Download Identification of a C-terminal Poly(A)-binding Protein (PABP)

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 278, No. 47, Issue of November 21, pp. 46357–46368, 2003
Printed in U.S.A.
Identification of a C-terminal Poly(A)-binding Protein (PABP)-PABP
Interaction Domain
ROLE IN COOPERATIVE BINDING TO POLY(A) AND EFFICIENT CAP DISTAL
TRANSLATIONAL REPRESSION*
Received for publication, July 15, 2003, and in revised form, August 25, 2003
Published, JBC Papers in Press, September 2, 2003, DOI 10.1074/jbc.M307624200
Eduardo O. Melo‡§, Rafael Dhalia‡¶, Cezar Martins de Sa‡, Nancy Standart储,
and Osvaldo P. de Melo Neto¶**
From the ‡Departamento de Biologia Celular, Universidade de Brasilia, Brasilia DF 70910-900, Brazil, §Embrapa
Recursos Genéticos e Biotecnologia, Parque Rural, Final W5, Asa Norte, Brası́lia DF 70770-900, Brazil, ¶Centro de
Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Campus da Universidade Federal de Pernambuco, Avenida
Moraes Rego s/n, Recife PE 50670-420, Brazil, and the 储Department of Biochemistry, University of Cambridge, Tennis
Court Road, Cambridge CB2 1GA, United Kingdom
* This work was supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico, Coordemação de Aperfeiçoamento de
Pessoal de Nı́vel Superior, and the British Council. The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel.: 55-81-33012568; Fax: 55-81-3453-2449; E-mail: [email protected].
This paper is available on line at http://www.jbc.org
The poly(A)-binding protein (PABP)1 bound to the 3⬘ poly(A)
tail of eukaryotic mRNAs is now recognized as an important if
not an essential component of the apparatus required for
mRNA translation. Evidence from yeast, plant, and animal
models has shown a role for the PABP-poly(A) complex in
translation initiation and points to a synergism between the 3⬘
and 5⬘ ends of the mRNAs (through the cap- and poly(A)associated proteins) in promoting translation (reviewed in
Refs. 1– 4). The demonstration of an interaction between the
translation initiation factor eIF4G and PABP provides an elegant explanation as to how this synergism occurs (5). Simultaneous binding of eIF4G to both PABP and eIF4E (the capbinding protein) leads to a circularization of the mRNA, as seen
by atomic force microscopy, which may enhance its ability to
undergo re-initiation (6). The PABP-poly(A) complex has also
been implicated in the control of mRNA stability in yeast (reviewed in Refs. 7 and 8) and in mammals (9, 10).
PABP consists of a highly conserved N terminus containing
four tandem RNA recognition motifs (RRM) followed by a more
variable C terminus (for review, see Ref. 4). The first two RRMs
are sufficient for specific poly(A)-binding (11–14). In addition,
RRM2 promotes the interaction between PABP and eIF4G
(15–17). RRM4 is responsible for most of the nonspecific RNA
binding of PABP (12, 13); in yeast the first half of RRM4 is
sufficient to confer viability to cells depleted of the normal
PABP gene (11). In Xenopus oocytes, RRMs can stimulate
translation when tethered to reporter mRNA (18). As to the C
terminus, although it does not bind RNA, it enables PABP to
multimerize on poly(A) (12). Recently it was shown to include a
domain responsible for binding to the PABP-interacting proteins Paip1 and Paip2 as well as to the release factor eRF3 (19).
This 74-residue domain (called PABC) is conserved in all
PABPs described so far (from protists to vertebrates) and is
also present in the hyperplastic discs protein (HYD) family of
ubiquitin ligases (20). The structures of both human PABC and
its HYD homologue confirmed it as a novel phylogenetically
conserved domain responsible for protein-protein interactions
(19, 20). The C terminus can contribute to mRNA stabilization
1
The abbreviations used are: PABP, poly(A)-binding protein; RRM,
RNA recognition motif; CAT, chloramphenicol acetyltransferase; nt,
nucleotide(s); UTR, untranslated region; IRE, iron regulatory element;
IRP, iron regulatory protein; GST, glutathione S-transferase; BB, binding buffer; BSA, bovine serum albumin; Ni-NTA, nickel-nitrilotriacetic
acid; DMEM, Dulbecco’s modified Eagle’s medium.
