From INSERM U511, Hôpital La
Pitié-Salpêtrière, 75643 Paris Cedex 13, France,
¶ Unité des Hépacivirus, Institut Pasteur, 75724 Paris
Cedex 15, France, the
Molecular Oncology Group/McGill
AIDS Centre, Lady Davis Institute for Medical Research, 3755 Côte
Ste Catherine, Montréal, Québec H3T 1E2, Canada, and the
Department of Medicine and Microbiology
& Immunology, McGill University, Montréal, Québec H3A 2B4,
Canada
Received for publication, September 3, 2002, and in revised form, December 4, 2002
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ABSTRACT |
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TRBP (HIV-1 transactivating
response (TAR) RNA-binding protein) and PKR, the interferon-induced
dsRNA-regulated protein kinase, contain two dsRNA binding domains.
They both bind to HIV-1 TAR RNAs through different sites.
Binding to dsRNA activates PKR that phosphorylates the
eukaryotic initiation factor eIF-2 TRBP1 (the HIV-1
transactivating response (TAR) RNA-binding protein) is a cellular
protein, which was initially isolated from a HeLa cell expression
library using TAR RNA as a probe (1). Two cDNAs have been isolated
and have been named TRBP1 (originally TRBP) and TRBP2. They are
identical but for an alternative first exon that arises from the use of
a second internal promoter in the first intron of TRBP1. TRBP2 is the
longer isoform and has 21 additional amino acids at its N terminus
(2-4). TRBP1 and TRBP2 each contain two double-stranded RNA binding
domains (dsRBD), although dsRBD1 binds RNA with low affinity whereas
dsRBD2 binds TAR with high affinity because of the presence of a KR
helix motif (5). TRBPs, its dsRBD2, and a 24-amino acid TRBP peptide
corresponding to the KR helix motif bind to two different sites of the
highly ordered RNA structures of TAR. One site of high affinity is
located between the bulge and the loop, and a second site of low
affinity is located within the stem structure (1, 5-7). The dsRBDs have also been found in several other proteins (8, 9).
The interferon-induced, dsRNA-regulated protein kinase, PKR, a cellular
serine/threonine kinase activated upon binding to dsRNA, induces
inhibition of protein synthesis by phosphorylating the TRBP has been reported to behave as an inhibitor of PKR. By
cotransfection analysis, TRBP was found to enhance the translational efficiency of DHFR mRNAs and to restore vaccinia protein synthesis in E3L-vaccinia mutant-infected cells (15). Yeast two-hybrid assays as
well as Far-Western techniques showed heterodimerization between PKR
and TRBP and conveyed the idea that PKR and TRBP need dsRNA as a bridge
for their mutual interaction (16). The ability of TRBP to inhibit PKR
activity was confirmed by overexpression of TRBP cloned into the
nef gene of the HIV-1 genome. This allowed the virus to
escape the PKR-mediated inhibition of virus replication. In addition,
this work demonstrated that PKR and TRBP can heterodimerize in the
absence of dsRNA (17). Furthermore, recent data have shown that TRBP
reverses the PKR-induced HIV-1 LTR inhibition of expression (18, 19).
The binding sites between TRBP and PKR have been mapped in each dsRBD
of TRBP and are independent of the KR helix motif that mediates major
RNA binding activity in TRBP (19).
TRBP function has been mainly studied in the context of viral
infection. These activities include its binding to HIV-1 TAR RNA and
PKR inhibition and activation of HIV LTR expression. Recent data
indicate a direct correlation between a weak expression of TRBP and a
low HIV replication in astrocytoma cells, which suggests a major role
in viral expression (4). PRBP, the murine homologue of TRBP, binds the
3'-UTR of Prm1 protamine RNA, regulates its translation and
plays a physiological role in spermatogenesis (20, 21). Mice that carry
a targeted disruption of the tarbp2 gene have a growth
defect, are sterile, and severely oligospermic. Xlrbpa, the
Xenopus homologue of TRBP, is associated with ribosomes and
hnRNPs (22). These data point to a stimulatory role of TRBP on
translation by a direct activation through its RNA binding properties
and/or through its inhibitory effect on PKR.
Here, we have analyzed the effect of TRBP on the in vitro
translation of CAT RNAs with or without the HIV-1 TAR structure at
their 5'-end. The ability of TRBP to inhibit PKR was also monitored by
analyzing the phosphorylation state of the endogenous eIF-2 Antibodies--
Rabbit polyclonal antibodies directed against a
synthetic 13-residue phosphorylated rat eIF-2 Cell Cultures--
PKR-deficient murine embryo fibroblasts
(MEFs) were provided by B. R. G. Williams (23). They were cultured in
Glutamax-1 Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 5 µg of penicillin-streptomycin per ml and
containing 10% fetal calf serum.
