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J. Biol. Chem., Vol. 277, Issue 43, 40462-40471, October 25, 2002
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From the Department of Medical Biochemistry and Microbiology,
Biomedical Center, Uppsala University, 751 23 Uppsala, Sweden
Received for publication, June 14, 2002, and in revised form, July 25, 2002
The human papillomavirus type 1 (HPV-1) late
mRNAs contain a 57-nucleotide adenosine- and uridine-rich RNA
instability element termed h1ARE in their late 3' untranslated regions.
Here we show that five sequence motifs in the h1ARE (named I-V) affect
the mRNA half-life in an additive manner. The minimal inhibitory
sequence in motifs I and II was mapped to UAUUUAU, and the minimal
inhibitory sequence in motifs III-V was mapped to UAUUUUUAU. We also
provide evidence that the same motifs in the AU-RNA instability element inhibit mRNA translation, an effect that was entirely dependent on
the presence of a poly(A) tail on the mRNA. Additional experiments demonstrated that the h1ARE interacted directly with the
poly(A)-binding protein, suggesting that the h1ARE inhibits translation
by interfering with the function of the poly(A)-binding protein.
Human papillomaviruses
(HPVs)1 are a group of
non-enveloped, double-stranded DNA tumor viruses with tropism for
epithelial cells (1, 2). Expression of the late mRNAs is restricted to the terminally differentiated cells in the upper layers of the
epithelium and at least four papillomaviruses (bovine papillomavirus type 1 (BPV-1), HPV-1, -16, and -31) have been shown to contain cis-acting inhibitory RNA elements located in the late 3' UTR (reviewed
in Refs. 3-6). In addition, negative RNA elements have been identified
in the HPV-16 L1 and L2 open reading frames (4, 7, 8).
We have previously identified and characterized an inhibitory AU-rich
element (ARE) located in the HPV-1 late 3' UTR region named h1ARE
(4-6, 9) (Fig. 1). Using actinomycin D we showed that the presence of
the h1ARE reduced the mRNA half-life (10). The minimal inhibitory
sequence termed XB spans 57 nucleotides (nt) and contains 93% A and U. The element contains two AUUUA- and the three UUUUU-containing
sequences (9, 10). Replacing two uridines (U) with cytidines (C) in
each motif inactivated the h1ARE (10). The h1ARE interacts with
cellular factors (11, 12), which bind to the c-fos
ARE (10). Two of the h1ARE binding factors interacted with the wild
type h1ARE but not with a functionally inactive mutant of the h1ARE
(10). These proteins were identified as HuR and hnRNP C (10, 13), and
we later showed that binding of the HuR protein correlates with
inhibitory activity of a panel of h1ARE mutants (13). While HuR binds
to both AUUUA- and UUUUU-motifs (13), hnRNP C binds exclusively to the
UUUUU-motifs (14). The role of hnRNP C in HPV-1 late gene expression is
unclear. The HuR protein shuttles between the nucleus and the cytoplasm (15), and we observed that there was an inverse correlation between the
levels of HuR in the cell cytoplasm and the inhibitory activity of the
h1ARE (16), suggesting that the presence of high levels of HuR in the
cytoplasm antagonizes the inhibitory effect of the h1ARE, whereas a
primarily nuclear association of HuR is associated with inhibition of
HPV-1 late gene expression. Interestingly, the HIV Rev and RRE,
and the SRV-1 CTE can overcome the inhibition (9), suggesting that the
h1ARE traps the HPV-1 late mRNAs in the nucleus and that this may
lead to rapid mRNA degradation. Interestingly, the inhibitory
effect of the h1ARE was greater at the protein level than at the
mRNA level, suggesting that the h1ARE also inhibited the
utilization of the mRNA. Here we present results of a mutational
analysis of the h1ARE, and we provide evidence that the h1ARE inhibits
mRNA translation.
Plasmid Constructions
CMV Promoter-driven Plasmids--
All the eukaryotic expression
plasmids containing the h1ARE mutants are derived from p Bacteriophage T7 Promoter-driven Plasmids--
To generate
pCC In Vitro Transcription and Transfections
DNA transfections were performed with FuGENE (Roche Molecular
Biochemicals) as described previously (10). Transfections were
performed in triplicates, and mean values and standard deviations are
displayed in the figures. For RNA analysis triplicate samples were
pooled and analyzed by Northern blot. Each plasmid was analyzed in at
least three independent transfection experiments. RNA synthesis and
transfections were performed as described previously (18).
To generate RNAs with a poly(A) tail of fixed length, two PCR fragments
were first amplified with the following primer pairs: T7CATS
(5'-GTAATACGACTCACTATAGGGTACTGCGATGAGTGGCAGGG-3') and HPV1ANTIPA (5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCACACTTGTGTATAATGCACCGG-3') (which encodes a A60 tail) or T7CATS and HPV1ALW
(5'-CACACTTGTGTATAATGCACCGG-3'). The PCR fragments that were generated
from plasmid p CAT-ELISA and hGH-ELISA
To monitor CAT protein levels, RNA-transfected HeLa cells were
harvested as described previously (8) at indicated time points. For DNA
transfections, cells were harvested at 40 h posttransfection. The
levels of CAT, hGH, and Primer Extension and Northern Blotting
Cytoplasmic RNA extraction and primer
extension were performed as described previously (8). To perform
Northern blotting, total cytoplasmic RNA was extracted at various times
posttransfection as previously described (18). Northern blot analysis
was performed as described (10). Briefly, 10 µg of total or
cytoplasmic RNA was separated on 1% agarose gels containing 2.2 M formaldehyde, followed by transfer to a nitrocellulose
filter and hybridization. Random priming of the DNA probe was performed
using a DECAprime kit (Ambion) according to the manufacturer's instructions.