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The poly(A)-binding protein (PABP), bound to the 3ⴕ
poly(A) tail of eukaryotic mRNAs, plays critical roles in
mRNA translation and stability. PABP autoregulates its
synthesis by binding to a conserved A-rich sequence
present in the 5ⴕ-untranslated region of PABP mRNA
and repressing its translation. PABP is composed of two
parts: the highly conserved N terminus, containing 4
RNA recognition motifs (RRMs) responsible for poly(A)
and eIF4G binding; and the more variable C terminus,
which includes the recently described PABC domain,
and promotes intermolecular interaction between
PABP molecules as well as cooperative binding to
poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20
or more A residues in their 5ⴕ-untranslated regions and
remains effective as a repressor when an A61 tract is
placed at different distances from the cap, up to 126
nucleotides. Deletion of the PABP C terminus, but not
the PABC domain alone, significantly reduces its ability
to inhibit translation when bound to sequences distal to
the cap, but not to proximal ones. Moreover, cooperative
binding by multiple PABP molecules to poly(A) requires
the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction
between PABP molecules, mediated by the C terminus,
does not require the PABC domain and is enhanced by
the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat
protein MS2 have an enhanced ability to prevent the
expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal
distances from the cap. Altogether, our results identify a
proline- and glutamine-rich linker located between the
RRMs and the PABC domain as being strictly required
for PABP/PABP interaction, cooperative binding to
poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.
46358
Identification of a C-terminal PABP-PABP Interaction Domain
EXPERIMENTAL PROCEDURES
Cyclin Reporter Plasmids—The cyclin mRNAs described in this
study were based on the Xenopus cyclin A cDNA in the plasmid pTZ18R,
which contained a tract of 61 A residues, located 41 nucleotides from the
5⬘ end of cyclin mRNA (25). In this and in the following constructs, the
cyclin cDNA is missing the end of its 3⬘-UTR (⬃80 nt) and it lacks a 3⬘
poly(A) tail (we have seen that the presence or absence of such a tail
does not affect translation inhibition/stimulation by poly(A)). To reduce
the A tract length, this plasmid was digested with StuI and XbaI and
the longer fragment, devoid of the original A tract, was ligated to pairs
of oligonucleotides designed to reduce the number of A residues to 20 or
10 nucleotides (5⬘ primers: 5⬘-CCTA20T-3⬘ and 5⬘-CCTA10T-3⬘). To reduce the distance between the transcription initiation site and the start
of the A tract, the original plasmid was digested with EcoRI and StuI,
treated with T4 DNA polymerase, and religated (distance cap/A tract of
10 nucleotides). To increase the distance, the starting plasmid was
digested with KpnI and BamHI and ligated with the 62-bp KpnI/
BamHI fragment from plasmid Bluescript II KS⫹ from Stratagene.
This plasmid was then digested with XhoI and ClaI, treated with T4
DNA polymerase, and then either religated (distance 86) or ligated with
either one or two copies of the 40-nt StuI/SacII fragment obtained from
the human PABP cDNA 5⬘-UTR (distances 126 and 166). All plasmid
constructions were confirmed by sequencing.
In Vitro Transcription and Translation—Transcriptions were performed as described previously (25), using T7 RNA polymerase with
SalI (cyclin) or BamHI (PABP) linearized plasmid DNAs. For the translation reactions, the rabbit reticulocyte cell-free system (Promega) was
treated with micrococcal nuclease to render it mRNA-dependent. In
vitro transcribed RNAs were translated at the final concentrations
indicated in the figures. Protein synthesis was assayed at 30 °C with
[35S]methionine (Amersham Biosciences) and the radiolabeled products
analyzed by 15% SDS-polyacrylamide gel electrophoresis and autoradiography. When used, polynucleotides were pre-incubated with the
reticulocyte mix for 15 min on ice, prior to addition of mRNA, followed
by incubation at 30 °C for 1 h. For the translations with the added
proteins, mRNAs were pre-incubated with the GST fusion proteins and
ribonuclease inhibitor (final concentration of ⬃200 units/ml in the
translation reaction) on ice for 15 min in the presence of Escherichia coli
rRNA (2.5 mg/ml) and poly(G) or poly(C) (at 2 mg/ml final) as nonspecific competitors, prior to the addition of the translation extract. All the
translation results shown are representative of various experiments
performed with different batches of mRNAs and proteins, and the data
were highly reproducible in trend from experiment to experiment. Repeating the experiments with the same mRNA/protein batches produced identical results.
