Inhibition of translation by UAUUUAU and UAUUUUUAU motifs ... · C). To investigate if the 5th...

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Inhibition of translation by UAUUUAU andUAUUUUUAU motifs of the AU-rich RNA

instability element in theHPV-1 late 3’ UTR

Lisa Wiklund, Marcus Sokolowski, Anette Carlsson, Margaret Rush and Stefan Schwartz*

Department of Medical Biochemistry and Microbiology, Biomedical Center,Uppsala University, 751 23 Uppsala, Sweden.

Running title: Inhibition of translation by the HPV-1 ARE.

*Corresponding authorStefan Schwartz, PhDDepartment of Medical Biochemistry and MicrobiologyBiomedical Center, Uppsala UniversityHusargatan 3, Box 582,751 23 UppsalaSwedenPhone: 4618 471 4239Telefax: 4618 509 876e-mail: [email protected]

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 29, 2002 as Manuscript M205929200 by guest on Septem

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SUMMARY

The human papillomavirus type 1 (HPV-1) late mRNAs contain a 5 7

nucleotide adenosine- and uridine-rich RNA instability element termed

h1ARE in their late 3´ untranslated region. 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 were mapped to

UAUUUAU and the minimal inhibitory sequence in motifs III-V were

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 polyA tail on the mRNA. Additional

experiments demonstrated that the h1ARE interacted directly with the polyA

binding protein, suggesting that the h1ARE inhibits translation by interfering

with the function of the polyA binding protein.

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INTRODUCTION

Human papillomaviruses (HPVs) 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 (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 characterised 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), the same factors that 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 antagonises 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, ad the SRV-

1 CTE can overcome the inhibition (9), suggesting that the h1ARE traps the HPV-1 late


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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 utilisation of the mRNA. Here we present

results of a mutational analysis of the h1ARE and we provide evidence that the h1ARE

inhibits mRNA translation.


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EXPERIMENTAL PROCEDURES

Plasmid constructions

CMV promoter driven plasmids

All the eukaryotic expression plasmids containing the h1ARE mutants are derived

from pSKX (11) (Fig. 1B). Oligonucleotides were annealed and cloned into digested with

KpnI and XbaI that resulted in the insertion in pSnX of the wild type and mutant h1ARE

sequences displayed in the Figures. All mutants were sequenced. pCMVlacZ was constructed

by replacing the CAT gene in pSKX by the lacZ gene.

Bacteriophage T7 promoter driven plasmids

To generate pCCS that lacks the h1ARE, and its derivatives, a fragment containing

the first 75 nts that are transcribed from the CMV promoter, the chloramphenicol

acetyltransferase (CAT) gene and HPV-1 late 3´ UTR sequences spanning nt 7184 to nt 7447

was first amplified from pSXKb 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 pS. pS was digested with ApaI and

EcoRV, filled-in with T4 DNA polymerase and religated in order to remove polylinker

sequences in-between the T7 promoter and the cloned PCR fragment, generating pCCS.

pCCS also lacks the downstream polylinker sequences between KpnI and XhoI that 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


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SacI-XhoI fragment from the pCR2.1-based intermediate to pCCS, resulting in pCC. To

generate pCC(A) that 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). Radiolabelled RNA for UV cross

linking were produced from pKSXB, pKSAUM/UM, pKSB2 and pKSC1 (10).

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 analysed by Northern blot. Each plasmid was analysed in at least three

independent transfection experiments. RNA synthesis and transfections were performed as

described previously (18).

To generate RNAs with a polyA tail of fixed length, two PCR fragments were first

amplified with the following primer pairs: T7CATS

(5'-GTAATACGACTCACTATAGGGTACTGCGATGAGTGGCAGGG-3') and

HPV1ANTIPA

(5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

TTCACACTTGTGTATAATGCACCGG-3') (which encodes a 60A tail) or T7CATS and

HPV1ALW (5'- CACACTTGTGTATAATGCACCGG -3'). The PCR fragments that were


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generated from plasmid pSXKb, or pSXKb containing the 57nt h1ARE fragment, using the

two primer pairs described above, were gel purified and used for synthesis of capped RNAs,

with and without the 60A tail.

Plasmids or RNA encoding chloramphenicol acetyltransferase (CAT)-, human growth

hormone (hGH)- or ß-galactosidase were included in all transfections to monitor the

transfection efficiency.

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 20 h posttransfection. The levels of chloramphenicol acetyltransferase (CAT)-,

human growth hormone (hGH)- and ß-galactosidase proteins were quantified using CAT,

hGH and ß-galactosidase antigen capture enzyme-linked immunosorbent assays (ELISA;

Roche Molecular Biochemicals), respectively. All CAT quantitations were normalised to the

protein concentration of the cell extract, as determined by the Bradford method.

