From the Office of Clinical Research and Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, January 24, 2001, and in revised form, March 8, 2001
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ABSTRACT |
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The CCCH family of tandem zinc finger proteins
has recently been shown to promote the turnover of certain mRNAs
containing class II AU-rich elements (AREs). In the case of one member
of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor Tristetraprolin (TTP;1
also known as TIS11, Nup475, and G0S24) (1-5) is the prototype of a
small family of zinc finger proteins characterized by a pair of closely
spaced zinc fingers of the CCCH type. Although TTP was originally
thought to be a probable transcription factor, we have recently shown
that TTP and related proteins can promote the turnover of certain
mRNAs containing AU-rich elements (AREs) in their 3'-untranslated
regions (UTRs). Using cells derived from TTP-deficient mice, we
showed that TTP is a physiological inhibitor of the secretion of tumor
necrosis factor TTP and its related proteins bind to the ARE of these mRNAs and
then appear to promote their initial destruction from the 3'-end. This
results in a decrease in total hybridizable mRNA levels as well as
the formation of what appear to be mRNA bodies missing their
poly(A) tails (9). However, it is unclear whether TTP achieves this
effect by stimulating the initial 3' to 5' processive deadenylation of
these mRNAs, by stimulating another 3'-exonuclease, or by some
other mechanism (e.g. an ARE-dependent
endonuclease activity).
The current view of ARE-dependent destruction of mRNAs
containing so-called class II AREs is that there is first processive deadenylation of the poly(A) tail, presumably by one or more
poly(A)-specific exonucleases, followed by further exonucleolytic and
perhaps endonucleolytic degradation of the mRNA body after removal
of the protective poly(A) tail (10). Many proteins have been found to
be capable of forming protein-ARE complexes in cell-free assays (for
reviews, see Refs. 11 and 12); however, few ARE-binding proteins have
been shown to participate in the destabilization of class II
ARE-containing mRNAs in intact cells. TTP and its related proteins
cMG-1 and TIS11d can all stimulate the degradation of class II
ARE-containing mRNAs in cell transfection studies (9).
In an attempt to further elucidate the mechanism by which the CCCH
proteins destabilize ARE-containing mRNAs, we have tested the
hypothesis that TTP can promote degradation of class II ARE-containing mRNAs that do not have a poly(A) tail. We show here that TTP can stimulate the degradation of a nonpolyadenylated histone mRNA when
a class II ARE is inserted into this mRNA and that TTP can stimulate the breakdown of mRNAs that normally contain a class II
ARE but in which a histone 3'-end-processing sequence has replaced the
normal poly(A) tail.
Plasmid Constructs
Full-length TNF
CMV.mGM-CSF was constructed by RT-PCR using total cellular RNA from RAW
264.7 cells treated for 4 h with 1 µg/ml
lipopolysaccharide as the template for RT. The 5' primer for PCR
amplification was 5'-gtcgacACTCAGAGAGAAAGGCTAAGG-3', and the 3' primer
was 5'-tctagaAAAGTTTTAATAATTTATTATTCTGG-3'. The capital letters in the
5' primer contain the beginning of exon I of the mouse GM-CSF gene (bp
1136-1156 of GenBankTM accession number X03020), and those
in the 3' primer contain the end of exon IV (bp 3481-3506 of
GenBankTM accession number X03020). The lowercase letters
in the primers indicate the restriction sites for SalI and
XbaI, respectively. The resulting PCR product is a 775-bp
mouse GM-CSF cDNA.
The IL-3 expression vector CMV.mIL-3 was prepared by RT-PCR
using total cellular RNA from WEHI-3 cells as the template for RT. The
5' primer for PCR amplification was 5'-gtcgacAGAACCCCTTGGAGGACCAG-3', and the 3' primer was 5'-ccgcggTGTTCAAAGTTATTTATTTCC-3'. The capital letters in the 5' primer contain the 5'-end of exon I of the mouse IL-3
gene (bp 695-714 of GenBankTM accession number K03233),
and those in the 3' primer contain the 3'-end of exon V (bp 2869-2889
of GenBankTM accession number K03233). The lowercase
letters in the primers indicate the restriction sites for
SalI and SstII, respectively. The resulting PCR
product is an 851-bp mouse IL-3 cDNA.
The resulting PCR products of these full-length cDNAs were digested
with SalI and XbaI (for mTNF Full-length and Modified Histone mRNA (Fig. 1D)--
Plasmid
H2a-614, which contains sequence corresponding to bp 1-1645 of
GenBankTM accession number X16148 (mouse histone H2a gene;
Ref. 13), was purchased from ATCC (Manassas, VA). To create plasmid
CMV.H2a, plasmid H2a-614 was used as a template. A 549-bp fragment (bp 814-1362 of X16148) containing the H2a precursor RNA (bp 814-1328 of
X16148) and the sequence of the histone downstream element (bp
1342-1355 of X16148) was PCR-amplified with the following primers: 5'
primer, 5'-gtcgacCGTTTCCTGGTTGTGGCC-3'; 3' primer, 5'-ccgcggCTACCGTGACACAACTC-3'. The lowercase letters in the primers denote restriction sites for SalI and SstII,
respectively. This PCR product was then inserted into the same sites of
plasmid vector CMV/SK Modified IL-3/Histone (See Fig. 6C) and TNF
In the mTNF Transfection of HEK 293 Cells, Northern Analysis, and RNase H
Assays
HEK 293 cells were maintained, and transient transfection of
1.2 × 106 cells with plasmid constructs in
calcium-phosphate precipitates was performed as described (9). In some
experiments, pXGH5 (Nichols Institute Diagnostics, San Juan Capistrano,
CA) was co-transfected to monitor transfection efficiency. Assays of
released human growth hormone (HGH) were performed as described (14).
In most experiments, the cells were co-transfected with various CCCH
protein-expressing plasmids. Details of the construction of these
plasmids have been described (8, 9).
Twenty-four h after the removal of the transfection mixture, aliquots
of cell culture medium were analyzed for HGH according to the
manufacturer's protocol. Total cellular RNA was then harvested from
the HEK 293 cells using the RNeasy system (Qiagen, Valencia, CA).
Northern blots were prepared as described (1). Blots were hybridized as
indicated with random-primed, RNase H assays were performed by annealing total cellular RNA (5-10
µg) and the appropriate synthetic oligonucleotide (0.5-1 µg) in 10 µl of 50 mM KCl for 5 min at 50 °C, followed by an
additional 10 min at 22 °C. The mixture was incubated further at
37 °C for 40 min in a buffer (4 mM HEPES-KOH (pH 8), 50 mM KCl, 2 mM MgCl2, 0.2 mM dithiothreitol, and 1 µg/µl bovine serum albumin)
containing 1 unit of RNase H (Promega, Madison, WI) in a final volume
of 25 µl. The reaction mixture was then precipitated with sodium acetate and ethanol, and the resulting RNA was subjected to Northern analysis as described above.
The Effect of Co-transfection of TTP and Related CCCH Proteins on
Naturally Occurring ARE-containing Full-length mRNAs--
Our
previous transfection experiments in 293 cells examined the effects of
TTP and related proteins on the turnover of a synthetic TNF
When these three CCCH proteins were each individually co-expressed with
a human TNF
When the identical experiment was repeated with a 5'-truncated version
of TNF
The same experiment was performed with a TNF
We concluded that the truncated form of TNF
Similar experiments were performed with full-length cDNAs encoding
GM-CSF and IL-3. In the case of GM-CSF, we have demonstrated in
experiments with wild-type and TTP-deficient mice that turnover of this
mRNA is regulated by TTP in bone marrow stromal cells (7); however,
no comparable physiological function of TTP has been demonstrated to
date in the regulation of IL-3 mRNA stability. In the case of
GM-CSF mRNA, low concentrations of transfected TTP, cMG1, and
XC3H-3 DNA (Fig. 3A,
lanes 2, 5, and 8) all
stimulated a decrease in mRNA steady state levels, whereas higher
DNA concentrations again resulted in the accumulation of at least two
smaller species of GM-CSF mRNA. Cleaner results were obtained when
the cells were treated for 1-8 h with actinomycin D to inhibit ongoing
transcription (Fig. 3B). This experiment compared the
effects of no TTP, the genomic TTP construct H6E (5 µg DNA), and low
concentrations of the TTP expression construct CMV.hTTP.tag (0.1 µg
of DNA) on the levels of GM-CSF mRNA species without
(lanes 1-3) or with actinomycin D treatment for
various times. The expression of GAPDH mRNA is shown in the
middle panel, and the expression of TTP mRNA
in the same samples is shown in the bottom panel.