UV Cross-linking and Preparation of Recombinant Protein
UV cross-linking and synthesis of radiolabeled RNA were
performed as previously described (11, 19). GST-PABP, GST-HuR, and
GST-PCBP were purified on GS beads according the manufacturers recommendations (Amersham Biosciences).
Mutational Analysis of the h1ARE--
To study the HPV-1 late 3'
UTR element named h1ARE (Fig.
1A) further, the 57-nt minimal
element (XB) (Fig. 1B) or a functionally inactive mutant
thereof (AUM/UM) (Fig. 1B) was inserted between the CAT
reporter gene, driven by the human cytomegalovirus promoter, and the
late HPV-1 poly(A) signals resulting in pXB and pAUM/UM (Fig.
1B). As controls, we used p
Analysis of CAT production in transient transfections of HeLa cells and
calculation of fold difference between pCCKH1(A)/pCCKH1 and pXB/pAUM/UM
revealed that the fold difference between pCCKH1(A)/pCCKH1 and
pXB/pAUM/UM were similar and demonstrated that the 57-nt XB contains
the major inhibitory sequence (data not shown). The mRNAs containing the 57-nt XB fragment have a short half-life (Fig. 1C). In agreement with our previous findings, the presence
of the h1ARE on the mRNA also results in a higher ratio of nuclear versus cytoplasmic mRNA (data not shown). We concluded
that pXB and pAUM/UM could be used for further studies of the AU-rich element.
The XB sequence contains two AUUUA motifs and three AUUUUUA motifs that
were numbered I-V (Fig. 2A).
These motifs were all mutated in AUM/UM (Fig. 2A). To
investigate if all motifs were required for inhibition, the motifs were
mutated one by one (Fig. 2A). However, there was only a
modest increase in CAT RNA and protein levels (Fig. 2, B and
C) for each mutant. To obtain a clearer answer on the
importance of each motif, consecutive mutations were introduced in the
motifs (Fig. 3A). The results
revealed that higher expression levels were obtained as more motifs
were mutated (Fig. 3, B and C), demonstrating
that each motif contributed to inhibition. However, mutations in four
of the motifs (pM4) yielded as high expression levels as mutations in
all five motifs (pAUM/UM) (Fig. 3, B and C). To
investigate if the fifth motif contributed to inhibition, it was
mutated in pM2 (Fig. 3A), resulting in pM2V (Fig.
3A). The results revealed that pM2V expressed higher RNA and
protein levels than did pM2. These levels were similar to those
produced from pM3 (Fig. 3, B and C), thereby
demonstrating that also the fifth motif contributed to inhibition.
Interestingly, the effect was greater at the protein level than at the
RNA levels, for all analyzed mutants (Fig. 3, B and
C). We concluded that all five motifs contributed to
inhibition in an additive manner.
Determination of the Minimal Inhibitory Sequence of Each Sequence
Motif within the h1ARE--
To determine the minimal inhibitory
sequence of the AUUUA-containing motifs, point mutations were
introduced in motifs I and II (Fig.
4A). Mutations in the tri-U
nucleotides, the flanking As or the Us immediately flanking the As were
not well tolerated (Fig. 4, B and C). However,
mutations in the second nucleotide position outside the As (Fig.
4A), did not significantly affect the inhibitory activity of
these motifs (Fig. 4, B and C), indicating that
the smallest inhibitory motif was UAUUUAU. The two UAUUUAU were
separated by a four-nucleotide spacer sequence (Fig. 4A). Substituting this sequence with four Cs did not affect the inhibitory activity of the h1ARE (Fig. 4, B and C),
indicating that this spacer sequence did not contribute to the
inhibitory activity.
To determine the minimal inhibitory sequence of the penta-U motifs, the
nucleotides flanking the penta-Us were mutated (Fig. 5A). The results revealed that
mutations in both the As flanking the penta-Us and the Us flanking the
As resulted in higher CAT protein and RNA expression levels (Fig. 5,
B and C). Substituting two Us in the penta-U
sequence with two Cs had the strongest effect, whereas mutations in the
Us flanking the As had the smallest effect on the inhibitory activity
(Fig. 5, B and C). Therefore, the minimal motif
was UAUUUUUAU.
UAUUUAU and UAUUUUUAU Motifs Can Functionally Substitute for One
Another--
The h1ARE may be divided into the B2 region with the two
UAUUUAU motifs and the C1 region with the three UAUUUUUAU motifs (Fig.
6A). The B2 region, although
less inhibitory than the XB sequence, as expected, inhibited CAT
production and reduced mRNA levels to the same extent as the did C1
region (Fig. 6, B and C). Two B2 or two C1
regions were as inhibitory as the entire XB (Fig. 6, B and
C), demonstrating that one type of motif (UAUUUAU or
UAUUUUUAU) could substitute for one another. Furthermore, if the two
AUUUA motifs were extended to two AUUUUUA motifs by insertion of two Us
in each motif, resulting in pAUUUUUA (Fig.
7A), or if the three penta-U
motifs were all shortened to contain only three Us, as in pAUUUA (Fig.
7A), the resulting inhibitory elements were nearly as
inhibitory as the wild type h1ARE (Fig. 7, B and C). Both B2 regions and C1 regions acted by reducing
mRNA steady state levels and protein production. Inhibition was
also greater at the protein level than at the mRNA level. Taken
together, the results demonstrated that both motifs acted in a similar
manner.