GST-PABP Expression Plasmids—The plasmid encoding human
PABP (construct P10 – 636, missing the first nine codons), cloned into
the BamHI site of pGEX2T, has been described (25). Clones encoding
the PABP variants P10 –584, P10 –370, and P372– 636 were obtained by
PCR using an annealing temperature of 50 °C for 30 cycles. As 5⬘
primer for variants P10 –584 and P10 –370, the oligonucleotide 6291
(5⬘-CGGGATCCCTGCGGGCAGCCG-3⬘), which anneals immediately
upstream of the PABP translation initiation codon, was used. The 3⬘
oligonucleotides, 3771 and 3772, which are complementary to sequences coding for conserved motifs present at the end of the fourth
RRM (3⬘ end of P10 –370) and in the second half of the C terminus (3⬘
end of P10 –584), have been described previously (39). To clone fragments P10 –584 and P10 –370 into the pGET2T expression vector, the
PCR fragments were digested with NcoI (which cuts on the second
methionine at amino acid position 10) and BamHI, prior to in-filling and
ligation into the in-filled BamHI-cut pGEX2T vector (Amersham Biosciences). The P372– 636 PCR fragment was amplified with the oligonucleotides 7736 (5⬘-CCGGGATCCGAAGAGCGCCAGGCT-3⬘) and
7737 (5⬘-CGGAATTCTAGATATTTTTCTTCGGTG-3⬘) and inserted into
the BamHI/EcoRI sites of the pGEX2T vector. The P10 –370 plasmid
was further digested with EcoRI and the larger fragment gel religated
to yield the P10 –190 plasmid. P237– 636 was the result of digesting the
plasmid coding for wild type PABP with NcoI/HindIII followed by infilling and religation of the plasmid-containing fragment. P10 –542 was
obtained by site directed mutagenesis of the P10 – 636 plasmid to replace the glutamic acid at position 543 with a stop codon. Mutagenesis
was performed with the QuikChangeTM site-directed mutagenesis kit
(Stratagene). Likewise, P237–542 was obtained from P237– 636 by the
introduction of the same mutation. All plasmid constructions were
confirmed by sequencing. Protein expression and purification has been
described previously (25).
Band-shift Assays—The RNA probes used in the band-shift assays
were synthesized from linearized plasmids using T7 RNA polymerase
and [␣-32P]ATP (Amersham Biosciences). Band-shifts were performed
as described by Walker et al. (40) with minor modifications. Briefly,
10-␮l reactions were carried out in microtiter plates. The labeled probes
were initially denatured by incubation at 95 °C for 3 min and then,
following rapid cooling, were incubated with different concentrations of
the recombinant proteins for 15 min on ice in the presence of binding
buffer (5 mM Hepes, pH 7.2, 1.5 mM MgCl2, 2.5% glycerol, and 0.5 mM
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(21) and also plays a role in the nuclear export of PABP bound
to newly synthesized poly(A)-containing RNA (22).
PABP expression is regulated at the translational level.
PABP mRNA is a member of the 5⬘-terminal oligopyrimidine
tract mRNA family (23), which includes mRNAs encoding components of the protein synthesis machinery. Translation of
these mRNAs is linked to the growth status of the cell, and this
regulation strictly requires a short sequence of polypyrimidines
at their 5⬘ end (reviewed in Ref. 24). Control of PABP expression is also achieved through an autoregulatory mechanism
whereby PABP binds to an A-rich tract present in the 5⬘-UTR
of its mRNA and represses its own synthesis, when in excess
over poly(A)⫹ RNA (25–28). The A-rich tract, 50 –70 nt long, is
found in almost all PABP mRNAs (25, 29, 30). In the human
mRNA, the 61-nt-long tract contains sets of 5– 8 A residues
interspersed by 3– 6 pyrimidine residues and is located 73
nucleotides from the cap (23). Repression by PABP may be
relieved when there is an increase in the intracellular poly(A)⫹
mRNA, or an increase in the length of pre-existing poly(A) tails.