The 8-h actinomycin D samples showed that, in the cells expressing
vector alone (lane 13), the GM-CSF mRNA
appeared as a large, diffuse band. The amount of this species was
greatly decreased in cells expressing TTP (lanes
14 and 15). In cells expressing the genomic TTP
clone, most of the hybridizing GM-CSF species appeared to be
full-length, whose levels were markedly decreased compared with
control (compare lane 14 with lane
13). Also detectable were low levels of two smaller forms of
GM-CSF mRNA when this TTP construct was expressed (see
lanes 2, 5, 8,
11, and 14). However, when the TTP cDNA was expressed, there was a marked decrease in total hybridizable GM-CSF mRNA, and the remaining mRNA was in the form of the shortest of the three hybridizing bands. RNase H and oligo(dT) experiments demonstrated that the single band observed in lane
15 was considerably shorter than the completely deadenylated
form of the full-length mRNA (not shown), again consistent with a
GM-CSF mRNA that was truncated in a 3' to 5' direction to
approximately the position of the ARE.
Parallel experiments were performed with full-length IL-3 mRNA.
This mRNA is considerably smaller than the 18 S ribosomal RNA,
making the results of Northern analysis easier to interpret. As shown
in Fig. 4A (top
panel), the IL-3 mRNA was expressed as a single diffuse
band that appeared as a tightly spaced pair of bands following a
shorter autoradiographic exposure (lane 1). Steady state levels of this mRNA were markedly decreased by TTP co-expression, either from the genomic clone H6E (5 µg of DNA; lane 2) or from low concentrations of the
expression construct CMV.hTTP.tag (0.1 µg of DNA; lane
3). Levels were also markedly reduced by co-expression of
higher concentrations of either cMG1 (1 µg of DNA; lane
4) or XC3H-3 (1 µg of DNA; lane 5).
In all cases in which the CCCH proteins were expressed, but most
obviously with cMG1, there was a decrease in the intensity of the upper two bands and the appearance of a third band. These changes were more
obvious after transcription was inhibited with actinomycin D. For
example, the tightly spaced doublet of IL-3 mRNA from cells transfected with vector (lane 16) largely
disappeared with expression of the CCCH proteins, with the appearance
of a smaller stable intermediate being apparent in the cMG1 lanes
(lanes 4, 9, 14, and
19).
To determine the polyadenylation status of these forms of the IL-3
mRNA, total cellular RNA samples from this experiment were incubated with an oligo(dT) primer and RNase H (Fig. 4B).
The samples shown in lanes 1-4 correspond to
lanes 16-20 of Fig. 4A; the samples
shown in lanes 5-8 represent the same samples
after oligo(dT) and RNase H treatment. These results indicate that the smaller of the two tightly spaced bands in the IL-3 doublet was the
deadenylated form of the mRNA (dA), while the upper of the two
bands is the full-length polyadenylated form (FL; compare lane 1 with lane 5).
However, the still smaller band that was most evident in the cMG1
samples (lane 3) was not further reduced in size
by the oligo(dT)/RNase H treatment (P; lane
7) and represents a further 3'-truncated mRNA. That this
was not a 5' truncation of the mRNA was shown by similar RNase H
experiments using an internal primer complementary to bp 868-889 of
accession number K03233. In this case, identical 173-base mRNA
fragments corresponding to the 5'-end of the IL-3 mRNA were found
in both samples from control and TTP-expressing cells (data not shown).
As with the TNF
To confirm the ARE-dependence of the TTP-stimulated degradation of the
full-length IL-3 mRNA, we co-transfected cells with genomic and
cDNA TTP vectors in the presence of IL-3 mRNA that contained
(Fig. 4C, lanes 1-6) or did not
contain (lanes 7-12) the ARE. As before, both
TTP expression vectors resulted in a concentration-dependent decrease in IL-3 mRNA
expression, with the higher concentrations of TTP achieved with the
cDNA expression vector resulting in accumulation of the lowest of
the three IL-3 mRNA bands. When these experiments were repeated
with the IL-3 mRNA lacking its ARE, the co-expression of TTP had no
effect on the total level of IL-3 mRNA and did not stimulate the
formation of the lowest mRNA band. In this experiment, the
corresponding amounts of TTP mRNA are shown in the
bottom panel of Fig. 4C, and the
levels of GAPDH mRNA in the same samples are shown in the
middle panel.
These experiments indicated that TTP and its related proteins cMG1 and
XC3H-3 could stimulate the breakdown of three naturally occurring class
II ARE-containing mRNAs and cause the accumulation of an mRNA
fragment that was truncated in a 3' to 5' direction past the 5'-end of
the poly(A) tail to approximately the location of the ARE itself.
However, they do not distinguish between a stimulated poly(A) or other
3'-exonuclease activity, with "protection" at the ARE, and a
hypothetical CCCH protein-stimulated endonuclease activity.
Effect of TTP on an ARE-containing Histone mRNA Lacking a
Poly(A) Tail--
To address the question of whether TTP could
stimulate the breakdown of ARE-containing mRNAs that lacked poly(A)
tails, we took two approaches: inserting an ARE into the sequence of a
nonpolyadenylated histone mRNA and replacing the IL-3 and TNF
To control for the possibility that insertion of 70 bp of any random
DNA sequence into the histone mRNA might affect its susceptibility to TTP-induced mRNA turnover and to shift the resulting hybrid transcript away from the endogenous histone mRNA on the gels, a
75-bp multiple cloning site fragment from the plasmid cloning vector
Bluescript pSK
These studies confirmed that the ability of TTP to decrease histone-ARE
expression was not just a function of disrupting the normal histone
mRNA with 70 bp of foreign sequence: In addition, the effect of TTP
was more obvious when the expressed histone transcripts were
physically separated from the endogenous histone mRNA bands.
To confirm that the transfected histone gene was appropriately
processed and was not polyadenylated in this transient expression system, RNA samples were digested with RNase H in the presence of
oligo(dT). Incubation with RNase H and oligo(dT) did not affect the
levels or size of endogenous histone mRNA (Fig. 5C,
lanes 1 and 2). Furthermore, this
treatment did not result in the disappearance or shortening of a
significant amount of histone mRNA expressed by the transfected
constructs, whether this was wild-type histone mRNA
(lanes 3-6) in the absence (lanes
3 and 4) or presence (lanes 5 and 6) of TTP (5 µg of DNA); TNF Effect of TTP on IL-3 and TNF
As shown in Fig. 6A, co-transfection of cells with TTP
(1-100 ng of DNA) and the wild-type IL-3 construct resulted in a
decrease in total hybridizable IL-3 transcripts and the appearance of
the characteristic three bands of IL-3 mRNA discussed above: the
fully polyadenylated transcript (top arrow
indicating the top band in lanes
1-5 of Fig. 6A, upper
panel), the deadenylated transcript (middle
arrow indicating the lower bands in
lanes 1-4 and the middle
band in lane 5), and the further
3'-truncated transcript (lowest arrow indicating
the lowest band in lane 5).
In contrast to cells not transfected with TTP, which expressed IL-3 as
a broad band consisting of two major transcripts (fully polyadenylated and deadenylated; lane 1, upper
panel), the IL-3 histone chimeric message was expressed as a
single band (lane 6) of approximately the same
size as the deadenylated form of native IL-3 mRNA. However, when
these cells were also transfected with the TTP-expressing plasmid
(1-100 ng of DNA), there was a decrease in the amount of full-length
chimeric IL-3 mRNA (lanes 7-10). At the
highest TTP concentration used (100 ng/plate), a truncated transcript accumulated (lane 10), whose size was
approximately the same as the shortest transcript seen after the
expression of full-length IL-3 mRNA and the same concentration of
TTP (lane 5). The expression of TTP mRNA in
this experiment is shown in the lower panel of Fig. 6A. Similar results were obtained from several
experiments, which also revealed that the concentration dependence of
the TTP effect was similar with both the native IL-3 mRNA and the
chimeric IL-3-histone transcript.
To confirm that the hybrid IL-3-histone mRNA was not polyadenylated
in these experiments, the same RNA samples were digested with RNase H
in the presence of oligo(dT). As indicated in Fig. 6B,
lanes 1-4, this experiment confirmed that the
upper of the two normal IL-3 transcript bands was the fully
polyadenylated species, whereas the lower of the two bands was the
deadenylated species. When the hybrid mRNA formed by the
transfected IL-3-histone plasmid was treated in this way, its migration
was unaffected, confirming that no significant poly(A) tail was present
(lanes 5-8). These data indicate that the
primary transcript formed by the expression of the IL-3-histone
cDNA was completely resistant to the effects of RNase H and
oligo(dT), confirming that it was not polyadenylated under the
conditions of these experiments.