Plasmids--
The different TAR constructs (wild type and
mutants) have been previously described (1). These pUC18-T7-TAR-CAT
plasmids contain the CMV promoter (700 bp), T7 RNA polymerase binding
site (20 bp), TAR (80 bp), and the chloramphenicol acetyltransferase (CAT) gene (HindIII-BamHI insert of 1600 bp). The
plasmid T7-CAT was constructed by removing the CMV, T7, and TAR
sequences (NdeI-HindIII) and inserting the
T7-promoter as an oligonucleotide (5'-TATGTAATACGACTCACTATAGGGA-3' and
5'-AGCTTCCCTATAGTGAGTCGTATTACA-3') in
NdeI-HindIII sites of the same plasmid. The
plasmid pGl2-TAR-Luc and its derivatives were constructed as follows:
an 800-bp region containing the CMV and the T7 promoters followed by
TAR were excised from the pUC18-T7-TAR-CAT plasmids by
HindIII digestion and inserted into the pGL2 basic vector at
the HindIII site upstream of the luciferase coding sequence. The corresponding plasmid pGl2-Luc deprived of TAR was constructed as
follows: the CMV-T7 sequence was PCR-amplified from the pGl2-TAR-Luc plasmid using GAGCTCTTACGCGTGCTAGCT as 5' primer upstream of the HindIII restriction site and CCCAAGCTTATAGTGAGTCGTA as 3'
primer corresponding to the 3'-end of the T7 sequence. After subcloning in pCR2.1-TOPO (Invitrogen), the 720-bp PCR product was cut by HindIII and inserted in pGl2 basic vector. All constructs
were confirmed by sequence analysis. The pMAL-TRBP2 plasmid in which TRBP2 was fused in-frame with the maltose-binding protein (MBP) has
been described previously (7). TRBP2 cDNA (3) was inserted in the
pcDNA1/AMP vector at the BamH1 site to make
pcDNA1-TRBP2. The pcDNA3 expressing TR-A, TR-B, and TR-C have
been described previously (19). The pcDNA3-TR-AB was constructed by
amplification of the AB part from pBS-TRBP2 by PCR and insertion into
pcDNA3 BamH1-XbaI sites.
In Vitro Transcription and Translation--
For in
vitro transcription, the pUC18-T7-CAT or pUC18-T7-TAR-CAT plasmids
were linearized with BamHI. Transcription was performed at
37 °C for 2 h in a total volume of 25 µl containing 5 µg of linearized DNA and 10 units of T7 RNA polymerase according to the
manufacturer's protocol (Promega). The DNA template was removed by
DNase treatment for 20 min at 37 °C (1 unit of DNase/µg of DNA),
and the transcribed RNA was purified by phenol extraction and
resuspended in diethyl pyrocarbonate-treated distilled water. The
integrity of all RNAs was confirmed by electrophoresis on a 1% agarose
gel and visualized by fluorography. The concentration of the RNA
preparations was estimated by spectrophotometry using known amounts of
DNA as standards (Smart Ladder, Eurogentech). 25 ng of the in
vitro transcribed RNAs were translated at 30 °C for 45 min as
previously described (24), unless otherwise indicated. 5-µl aliquots
were analyzed by SDS, 12.5% PAGE.
Fusion Protein Production and Purification--
Plasmid DNA of
the empty pMAL-c vector (New England Biolabs) and pMAL-TRBP constructs
were transformed in Escherichia coli DH5 Immunoblotting--
The reticulocyte lysates were adjusted with
an equal amount of lysis buffer (20 mM Tris-HCl, pH 7.6, 50 mM KCl, 400 mM NaCl, 1 mM EDTA, 1%
Triton X-100, 5 mM 2-mercaptoethanol, 0.005% aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 20% glycerol)
supplemented with phosphatase inhibitors (5 mM sodium
fluoride, 10 mM paranitrophenylphosphate, 1 mM
sodium orthovanadate, and 10 mM Reporter Assay by Microtransfection--
The PKR-deficient MEFs
(23) were seeded at 20,000 cells per well in 96-well plates. They were
transfected, by a calcium phosphate precipitation-glycerol shock
technique as previously described (18, 25), with the same amounts of
plasmids by addition of the empty vector pcDNA1/Amp.
Preparation of Total RNA and RT-PCR--
PKR-deficient MEFs (23)
cultured in 10-cm Petri dishes (seeded at 2 × 106
cells per dish) were transfected by a calcium phosphate
precipitation-glycerol shock technique with 10 µg of plasmid pGl2
CMV-T7 TAR-Luc in the absence or in the presence of increasing amounts
of pcDNA1-TRBP2. Total RNA was extracted 24 h
post-transfection with Trizol isolation reagent (Invitrogen) and
treated by DNaseI (Amersham Biosciences). Luciferase and GAPDH
cDNAs were reverse-transcribed from 5 µg of total RNA using 5 pmol of 3'-UTR SV40 antisense (5'-GGAGGAGTAGAATGTTGAGAGTCA-3') and
GAPDH antisense-specific primer (5'-CCAAAGTTGTCATGGATGACC-3') in a
25-µl reaction containing 30 units of RNasin (Amersham Biosciences), 1 mM dNTP, 10 mM dithiothreitol and 300 units
of Superscript II (Invitrogen). Incubation was performed at 42 °C
for 1 h and 5 µl of the resulting reaction containing the single
strand cDNA template were used for PCR amplification. Conditions
for luciferase and GAPDH amplifications were 95 °C for 5 min, 20 cycles of 95 °C for 1 min, 58 °C for 1 min, and 72 °C for 2 min, followed by a 5-min incubation at 72 °C. PCR amplifications
were performed in a 100-µl reaction mixture containing 250 ng of each
GAPDH sense (5'-CCTTCATTGACCTCAACTACAT-3') or 5' luc 1600 primer
(5'-CCGCGAAAAAGTTGCGCGGAGGA-3'), 2.5 units of TaqDNA
Polymerase (Invitrogen), 1.5 mM MgCl2, 0.2 mM dNTP, and 1× Taq buffer (Invitrogen). The
products were fractionated on a 1.5% agarose gel. The luciferase set
of primers identifies a 497-nt band amplified by RT-PCR from the
mRNA and a 561-nt band amplified from the DNA when the DNase
treatment is not complete (25).