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, 10µg of total or cytoplasmic RNA was separated on

1% agarose gels containing 2.2M formaldehyde, followed by transfer to a nitrocellulose filter

and hybridisation. 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


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UV cross-linking and synthesis of radiolabeled RNA was performed as previously

described (11, 19). GST-PABP, GST-HuR and GST-PCBP were purified on GS-beads

according the manufacturers recommendations (Pharmacia Biotech).


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RESULTS

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 polyA signals resulting in pXB and pAUM/UM (Fig. 1B). As

controls, we used pSKX (which lacks the major part of the late 3’ UTR), pCCKH1 (contains

the entire late 3’ UTR) and pCCKH1(A) (contains the region of the late 3’ UTR containing

the XB sequence in antisense orientation). HeLa cells were transfected in triplicates, the CAT

production was monitored in each plate and mean values and standard deviations are

displayed in the figures. pCMVlacZ was inserted as internal control in all transfections and

the variation was less than 20% between the samples in the triplicates. For RNA extraction,

cytoplasmic extract from the triplicates were pooled and subjected to RNA extraction and

Northern blotting.

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 57nt 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

ration 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, they were mutated one by one (Fig. 2A).

However, there was only a modest increase in CAT RNA and protein levels (Fig. 2B and C)

for each mutant. To obtain a clearer answer on the importance of each motif, consecutive


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mutations were introduced in the motifs (Fig. 3A). The results revealed that higher

expression levels were obtained as more motifs were mutated (Fig. 3B 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. 3B and

C). To investigate if the 5th motif contributed to inhibition, the 5th motif was mutated in pM2

(Fig. 3A), resulting in pM2V (Fig. 3A). The results revealed that pM2V expressed higher

RNA and protein levels than pM2. These levels were similar to those produced from pM3

(Fig. 3B and C), thereby demonstrating that also the 5th motif contributed to inhibition.

Interestingly, the effect was greater at the protein level than at the RNA levels, for all

analysed mutants (Fig. 3B 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. 4B 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. 4B 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. 4B 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. 5B and C). Substituting two Us in the penta-U sequence with two Cs


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had the strongest effect whereas mutations in the Us flanking the As had the smallest effect

on the inhibitory activity (Fig. 5B 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 C1 region (Fig. 6B and C). Two B2 or two C1 regions were

as inhibitory as the entire XB (Fig. 6B and C), demonstrating that (or one type of motif,

UAUUUAU or UAUUUUUAU) could substitute for the 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 two contain only three Us, as in pAUUUA (Fig. 7A), the resulting inhibitory

elements were nearly as inhibitory as the wild type h1ARE (Fig. 7B 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. 2BC, Fig. 3BCD, Fig. 4BC,

Fig. 5BC, Fig. 6BC and Fig. 7BC). In other words, the mRNAs that contained the h1ARE,

or partially active mutants thereof were utilised less efficiently by the translation machinery

than mRNAs lacking the h1ARE or mRNAs containing functionally inactive mutants. To


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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 were 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. 8B and

C) and the fold inhibition was greater at the protein level than at the RNA level (Fig. 8B and

C). Extrapolation of the data in a semilog plot allowed a better estimate of the inhibitory

activity of one single XB sequence, at the protein level and at the RNA level (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 utilised for translation.

Deadenylation (20,21) could potentially cause the inhibition of translation observed

here. To determine the polyA 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 polyA 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.

In order 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) that we had used previously to study the h1ARE. These cloning

steps resulted in pCC(A) and pCC (Fig. 9A) that were linearised with XhoI and used as


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templates for in vitro synthesis of capped and polyadenylated CC(A) and CC mRNAs as

described in 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

20h posttransfection were quantified by using a CAT ELISA. The results revealed that CC

mRNAs that contain the h1ARE in sense orientation produced approximately 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 results 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 utilised 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


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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 synthesised 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. Figure 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 to 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 23h 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. Figure 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 Fig. 10D and 9C). The results confirmed that the


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h1ARE acts primarily by reducing mRNA translation in the RNA transfection experimnets

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 polyA tail.