Similar experiments were performed using a chimeric TNF
To confirm that the chimeric TNF Previous experiments in TTP knockout mice and cells derived from
them have shown that TTP is a physiologically important regulator of
the stability of mRNAs encoding two important cytokines, TNF In contrast, the time course of GM-CSF mRNA induction and
disappearance following lipopolysaccharide or TNF However, parallel experiments conducted with bone marrow-derived
stromal cells from the TTP-deficient mice revealed a strikingly different pattern (7). First, GM-CSF mRNA stability was increased to the point that no half-life could be calculated. In addition, there
was a greatly increased accumulation of the fully polyadenylated GM-CSF
mRNA relative to the deadenylated species, so that the latter was virtually undetectable in most Northern blots. In other words, TTP deficiency not only led to stabilization of the full-length GM-CSF mRNA but also greatly inhibited the formation of the
deadenylated mRNA body from the polyadenylated full-length mRNA.
These data strongly support a model in which TTP deficiency inhibits
the first important step in class II ARE-containing mRNA degradation, the processive removal of the poly(A) tail by 3' to 5'
exonuclease activity. Conversely, they also support a model in which
TTP binding to the ARE in some way increases the rate of deadenylation
of the full-length GM-CSF mRNA.
In the current studies, we attempted to gain further insight into the
importance of the poly(A) tail in CCCH protein-stimulated turnover of
mRNAs containing class II AREs. To do this, we performed three
types of experiments: 1) co-expression of full-length transcripts for
TNF The first group of studies, using full-length mRNAs for TNF In the experiments in which the CCCH proteins stimulated
the breakdown of full-length TNF Whatever the mechanism of this effect, several factors suggest that
this accumulation of a 3'-truncated intermediate that is even shorter
than the deadenylated mRNA body is an artifactual consequence of
the expression of high concentrations of the CCCH proteins. First, as
noted above, this only occurs at concentrations of expressed CCCH
proteins that are at least 10-fold greater than the concentration
required to promote maximum disappearance of the ARE-containing
mRNAs (8). Even when the concentration dependence of the TTP effect
was assessed in the presence of actinomycin D, there was a striking
effect of TTP to decrease native IL-3 mRNA accumulation at 5 ng of
DNA/plate, whereas the first accumulation of the further 3'-5'
truncated intermediate did not occur until ~100 ng of TTP
DNA.2 Second, when even
higher concentrations of nonbinding tandem zinc finger domain mutants
have been co-transfected with ARE-containing mRNAs, we have never
seen accumulation of the 3'-5'-most truncated intermediate (8). Third,
when only the tandem zinc finger domain of TTP was transfected, the
3'-5'-most truncated intermediate was readily seen, although the
overall decrease in full-length ARE-containing mRNA was less than
that seen with full-length TTP (9). Fourth, although we have looked
hard for the formation of such an intermediate derived from TNF However, an interesting study by Yang et al. (21) detected a
degradation intermediate in IL-11 mRNA in PU-34 cells, a primate bone marrow stromal cell line. The IL-11 mRNA contains several class II ARE-like sequences in its 3'-UTR (see GenBankTM
accession number M57766.1). These authors established that the
intermediate identified in these cells was truncated from the 3'-end to
approximately base 790 (corresponding to approximately base 700 in
accession number M57766.1), a position within 7-24 bases of the
longest ARE-like sequence (UUAUUUAUUUAUUU; bases 707-724 in M57766.1).
They showed that deletion of this sequence did not affect the stability
of the IL-11 mRNA in transfection studies in this cell type, and
they were unable to determine whether the intermediate was formed by an
endonuclease or a 3'-5'- exonuclease activity. It will be of interest
to determine whether this ARE sequence can bind CCCH proteins and, more
importantly, whether it contributes to TTP-mediated IL-11 mRNA
instability in bone marrow-derived stromal cells or other cell types
derived from wild-type and TTP-deficient mice.
To evaluate the requirement for a poly(A) tail in TTP activity, we took
advantage of the fact that some mammalian histone mRNAs do not
contain poly(A) tails. Instead, they rely on the processing of a
characteristic 3' stem-loop-containing sequence to permit export of the
mature mRNA from the nucleus (17, 22). We used this information to
construct two types of expressed mRNAs. In the first type, we
inserted class II ARE sequences from the TNF These experiments showed that, in all cases, the co-expression of
wild-type TTP, but not of a nonbinding mutant, led to the increased
destruction of the ARE-containing mRNAs, although no poly(A) tails
were present in these mRNAs. In the case of the IL-3/histone
chimeric mRNA, there was accumulation of a stable intermediate in
the TTP-treated samples, in which the mRNA was truncated at the
3'-end; as seen with the wild-type IL-3 transcript, the 3'-end of the
stable intermediate appeared to correspond approximately to the site of
the naturally occurring IL-3 ARE and was only seen at the highest
concentration of transfected TTP DNA used. In the case of the
ARE-containing histone mRNA and the TNF How can we explain this effect of TTP and its related CCCH proteins, in
light of the data described above, using cells derived from TTP
knockout mice, in which the predominant initial effect of TTP appears
to be stimulation of deadenylation? One possibility is that TTP could
recruit or activate one or more endonucleases to act on the
mRNA, perhaps at a site at or near the ARE itself. For example,
Schmid and colleagues (23) have recently described a proteasomal
endonuclease that can cleave RNA at class II ARE sequences. However,
the sine qua non for implicating endonuclease activity is
the demonstration of both 5' and 3' cleavage fragments; these have not
been detected in our studies, either in cell transfection experiments
or in naturally occurring, TTP-expressing cells. A second possibility
is that TTP could functionally activate a 3' to 5' exonuclease activity
toward the mRNA, even in the absence of a poly(A) tail but in the
presence of a histone mRNA 3'-end-terminal sequence. This could be
achieved by recruiting a protein or protein complex containing
exonuclease activity to the 3'-end of the transcript, by activating
such an activity, or by displacing ARE-protecting or 3'-end-protecting
proteins such as the poly(A)-binding protein. The effect on the
transcripts containing histone 3' sequences might be mediated by the
same type of displacement of protecting proteins (e.g. the
histone stem loop-binding protein (see Ref. 22 for review)). Whatever
model of TTP action is eventually proved to be correct, it must be able
to explain the ability of TTP and its related CCCH proteins to
stimulate the turnover of mRNAs lacking poly(A) tails.
(TNF
) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its
related CCCH proteins into 293 cells with vectors encoding full-length
TNF
, granulocyte-macrophage colony-stimulating factor, and
interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the
rapid turnover of these ARE-containing mRNAs and also promoted the
accumulation of stable breakdown intermediates that were truncated at
the 3'-end of the mRNA, even further 5' than the 5'-end of the
poly(A) tail. To determine whether an intact poly(A) tail was necessary
for TTP to promote this type of mRNA degradation, we inserted the
TNF
ARE into a nonpolyadenylated histone mRNA and also attached
a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated
interleukin-3 and TNF
mRNAs. In all three cases, TTP stimulated
the turnover of the ARE-containing mRNAs, despite the demonstrated
absence of a poly(A) tail. These studies indicate that members of this
class of CCCH proteins can promote class II ARE-containing mRNA
turnover even in the absence of a poly(A) tail, suggesting that the
processive removal of the poly(A) tail may not be required for this
type of CCCH protein-stimulated mRNA turnover.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF
) (6) and granulocyte-macrophage
colony-stimulating factor (GM-CSF) (7), an effect mediated by TTP's
ability to destabilize the mRNAs encoding both proteins. TTP
mediates this effect after initially binding to the ARE in the
3'-UTRs of these mRNAs (6-8). In transfection experiments, the
known vertebrate TTP-related proteins had similar effects to promote
the breakdown of these ARE-containing mRNAs (9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, GM-CSF, and Interleukin (IL)-3 mRNAs
(Fig. 1,
A-C)--
CMV.mTNF
-(127-1325), containing a
NarI-XbaI fragment spanning bp 127-1325 of a
mouse TNF
cDNA sequence (GenBankTM accession number
X02611), was made as described (8). The mTNF
cDNA clone,
provided by Dr. B. Beutler (University of Texas Southwestern Medical
Center, Dallas, TX), contained an incomplete 3'-UTR that ended at bp
1325 of GenBankTM accession number X02611, with 33 adenylate residues attached to the last T. This sequence is shown in
Fig. 1 of Ref. 9. To make CMV.mTNF
-(1-1627), a full-length
cDNA coding for the mouse TNF
mRNA sequence was prepared by
RT-PCR using total cellular RNA from RAW 264.7 cells treated for 4 h with 1 µg/ml of lipopolysaccharide as the template for RT.
This TNF
cDNA spanned bp 1-1627 of GenBankTM
accession number X02611 and contained the naturally occurring polyadenylation signal at bp 1614-1619. The 5' primer for PCR amplification was 5'-gtcgacCTCAGCGAGGACAGCAAGG-3', and the 3' primer
was 5'-tctagaAGCGATCTTTATTTCTCTC-3'. The capital letters in the 5'
primer contain the 5'-end of the TNF
mRNA, and the capital
letters in the 3' primer contain the 3'-end of the mRNA. The
lowercase letters in the primers indicate the restriction sites for
SalI and XbaI, respectively.