RNA Structure Analysis--
The 5'-UTR RNAs plus the AUG were
folded using the Mfold software version 3.1 (26, 27) on the Mfold
server using the standard parameters at 37 °C
(bioinfo.math.rpi.edu/~mfold/rna/form1.cgi). When single or double UG
type pairs were shown as 1 × 1 or 2 × 2 interior loops on
the folding structure, they were considered as non-canonical base pairs
and adjusted accordingly.2
The sequences were downloaded as Mac ct files and drawn with LoopDloop
software
(iubio.bio.indiana.edu/soft/molbio/java/apps/loops/loopDloop-doc.html).
Alteration of the HIV-1 TAR Structure Affects the Efficiency of
Translation of TAR-CAT Transcripts--
We have first analyzed whether
the structure of the TAR element could affect the translation of a
downstream CAT reporter RNA. Different CAT RNA transcripts either with
or without a TAR structure at their 5'-end were used for in
vitro transcription. These TAR RNA structures were previously
described (1) and are either wild type TAR (TAR) or TAR mutated in the
loop (TAR L135), in the bulge (TAR B123), between the bulge and loop
(TAR BL234), or deleted from nucleotides 3 to 11 (S TRBP Stimulates the Translation of TAR CAT Transcript--
TRBP
was initially isolated through its ability to bind to TAR RNA (1). We
have analyzed the effect of TRBP on the translation of CAT or TAR-CAT
RNAs in reticulocyte lysates. MBP, MBP-TRBP fusion protein (5) were
produced and purified in E. coli (Fig. 2). These proteins were added in
translation assays performed with CAT and TAR-CAT used as RNA
representatives of efficiently or poorly translated mRNAs,
respectively.
As previously, translation of CAT was much more efficient than
translation of TAR-CAT RNA (Fig. 3,
lane 1). Increasing concentrations of the MBP-TRBP protein
(50-5000 nM) progressively enhanced the efficiency of
translation of the TAR-CAT RNA (Fig. 3, lanes 6-8, bottom) with the best efficiency obtained for 1 µM of MBP-TRBP (lane 8). In comparison,
addition of the fusion protein did not increase the translation of CAT
RNA at low concentration, and inhibition was observed at high
concentrations (lanes 6-9, top). In contrast,
addition of same concentrations of the MBP control protein had no
effect on translation of CAT or TAR-CAT RNAs (Fig. 3, lanes
2-5). In agreement with previous results using TRBP in gene
expression experiments (2, 19), high concentrations inhibited the
translation of both RNAs (Fig. 3, lanes 8-9,
top; lane 9, bottom). These results
clearly indicate a positive effect of TRBP on the translation
efficiency of TAR-CAT RNA.
The ability of TRBP to selectively increase translation
efficiency of TAR containing RNAs by comparison with CAT RNAs was further confirmed in a kinetic experiment using 250 nM of
MBP or MBP-TRBP. The kinetics of TAR-CAT translation was identical to
CAT translation, increasing markedly after 15 min and with the highest
amount of product synthesized at 30 min. In agreement with the previous
results, the efficiency of translation of TAR-CAT RNA was
dependent upon the addition of MBP-TRBP (Fig.
4; lower panel, compare
lanes 5-8 to 1-4). Taken together, these
results demonstrate the ability of TRBP to increase the
efficiency of translation of TAR-containing RNAs and show that this
increase is not due to a change in the kinetics of translation.
eIF-2 Stimulation of TAR-CAT Translation by TRBP Cannot Be Solely
Attributed to PKR Inactivation--
We next explored whether the
ability of TRBP to increase the translation efficiency of TAR-CAT RNA
could be attributed to its ability to inhibit PKR activity and eIF-2
Therefore, the ability of TRBP to behave as a strong inhibitor for PKR,
is confirmed in the presence of rabbit endogenous PKR. Because TRBP
inhibits eIF-2 TRBP Stimulates Expression of TAR-containing Transcripts in
PKR-deficient MEFs--
TRBP has been reported to act as an inhibitor
of PKR in several assays (17-19) suggesting a major function in
cellular response to infection. Another function of TRBP in normal
cells, is to bind dsRNA segments of mRNAs to form
ribonucleoproteins with highly ordered structures (7, 20). Therefore,
it is likely that TRBP can stimulate protein synthesis through two
different mechanisms, one being by heterodimerization with and
inhibition of PKR, the other by favoring access of highly structured
RNAs to the ribosomal and translational machinery. If this hypothesis
is correct, one would expect TRBP to stimulate translation of
TAR-containing RNAs in the absence of PKR. To test this, we compared
the effect of TRBP on a luciferase reporter gene expressed under the
control of a CMV promoter and containing or not various TAR structures at its 5'-end. Indeed, the effect of TRBP2 was analyzed on some of the
TAR constructs described in Fig. 1, chosen to contain authentic (TAR),
partially disrupted (BL234), extensively disrupted (S
We then assessed if a specific domain in TRBP mediates the increased
expression of TAR-containing RNAs. TRBP has been previously divided
into fragment A (TR-A), which contains dsRBD1, fragment B (TR-B), which
contains dsRBD2, and fragment C (TR-C), which contains a basic region
in its C-terminal end but has no RNA or PKR-binding properties. TR-AB
has both dsRBDs but not the C-terminal domain. The dsRBD1 binds TAR RNA
with low affinity whereas dsRBD2 binds with high affinity (5). We
cotransfected PKRo/o MEFs cells with the CMV-TAR-Luc plasmid and
pcDNA3 vectors expressing either TRBP2 or fragments TR-AB, TR-A,
TR-B, and TR-C (Fig. 6B). In all cases, we observed an
increase of the luciferase expression except with TR-C that was
inhibitory or had no effect. We therefore concluded that each dsRBD
will likely mediate PKR independent activation of TAR containing
transcripts with AB and A having the highest activity.