Multiple Copies of the HPV-1 AU-rich Element Inhibits Protein
Production >99%--
For all mutants that retained inhibitory
activity, we observed that the inhibitory effect was greater at the
protein level than at the mRNA level (see Fig. 2, B and
C, Fig. 3, B-D, Fig. 4, B and
C, Fig. 5, B and C, Fig. 6,
B and C, and Fig. 7, B and C). In other words, the mRNAs that contained the h1ARE,
or partially active mutants thereof were utilized less efficiently by
the translation machinery than mRNAs lacking the h1ARE or mRNAs
containing functionally inactive sequences. To better compare the
effect on protein production and the effect on the mRNA levels,
multiple XB sequences were inserted into the reporter plasmid pXB,
resulting in p2XB, p3XB, and p4XB (Fig.
8A), and the fold inhibition
at the protein and mRNA levels was separately plotted against the
number of XBs on the mRNA. As can be seen, there is a gradual
decrease in CAT protein and mRNA levels for every inserted copy of
the XB fragment (Fig. 8, B and C), and the fold
inhibition was greater at the protein level than at the RNA level (Fig.
8, B and C). Extrapolation of the data in a
semilog plot allowed a better estimate of the inhibitory activity of
one single XB sequence at protein and RNA levels (Fig. 8D)
within the context of these plasmid constructs. The results clearly
demonstrated that XB had a greater effect at the protein level than at
the RNA level (Fig. 8D). On average from multiple experiments, we found that CAT protein levels were reduced 3.7-fold per
XB and RNA levels 1.4-fold per XB in the context of the mRNAs with
multiple XBs (Fig. 8D). We concluded that in addition to the
effect on mRNA half-life, mRNAs carrying the h1ARE are
inefficiently utilized for translation.
Deadenylation (20, 21) could potentially cause the inhibition of
translation observed here. To determine the poly(A) tail length of the
mRNAs shown in Fig. 8C, they were subjected to
oligonucleotide-directed RNaseH cleavage with the "RNaseH oligo"
shown in Fig. 8A followed by Northern blot using a probe
located downstream of the RNaseH oligo. The results revealed that all
mRNAs contained poly(A) tails of similar length (Fig.
8E), demonstrating that inhibition of translation was not a
result of deadenylation.
The HPV-1 AU-rich Element Inhibits Translation of the Transfected
mRNAs--
To study the effect of the h1ARE on translation
further, we replaced the CMV immediate-early promoter with the
bacteriophage T7 promoter and the cleavage and polyadenylation signal
with the XhoI restriction site in the reporter constructs
pCCKH1(A) and pCCKH1 (Fig. 1B) used previously to study the
h1ARE. These cloning steps resulted in pCC(A) and pCC (Fig.
9A) that were linearized with
XhoI and used as templates for in vitro synthesis
of capped and polyadenylated CC(A) and CC mRNAs as described under
"Experimental Procedures." These mRNAs contained the same
sequences as the mRNAs produced by the pCCKH1 and pCCKH1(A)
plasmids in the nuclei of transfected cells (Fig. 1B).
Capped and polyadenylated CC and CC(A) mRNAs were transiently
transfected in triplicates into HeLa cells by electroporation, and the
CAT levels produced at 20 h posttransfection were quantified by
using a CAT ELISA. The results revealed that CC mRNAs that contain
the h1ARE in sense orientation produced ~15-fold lower CAT protein
levels than the CC(A) mRNAs, which contain the h1ARE in antisense
orientation (Fig. 9A). Cotransfected hGH-encoding mRNAs
included in all samples as an internal control produced similar hGH
protein levels (Fig. 9A).
Next, aliquots of electroporated cells were harvested at different time
points, and the levels of CAT protein were monitored and plotted
against time (Fig. 9B). Two interpretations of the results
shown in Fig. 9B were appropriate: either the mRNAs with the HPV-1 late 3' UTR were rapidly degraded, preventing further CAT
protein synthesis, or the mRNAs were not available for further rounds of translation as a result of a direct inhibition of
translation, presumably by factors binding to the h1ARE.
To investigate if the half-lives of the transfected mRNAs were
reduced by the presence of the HPV-1 late 3' UTR, CC and CC(A) mRNA
levels at 1, 3, 5, and 23 h posttransfection were determined by
primer extension (Fig. 9C). CC and CC(A) mRNAs decayed
at similar rates, as detected at 1, 3, and 5 h posttransfection
(Fig. 9C). Longer exposures also detected similar amounts of
CC and CC(A) mRNA at 23 h posttransfection (data not shown).
The mRNA half-lives were calculated to be 2.6 h for CC
mRNAs and 2.7 h for CC(A) mRNAs. A number of control
experiments were performed using CC(A) and/or CC mRNA. These
experiments verified that electroporated RNAs were not sticking to the
outside of the cell and that the majority of the transfected mRNAs
are normally utilized by the translation machinery (data not shown). We
concluded that the inhibitory effect on the transfected mRNAs,
mediated by the HPV-1 late 3' UTR, was not a result of reduced mRNA
half-life in the cytoplasm and that the HPV-1 late 3' UTR acted by
inhibiting mRNA translation. In addition, the previously observed
effect on the mRNA half-life was dependent on a nuclear experience
of the mRNA and was not seen when in vitro synthesized
mRNAs were transfected into cells.