View larger version (13K):
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Fig. 1.
Schematic representation of plasmid
constructs. Constructs were made as described under
"Experimental Procedures." A, CMV.mTNF -(1-1627)
contains the full-length mouse TNF
cDNA. The ARE is located at
bp 1302-1332 of GenBankTM accession number X02611 as
indicated. B, CMV.mGM-CSF contains a 775-bp full-length
mouse GM-CSF cDNA. The ARE is located at bp 668-722 of the
cDNA. The numbers in parentheses are the
corresponding bp numbers of the mouse GM-CSF gene
(GenBankTM accession number X03020). C,
CMV.mIL-3 contains an 851-bp full-length mouse IL-3 cDNA. The ARE
is located at bp 700-732 of the cDNA. The numbers in
parentheses are the corresponding bp numbers of the mouse
IL-3 gene (GenBankTM accession number K03233).
D, CMV.H2a contains a 549-bp mouse histone H2a sequence that
corresponds to bp 814-1362 (in parentheses) of
GenBankTM accession number X16148. An EcoRV site
was created at bp 352 (bp 1145 in GenBankTM accession
number X16148).
-(1-1627) and
mGM-CSF) or with SalI and SstII (for mIL-3) and
cloned into the SalI and XbaI sites or the
SalI and SstII sites of the vector pSK
, which
contains the human CMV promoter-enhancer (9) (Fig. 1, A-C).
The correct sequences of the 1627-bp mouse TNF
, the 775-bp mouse
GM-CSF, and the 851-bp mouse IL-3 cDNA inserts were
confirmed by dRhodamine Terminator Cycle Sequencing (PerkinElmer Life Sciences).
(8) (Fig. 1D). The correct sequence
of the PCR product and the correct orientation of H2a sequence in
relationship to the CMV promoter were verified by dRhodamine Terminator
Cycle Sequencing. To create plasmid CMV.H2a (EN),
b1166 of X16148 was mutated from C to A, and
b1167 was mutated from G to T, to make an EcoRV
restriction site in the H2a sequence using the PCR primer-overlapping
mutagenesis technique. Plasmids CMV.H2a (mTNF
) (containing bp
1281-1350 of GenBankTM accession number X02611), CMV.H2a
(mGM-CSF) (containing bp 3399-3467 of GenBankTM accession
number X03020), and CMV.H2a (mIL-3) (containing bp 2738-2770 of
GenBankTM accession number K03233) were constructed by
inserting the sequences coding for the AREs of these mRNAs into the
EcoRV site of plasmid CMV.H2a (EN). Plasmid CMV.H2a (AX75)
was constructed by inserting a blunt-ended 75-bp fragment containing
the multiple cloning site sequence (from the Asp718 to
XbaI sites) from plasmid vector SK- into the
EcoRV site of CMV.H2a (EN).
/Histone mRNAs
(See Fig. 7C)--
The mouse IL-3 expression vector CMV.mIL-3 (H2aSL),
in which the polyadenylation signal was replaced by the 3'-end
stem-loop sequence of mouse histone H2a, was created as follows. Using
the PCR primer-overlapping mutagenesis technique (14), the sequence in
the 3'-UTR of mIL-3, ggaAATAAAtaa, which contained the normal IL-3
mRNA polyadenylation signal (in capital letters), was mutated to
ggaAAGATAtca (mutated bases in italic
type) to include an EcoRV restriction site (gatatc). A 60-bp
double-stranded oligonucleotide, flanked by the EcoRV
sequence, which encoded the histone H2a stem-loop and histone
downstream element (bp 1303-1362 of GenBankTM accession
number X16148), was inserted into this newly created EcoRV
site in CMV.mIL-3.
expression vector CMV.mTNF
-(127-1325 H2aSL), the 33 adenylate residues attached to the last T (bp 1325 of
GenBankTM accession number X02611) of CMV.mTNF
were
deleted using the PCR primer-overlapping mutagenesis technique. An
XbaI restriction site was also created in the same process.
The 60-bp double-stranded oligonucleotide coding for the histone H2a
stem-loop and histone downstream element was inserted into this newly
created XbaI site at the end of CMV.mTNF
-(127-1325). The
correct orientation of the histone sequence was confirmed by dRhodamine
terminator cycle sequencing.
-32P-labeled cDNA
probes coding for various CCCH zinc finger proteins, including mouse
TTP (1), Xenopus XC3H-3 (15), or rat cMG1 (16). Blots were
also hybridized as indicated with a ~1-kb
NarI-BglII fragment of a mTNF
cDNA (8), a
422-bp SalI-EcoRV 5' fragment of mouse GM-CSF
cDNA (from plasmid CMV.mGM-CSF), and a 468-bp SalI-XbaI 5' fragment of mouse IL-3 cDNA
(from plasmid CMV.mIL-3). Some blots were also hybridized to an
-32P-labeled GAPDH cDNA probe (6) to monitor gel loading.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mRNA
construct, in which 33 A residues encoded by the vector were attached
to the middle of the ARE. In the present experiments, we wished to
confirm that TTP and related proteins would exert similar effects on
three naturally occurring mRNAs that contain class II AREs in their
3'-UTRs: TNF
, GM-CSF, and IL-3. As in our previous studies, we used
human genomic and cDNA clones encoding TTP, the rat cMG1 cDNA
as the representative of the cMG1/TIS11b/ERF1/Berg36 protein, and the
Xenopus XC3H-3 cDNA as the representative of the
TIS11d/ERF2 protein.
cDNA that was truncated at both the 5'- and 3'-ends
(bases 127-1325 of accession number X02611) in 293 cells, all three
proteins caused a decrease in steady state levels of TNF
mRNA at
low concentrations (9). A characteristic finding was that increasing
concentrations of transfected DNA encoding these CCCH proteins resulted
in the accumulation of a truncated species of the TNF
mRNA,
which we have shown previously to be truncated at the 3'-end. An
example of this type of experiment using the TNF
clone truncated at
both ends is shown in the upper panel of Fig.
2A, lanes
1-5; the corresponding levels of CCCH protein mRNAs are
shown in the lower panel. Compared with
lane 1, which shows the steady state level of
TNF
mRNA in cells transfected with vector DNA instead of the
CCCH protein DNA, lane 2 shows the characteristic
decrease in total hybridizable TNF
mRNA that occurs with low
level expression of TTP, achieved in this case by the co-transfection
of the genomic human TTP clone (5 µg DNA); the low level of TTP
mRNA expression achieved with this concentration of the genomic
clone DNA is shown in lane 2, lower
panel. At higher levels of CCCH mRNA expression, TTP,
cMG1, and XC3H-3 (at 1 µg of DNA/plate) all caused the characteristic
disappearance of the upper band of TNF
mRNA and the accumulation
of the lower band that we previously identified as a deadenylated
species.
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Fig. 2.
Effect of TTP and related proteins on the
stability of full-length and truncated TNF
mRNA. Expression constructs containing different lengths
of the mouse TNF
cDNA were co-transfected into 293 cells with
TTP or other CCCH zinc finger protein expression constructs or with
vector alone, as indicated. Total cellular RNA was harvested as
described under "Experimental Procedures." Each gel lane was loaded
with 10 µg of total cellular RNA. Electrophoresis and Northern
hybridization were performed as described under "Experimental
Procedures." A, upper panel,
lanes 1-5, RNA from 293 cells co-transfected
with a CMV.mTNF
construct encoding bases 127-1325 of
GenBankTM accession number X02611; lanes
6-10, RNA from 293 cells co-transfected with a CMV.TNF
construct encoding bases 127-1627; lanes 11-15,
RNA from 293 cells co-transfected with a CMV.TNF
construct encoding
bases 1-1627. Vector or CCCH zinc finger protein expression constructs
were co-transfected as follows. Lanes 1,
6, and 11, vector alone (BS+; 5 µg/plate);
lanes 2, 7, and 12,
H6E.HGH3' (5 µg/plate); lanes 3, 8,
and 13, CMV.hTTP.tag (1 µg/plate); lanes
4, 9, and 14, CMV.cMG1.tag (1 µg/plate); lanes 5, 10, and
15, CMV.XC3H-3.tag (1 µg/plate). For lanes
3-5, 8-10, and 13-15, vector DNA
was also added to make the total co-transfected plasmid DNA equal to 5 µg/plate. The Northern blot was probed with a 32P-labeled
mTNF
cDNA probe (bp 127-1200). The position of the 18 S
ribosomal RNA is indicated. The arrows labeled
TNF
indicate the two or three species of TNF
mRNA
discussed. In the lower panel of A,
identical RNA samples as in the upper panel were
blotted and probed simultaneously with 32P-labeled mouse
TTP, rat cMG1, and Xenopus XC3H-3 cDNAs, and the
corresponding mRNAs are shown in the lower
panel; the positions of these expressed CCCH mRNAs are
indicated by the arrow. The position of the 18 S ribosomal
RNA is indicated. B, some of the same RNA samples described
in A, as indicated by the lane
numbers, were probed with a 32P-labeled mTNF
cDNA probe that spans only from base 1370 to 1627 of the mRNA,
between the ARE and the poly(A) tail. The position of the 18 S
ribosomal RNA is indicated. The TNF
mRNA species indicated by
the two arrows are the same as those indicated by
the upper two arrows in
lanes 6-15 of A. Note that the TNF
species indicated by the two bands in
lanes 1-5 of A and by the
lowest band in lanes 6-15
of A are not detected by this extreme 3' probe.