TRBP Directly Increases Translation Rather Than RNA
Stability--
The increase of the expression of TAR-containing RNAs
in PKRo/o MEFs cells (Fig. 6) and in in vitro translation
suggests that TRBP increases luciferase expression by enhancing, either
mRNA stability and/or translation but not transcription efficiency. To determine which hypothesis is correct, PKR-deficient MEFs were transfected with the CMV-TAR-Luc and the TRBP2 plasmids as in Fig. 6.
Semiquantitative RT-PCR assays were performed in parallel with
luciferase measurements (Fig. 7). Whereas
the luciferase activity increased with the first two TRBP
concentrations (lanes 2 and 3), the level of
luciferase mRNA remained unchanged, and even slightly decreased at
high concentrations of TRBP. In all points, the ratio between the
luciferase and the endogenous GAPDH mRNAs remained constant. These
results show that the TRBP-mediated increase in luciferase activity
cannot be ascribed to an enhanced level of the corresponding mRNA
transcripts. We therefore concluded that the enhanced luciferase
expression of TAR-containing RNAs by TRBP in the absence of PKR is due
to a direct activity on translation.
Secondary structure of the 5'-UTR RNAs as well as cellular
proteins influence the rate of translation of a given transcript (30).
In this study, by using a reticulocyte lysate-based system of
translation, we have shown that the secondary structure of TAR RNA
affects the translation efficiency of the corresponding transcripts.
RNA transcripts, starting with an authentic TAR or with TAR in which
the dsRNA stem structure is retained (B123, L135), were poorly
translated when compared with transcripts without TAR. In contrast, TAR
mutants, in which the dsRNA structure was disrupted (S The ability of TAR-CAT transcripts to induce eIF-2 TRBP interacts with both the TAR dsRNA structure present at the 5'-end
of all HIV-1 mRNAs and with PKR. In the latter case, TRBP has been
shown to directly bind PKR in the absence of dsRNA and to reverse the
inhibitory effect of PKR on HIV replication (17). In agreement with
that, we have recently identified TRBP as a partner for PKR in the
screening of a human cDNA library with PKR using the yeast
two-hybrid assay.3 TRBP
reverses the inhibitory effect of PKR on HIV-1 replication (17) and on
HIV-1 LTR expression in a reporter assay (18). TRBP can also reverse
the PKR-mediated control of yeast growth (19). The ability of TRBP to
inhibit PKR is believed to occur by antagonizing the translational
block imposed by PKR, but no direct evidence has been demonstrated in
an in vitro translation assay. Our initial aim was to study
the effect of TRBP on the translation of TAR-containing RNA, in the
context of dsRNA-mediated PKR activation. TRBP increased specifically
translation of TAR-CAT RNA but not the translation of CAT RNA, which
proves its ability to antagonize a translational block. In this
context, incubation of the reticulocyte lysate with TAR-CAT or CAT RNAs
led to similar increase in eIF-2 To verify this hypothesis, the activity of TRBP was assayed in a
PKR-free context. We performed reporter gene assays in PKR-deficient MEFs (23), to demonstrate that TRBP has a direct stimulatory effect on
the expression of the TAR-containing RNAs (Fig. 6). We measured the
expression of CMV-Luc plasmids containing the authentic TAR, TAR
mutated in the upper stem-loop region (BL234), TAR deleted in the
bottom stem (S leading to protein synthesis
inhibition. TRBP and PKR can heterodimerize, which inhibits the kinase
function of PKR and has a positive effect on HIV-1 expression.
In this study, an in vitro reticulocyte assay revealed the poor expression of TAR containing CAT RNAs compared with
CAT RNAs. Addition of TRBP restored translation efficiency of TAR-CAT
RNA and decreased the phosphorylation status of eIF-2
, confirming
its role as a PKR inhibitor. Unexpectedly, eIF-2
was phosphorylated
in the presence of TAR-CAT as well as CAT RNA devoid of the TAR
structure. TRBP inhibited eIF-2
phosphorylation in both cases,
suggesting that it restores the translation of TAR-CAT RNA
independently and in addition to its ability to inhibit PKR. TRBP
activity on gene expression was then analyzed in a PKR-free environment
using PKR-deficient murine embryo fibroblasts. In a transient reporter
gene assay, TRBP stimulated the expression of a TAR-containing
luciferase 3.8-fold whereas the reporter gene with mutated TAR
structures or devoid of TAR was stimulated 1.5- to 2.4-fold. Overall,
the activity of TRBP2 was higher when the 5'-end of the mRNA was
structured and was mediated independently by each dsRBD in TRBP.
Increasing concentrations of TRBP showed no significant modification of
the luciferase RNA levels, suggesting that TRBP stimulates translation
of TAR-containing RNAs. Therefore, TRBP is an important cellular
factor for efficient translation of dsRNA containing transcripts, both
by inhibiting PKR and in a PKR-independent pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit of
eukaryotic initiation factor 2 (eIF-2
). Once phosphorylated,
eIF-2
prevents translation initiation (10, 11). As a dsRNA-binding
protein, PKR, also binds TAR RNA, which leads to its activation as a
kinase (12). The PKR/TAR interaction can be displaced by the addition
of a 60-amino acid synthetic peptide corresponding to residues of the
first dsRBD present in PKR (13). The integrity of the stem structure of
TAR is required for its interaction with PKR. TAR mutants whose
structure is disrupted are no longer able to bind to and activate PKR
(14).
present
in the reticulocytes used for the translation. Finally, the effect of
TRBP and its different domains on the expression of different TAR
transcripts was analyzed in a PKR-free environment. Our results lead us
to propose that TRBP can directly stimulate translation of
TAR-containing RNAs, independently, and in addition to its ability to
suppress the block imposed by PKR.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
peptide were obtained
from Research Genetics (Huntsville, AL). Mouse polyclonal antibodies directed against eIF2 were a gift of C. Proud (University of Dundee). Anti-mouse and anti-rabbit polyclonal antibodies conjugated to horseradish peroxidase were obtained from Amersham Biosciences.