Inhibition of Translation by the h1ARE Requires
Intact UAUUUAU or UAUUUUUAU Motifs in the h1ARE--
To confirm that
the 57-nt minimal XB sequence containing the two UAUUUAU and the three
UAUUUUUAU motifs inhibited translation in the RNA transfection
experiments, CCXB and CCAUM/UM mRNAs (Fig. 10A) were transfected into
HeLa cells in parallel. The CCAUM/UM mRNAs produced 41-fold higher
levels of CAT than the CCXB mRNAs (Fig. 10B). hGH
protein levels produced from the internal control mRNAs were
similar in both samples (Fig. 10B). Next, capped and polyadenylated CCXB, CCAUM/UM, CCB2, and CCC1 mRNAs (Fig.
10A) were electroporated into HeLa cells, and the levels of
CAT protein produced at 3, 6, 23, and 47 h posttransfection were
quantified. hGH mRNAs were included in all samples as an internal
control. Fig. 10C shows that XB-containing mRNAs
produced lower CAT protein levels than the AUM/UM-containing mRNAs
as expected, whereas B2- and C1-containing mRNAs showed similar
intermediate inhibition of CAT protein production compared with AUM/UM-
and XB-containing mRNAs (Fig. 10C). hGH protein
accumulation in the cell culture medium was similar in all transfected
samples at each time point (Fig. 10C).
The CAT protein production peaked at the 23 h time point for all
four mRNAs, after which the CAT protein levels decreased. This is
the expected result if the mRNAs have similar half-lives. The
mRNA decay rates in the cells transfected with CCXB and CCAUM/UM mRNA were determined by primer extension on RNA extracted from the
transfected cells. Fig. 10D shows that the levels of CCXB
and CCAUM/UM mRNAs were similar at 1 and 2.5 h
posttransfection and decayed with a rate comparable to that observed
for CC and CC(A) mRNAs (compare Figs. 10D and
9C). The results confirmed that the h1ARE acts primarily by
reducing mRNA translation in the RNA transfection experiments
performed here and demonstrated that both the two UAUUUAU motifs and
the three UAUUUUUAU motifs inhibit translation.
The h1ARE Inhibits Translation of mRNAs Carrying a Cap and a
poly(A) Tail--
The poly(A) tail is required for efficient mRNA
translation (23). We therefore wished to investigate if the HPV-1
AU-rich element interfered with the function of the poly(A) tail. We
transfected polyadenylated and unpolyadenylated capped CC and CC(A)
mRNAs and monitored the levels of CAT protein in the cells at
24 h posttransfection. Fig.
11A shows that the
polyadenylated CC mRNA, which contains the HPV-1 AU-rich element in
sense orientation, produced 56-fold lower CAT protein levels than
polyadenylated CC(A) mRNA, which contains the HPV-1 AU-rich element
in antisense orientation. In contrast, the presence of the h1ARE on the
unpolyadenylated CC mRNA did not inhibit CAT production
significantly (Fig. 11A). The stimulatory effect of the
3'-poly(A) tail, when added to the capped CC mRNA, which contains
the h1ARE, was only 1.5-fold, compared with 58-fold when added to the
CC(A) mRNA. Translation of co-transfected hGH mRNA was similar
in all samples (Fig. 11A). Similar results were obtained in
a time course experiment using the XB sequence and the inactive AUM/UM
mutant (Fig. 11B), demonstrating a connection between the
57-nt XB sequence and the poly(A) tail. Taken together, the results
demonstrated that inhibition of CAT production by the h1ARE was
dependent on a 3'-poly(A) tail. Therefore, the results showed that the
stimulatory effect on translation mediated by the 3'-poly(A) tail on
cap-dependent translation was perturbed by the h1ARE.
Also here could the inhibitory effect of the AU-rich RNA element on
translation be indirect through deadenylation. To investigate if the
h1ARE promoted deadenylation of the in vitro synthesized mRNAs that were transfected into the HeLa cells, mRNAs with a poly(A) tail of fixed length were transfected into the cells and the
length of the poly(A) tail was determined at 1 or 4 h
posttransfection by Northern blotting. Capped mRNAs with and
without the h1ARE or poly(A) tail were analyzed. The results revealed
that the mRNAs were not deadenylated at 1 h posttransfection
(Fig. 11C). In addition, analysis of the polyadenylated
mRNAs containing the h1ARE at 4 h posttranfection showed that
the poly(A) tails remained intact (Fig. 11C). Therefore,
inhibition of translation was not caused by deadenylation.
The h1ARE Interacts with the Poly(A)-binding Protein--
Having
established that the h1ARE inhibited the function of the poly(A) tail,
it was reasonable to speculate that the h1ARE, or h1ARE binding
factors, interact directly with the poly(A)-binding protein (PABP). We
therefore tested if PABP bind directly to the XB RNA. GST-PABP was UV
cross-linked to XB RNA or AUM/UM RNA. UV cross-linking of GST-PABP
revealed that GST-PABP bound strongly to the XB RNA but only weakly to
the AUM/UM RNA (Fig. 12A);
GST-PABP did not bind to an unrelated RNA derived from the L1 coding
region in HPV-16 (Fig. 12A). The GST-HuR protein was used as
positive control and interacted only with XB, as expected (13), and
GST-PCBP did not bind to any of the RNA sequences (Fig.
12A). To confirm that the binding of GST-PABP to the XB
sequence was sequence specific, a competition experiment was performed.
The XB RNA competed efficiently with the RNA probe for binding to
GST-PABP, whereas the HPV-16 L1-derived RNA did not (Fig.
12B), demonstrating that the interaction with XB was
sequence-specific. Analysis of the deletion mutants B2 and C1 that were
shown to inhibit translation to a similar extent also interacted with
the PABP (Fig. 12A). Therefore, binding of PABP to the
different mutants (B2, C1, and AUM/UM)) correlated with their
inhibitory effect on translation.