that contained the full 3'-end, the pattern of TNF
hybrizidation was somewhat different. In cells transfected with vector
alone and no CCCH protein constructs (Fig. 2A,
lane 6, upper panel), the
TNF
mRNA appeared as two closely spaced bands; the upper band,
representing the expressed full-length cDNA, was partially obscured
by the 18 S ribosomal RNA. The lower of the two bands was shown by
RNase H experiments using oligo(T) primers to be the deadenylated form
of the mRNA (data not shown). In the presence of expressed CCCH
proteins, however (lanes 8-10), a third band
appeared that was of identical size to the 3'-truncated mRNA shown
in lanes 3-5. Because the 5'-end of this
construct was the same as that of the construct used for
lanes 1-5, we conclude that the 3'-truncation of
the TNF
mRNA caused by expression of the CCCH proteins was to
approximately the same location as that seen in the truncated mRNA
used in lanes 1-5.
construct that was
full-length in both the 5'- and 3'-ends, bases 1-1627 of accession
number X02611 (lanes 11-15 of the
upper panel, Fig. 2A). In this case,
the full-length TNF
mRNA in the control lane (lane
11) was obscured by the ribosomal RNA. Nonetheless, it was still possible to detect a decrease in the amount of the full-length TNF
mRNA when co-expressed with the CCCH proteins, with
accumulation of a 3'-truncated band of larger size than that seen with
the other two TNF
constructs, compatible with its 127-base increase in length.
seen after expression of
each of the three CCCH proteins was likely to be truncated to
approximately position 1325 in the TNF
clone (i.e. at the middle of the class II ARE). To confirm that the effect of the co-transfected CCCH proteins was to remove this 3'-end, several of the
RNA samples shown in Fig. 2A were hybridized with a probe spanning bases 1370-1627 of accession number X02611 (Fig.
2B). This probe represents a sequence entirely 3' of the ARE
(which occurs between bases 1299 and 1332) and includes the
polyadenylation signal (bases 1614-1619). As expected, this probe did
not hybridize to any TNF
mRNA species when the 127-1325 TNF
clone was transfected, either in the absence (lane
1) or the presence (lane 2) of
co-transfected TTP. However, the probe readily hybridized to the
two bands representing the longer TNF
mRNA species, either
127-1627 (lanes 6-10) or 1-1627
(lanes 11-15). This result confirms that the
shortest of the three major TNF
species generated in response to
CCCH protein expression in Fig. 2A represents a 3'-truncated
form of TNF
mRNA that is missing not only the poly(A) tail but
also sequences between the poly(A) tail and the ARE.
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Fig. 3.
Effect of TTP and related zinc finger
proteins on the stability of GM-CSF mRNA. Cell transfection,
total cellular RNA preparation, electrophoresis, and Northern
hybridization were performed as described under "Experimental
Procedures." Each lane was loaded with 10 µg of total
cellular RNA. A, upper panel, effect
of CCCH proteins on GM-CSF mRNA stability. CMV.mGM-CSF (1 µg/plate) was co-transfected into 293 cells with TTP, cMG1, or XC3H-3
expression constructs or with vector alone. Lane
1, vector alone (BS+; 5 µg of DNA/plate); lanes
2-4, CMV.hTTP.tag (0.1, 1, and 5 µg/plate, respectively);
lanes 5-7, CMV.cMG1.tag (0.1, 1, and 5 µg/plate, respectively); lanes 8-10,
CMV.XC3H-3.tag (0.1, 1, and 5 µg/plate, respectively). For
lanes 2, 3, 5,
6, 8, and 9, vector was also added to
make the total amount of co-transfected DNA 5 µg/plate. B,
upper panel, relative stability of GM-CSF
mRNA in the presence of TTP and actinomycin D. CMV.mGM-CSF (1 µg/plate) was co-transfected into 293 cells with hTTP expression
constructs or vector alone (BS+). Lanes 1,
4, 7, 10, and 13, 5 µg/plate of vector was co-transfected (BS+). Lanes
2, 5, 8, 11, and
14, 5 µg/plate of H6E.HGH3' was co-transfected.
Lanes 3, 6, 9,
12, and 15, 0.1 µg/plate of CMV.hTTP.tag was
co-transfected, together with 4.9 µg/plate of vector DNA. Total
cellular RNA was harvested after the addition of actinomycin D to a
final concentration of 10 µg/ml for 1, 2, 4, or 8 h or buffer
alone for 8 h ( ), as described under "Experimental
Procedures." The Northern blots shown in the upper
panels of both A and B were probed
with a 32P-labeled mGM-CSF cDNA, and the three major
species of GM-CSF mRNA present are indicated by the
arrows. After autoradiography, the blots were stripped and
reprobed with a 32P-labeled GAPDH probe, and the results
are shown in the middle panels of both
A and B. Identical RNA samples as in the
upper panels were blotted and probed
simultaneously with 32P-labeled mouse TTP cDNA, rat
cMG1 cDNA, and Xenopus XC3H-3 cDNA (A) or
with the TTP cDNA alone (B), and the results are shown
in the lower panels of both A and
B. The position of the 18 S ribosomal RNA is indicated in
each case. The expressed GAPDH, TTP, and other CCCH zinc finger protein
mRNAs are also indicated by arrows.
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Fig. 4.
Effect of TTP and related proteins on the
stability of IL-3 mRNA. Cell transfection, total cellular RNA
preparation, electrophoresis, and Northern hybridization were performed
as described under "Experimental Procedures." Each lane
was loaded with 10 µg of total cellular RNA. A,
upper panel, relative stability of IL-3 mRNA
in the presence of TTP, cMG1, or XC3H-3. CMV.mIL-3 (1 µg/plate) was
co-transfected into 293 cells with CCCH protein expression constructs
or vector alone. Lanes 1, 6,
11, and 16, 5 µg/plate of vector was
co-transfected (BS+). Lanes 2, 7,
12, and 17, 5 µg/plate of H6E.HGH3' was
co-transfected. Lanes 3, 8,
13, and 18, 0.1 µg/plate of CMV.hTTP.tag
together with 4.9 µg/plate of vector BS+ was co-transfected.
Lanes 4, 9, 14, and
19, 1 µg/plate of CMV.cMG1.tag was co-transfected.
Lanes 5, 10, 15, and
20, 1 µg/plate of CMV.XC3H-3.tag was co-transfected. For
plates co-transfected with cMG1 or XC3H-3 expression constructs, 4 µg/plate of BS+ was also added. Total cellular RNA was harvested
after the addition of actinomycin D to a final concentration of 10 µg/ml (+ActD) for 1, 2, or 4 h or buffer alone
( ActD), as described under "Experimental Procedures."
B, evidence that one of the smaller species of IL-3 mRNA
formed in the presence of CCCH proteins is a deadenylated intermediate.
Cells were co-transfected with CMV.mIL-3 (1 µg/plate) and either
vector alone or zinc finger expression constructs as follows.