. An overnight
culture from a single colony was diluted 1:100 (v/v) and grown to an
OD600 of 0.4. After 4 h of induction in 0.3 mM of isopropyl-
-D-thiogalactopyranoside
(IPTG), the bacteria were harvested and resuspended in cold sonication
buffer A (20 mM Tris-HCl, pH 7.6, 200 mM NaCl,
1 mM EDTA, and 100 µg/ml lysozyme containing protease
inhibitors (1 mM phenylmethylsulfonyl fluoride, 100 µg/ml
leupeptin, 10 µg/ml pepstatin). The suspension was sonicated and
clarified by centrifugation at 10,000 × g for 30 min
at 4 °C. The fusion proteins were purified to near homogeneity by
amylose column chromatography as described by the manufacturer (New
England Biolabs). Protein concentration was determined using the BioRad dye reagent with bovine serum albumin as a standard. For each protein
preparation, increasing concentrations (50-5000 nM) of MBP
or MBP-TRBP proteins were added in a preliminary in vitro translation assay, to determine the best concentrations to use.
-glycerophosphate).
After 5 min of incubation on ice, the samples were diluted twice with 2× protein electrophoresis buffer, and the proteins were separated by
SDS, 12.5% PAGE. The proteins were transferred to immobilon polyvinylidene difluoride membranes (Millipore), and the membranes were
processed for immunoblotting and for incubation with the primary and
secondary antibodies as described previously (17).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3-11) or 3 to 17 (S
3-17) in the stem region. To determine the most likely secondary structures of these RNAs, the corresponding 5'-untranslated RNAs plus
the starting AUG were folded using the Mfold software 3.1 (26-28). The
different structures and free energies (
G) indicate that TAR and TAR
mutants L135, B123 have the highest stability. BL234 has the overall
similar structure but a slightly decreased stability. S
3-11 and
S
3-17 have a less stable stem-loop at their 5'-end and a different
overall structure whereas the CAT UTR alone is not well structured
(Fig. 1A). All
the transcripts were found to migrate as single bands at the expected
size (0.9 kb) by gel electrophoresis (Fig. 1B). Equal
amounts of the various in vitro transcribed RNAs were
translated in a reticulocyte lysate cell-free system, and the
synthesized products were analyzed by SDS-PAGE (Fig. 1C).
The in vitro synthesis of the CAT protein markedly decreased
when translation was performed with TAR CAT transcript, and with the
TAR B123 CAT and TAR L135 CAT RNAs (Fig. 1C,
lanes 2, 3, and 5, respectively)
compared with its synthesis from the CAT RNA (lane 1). In
contrast, translation of TAR BL234 CAT (lane 4) was more
efficient than the translation of the other TAR CAT constructs and
translation products from TAR S
3-11 CAT and TAR S
3-17 CAT RNAs
(lanes 6 and 7) had equal intensity as that from CAT RNA. Overall, the translation efficiency of each RNA is in good
agreement with its calculated free energy (Fig. 1). Therefore, the
in vitro translation data indicate that the presence of the TAR dsRNA structure at the 5'-end of mRNAs severely affects their translation efficiency. In contrast, attenuation or disruption of this
structure leading to a decreased thermodynamic stability increases
translation of the corresponding RNAs.
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Fig. 1.
The presence or absence of different TAR
structures in the 5'-UTR, determine the efficiency of translation of
the CAT transcripts. A, secondary structure and
thermodynamic stability of the different 5'-UTR RNAs used in the
in vitro translation assay. The 5'-UTR of the transcripts
including the wild type TAR sequence (TAR) and different TAR mutants
previously described (1) were folded up to the AUG start codon using
the Mfold software version 3.1 (bioinfo.math.rpi.edu/~mfold/rna/form1.cgi). The most stable folded
structures are represented, and their calculated free energy is
G =
54.7 (TAR-CAT) and (B123),
55.2 (L135),
50.1 (BL234),
40.7 (S
3-11),
34.2 (S
3-17) and
16.9 (CAT)
kcal/mol. The dots in the BL234, B123, and the L135
structures indicate that the structure that follows is identical to TAR
CAT. The base changes in the TAR mutants L135, B123, and BL234 are
shown in bold letters. B, ethidium bromide
staining of in vitro transcribed TAR- and TAR-mutant-CAT RNAs. Each of the RNA expressing CAT either alone
or preceded by one of the TAR sequence shown above was transcribed
in vitro by the T7 RNA polymerase after linearization of
each corresponding template plasmid (pUC) with BamHI. Each
RNA migrates as a 0.9-kb band in a 1% agarose gel. An inverted image
of the fluorogram is presented. C, translation of the
TAR and mutant-CAT transcripts. The CAT or TAR-CAT RNAs were translated
in vitro in rabbit reticulocyte lysates. Lane 1,
CAT; lane 2, TAR CAT; lane 3, TAR B123
CAT; lane 4, TAR BL234 CAT; lane 5, TAR
L135 CAT; lane 6, TAR
S3-11 CAT; lane
7, TAR
S3-17 CAT. In the absence of RNA, no synthesized
product was detected (data not shown). Apparent molecular weight is
indicated on the right.
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Fig. 2.