Addition of exogenous poly(A) mimics this effect and specifically stimulates PABP synthesis in vitro in the absence of
mRNA synthesis (25), whereas overexpression of PABP in vivo
leads to the translational repression of endogenous PABP
mRNA (27, 28).
The prototype mechanism for translational repression of
mRNAs by 5⬘-UTR-bound proteins is that described for ferritin
mRNA. Regulation of ferritin mRNA translation is mediated by
a stem-loop structure called the IRE (iron regulatory element)
and its binding factor, the iron regulatory protein (IRP; for
reviews, see Refs. 31 and 32). The IRE-IRP complex represses
ferritin mRNA translation by precluding the recruitment of the
small ribosomal subunit (33). To be effective in vivo and in the
rabbit reticulocyte lysate cell-free system, the IRE motif must
be located within 60 nucleotides of the mRNA cap structure
(34, 35). When placed further from the cap, the IRE-IRP complex delays productive scanning of the ribosomal subunit along
the mRNA 5⬘-UTR, but without greatly compromising overall
translation (36). In contrast, in plant or yeast extracts, which
lack endogenous IRP, a cap-distal IRE-IRP complex can arrest
ribosomal scanning and efficiently inhibit translation (36, 37).
As to PABP-mediated translational repression, recent evidence
indicates that, in HeLa cells, PABP bound to the A-rich tract in
the reporter mRNA 5⬘-UTR, up to 200 nt from the 5⬘ cap, stalls
the migration of the 40 S ribosomal subunit along the mRNA,
preventing it from reaching the AUG and forming the elongating 80 S complex (38).
In this study, we examined the requirements of the PABP
molecule to act as a translational repressor of reporter mRNAs
containing A-tracts in their 5⬘-UTR. We confirm the lack of
positional requirement for effective inhibition by wild type
PABP (38). Moreover, we observe that PABP variants lacking
the C terminus are much less efficient in repressing the translation of mRNAs carrying cap-distal A-tracts. These PABP
variants have affinities for poly(A) similar to that of the fulllength protein, but have a reduced ability to associate cooperatively to poly(A). We go on to show a correlation between the
presence of the fourth RRM and part of the C terminus, but not
including the PABC domain, and the ability of PABP to associate cooperatively to poly(A), multimerize, and enhance PABPmediated translational repression.
Identification of a C-terminal PABP-PABP Interaction Domain
15 min at 37 °C, and the clarified supernatant was assayed for CAT and
␤-galactosidase activities using standard methods (44).
For the RNA expression analysis, 5-fold transfection reactions were
carried out using the MSC-91 reporter plasmid with the highest concentration of effector plasmid tested. Approximately 15% of the transfected cells were processed and assayed for CAT and ␤-galactosidase.
The remaining transfected cells were extracted with TRIzol (Invitrogen), and the total RNA was digested with RNase-free DNase I (Amersham Biosciences) and then run on denaturing formaldehyde gels prior
to Northern blotting. The following DNAs were used as probes: the
0.7-kb XbaI/HindIII fragment of pET-MS2, the 1.6-kb HindIII/BamHI
fragment of pSV2-CAT, the 2.4-kb NcoI/SmaI fragment of pGEX2TPABP, and the 3.5-kb ScaI/NotI fragment of pCMV␤ containing the
␤-galactosidase gene. Probes were labeled with [␣-32P]dCTP using the
Megaprime kit (Amersham Biosciences).
RESULTS
Requirements for Translational Regulation of Reporter
mRNAs Containing 5⬘-UTR A-tracts—To study the role of the
A-rich region of the PABP mRNA 5⬘-UTR in PABP-mediated
translation repression, we previously used mRNA reporters
containing A-rich sequences followed by the coding region of
Xenopus cyclin A (25). Here, we first examined the effect of
varying the length of the A-tracts upstream of the cyclin reporter mRNAs, by comparing the effect of 10, 20, and 61 A
residues in the 5⬘-UTR. The longest tract is the same length as
the entire A-rich region in the human PABP mRNA leader
region. The assay is based upon the ability of endogenous
reticulocyte PABP to repress translation of the A-tract containing reporter mRNAs. We have shown that the presence of the
5⬘-UTR A-tract is associated with a reduced translation efficiency of the reporter mRNAs, as a result of bound PABP, as
compared with control RNAs devoid of the adenylate sequence.