Lanes 1 and 5, vector alone (5 µg/plate; BS+); lanes 2 and 6,
CMV.hTTP.tag (0.1 µg/plate); lanes 3 and
7, CMV.cMG1.tag (1 µg/plate); lanes
4 and 8, CMV.XC3H-3.tag (1 µg/plate). 5 µg of
RNA was loaded into each gel lane. Oligonucleotide
(dT)12-18 (1 µg) was added to samples 5-8. As
indicated, the RNA samples were treated with (+) or without (
) 1 unit
of RNase H as described under "Experimental Procedures." The
Northern blots were probed with a 32P-labeled mouse IL-3
cDNA. The position of the 18 S ribosomal RNA is indicated. The
three arrows indicate IL-3 mRNA species that
contained (FL) or did not contain (dA) their
poly(A) tails or were further truncated in a 3'-5' direction
(P). C, upper panel, effect
of TTP on the stability of wild-type and ARE-deleted mIL-3 mRNA. 1 µg/plate of CMV.mIL-3 or CMV.mIL-3 (dARE) was co-transfected into 293 cells with TTP expression constructs or vector alone. Lanes
1-6, wild-type CMV.mIL-3; lanes
7-12, CMV.mIL-3 (dARE), in which ARE-containing sequence bp
2738-2771 was deleted. Lanes 1 and 7,
5 µg/plate of vector (BS+) was co-transfected; lanes
2, 3, 8, and 9, H6E.HGH3'
was co-transfected in the indicated amounts (µg/plate);
lanes 4-6 and 10-12, CMV.hTTP.tag
was co-transfected in the indicated amounts (µg/plate). For
lanes 2, 4-6, 8, and
10-12, vector DNA was also added to make the total
co-transfected plasmid DNA equal to 5 µg/plate. The Northern blots
shown in the upper panels of both A
and C were probed with a 32P-labeled mIL-3
cDNA. After autoradiography, these blots were then stripped and
reprobed with a 32P-labeled GAPDH probe, and the results
are shown in the middle panels of A
and C. Identical RNA samples as in the upper
panels were blotted and probed simultaneously with
32P-labeled mouse TTP, rat cMG1, and Xenopus
XC3H-3 cDNAs (A) or the TTP cDNA alone
(C), and the results are shown in the lower
panels of A and C. The position of the
18 S ribosomal RNA is indicated. The three arrows
in the upper panels of A and
C indicate the three species of IL-3 mRNA discussed. The
expressed GAPDH, TTP, or other CCCH zinc finger protein mRNAs are
also indicated by arrows.
and GM-CSF data, these results are compatible with a
CCCH protein-mediated 3'-truncation of the mRNA that was greater
than that seen with deadenylation alone. All three further truncations appeared to be approximately at the location of the ARE in these mRNAs.
poly(A) tail with a histone 3'-end-processing sequence (for a review,
see Ref. 17). In the first series of experiments, we used a histone H2a
precursor RNA coding sequence from a mouse genomic clone for
H2A and H3 (accession number X16148) driven by
the CMV promoter and containing the normal histone 3'-processing
sequences without an added polyadenylation signal or poly(A)
tail. As shown in Fig. 5A
(lane 1), the mouse histone cDNA probe
hybridized to an endogenous human histone transcript of ~0.5 kb in
mock-transfected 293 cells. When the histone genomic construct was
co-transfected into cells with vector alone, a prominent 0.5-kb band of
histone mRNA was detected whose levels were ~3 times greater than
that of the endogenous histone, as determined by PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA) analysis (lane 2). Co-transfection with TTP had little or
no effect on the histone mRNA expression at 5 and 10 ng of
co-transfected DNA (lanes 3 and 4);
however, at 100 ng of TTP DNA, a decrease in expression of histone
mRNA was apparent (lane 5). The zinc finger TTP mutant C124R had no effect on histone mRNA expression at 10 and
100 ng of the mutant DNA (lanes 6 and
7). When an otherwise identical histone construct was
expressed that contained the 70-bp TNF
ARE, the resulting
steady-state level of mRNA was approximately the same as that seen
with the wild-type histone (lane 8). In addition,
the added sequence allowed the mRNA expressed from the transfected
DNA to be separated from the endogenous histone mRNA on the blot
(lane 8). However, when TTP was co-transfected at 5, 10, and 100 ng of DNA, there was a marked decrease in the
accumulation of histone mRNA (lanes 9-11),
with minimal effect on endogenous histone mRNA levels. The zinc
finger mutant of TTP had little or no effect on the histone-ARE
mRNA levels (lanes 12 and 13). Otherwise identical experiments were performed with the wild-type histone sequence into which a EcoRV restriction site had
been made by mutating bp 1167 and 1168 of accession number X16148 from
C to A, and G to T, respectively. Except at the highest concentration of transfected TTP (100 ng of DNA; lane 17), both
wild-type and mutant TTP had little effect on the expression of the
modified histone mRNA (lanes 14-19).
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Fig. 5.
Effect of TTP on the stability of
ARE-containing histone mRNA. Cell transfection, total cellular
RNA preparation, electrophoresis, and Northern hybridization were
performed as described under "Experimental Procedures." Each lane
was loaded with 10 µg of total cellular RNA. A,
upper panel, effect of TTP on H2a (TNF )
mRNA stability. Histone H2a expression constructs with or without
the TNF
ARE insertion were co-transfected into 293 cells with
CMV.hTTP.tag, its zinc finger mutant C124R, or vector alone (BS+).
Lane 1 (mock), vector BS+ (5 µg/plate) alone
was transfected. Lanes 2-7, 1 µg/plate of
CMV.H2a was transfected. Lanes 8-13, 1 µg/plate of CMV.H2a (TNF
) was transfected. Lanes
14-19, 1 µg/plate of CMV.H2a (EN) was transfected.
Lanes 2, 8, and 14, 4 µg/plate of vector was co-transfected (BS+). The following
lanes are from plates co-transfected with the
CMV.hTTP.tag. Lanes 3, 9, and
15, 5 ng/plate; lanes 4,
10, and 16, 10 ng/plate; lanes
5, 11, and 17, 100 ng/plate. The
following lanes are from plates co-transfected with the
CMV.hTTP.tag mutant C124R. Lanes 6,
12, and 18, 10 ng/plate; lanes
7, 13, and 19, 100 ng/plate. For
plates co-transfected with the TTP expression constructs, vector BS+
was also added to make the total transfected plasmid DNA 5 µg/plate.
B, upper panel, effect of TTP on the
stability of ARE-containing H2a mRNA. Histone H2a expression
constructs (1 µg/plate) containing ARE (lanes
1-4, CMV.H2a (TNF
ARE); lanes
5-8, CMV.H2a (mIL-3 ARE); lanes
9-12, CMV.H2a (mGM-CSF ARE)); lanes
13-16, random sequence (CMV.H2a (AX75)) insertions were
co-transfected into 293 cells with CMV.hTTP.tag or vector alone.
Lanes 1, 5, 9, and
13, vector (4 µg/plate; BS+) was transfected. The
following lane numbers indicate the amounts of
co-transfected CMV.hTTP.tag DNA. Lanes 2,
6, 10, and 14, 1 ng/plate;
lanes 3, 7, 11, and
15, 5 ng/plate; lanes 4, 8,
12, and 16, 10 ng/plate. For plates
co-transfected with the TTP expression construct, vector BS+ was also
added to make the total transfected plasmid DNA 5 µg/plate. The
Northern blots shown in the upper panels were
probed with a 32P-labeled mouse H2a fragment (bp 814-1362
of X16148). The arrow labeled endo indicates
endogenous histone mRNA. In both A and B,
identical RNA samples as in the upper panels were
blotted and probed with 32P-labeled mouse TTP cDNA, and
the results are shown in the lower panels, in
which the TTP mRNA is indicated by an arrow. The
position of the 18 S ribosomal RNA is indicated. C, evidence
that the transfected and expressed H2a mRNA did not contain a
poly(A) tail, either in the absence or presence of TTP. Cells were
co-transfected with plasmids CMV.H2a (EN) (1 µg/plate;
lanes 3-6), CMV.H2a (TNF
ARE) (1 µg/plate;
lanes 7-10), or CMV.H2a (AX75) (1 µg/plate;
lanes 11-14) together with CMV.hTTP.tag or with
vector (BS+) alone. Lanes 1 and 2 (mock), vector (5 µg/plate; BS+) was transfected; lanes
3, 4, 7, 8, 11, and 12, vector (4 µg/plate; BS+) was co-transfected; lanes 5,
6, 9, 10, 13, and
14, CMV.hTTP.tag (5 ng/plate) and vector BS+ (4 µg/plate)
were co-transfected. 10 µg of RNA was loaded into each gel lane.
Oligonucleotide (dT)12-18 (1 µg) was added to samples 2, 4, 6, 8, 10, 12, and 14, as indicated by the plus
sign. All RNA samples were treated with 1 unit of RNase H as
described under "Experimental Procedures." The Northern blots were
probed with a 32P-labeled mouse H2a fragment (bp 814-1362
of X16148). The endogenous histone mRNA is indicated by the
arrow labeled endo in lane
1.