The mutational analysis presented here revealed that the HPV-1
AU-rich element consists of the two UAUUUAU heptamers and the three
UAUUUUUAU nonamers. Previous studies on the c-fos ARE
led to the conclusion that the UAUUUUAU motif was sufficient for
mRNA destabilization but not optimal, and that multiple copies were needed for significant destabilization (24). This is in agreement with
the results presented here. Based on a mutational analysis and sequence
alignments, these authors concluded that the minimal motif may be
UUAUUUA(U/A)(U/A). In another article on the c-fos ARE, a deletion analysis of the c-fos ARE led to the
conclusions that the UUAUUUAUU nonamer, and not the UAUUUAU heptamer,
was the shortest destabilizing motif (25). These authors also found that mRNAs containing multiple copies of the nonamer are degraded more rapidly than mRNAs with only one copy. Using the HPV-1 ARE, we
also found that multiple copies of the motif were more inhibitory than
one copy. However in our system, mutations outside of the heptamer
UAUUUAU did not affect its ability to reduce mRNA levels. In the
context of the HPV-1 ARE, the UAUUUAU was the shortest motif with
inhibitory activity, indicating that the sequence context may affect
the potency of an AU-rich element. In contrast to
the HPV-1 AU-rich element that contains two UAUUUAU and three UAUUUUUAU motifs, the AU rich element on the IL-3 mRNA contains six AUUUA motifs. Mutations in three of these motifs had the same effect as
deleting the entire element (26). The HPV-1 AU-rich element contains
five motifs, and we show that all five contribute to inhibition in an
additive manner. Similarly, mutations in all three AUUUA motifs in the
c-fos ARE resulted in mRNA stabilization (27). It
appears that multiple copies of the "AUUUA"-related motifs are
required for full function of the various AU-rich RNA elements.
We have previously shown that the mRNA half-life is reduced by the
presence of the h1ARE when using DNA transfections in which the
mRNAs are synthesized in the cell nuclei (9, 10). In contrast, when
the same mRNAs were introduced directly into the cytoplasm as
described here, bypassing the nucleus, there was no effect on the
mRNA half-life by the h1ARE. These results indicate that a nuclear
experience of the h1ARE-containing mRNAs is necessary for rapid
mRNA degradation, suggesting that the mRNAs are either modified
in the nuclei or interact with nuclear factors that induce premature
mRNA degradation. Recent results on the ARE-containing c-fos mRNA showed that HuR mediates nuclear
export of c-fos mRNAs (28). HuR also increases the
c-fos mRNA half-life, suggesting that nuclear export and
mRNA stability are connected and that inefficient mRNA export
leads to premature degradation. In a previous article, we reported that
the HIV-1 mRNA export factor Rev in combination with RRE or the
SRV-1 CTE could overcome the inhibitory effect of the h1ARE (9),
demonstrating that export of the h1ARE-containing mRNAs through an
alternative, productive pathway overcomes inhibition and results in
high expression. These results also demonstrated that the h1ARE has an
inhibitory function in the nucleus, in addition to its inhibitory
effect of translation in the cytoplasm described here.
The h1ARE binds PABP and may inhibit the interaction between PABP and
eIF4G, thereby preventing circularization of the mRNA and the
subsequent loading of ribosomes on the mRNA. This is not without
precedent since it was recently shown that the rotavirus mRNA
3'-end-binding protein NSP3 interacts with eIF-4G and that NSP3
competes with the PABP for the eIF4G (29). Alterations of either the
poly(A)-PABP or cap-eIF4E complexes lead to access to the poly(A) tail
and cap by the poly(A) ribonuclease (PARN/DAN), resulting in
deadenylation (30). Deadenylation was not observed here. It has also
been proposed that the shuttling elav-like proteins are involved
in mRNA translation directly (31, 32). Similarly, redistribution of
HuR protein from the nucleus to the cytoplasm is associated with
increased protein production from mRNAs containing AREs with HuR
binding sites (33, 34). Therefore, HuR may act similarly to HuB and
promote polysomal loading of ARE-containing mRNAs. HIV-1 Rev
protein that overcomes the inhibitory effect of the h1ARE in HeLa cells
also induces polysomal loading of target mRNAs (35, 36). Perhaps
elav-like proteins such as HuR lead mRNAs onto a productive pathway
that includes efficient nuclear export and polysomal loading. The role
of the h1ARE, HuR, and the PABP in the HPV-1 life cycle remains to be determined.
We are grateful to H. Furneaux for GST-HuR
plasmid, J. Bag for GST-PABP, H. Leffers for GST-PCBP, and A. Grynfeld
for critically reading the manuscript.
*
This work was supported by the Swedish Medical Research
Council, the Swedish Cancer Society, and the Swedish Society for
Medical Research.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: Dept. of Medical
Biochemistry and Microbiology, Biomedical Center, Uppsala University, Husargatan 3, Box 582, 751 23 Uppsala, Sweden. Tel.: 4618-471-4239; Fax: 4618-509-876; E-mail: Stefan.Schwartz@imbim.uu.se.
Published, JBC Papers in Press, July 29, 2002, DOI 10.1074/jbc.M205929200
The abbreviations used are:
HPV(s), human papillomavirus(es);
UTR, untranslated region;
ARE, AU-rich element;
nt, nucleotides;
CAT, chloramphenicol acetyltransferase;
CMV, cytomegalovirus;
hGH, human growth hormone;
ELISA, enzyme-linked
immunosorbent assay;
GST, glutathione S-transferase;
PABP, poly(A)-binding protein.