The polyA 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 polyA 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. Figure 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,

that 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). Or, 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 to 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

or the inactive AUM/UM mutant (Fig. 11B), demonstrating a connection between the 57nt

XB sequence and the polyA 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. In order to investigate if the h1ARE promoted deadenylation

of the in vitro synthesised mRNAs that were transfected into the HeLa cells, mRNAs with a

polyA tail of fixed length were transfected into the cells and the length of the polyA tail was

determined at 1 h or 4 hrs post transfection by Northern blotting. Capped mRNAs with and


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without the h1ARE or polyA tail were analysed. 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 hrs posttranfection, showed that the

polyA 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 polyA tail, it was

reasonable to speculate that the h1ARE, or h1ARE binding factors, interact directly with the

polyA 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 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.


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DISCUSSION

The mutational analysis presented here revealed that the HPV-1 AU-rich element

consisted 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 destabilisation, but not optimal, and that multiple copies were needed

for significant destabilisation (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 destabilising 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 hepatmer 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 stabilisation (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 synthesised 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


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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 circularisation 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 polyA-PABP or cap-eIF4E complexes

result in access of the polyA tail and cap to the polyA 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


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polysomal loading. The role of the h1ARE, HuR and the PABP in the HPV-1 life cycle

remains to be determined.


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ACKNOWLEDGEMENTS

We tare 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. M. Sokolowski received a fellowship from the Emil

and Ragna Börjessons Minnesfond.


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FIGURE LEGENDS

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

polyA 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

polyA 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 that are believed to be inhibitory 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 actinomycinD at 20h posttransfection for the indicated number of

hours. The RNAs were analysed by Northern blotting (inset) and RNA levels were quantified

by phosphoimager. lg(%RNA); lg %RNA remaining after time point 0.

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 triplicates as described in

Experimental procedures. The cells were harvested, cytoplasmic extract 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

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 phosphoimager quantitation (upper panel). The

RNA levels are displayed as percent of RNA compared to pAUM/UM. Pooled RNAs from

triplicate experiments are shown.


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indicated plasmids were transiently transfected into HeLa cells in triplicates 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 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 phosphoimager

quantitation (upper panel). The RNA levels are displayed as percent of RNA compared to

pAUM/UM. Pooled RNAs from triplicate experiments are shown.





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 phosphoimager quantitation

(upper panel). The RNA levels are displayed as percent of RNA compared to pAUM/UM.

Pooled RNAs from triplicate experiments are shown.







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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 phosphoimager quantitation (upper

panel). The RNA levels are displayed as percent of RNA compared to pAUM/UM. Pooled

RNAs from triplicate experiments are shown.






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 phosphoimager quantitation (upper

panel). The RNA levels are displayed as percent of RNA compared to pAUM/UM. Pooled

RNAs from triplicate experiments are shown.


motifs in XB are underlined and numbered. Introduced mutations are underlined. Brackets

mark the deletions. (B) The indicated plasmids were transiently transfected into HeLa cells in

triplicates 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

phosphoimager quantitation (upper panel). The RNA levels are displayed as percent of RNA

compared to pAUM/UM. Pooled RNAs from triplicate experiments are shown.


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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

polyA signals. (B) The indicated plasmids were transiently transfected into HeLa cells in

triplicates 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 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 phosphoimager quantitation (upper panel). The RNA levels are displayed as percent of

RNA compared to 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 polyA tails as the mRNAs containing

the functionally inactive AUM/UM sequence. Pooled RNAs from triplicate experiments are

shown.

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


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unique XhoI site replaces the polyA signal at nt 7426 and is used for linearisation 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

11h 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 post

transfection as indicated. The arrow indicates the specific extension products of the

transfected mRNAs.

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 utilised for linearisation 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


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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 post transfection as indicated. The arrow

indicates the specific extension products of the transfected mRNAs.

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 polyA 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 polyA 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 synthesised in the absence (-) or presence of a polyA tail (+) of fixed

length (60A). The in vitro synthesised RNAs were transfected into HeLa cells, total

cytoplasmic RNA were harvested at 1h or 4hrs posttransfection and the RNAs were analysed

by Northern blotting in order to investigate if h1ARE-containing mRNAs were rapidly

deadenylated. U, RNA from untransfected cells.

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

pos.5732 to 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.