(Stratagene; bp 653-731) was inserted into the
EcoRV site of the modified CMV.H2a histone sequence. The
effect of TTP on the accumulation of this hybrid mRNA was assessed
along with its effect on the ARE-containing histone mRNA. This
experiment used lower concentrations of TTP DNA (1.0-10 ng) to prevent
the apparent nonspecific effects of higher concentrations of TTP on histone mRNA levels. As shown in Fig. 5B
(upper panel), TTP (5 and 10 ng of DNA) again
appeared to decrease the accumulation of histone-ARE mRNA at
(lanes 1-4). We also tested whether the AREs
from GM-CSF and IL-3, as well as TNF
, could make the histone mRNA susceptible to TTP-induced degradation. As shown in Fig. 5B (upper panel), insertion of the
AREs from IL-3 (lanes 5-8) and GM-CSF
(lanes 9-12) all conferred TTP sensitivity on
the histone mRNA, at similar TTP concentrations. However, the
control histone sequence that contained the insert of similar size from
pSK
was unaffected by the increasing amounts of co-transfected TTP
(lanes 13-16). The low concentrations of TTP DNA
used in this experiment had minimal nonspecific effects on histone gene
transcription, as confirmed by the lack of effect on the
pSK
-containing histone (lanes 13-16). The low
concentration of TTP required for mRNA degradation is illustrated
by a comparison of the upper and lower panels of Fig. 5B. Identical cpm of the histone
probe (upper panel) and TTP probe
(lower panel) were added to two identical blots in Fig. 5B; however, the autoradiographic exposure of the
blot shown in the upper panel was 45 min at
70 °C, whereas the exposure of the blot shown in the
lower panel was 16 h at the same temperature.
ARE-containing histone mRNA (lanes 7-10) in
the absence (lanes 7 and 8) or
presence (lanes 9 and 10) of TTP; or
the histone mRNA modified with the 75-bp pSK
insert
(lanes 11-14) in the absence (lanes
11 and 12) or presence (lanes
13 and 14) of TTP. These data confirm that the transfected histone pre-mRNA was appropriately 3'-processed and that spurious poly(A) tail addition did not take place in the presence
or absence of TTP.
mRNAs Containing Histone
3'-End-processing Sequences--
To further test the hypothesis that a
poly(A) tail was not necessary for TTP activity, we replaced the
naturally occurring polyadenylation signal in the IL-3 mRNA and the
33 adenine residues in the TNF
expression construct with the histone
3'-end-processing sequence (see schematic illustrations of these
constructs in Figs. 6C and
7C). These constructs were
transfected into 293 cells in the presence or absence of TTP.
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Fig. 6.
Effect of TTP on an IL-3 mRNA containing
a histone 3'-end-processing sequence. Cell transfection, total
cellular RNA preparation, electrophoresis, and Northern hybridization
were performed as described under "Experimental Procedures."
A, each lane was loaded with 10 µg of total cellular RNA.
Upper panel, stability of IL-3 mRNA expressed
with or without a poly(A) tail in the presence of TTP. CMV.mIL-3 or
CMV.mIL-3 (H2aSL) (1 µg/plate, indicated as Control or
H2aSL) was co-transfected into 293 cells with CMV.hTTP.tag
or vector alone. Lanes 1 and 6, 5 µg/plate of vector was transfected (BS+). The following
lanes are from plates co-transfected with the CMV.hTTP.tag.
Lanes 2 and 7, 1 ng/plate;
lanes 3 and 8, 5 ng/plate;
lanes 4 and 9, 10 ng/plate;
lanes 5 and 10, 100 ng/plate. For
plates co-transfected with the TTP expression constructs, vector BS+
was also added to make the total transfected plasmid DNA 5 µg/plate.
The Northern blot was probed with a 32P-labeled mIL-3
cDNA. Identical RNA samples as in the upper
panel were blotted and probed simultaneously with
32P-labeled mouse TTP cDNA, and the results are shown
in the lower panel of A. The position
of the 18 S ribosomal RNA is indicated. The three
arrows indicate the three species of IL-3 mRNA
discussed. The expressed TTP mRNA is also indicated by an
arrow in the lower panel.
B, evidence that the species of IL-3 mRNA expressed from
transfected CMV.mIL-3 (H2aSL) did not contain a poly(A) tail.
Lanes 1-4, cells were transfected with CMV.mIL-3
(1 µg/plate, Control). Lanes 5-8,
cells were transfected with CMV.mIL-3 (H2aSL) (1 µg/plate,
H2aSL). The cells were co-transfected with either vector
alone (4 µg/plate; BS+; lanes 1, 2,
5, and 6) or CMV.hTTP.tag (10 ng/plate together
with 4 µg/plate of vector; lanes 3,
4, 7, and 8). 5 µg of RNA was loaded
into each gel lane. Oligonucleotide (dT)12-18 (1 µg) was
added to samples 2, 4, 6, and 8; all RNA samples were treated with 1 unit of RNase H as described under "Experimental Procedures." The
Northern blot was probed with a 32P-labeled mouse IL-3
cDNA. The two arrows indicate IL-3 mRNA
species that contained or did not contain poly(A) tails. C,
schematic representation of the CMV.mIL-3 (H2aSL) construct. A 3'
portion of the mouse histone H2a sequence containing the stem-loop
(underlined, in capital letters) and
histone downstream element (HDE; in capital
letters) was inserted into an EcoRV site that was
created to replace the polyadenylation signal in the mIL-3 cDNA in
the CMV.mIL-3 expression construct. The 3'-end of the H2a mRNA
(C) is indicated by italic type.
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Fig. 7.
Effect of TTP on a TNF
mRNA containing a histone 3'-end-processing sequence.
Cell transfection, total cellular RNA preparation, electrophoresis, and
Northern hybridization were performed as described under
"Experimental Procedures." A, each lane was
loaded with 10 µg of total cellular RNA. Upper
panel, stability of TNF
mRNA expressed with or
without a poly(A) tail in the presence of TTP. TNF
expression
constructs (1 µg/plate) were co-transfected into 293 cells with
CMV.hTTP.tag or vector alone. Lanes 1-3
(Control), CMV.TNF
-(127-1325); lanes
4-6 (H2aSL), CMV.TNF
-(127-1325 H2aSL), in
which the 33 3'-terminal adenylate residues were replaced with the
histone stem loop and histone downstream element as described under
"Experimental Procedures." Lane 7 (dA), CMV.TNF
-(127-1325), in which the 33 adenylate
residues attached to the 3'-most bp of the TNF
sequence were
deleted. Lanes 1, 4, and 7,
4 µg/plate of vector (BS+) was co-transfected; lanes
2, 3, 5, and 6,
CMV.hTTP.tag was co-transfected in the indicated amount (ng/plate). For
lanes 2, 3, 5, and
6, vector DNA was also added to make the total transfected
plasmid DNA 5 µg/plate. The Northern blot was probed with a
32P-labeled TNF
cDNA. Identical RNA samples as in
the upper panel of A were blotted and
probed simultaneously with 32P-labeled mouse TTP cDNA,
and the results are shown in the lower panels of
A. The position of the 18 S ribosomal RNA is indicated. The
two arrows indicate the two species of TNF
mRNA discussed. The expressed TTP mRNA is also indicated by an
arrow in the lower panel.
B, evidence that the species of TNF
mRNA expressed
from transfected CMV.TNF
-(127-1325 H2aSL) did not contain a poly(A)
tail. Lanes 1-3 (Control), cells were
transfected with CMV.TNF
-(127-1325) (1 µg/plate).
Lanes 4-6 (H2aSL), Cells were
transfected with CMV.TNF
-(127-1325 H2aSL) (1 µg/plate). The cells
were co-transfected with vector BS+ (4 µg/plate). 5 µg of RNA was
loaded into each gel lane. Oligonucleotide (dT)12-18
(T; 1 µg) was added to samples 2 and 5. An oligonucleotide
that was complementary to the stem-loop sequence of histone H2a
(SL; 1 µg) was added to lanes 3 and
6. All RNA samples were treated with 1 unit of RNase H as
described under "Experimental Procedures." The Northern blot was
probed with a 32P-labeled TNF
cDNA. C,
schematic representation of the CMV.TNF
-(127-1325) constructs.
Control, CMV.TNF
-(127-1325) in which the 33 adenyl
residues were directly attached to the 3'-most bp of the TNF
sequence. H2aSL, CMV.TNF
-(127-1325 H2aSL) in which the
33 adenyl residues were deleted and a 3' portion of the mouse histone
H2a sequence containing the stem-loop (capital
letters, underlined) and histone downstream
element (HDE; in capital letters) was
inserted immediately after the TNF
sequence. The 3'-end of the H2a
mRNA (C) is indicated by italic
type.