Inhibition of Translation by UAUUUAU and UAUUUUUAU Motifs of the
AU-rich RNA Instability Element in the HPV-1 Late 3' Untranslated
Region*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
KX (11)
(Fig. 1B). Oligonucleotides were annealed and cloned
into pKX digested with KpnI and XbaI, which
resulted in the insertion in pKX of the wild type and mutant h1ARE
sequences displayed in the figures. All mutants were sequenced. pCMVlacZ was constructed by replacing the chloramphenicol
acetyltransferase (CAT) gene in pKX by the lacZ gene.
that lacks the h1ARE and its derivatives, a fragment containing
the first 75 nts that are transcribed from the CMV promoter, the CAT
gene and HPV-1 late 3' UTR sequences spanning nt 7184-7447 was first
amplified from pKX using oligonucleotides HCMV-S
(5'-CGAGCTCTCAGATCGCCTGGAGACGCC-3') and XhoIpA (9), introducing unique 5'-SacI and 3'-XhoI sites. The
PCR fragment was ligated to pCR2.1 (Invitrogen), downstream of the T7
RNA polymerase promoter, generating p. p was digested with
ApaI and EcoRV, filled in with T4 DNA polymerase,
and religated to remove polylinker sequences between the T7 promoter
and the cloned PCR fragment, generating pCC. pCC also lacks the
downstream polylinker sequences between KpnI and
XhoI, which are replaced by a unique NsiI site. To generate pCC, a PCR fragment was first amplified from pCCKH1 (11)
(Fig. 1B) by using oligonucleotides HCMV-S (see above) and
XhoIpA (9) followed by insertion into pCR2.1 (Invitrogen). This step was followed by transfer of a SacI-XhoI
fragment from the pCR2.1-based intermediate to pCC, resulting in
pCC. To generate pCC(A), which contains the h1ARE in the
antisense orientation, a PCR fragment amplified from pCCKH1 by using
oligonucleotides H1KPNI-A (5'-GGTACCGAACACTACTGTAGAATATGTG-3') and
H1XBA-S (5'-TCTAGAGCTACTAGTTCCACCACAAAGCGC-3') was inserted into
EcoRV-digested pBlueScript (Stratagene) generating pKS-H1XK. This was followed by transfer of a
KpnI-XbaI fragment from pKS-H1XK to pCC,
resulting in pCC(A). Plasmids pCCXB, pCCAUM/UM, pCCAUM, pCCUM, pCCB2,
and pCCC1 were generated by transfer of KpnI-XbaI
fragments from the previously described plasmids pKSXB, pKSAUM/UM,
pKSAUM, pKSUM, pKSB2, and pKSC1 (10), respectively, to pCC digested
with KpnI and XbaI. pCMVhGH has been described previously (17). Radiolabeled RNAs for UV cross-inking were produced
from pKSXB, pKSAUM/UM, pKSB2, and pKSC1 (10).
KX or pKX containing the 57-nt h1ARE fragment using
the two primer pairs described above were gel-purified and used for
synthesis of capped RNAs with and without the A60
tail. Plasmids or RNA encoding CAT, human growth hormone (hGH), or
-galactosidase were included in all transfections to monitor the
transfection efficiency.
-galactosidase proteins were quantified using CAT, hGH and -galactosidase antigen capture ELISA (Roche Molecular Biochemicals), respectively. All CAT quantitations were normalized to the protein concentration of the cell extract as determined by the Bradford method.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
KX (which lacks the major part of the late 3' UTR), pCCKH1 (which contains the entire late 3' UTR),
and pCCKH1(A) (which contains the region of the late 3' UTR containing
the XB sequence in antisense orientation). HeLa cells were transfected
in triplicate, the CAT production was monitored in each plate, and mean
values and standard deviations are displayed in the figures. pCMVlacZ
was included as an internal control in all transfections, and the
variation was less than 20% between the samples in the triplicates.
For RNA analysis, cytoplasmic extracts from the triplicates were pooled
and subjected to RNA extraction and Northern blotting.
View larger version (13K):
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Fig. 1.
A, schematic illustration of the HPV-1
genome. The position of the HPV-1 AU-rich RNA element (h1ARE) in the
late 3' UTR is indicated (10). pAE and pAL, early
and late poly(A) signals, respectively. B, schematic
illustration of plasmid containing the human CMV immediate early
promoter, the CAT reporter gene, and the HPV-1 late 3' UTR and late poly(A) signals pAL1 and pAL2. Numbers refer to
nucleotide positions in the HPV-1a genomic clone (37). Plasmid names
are indicated on the left. The position of the h1ARE is
indicated. The sequence of the minimal inhibitory element XB and
AUM/UM, an inactive mutant thereof, is shown. The motifs under
investigation are numbered I-V, and the mutations in AUM/UM are
underlined. C, HeLa cells were transfected with
pAUM/UM and pXB and treated with actinomycin D at 20 h
posttransfection for the indicated number of hours. The RNAs were
analyzed by Northern blotting (inset), and RNA levels were
quantitated with a phosphorimager device. lg(%RNA), lg
%RNA remaining after time point 0.
View larger version (36K):
[in a new window]
Fig. 2.
A, HPV-1 sequences inserted into the
reporter plasmid are shown. The sequence motifs in XB are
underlined and numbered. Introduced mutations are
underlined. B, the indicated plasmids were
transiently transfected into HeLa cells in triplicate as described
under "Experimental Procedures." The cells were harvested,
cytoplasmic extract was prepared, and protein was removed for CAT ELISA
followed by pooling of the three samples and RNA extraction. An
internal control plasmid was included in all transfections.