Purification of MBP and MBP-TRBP after
expression in E. coli. A,
Coomassie Blue-stained gel of total proteins prepared either without
induction (lane 1), or after IPTG induction of E. coli cultures transformed with the control plasmid pMAL
(lane 2) or with the recombinant plasmid pMAL-TRBP
(lane 3). B, Coomassie Blue-stained gel of
purified MBP proteins. Lane 2, MBP; lane 3,
MBP-TRBP. Apparent molecular weights are indicated on the
right.
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Fig. 3.
TRBP increases the translation efficiency of
TAR-CAT RNA. The CAT (upper panel) or the TAR-CAT
(lower panel) RNAs were translated in the rabbit
reticulocyte lysate in the absence of additional protein (lane
1) or in the presence of 50 nM (lanes 2 and
6), 250 nM (lanes 3 and
7), 1000 nM (lanes 4 and
8), and 5000 nM (lanes 5 and
9) of MBP (lanes 2-5) or MBP-TRBP (lanes
6-9).
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Fig. 4.
Translation kinetics of CAT and TAR-CAT RNAs
in the presence of TRBP. The yield of CAT protein translated
either from the CAT (upper panel) or the TAR-CAT
(lower panel) transcript was monitored after 0 (lanes
1 and 5), 5 (lanes 2 and 6), 15 (lanes 3 and 7), and 30 (lanes 4 and
8) min of incubation in the presence of 250 nM
of MBP (lanes 1-4) or MBP-TRBP (lanes
5-8).
Phosphorylation Is Induced by CAT and TAR-CAT
mRNAs--
PKR is a ubiquitous protein present in a wide variety
of species and tissues, including rabbit reticulocytes. In the presence of dsRNA, PKR is activated and phosphorylates its substrate eIF-2
. A
concentration as low as 0.02 µg/ml of poly(rI)-poly(rC) is sufficient to activate the endogenous PKR from rabbit reticulocytes and to induce
eIF-2
phosphorylation (29) and Fig.
5B, lane 12. We therefore expected to observe the phosphorylated form of eIF-2
in
the presence of TAR-CAT RNA but not in the presence of CAT RNA. Kinetic
experiments were performed to explore the phosphorylation state of
eIF-2
in a translation assay. After the indicated incubation times,
the lysates were analyzed for eIF-2
phosphorylation by immunoblot
using antibodies directed against an eIF-2
peptide phosphorylated at
serine 51 and for total amounts of eIF-2
by using a mouse monoclonal
antibody directed against the full eIF-2 complex. Incubation of the
lysates in the presence of TAR-CAT RNA (Fig. 5A,
middle panel, lanes 9-14) resulted in eIF-2
phosphorylation, as expected from the dsRNA structure of TAR-CAT RNA
that activates PKR. Surprisingly, eIF-2
was also phosphorylated in
the presence of CAT RNA with the same kinetics (Fig. 5A,
middle, lanes 2-7). However, as already shown in
Fig. 1C, 3 and 4, translation of CAT
RNA was much more efficient than translation of TAR-CAT RNA (Fig.
5A, upper panel). We can therefore conclude that both RNAs activate PKR and that the phosphorylation of eIF-2
is not sufficient to explain the translation difference between CAT and TAR-CAT RNAs.
View larger version (65K):
[in a new window]
Fig. 5.
eIF-2
phosphorylation is not sufficient to inhibit translation and is
decreased by TRBP. A, eIF-2
phosphorylation is not
sufficient to inhibit mRNA translation. The kinetics of CAT
synthesis (upper panel) was compared with that of eIF-2
phosphorylation (middle panel). 50 µl of rabbit
reticulocyte lysates were incubated for 0 (lanes 1 and
8), 2 (lanes 2 and 9), 5 (lanes
3 and 10), 10 (lanes 4 and 11),
15 (lanes 5 and 12), 30 (lanes 6 and
13), and 45 (lanes 7 and 14) min with
either CAT RNA (lanes 1-7) or TAR-CAT RNA (lanes
8-14). 5 µl of the different translated products were analyzed
for CAT synthesis (upper panel). The rest of each sample was
diluted twice by addition of low salt buffer and incubated 5 min on
ice. An equal volume of 2× loading buffer was added, and the mixture
was separated on a 12.5% SDS-polyacrylamide gel. The proteins were
then analyzed by immunoblot for the presence of phosphorylated eIF-2
(middle panel) and for the presence of total eIF-2
(lower panel). B, TRBP decreases the
phosphorylation of elF-2
during translation of the CAT and the
TAR-CAT RNAs. 50 µl of rabbit reticulocyte lysates were incubated for
30 min in the absence (lane 1) and in the presence of CAT
(lanes 2-6), or TAR-CAT (lanes 7-11) RNAs.
Incubation was performed in the presence of 250 nM MBP
(lanes 3 and 8), 500 nM MBP
(lanes 4 and 9), 250 nM MBP-TRBP
(lanes 5 and 10), or 500 nM MBP-TRBP
(lanes 6 and 11). A positive control (lane
12) for eIF-2
phosphorylation was performed by incubation of
the lysate with 0.02 µg/ml of the synthetic dsRNA poly(I)-poly(C).
None represents incubation of lysate alone. The proteins were analyzed
by immunoblot for the presence of phosphorylated eIF-2
(upper
panel) and total eIF-2 (lower panel).
phosphorylation. Because TRBP has been shown to block PKR
autophosphorylation (17), we hypothesized that TRBP would prevent PKR
phosphorylation, which in turn will result in the non-phosphorylation
of its substrate eIF-2
. Therefore, the ultimate effect of increased
TRBP would be a decrease in eIF-2
phosphorylation and an increase in
translational initiation. The CAT and TAR-CAT RNAs were incubated for
30 min either in the absence or in the presence of two different
concentrations of MBP or MBP-TRBP (Fig. 5B). As in Fig.