-histone
construct (Fig. 7C). In this experiment, the co-transfection of two concentrations of TTP DNA (5 and 10 ng/plate) with the TNF
expression plasmid CMV.TNF
-(127-1325) resulted in the consistently observed decrease in total transcript amount as well as the formation of the deadenylated species (Fig. 7A, upper
panel, lanes 1-3). When the 33 adenylate residues in the CMV.TNF
-(127-1325) construct were
replaced with the histone 3'-processing sequence and then co-transfected with low concentrations of TTP vector, there was a
decrease in the levels of the expressed hybrid TNF
-H2a mRNA (Fig. 7A, upper panel,
lanes 4-6). In contrast to the effect of TTP
co-transfection on the TNF
mRNA expressed from construct CMV.TNF
-(127-1325), which resulted in two mRNA species that
were different in size (Fig. 7A, upper
panel, lanes 2 and 3), the
presence of TTP altered the amount but not the size of the hybrid
TNF
-H2a mRNA. The virtual absence of expression of a TNF
construct that contained neither the poly(A) tail nor the histone
3'-end-processing sequence is shown in Fig. 7A
(lane 7, upper panel).
-H2a mRNA was not polyadenylated
in these experiments, additional RNase H studies were performed on the
RNA samples shown in Fig. 7A (upper
panel, lanes 1 and 4). When
RNA from 293 cells transfected with the control plasmid CMV.TNF
-(127-1325) was treated with oligo(dT)12-18 and RNase H, TNF
mRNA decreased in size (Fig. 7B,
lanes 1 and 2). However, when RNA from
293 cells transfected with plasmid CMV.TNF
-(127-1325 H2aSL) was
treated with RNase H, there was no apparent difference in the size of
hybrid TNF
-H2a mRNA in the presence or absence of
oligo(dT)12-18 (Fig. 7B, lanes
4 and 5). This experiment confirmed that the
TNF
-H2a hybrid mRNA had not undergone polyadenylation under
these experimental conditions. When an oligonucleotide complementary to
the stem-loop sequence was used, the presence of this oligonucleotide slightly decreased the size of the hybrid TNF
-H2a mRNA (Fig. 7B, lane 6) while having no effect on
the size of the TNF
mRNA expressed from the control plasmid
CMV.TNF
-(127-1325) (Fig. 7B, lane
3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
GM-CSF. In cultured bone marrow-derived macrophages from normal mice,
the normal half-life of TNF
mRNA was about 40 min following the
addition of actinomycin D to the cells; in this situation, the mRNA
disappeared without the appearance of detectable degradation intermediates in Northern blots (6). In macrophages derived from TTP
knockout mice, the mRNA half-life was more than doubled, but again
no degradation intermediates were seen (6).
stimulation
of bone marrow-derived stromal cells revealed the presence of a
degradation intermediate (7). Specifically, following the induction of GM-CSF gene transcription by either agent, the first detectable transcript seen on Northern blots was approximately 1 kb in length, corresponding to the full-length mouse transcript containing a poly(A)
tail of ~200 residues. This full-length mRNA species was first
visible approximately 1 h after stimulation of the cells. However,
this was rapidly followed by the appearance of a smaller band of ~0.8
kb, which thereafter accounted for approximately half of the total
hybridizable GM-CSF mRNA. Both species then disappeared in a longer
time course or following actinomycin D treatment of the cells.
Importantly, the smaller band was demonstrated by RNase H analysis to
be the essentially completely deadenylated form of the mRNA; and in
the Northern blots from wild-type cells, a smear of mRNAs of
intermediate sizes could be detected between the two major transcripts
(7). The two major mRNA species have been seen before in Northern
blots of GM-CSF mRNA (18, 19); our studies firmly established that
these bands represent the fully adenylated and deadenylated forms of
the GM-CSF mRNA. These data fit the current model of class II
ARE-dependent mRNA degradation, in which processive
deadenylation leads to the usually transient accumulation of
deadenylated mRNA bodies (10).
, GM-CSF, and IL-3 with TTP and its related CCCH proteins cMG1
and XC3H-3; 2) insertion of class II ARE sequences into a normally
nonpolyadenylated histone transcript, followed by co-expression with
TTP; and 3) co-expression with TTP of two chimeric class II
ARE-containing transcripts that lacked a poly(A) tail but instead terminated in a histone 3' stem-loop sequence.
,
GM-CSF, and IL-3, demonstrated that co-transfection of normal TTP and
its related CCCH proteins, but not of a nonbinding mutant form of TTP,
could stimulate the breakdown of these class II ARE-containing mRNAs in this 293 cell transfection system. The effect of TTP to
stimulate the breakdown of the mRNAs is in keeping with our previous data concerning TNF
and GM-CSF mRNAs in cells derived from the TTP knockout mice (6, 7). This suggests that, at low
concentrations of transfected TTP DNA, TTP is stimulating the breakdown
of these ARE-containing mRNAs in 293 cells in a manner that is
directly related to its behavior in the normal physiology of
macrophages and bone marrow-derived stromal cells. We have not yet
shown that TTP deficiency results in stabilization of the IL-3 mRNA
in cells derived from TTP knockout mice, but the present results
suggest that this might be the case in an appropriate cell type. In
support of this idea is a recent report demonstrating that
co-expression of TTP destabilizes IL-3 mRNA in HT1080 cells
(20).
, GM-CSF, and IL-3 mRNAs, the
destabilization involves the 3'-end. This can be concluded, because in
all three cases, relatively high level CCCH protein expression resulted in the formation of stable breakdown products that were shown by RNase
H experiments to have normal 5'-ends but shortened 3'-ends. However, in
the cases of all three class II ARE-containing transcripts, at high
concentrations of transfected TTP, cMG1, and XC3H-3 DNA, stable
mRNA intermediates were formed that were even shorter than the
completely deadenylated mRNAs. These smaller 5' to 3' truncated fragments appeared to terminate at approximately the site of the ARE in
all three naturally occurring mRNAs. This phenomenon was also noted
in previous experiments using an artificially truncated TNF
mRNA
in which a 33-residue poly(A) "tail" was directly attached to the
middle of the ARE; in these studies, high concentrations of expressed
TTP, cMG1, and XC3H-3 resulted in the increased accumulation of a
deadenylated intermediate (8, 9). Although we speculated that at high
TTP concentrations the binding of TTP to the ARE might prevent further
3'to 5' exonuclease activity, the mechanism of this apparent
"protective" effect is not known; possibilities include physical
interference with a 3' to 5' exonuclease, sequestering an unknown
mRNA-destabilizing protein or factor from the ARE-associated complex, and others.
,
GM-CSF, and IL-3 mRNAs in Northern blots of macrophages, stromal
cells, mast cells, and fibroblasts from wild-type and TTP-deficient
mice, we have never seen them (6,
7).3 This is despite the fact
that the AREs in these three mRNAs are located considerably 5' from
the 3'-end of the mRNA body, which should make a significant
accumulation of these intermediates readily apparent on Northern blots.
Finally, as shown in the present study, even high concentrations of TTP
had no effect on the formation of either type of 3'-5' truncated
mRNA when the "target" mRNA was an IL-3 mRNA that
lacked an ARE. For all these reasons, we believe that the 3'-5'-most
truncated mRNA intermediate seen in these experiments is likely to
be an artifact seen only with high concentrations of transfected CCCH
protein DNA.
, GM-CSF, and IL-3
mRNAs into a wild-type histone gene. In the second type, we used
the native mouse IL-3 mRNA and a truncated TNF
mRNA, in
which the polyadenylation signals had been mutated or the poly(A) tail
removed and the genomic histone 3'-end-processing sequence had been
attached to the 3'-end of the mRNA. This should ensure that
histone-like cleavage of this 3'-element would be required for the
mRNA to be processed and released from the nucleus, but
polyadenylation would not occur. In both types of experiment, the
hybrid mRNAs were expressed with CCCH proteins in co-transfection studies in 293 cells, and the absence of polyadenylation in each case
was confirmed by RNase H analysis with an oligo(dT) primer.
-histone chimeric mRNA, no stable intermediates were evident after TTP-stimulated breakdown of the mRNA. Thus, it appears that TTP can stimulate the
breakdown of ARE-containing mRNAs even in the absence of poly(A) tails.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. William Marzluff for helpful advice and Drs. Dori Germolec and Jamie Bonner for helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a Cooperative Research and Development Agreement with AstraZeneca Pharmaceuticals, PLC.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.
To whom correspondence should be addressed: A2-05 NIEHS, 111 Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-4899; Fax: 919-541-4571; E-mail: black009@niehs.nih.gov.
Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M100680200
2 W. S. Lai and P. J. Blackshear, unpublished data.
3 E. Carballo and P. J. Blackshear, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TTP, tristetraprolin;
hTTP, human TTP;
TNF, tumor necrosis factor
;
mTNF
, mouse TNF
;
ARE, AU-rich element;
GM-CSF, granulocyte-macrophage colony-stimulating factor;
mGM-CSF, mouse
GM-CSF;
IL, interleukin;
mIL, mouse interleukin;
3'-UTR, 3'-untranslated region;
HGH, human growth hormone;
PCR, polymerase
chain reaction;
RT, reverse transcription;
bp, base pair(s);
kb, kilobase pair(s);
CMV, cytomegalovirus;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
![]() |
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