B, CAT levels were monitored by CAT ELISA, and the levels
are displayed as percent of CAT produced from pAUM/UM. Mean values and
standard deviations are shown. C, the RNA samples were
subjected to Northern blotting (lower panel) followed by
phosphorimaging quantitation (upper panel). The RNA levels
are displayed as percent of RNA compared with pAUM/UM. Pooled RNAs from
triplicate experiments are shown.
View larger version (31K):
[in a new window]
Fig. 3.
A, HPV-1 sequences inserted into the
reporter plasmid are shown. The sequence motifs in XB are
underlined and numbered. Introduced mutations are
underlined. B, the indicated plasmids were
transiently transfected into HeLa cells in triplicate, and protein was
removed for CAT ELISA followed by pooling of the three samples and
cytoplasmic RNA extraction. An internal control plasmid was included in
all transfections. B, CAT levels were monitored by CAT
ELISA, and the levels are displayed as percent of CAT produced from
pAUM/UM. Mean values and standard deviations are shown. C,
the RNA samples were subjected to Northern blotting (lower
panel) followed by phosphorimaging quantitation (upper
panel). The RNA levels are displayed as percent of RNA compared
with pAUM/UM. Pooled RNAs from triplicate experiments are shown.
View larger version (38K):
[in a new window]
Fig. 4.
A, HPV-1 sequences inserted into the
reporter plasmid are shown. The sequence motifs in XB are
underlined and numbered. Introduced mutations are
underlined. B, the indicated plasmids were
transiently transfected into HeLa cells in triplicate, and protein was
removed for CAT ELISA followed by pooling of the three samples and
cytoplasmic RNA extraction. An internal control plasmid was included in
all transfections. Mean values and standard deviations are shown.
B, CAT levels were monitored in CAT ELISA, and the levels
are displayed as percent of CAT produced from pAUM/UM. C,
the RNA samples were subjected to Northern blotting (lower
panel) followed by phosphorimaging quantitation (upper
panel). The RNA levels are displayed as percent of RNA compared
with pAUM/UM. Pooled RNAs from triplicate experiments are shown.
View larger version (36K):
[in a new window]
Fig. 5.
A, HPV-1 sequences inserted into the
reporter plasmid are shown. The sequence motifs in XB are
underlined and numbered. Introduced mutations are
underlined. B, the indicated plasmids were transiently
transfected into HeLa cells in triplicate, and protein was removed for
CAT ELISA followed by pooling of the three samples and cytoplasmic RNA
extraction. An internal control plasmid was included in all
transfections. B, CAT levels were monitored in CAT ELISA,
and the levels are displayed as percent of CAT produced from pAUM/UM.
Mean values and standard deviations are shown. C, the RNA
samples were subjected to Northern blotting (lower panel)
followed by phosphorimaging quantitation (upper panel). The
RNA levels are displayed as percent of RNA compared with pAUM/UM.
Pooled RNAs from triplicate experiments are shown.
View larger version (25K):
[in a new window]
Fig. 6.
A, HPV-1 sequences inserted into the
reporter plasmid are shown. The sequence motifs in XB are
underlined and numbered. Introduced mutations are
underlined. B, the indicated plasmids were
transiently transfected into HeLa cells in triplicate, and protein was
removed for CAT ELISA, followed by pooling of the three samples and
cytoplasmic RNA extraction. An internal control plasmid was included in
all transfections. B, CAT levels were monitored by CAT
ELISA, and the levels are displayed as percent of CAT produced from
pAUM/UM. Mean values and standard deviations are shown. C,
the RNA samples were subjected to Northern blotting (lower
panel) followed by phosphorimaging quantitation (upper
panel). The RNA levels are displayed as percent of RNA compared
with pAUM/UM. Pooled RNAs from triplicate experiments are shown.
View larger version (24K):
[in a new window]
Fig. 7.
A, HPV-1 sequences inserted into the
reporter plasmid are shown. The sequence motifs in XB are
underlined and numbered. Introduced mutations are
underlined. Brackets mark deletions. B, the
indicated plasmids were transiently transfected into HeLa cells in
triplicate, and protein was removed for CAT ELISA followed by pooling
of the three samples and cytoplasmic RNA extraction. An internal
control plasmid was included in all transfections. B, CAT
levels were monitored in CAT ELISA, and the levels are displayed as
percent of CAT produced from pAUM/UM. Mean values and standard
deviations are shown. C, the RNA samples were subjected to
Northern blotting (lower panel) followed by phosphorimaging
quantitation (upper panel). The RNA levels are displayed as
percent of RNA compared with pAUM/UM. Pooled RNAs from triplicate
experiments are shown.
View larger version (26K):
[in a new window]
Fig. 8.
A, the AUM/UM and XB sequences are
shown. Multiple XB sequences were inserted into the reporter plasmid
resulting in the indicated plasmids with one, two, three, or four XB
sequences. The names of the plasmids are indicated on the
left. pAL1 and pAL2, late poly(A)
signals. B, the indicated plasmids were transiently
transfected into HeLa cells in triplicate, and protein was removed for
CAT ELISA followed by pooling of the three samples and cytoplasmic RNA
extraction. An internal control plasmid was included in all
transfections. CAT protein levels were monitored by CAT ELISA, and the
levels are displayed as percent of CAT produced from pAUM/UM. Mean
values and standard deviations are shown. C, the RNA samples
were subjected to Northern blotting (lower panel) followed
by phosphorimaging quantitation (upper panel). The RNA
levels are displayed as percent of RNA compared with pAUM/UM. Pooled
RNAs from triplicate experiments are shown. D, lg% CAT RNA
or protein were plotted against the number of insert XB sequences. The
plotted numbers represent mean values from three different experiments.
