vitro system. The factor contains an essential nucleic acid component.
It will be of particular importance now to biochemically characterize
the trans-acting factors that are involved in the
iron-dependent degradation of TfR mRNA, in order to establish firmly
the role of polymerase III transcripts in the endonucleolytic cleavage
of this mRNA.