5A (middle panel, lanes 6 and
13), at 30 min after the onset of incubation, incubation of
the lysates in the presence of CAT or TAR-CAT RNAs (Fig. 5B, lanes 2 and 7) resulted in eIF-2
phosphorylation. In addition, incubation of both types of lysates
containing CAT RNA (lanes 2-6) or TAR-CAT RNA (lanes
7-11) resulted in a strong inhibition of eIF-2
phosphorylation
with MBP-TRBP (250 nM, lanes 5 and
10, and at 500 nM, lanes 6 and
11), whereas no inhibition was seen when extracts were
incubated in the presence of MBP (lanes 3, 4,
8, and 9). The difference in eIF-2
phosphorylation was only due to the addition of TRBP as the levels of
endogenous eIF-2
were similar in all samples (Fig. 5B,
lower panel).
phosphorylation in the presence of both RNAs but only
increases TAR CAT translation, we concluded that the ability of TRBP to
restore the translation of the TAR-CAT RNAs is at least in part,
independent from its ability to inhibit PKR phosphorylation.
3-17) or no
(
TAR) TAR structure (Figs. 1A and
6A). Analysis was performed by
transient transfection in PKR-deficient MEFs (23) with increasing amounts of pcDNA1-TRBP2. The results show that TRBP2 stimulates the
reporter gene expression in all cases, albeit with different efficiencies. The highest stimulation was observed with TAR (3.8-fold). It was decreased with BL234 (2.4-fold), S
3-17 (1.5-fold), and
TAR (2.3-fold). The TAR BL234 mutant has lost its high affinity binding site for TRBP but has retained its dsRNA stem structure (containing low affinity binding sites for TRBP) whereas the TAR S
3-17 and
TAR have completely lost the TAR structure (Fig.
1A). Therefore, these data demonstrate that TRBP exerts a
direct stimulation on the expression of TAR-containing RNAs in the
absence of PKR. This stimulation is directly correlated to the
calculated free energy of each 5'-UTR RNA. Hence, TRBP has a higher
activity if the 5'- UTR is more stable except for a mild activity in
the absence of TAR. Because TAR, TAR BL234, TAR S
3-17, and
TAR
are translated with increasing efficiencies (Fig. 1C), we
can conclude that TRBP is more active when the translation of a given
RNA is more difficult to achieve. In all cases, the stimulation was
dependent on the concentration of the plasmid encoding TRBP and
decreased with high-concentrations of TRBP as previously observed in
other cells (2, 19).
View larger version (38K):
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Fig. 6.
TRBP stimulates the luciferase expression of
TAR-containing transcripts in PKR-deficient MEFs. A,
TRBP2 stimulates expression of TAR-containing plasmids to a higher
extent than 5'-unstructured RNAs. 100 ng of plasmids pGL2 expressing
TAR-luciferase (TAR), TAR-BL234-luciferase (BL234),
TAR-S 3-17-luciferase (S
3-17), or luciferase alone (
TAR)
under the control of the CMV-T7 promoter were transfected in
PKR-deficient MEFs in the absence or in the presence of increasing
concentrations of pcDNA1-TRBP2 (10-300 ng). B,
each dsRBD in TRBP2 stimulates the expression of TAR-containing
plasmids. 100 ng of plasmid pGL2 expressing TAR-luciferase under the
control of the CMV promoter were transfected in PKR-deficient MEFs in
the absence or in the presence of 10-300 ng of pcDNA1-TRBP2,
pcDNA3-TR-AB, TR-A, TR-B, or TR-C (19). Each DNA sample was
adjusted to the same final DNA content with the empty pcDNA1/Amp
vector. The luciferase activity per microgram of protein (Luc Index)
was calculated as the means of the transfection in four different
wells. In Fig. 6, the data shown are expressed as fold stimulation of
luciferase index and are the means of three independent experiments (± S.E.).
View larger version (35K):
[in a new window]
Fig. 7.
TRBP increases translation rather than
mRNA level. PKR-deficient MEFs were transfected with 10 µg
of plasmid pGL2 CMV-T7 TAR-luciferase and 0 (lane 1), 1 (lane 2), 2 (lane 3), 5 (lane 4), 10 (lane 5), or 30 (lane 6) µg of
pcDNA1-TRBP2. After 24 h of incubation, cells were washed
twice in phosphate-buffered saline. One-tenth of the cells were used
for luciferase analysis (top), and the remainder was
processed for RNA extraction and RT-PCR analysis on both the
transfected luciferase and the endogenous GAPDH mRNAs as indicated
(bottom). The lower band in the luciferase RT-PCR
assay is amplified from the mRNA whereas the upper band
is amplified from the transfected plasmid.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3-11,
S
3-17), were translated as the controls without TAR structure (Fig.