The slope of each curve is shown as kCATPROT and
kCATRNA. E, the RNA samples shown in
Fig. 8C were subjected to RNaseH cleavage in the presence of
the RNaseH oligo indicated in Fig. 8A. The digested RNA
samples were subjected to Northern blotting using a probe located
downstream of the RNaseH oligo. The results demonstrate that mRNAs
containing multiple XB sequences display the same length distribution
of poly(A) tails as the mRNAs containing the functionally inactive
AUM/UM sequence. Pooled RNAs from triplicate experiments are
shown.
View larger version (17K):
[in a new window]
Fig. 9.
A, schematic illustration of plasmid
DNAs used as templates for in vitro synthesis of capped and
polyadenylated CC and CC(A) RNAs. Plasmid names are indicated on the
left. The T7 bacteriophage promoter, the CAT open reading
frame, and the HPV-1 late 3' UTR-containing sequences are indicated.
The arrow in the HPV-1 sequence in pCC(A) indicates the
antisense orientation of the HPV-1 sequence between nt position 6868 and 7184. The unique XhoI site replaces the poly(A) signal
at nt 7426 and is used for linearization of the plasmid prior to RNA
synthesis. Numbers refer to nt positions in the HPV-1a genomic clone
(37). The mean values and standard errors of the CAT levels produced in
HeLa cells transfected with capped and polyadenylated CC and CC(A)
mRNAs and the internal control hGH mRNA at 20 h
posttransfection are shown. B, graph showing CAT levels
produced at different time points posttransfection of HeLa cells with
capped and polyadenylated CC and CC(A) mRNAs. The levels of hGH
produced from the internal control hGH mRNA at 11 h
posttransfection is shown in the inset. C,
cytoplasmic CC and CC(A) mRNA levels detected by primer extension
in transfected HeLa cells at different time points posttransfection as
indicated. The arrow indicates the specific extension
products of the transfected mRNAs.
View larger version (12K):
[in a new window]
Fig. 10.
A, schematic illustration of plasmid
DNAs used as templates for in vitro synthesis of the capped
and polyadenylated mRNAs that are transfected into HeLa cells. The
T7 bacteriophage promoter, the CAT open reading frame, and the HPV-1
late 3' UTR sequences are indicated. The NsiI site utilized
for linearization of the DNA prior to in vitro transcription is indicated. The sequences of the minimal h1ARE
(XB), the mutant AUM/UM sequence, and the two deletion mutants of the
h1ARE (B2 and C1) are shown. Mutations in AUM/UM are
underlined. Plasmid names are indicated on the
left. The functionally important sequence motifs are
numbered and underlined. Numbers refer to
nucleotide positions in the HPV-1a genomic clone (37). B,
the histogram shows mean values and standard deviation of the
quantified CAT levels produced at 20 h posttransfection in HeLa
cells transfected with capped and polyadenylated CCXB and CCAUM/UM
mRNAs. A representative experiment is shown. Mean values and
standard deviation of the quantified hGH protein levels produced from
the hGH-encoding mRNA included as a internal control are shown
below the histogram. C, the graph shows quantified CAT
levels produced from capped and polyadenylated CCXB, CCAUM/UM, CCB2,
and CCC1 mRNAs at various time points posttransfection into HeLa
cells. The inset shows the hGH protein levels at the same
time points produced from the hGH-encoding mRNAs included as
internal control. D, cytoplasmic CCXB and CCAUM/UM mRNA
levels detected by primer extension in transfected HeLa cells at
different time points posttransfection as indicated. The
arrow indicates the specific extension products of the
transfected mRNAs.
View larger version (15K):
[in a new window]
Fig. 11.
A, the histogram shows mean values and
standard deviation of the CAT levels produced at 20 h
posttransfection in HeLa cells transfected with capped CC and CC(A)
mRNAs in the absence or presence of a poly(A) tail as indicated in
the figure. A representative experiment is shown. Mean values and
standard deviation of the quantified hGH protein levels produced from
the hGH-encoding mRNA included as a internal control are shown
below the histogram. B, the graph shows the CAT levels
produced from capped CCXB or CCAUM/UM mRNAs in the absence
( 
An) or presence (+An) of a poly(A) tail at
7.5, 24, and 32 h posttransfection into HeLa cells. C,
the presence of the h1ARE does not result in rapid deadenylation of the
transfected mRNAs. Capped RNAs with (+) or without () the 57-nt
XB h1ARE were synthesized in the absence () or presence of a poly(A)
tail (+) of fixed length (A60). The in
vitro synthesized RNAs were transfected into HeLa cells, total
cytoplasmic RNA were harvested at 1 or 4 h posttransfection, and
the RNAs were analyzed by Northern blotting to investigate if
h1ARE-containing mRNAs were rapidly deadenylated. U, RNA
from untransfected cells.
View larger version (47K):
[in a new window]
Fig. 12.
A, UV cross-linking of GST-PABP,
GST-HuR, and GST-PCBP to the XB probe, the AUM/UM probe, an unrelated
HPV-16 L1-derived RNA probe named L1 (nt 5732-5768 in the HPV-16R
genome) and UV cross-linking of GST-PABP to RNAs B2 and C1 (See Fig.
6A and 10A). B, GST-PABP was UV
cross-linked to XB RNA in the presence of serially diluted competitor
RNA. XB, serially diluted XB competitor; L1,
serially diluted unrelated HPV-16 L1-derived competitor RNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Received a fellowship from the Emil and Ragna Börjessons Minnesfond.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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