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E7

E1

E2

L1

E4

E5

L2

E6 NCR

3’UTR

HPV-1 genome

pAEpAL1

pAL2

Early genes Late genes

1A

h1ARE


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CMV CAT HPV-1

pAL2pAL1

pCCKH1

CMV CAT HPV-1

pAL2pAL1

XbaIKpnI

p∆KX

AUAUUUAUUAGUAGAUUAUUUAUUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

I II III IV V

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AUACCUAUUAGUAGAUUACCUAUUAUAUAUUCCUAUAUUCCUAUACUUCCUAUACUUAUM/UM

( )

h1ARE

KpnI XbaI

CMV CAT HPV-1

pAL2pAL1

pCCKH1(A)

h1ARE

1B

CMV CAT XBpXB HPV-1

pAL2pAL1

XbaIKpnI6868

7184

73807426


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0 1 2 4 0 1 2 4

pAUM/UM pXB

pXB

pAUM/UM

2.1

2.0

1.9

1.8

1.7

1.6

1.5

1.4

1.3

1.2

0 1 2 3 4

1C

t (h)

t (h):

lg (

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NA

)


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AUAUUUAUUAGTAGAUUAUUUAUUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

I II III IV V

pXB

AUACCUAUUAGUAGAUUACCUAUUAUAUAUUCCUAUAUUCCUAUACUUCCUAUACUUpAUM/UM

AUACCUAUUAGUAGAUUAUUUAUUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

AUAUUUAUUAGUAGAUUACCUAUUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

AUAUUUAUUAGUAGAUUAUUUAUUAUAUAUUCCUAUAUUUUUAUACUUUUUAUACU

AUAUUUAUUAGUAGAUUAUUUAUUAUAUAUUUUUAUAUUCCUAUACUUUUUAUACUU

AUAUUUAUUAGUAGAUUAUUUAUUAUAUAUUUUUAUAUUUUUAUACUUCCUAUACUU

pMI

pMII

pMIII

pMIV

pMV

pXB

pAUM/U

MpM

IpM

II

pMIII

pMIV

pMV

0

120

100

80

60

40

20

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120

100

80

60

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I II III IV V

pXB

AUACCUAUUAGUAGAUUAUUUAUUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

pAUM/UM

AUACCUAUUAGUAGAUUACCUAUUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

AUACCUAUUAGUAGAUUACCUAUUAUAUAUUCCUAUAUUUUUAUACUUUUUAUACUU

AUACCUAUUAGUAGAUUACCUAUUAUAUAUUCCUAUAUUCCUAUACUUUUUAUACUU

AUACCUAUUAGUAGAUUACCUAUUAUAUAUUCCUAUAUUCCUAUACUUCCUAUACUU

AUACCUAUUAGUAGAUUACCUAUUAUAUAUUUUUAUAUUUUUAUACUUCCUAUACUU

pM2

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pM1

pM2V

120

100

80

60

40

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120

100

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I II III IV V

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ACAUUUACUAGUAGAUCAUUUACUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

CUAUUUAUGAGUAGACUAUUUAUGAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

pAUM/UM AUACCUAUUAGUAGAUUACCUAUUAUAUAUUCCUAUAUUCCUAUACUUCCUAUACUU

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120

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pXB

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AUAUUUAUUAGUAGAUUAUUUAUUAUAUAUUCCUAUAUUCCUAUACUUCCUAUACUU

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120

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AUAUUUAUUAGUAGAUUAUUUAUUAUA

AUAUUUUUAUAUUUUUAUACUUUUUAUACUUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

AUAUUUAUUAGUAGAUUAUUUAUUAUAAUAUUUAUUAGUAGAUUAUUUAUUAUA

p2xC1

p2xB2

pC1

pB2

UAUAUUUUUAUAUUUUUAUACUUUUUAUACUU


120

100

80

60

40

20

0

pXB

p2xC1

p2xB2

pC1pB2

pAUM/U

M

%C

AT

pro

tein

120

100

80

60

40

20

0

pXB

p2xC1

p2xB2

pC1pB2

pAUM/U

M

%C

AT

RN

A

6A

6B 6C

pXB

p2xC1

p2xB2

pC1pB2

pAUM/U

M


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Dow

nloaded from

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I II III IV V

pXB

AUAUUUAUUAGTAGAUUAUUUAUUAUAUAUUUAUAUUUAUACUUUAUACUU

AUAUUUUUAUUAGTAGAUUAUUUUUAUUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

p5AUUUA

p5AUUUUUA


120

100

80

60

40

20

0

pXB

p5AUUUA

p5AUUUUUA

pAUM/U

M

%C

AT

pro

tein

120

100

80

60

40

20

0

pXB

p5AUUUA

p5AUUUUUA

pAUM/U

M

%C

AT

RN

A

7A

7B 7C

p5AUUUA

p5AUUUUUA

pAUM/U

MpXB

( ) ( ) ( )


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Dow

nloaded from

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AUAUUUAUUAGUAGAUUAUUUAUUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUUpXB

CMV CAT XB

CMV CAT XB

CMV CAT XB

CMV CAT XB

XB

XB XB

XB XB XB

pXB

p2XB

p3XB

p4XB

HPV-1

pAL2pAL1

HPV-1

HPV-1

HPV-1


KpnI XbaI

120

100

80

60

40

20

0

pXBp2X

Bp3X

Bp4X

B

pAUM/U

M

%C

AT

pro

tein

120

100

80

60

40

20

0

%C

AT

RN

A

pXBp2X

Bp3X

Bp4X

B

pAUM/U

M

8A

8B 8C

M/U

M pXBp2X

Bp3X

Bp4X

B

RNaseH oligo

100%

17%

5%1% 0.6%


http://ww

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Dow

nloaded from

http://www.jbc.org/

2.5

2.0

1.5

0.5

0.0

-0.5

1.0

CAT proteinCAT mRNA

0 1 2 3 4

8D

lg(%

)

Number of inserted XB

KCATPROT = -0.56

KCATRNA = -0.17


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

pAUM/U

M

pXBp4X

BpAUM

/UM

pCAT

pAn

8E


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Dow

nloaded from

http://www.jbc.org/

9.0±0.7

11±1.31.4±1.2

21±3.4

CAT

CAT HPV-1

XbaIKpnI

h1ARE

CAT HPV-1

h1ARE

XbaIKpnI6868

7184

pCC

pCC(A)

T7

T7

9A

XhoI

XhoI

hGH

time (h)

20

15

10

5

0

0 2 4 6 8 10 12

CA

T

CC mRNA

CC(A) mRNA

hGH

7.2

11

CC(A) mRNA

CC mRNA

9B


http://ww

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Dow

nloaded from

http://www.jbc.org/

9C

t (h): 3 5 23

CC CC(A)

CC CC(A)

CC CC(A)

CC CC(A)

1


http://ww

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Dow

nloaded from

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10A

AUAUUUAUUAGTAGAUUAUUUAUUAUAUAUUUUUAUAUUUUUAUACUUUUUAUACUUpCCXB

AUAUUUAUUAGUAGAUUAUUUAUUAUA

pCCC1

pCCB2

UAUAUUUUUAUAUUUUUAUACUUUUUAUACUU

pCCAUM/UM AUACCUAUUAGUAGAUUACCUAUUAUAUAUUCCUAUAUUCCUAUACUUCCUAUACUU

CAT HPV-1XB

74477184KpnIXbaI NsiI

pCCXB

I II III IV V

T7


http://ww

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Dow

nloaded from

http://www.jbc.org/

CCXBmRNA

CCAUM/UMmRNA

17±4.7

0.4±0.1

7.0±0.2 3.8±0.3

10B

10C

CA

T

hGH units:

0

5

10

15

20

25

CA

T

0

5

10

15

20

0 10 20 30 40 50

t (h)

CCAUM/UM

CCB2, CCC1

CCXB

hGH

t (h)

0

10

20

30

0 10 20 30 40 50


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Dow

nloaded from

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10D

t (h):

CCXBCCAUM

/UM

1 2.5

CCXBCCAUM

/UM


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Dow

nloaded from

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CCmRNA

CC(A)mRNA

CC(A)mRNA

CCmRNA

5.8±0.3 6.3±0.8 5.4±0.1 7.4±2.4

11B

-An +An

CA

T

0

5

10

15

20

25

30

35

hGH:

0.3+0.1 0.43+0.12 0.45+0.30

25+3.3

0

5

10

15

20

25

CA

T

t (h)

0 10 20 30 40

CCAUM/UM+An

CCAUM/U

M-A

n

CCXB+An

CCXB-An

11A


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Dow

nloaded from

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h1ARE:

pA tail:

+ -

1 2 3 4 5 6

+

+

- +

++ -- U

1h 4h

11C


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Dow

nloaded from

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XB AUM/UM L1

12A

PABPHuR

PCBPPABP

HuRPCBP

PABPHuR

PCBP

1x 9x 27x 1x 9x 27x XB

XB L1

12B

GST PABP

B1 C1

GST PABP

GST HuR


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nloaded from

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Lisa Wiklund, Marcus Sokolowski, Anette Carlsson, Margaret Rush and Stefan SchwartzRNA instability element in the HPV-1 late 3' UTR

Inhibition of translation by UAUUUAU and UAUUUUUAU motifs of the AU-rich

published online July 29, 2002J. Biol. Chem.

10.1074/jbc.M205929200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


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http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M205929200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2002/07/29/jbc.M205929200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2002/07/29/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2002/07/29/jbc.M205929200.citation

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Inhibition of translation by UAUUUAU and UAUUUUUAU motifs ... · C). To investigate if the 5th...

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Transcript of Inhibition of translation by UAUUUAU and UAUUUUUAU motifs ... · C). To investigate if the 5th...