1, A and C). A partial restoration of translation was observed with a mutant with a different TAR upper stem-loop structure (BL234). These data reinforce the role of the TAR element as
a translational inhibitor and point out the importance of the dsRNA
structure of transcripts in the efficiency of translation. It has been
previously reported that the secondary structure of HIV-1 TAR affects
translation efficiency by preventing the accessibility of the cap
structure (31), by activating the PKR-mediated phosphorylation of the
initiation factor eIF-2
(12, 32) or by both mechanisms (33, 34). Our
results indicate that the TAR secondary structure is a significant
impediment to translational initiation.
phosphorylation
(Fig. 5) was expected as a result of the activation of the reticulocyte
endogenous PKR by the dsRNA TAR structure (12, 32). Therefore, it was
surprising to observe that the CAT RNA transcript, that has no TAR
structure, was similarly capable of inducing eIF-2
phosphorylation
to a level comparable to that achieved with TAR-CAT RNA and with
similar kinetics (Fig. 5). The phosphorylation of eIF-2
may occur
from activation of other eIF-2
kinases such as the hemin-controlled
repressor HRI (35). Indeed, a measurable low level of phosphorylated
eIF-2
can be observed after incubation during 30 min of the control
lysates incubated in the absence of RNA transcript (Fig. 5B,
lane 1). It is also possible that the in vitro
transcript preparations contain traces of dsRNA (36). Indeed, very low
amounts of dsRNA are known to activate PKR in reticulocyte lysates, as
seen by the control experiment carried out with poly(I)-poly(C) (Fig. 5B, lane 12). Hence, since TAR-CAT and CAT
transcripts were prepared under similar conditions, this potential
dsRNA contamination cannot account for the difference in the
translational behavior of TAR and CAT transcripts. Another explanation
is that long RNAs most often have double-stranded regions in their
structure, which might be sufficient to activate PKR. Indeed, folding
of the first 500 nt of CAT and TAR-CAT RNAs on the Mfold server showed
18 and 12 alternative putative structures and the most stable of each
have several double-stranded regions besides TAR when present (data not
shown). These multiple structures probably can activate PKR and, as a
consequence, eIF-2
will become phosphorylated. Whatever the case,
our results indicate that translation of CAT RNA is not impaired by the
accumulation of phosphorylated eIF-2
and that the difference in
translation efficiency between the two RNAs must have a different
origin. The stable structure of TAR RNA located at the 5'-end of the
transcript is the most plausible explanation to a decreased
translation. The translational initiation complex may be more difficult
to assemble with this structure rather than with a less stable
structure and will require the activity of additional proteins to
increase the recruitment of ribosome subunits and translational
factors. The direct relationship between the RNA stability of the
5'-UTRs and the decrease in translation supports this hypothesis (Fig.
1).
phosphorylation, suggesting
identical activation of PKR (Fig. 5A). Furthermore, a
purified preparation of TRBP blocked the phosphorylation of eIF-2
to
the same extent, whether it was triggered by TAR-CAT or CAT messengers
(Fig. 5B). Finally, TRBP increased specifically the
translation efficiency of the TAR-CAT RNA but did not modify
translation of CAT transcripts (Figs. 3 and 4). Taken together, these
results strongly suggest that TRBP increases TAR-CAT translation
through a mechanism independent from PKR inhibition.
3-17) or in the absence of TAR (
TAR). These TAR
sequences are representative of the most stable, relatively stable and
weakly stable 5' RNA structure, respectively (Fig. 1). A better
stimulation effect of TRBP on TAR-Luc plasmid, followed by the BL234
mutant, by the S
3-17 mutant suggests that the TRBP-mediated
stimulation requires the dsRNA structure of the RNA. The small increase
in the absence of TAR remains unexplained and may be due to specific
RNA structure not seen in the folding or to a low TRBP activity on all
RNAs. Because luciferase assays represent all expression steps between
transcription and translation, we analyzed whether the mRNA level
was affected. An absence of variation of the Luc mRNA in an RT-PCR
assay showed that neither the transcription rate, nor the stability was
affected by TRBP. The remaining and most likely explanation is a direct
activity of TRBP on translation. This effect can be due to either an
unfolding of the TAR structure that increases its accessibility to
ribosomes or to a direct activity on the translational machinery. A
destabilization of the TAR RNA structure has been previously observed
with TRBP peptides that bind the RNA (6) but the similar activity of both dsRBDs (TR-AB, TR-A, and TR-B in Fig. 6B) suggests that
only low affinity binding is necessary to mediate this function.
Therefore, the overall effect of the protein may be a destabilization
of the stable RNA structure to allow efficient translation. These results demonstrate that the ability of TRBP to stimulate TAR RNA
containing structures is not solely mediated by its ability to inhibit
the translational block imposed by PKR, but also by a PKR-independent
activity on translation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Zuker for help in the folding outputs, D. Purcell for critically reading the article, and C. Boschet for contribution to the article.
![]() |
FOOTNOTES |
---|
* This work was supported by INSERM, by grants from the Agence Nationale de la Recherche sur le SIDA (to C. V. and E. F. M.), and by Grant HOP-38112 from the Medical Research Council of Canada (to A. G.).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.
§ Supported by Agence Nationale de Recherche sur le SIDA.
** Supported by a Canadian Institutes of Health Research postdoctoral fellowship.
§§ Research Scientist from the Fond de la Recherche en Santé du Québec.
¶¶ To whom correspondence may be addressed. Tel.: 33-1-40-77-81-11; Fax: 33-1-45-83-88-58; E-mail: vaquero@idf.ext.jussieu.fr (to C. V.) or Tel.: 33-1-45-68-87-77; Fax: 33-1-40-61-30-12; E-mail: emeurs@pasteur.fr (to E. F. M.).
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M208954200
2 M. Zuker, personal communication.
3 M. Bonnet and E. Meurs, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TRBP, HIV-1 transactivating response (TAR) RNA-binding protein; HIV, human immunodeficiency virus; eIF, eukaryotic initiation factor; dsRNA, double-stranded RNA; UTR, untranslated region; CMV, cytomegalovirus; CAT, chloramphenicol acetyltransferase; MBP, maltose-binding protein; nt, nucleotide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEF, murine embryo fibroblasts.
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