Addition of exogenous poly(A) stimulates the translation of
these mRNAs in the reticulocyte system by titrating the PABP
bound to their 5⬘-UTR (25). An internal control mRNA, which
lacks any regulatory elements in the leader region, ⌬(1–263)
PABP, is included in the translation assay (25).
Translation of cyclin mRNAs containing in their 5⬘-UTR
either 10, 20 or 61 A residues, at a fixed position relative to the
cap (41 nt), was assayed in the absence or presence of increasing amounts of added poly(A) (Fig. 1A). First, we noted that, in
the absence of poly(A) competitor, increasing the chain length
of A residues progressively reduced the efficiency of translation
of cyclin mRNAs, relative to the control mRNA, presumably
reflecting enhanced PABP binding. The 10-A-containing cyclin
mRNA was not affected by the addition of poly(A), except at the
highest doses, which inhibited the translation of the various
cyclin reporters and the control RNA. As has been described
previously (25, 45), inhibition of translation by poly(A) presumably occurs through a depletion of the lysate PABP, because
this inhibition can be reversed by addition of the purified
protein (45). The translation of both the 20- and 61-A-containing cyclin mRNAs was stimulated at intermediate poly(A) levels, with similar degrees of stimulation observed for both
mRNAs (Fig. 1B). These data are entirely consistent with the
finding that PABP can bind oligo(A) sequences as short as 12 nt
(11, 12) in vitro. Next, we investigated the importance of the
position of the adenylate tract, relative to the 5⬘ end of the
mRNA, to the translation of reporter mRNAs. Cyclin RNAs
were obtained, which contained 61 A nucleotides placed at
various distances from the cap, ranging from 10 to 166 nt.
Stimulation of translation by added poly(A) was observed in all
five distance reporter mRNAs, although the degree of enhancement was somewhat less efficient in the most cap-distal
mRNAs, with A-tracts at 126 and 166 nt from the cap (Fig. 1 (C
and D) and data not shown). This difference possibly reflects
the presence of additional sequences in the cap-distal mRNAs,
or a reduced efficiency of the translation of the mRNAs con-
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dithiothreitol), 250 ␮g/ml competitor E. coli rRNA, 100 mM KCl, 1
mg/ml BSA, and RNase inhibitor (2.5 units). Heparin was then added to
5 mg/ml followed by 2 ␮l of loading buffer (48.5% glycerol and 0.5% each
of bromphenol blue and xylene cyanol). Samples were then immediately
run on nondenaturing polyacrylamide gels (acrylamide/bisacrylamide
ratio of 60:1) in 0.5⫻ TBE, the gels having been previously pre-run for
15–30 min at 200 –300 volts. Electrophoresis was performed at 4 °C,
followed by autoradiography.
Pull-down Assays—The glutathione-Sepharose beads (Amersham
Biosciences) were initially pre-washed with binding buffer (BB: 150 mM
KCl, 5 mM MgCl2, 10 mM Hepes, pH 7.2, 0.2% Nonidet P-40) and
saturated with 10 mg/ml BSA in BB for 30 min at 4 °C, followed by two
more washes with BB. Approximately 20 ␮l of the beads were incubated
with 4 ␮g of the GST-PABP variants in a final volume of 100 ␮l for 30
min at room temperature. The beads were then washed two times with
BB containing 4 mg/ml BSA and incubated with 10 ␮l of translation
extract for 30 min at room temperature and washed twice again with
BB. Proteins bound to the beads were eluted by addition of SDS-PAGE
sample buffer and the samples run on 15% SDS-PAGE, followed by
Coomassie Blue staining and autoradiography. Labeled cyclin A and
PABP were synthesized in the TNT T7 coupled reticulocyte lysate system (Promega) using 5 ␮g/ml amounts of each plasmid. The labeled
PABP deletions were obtained by linearizations of the wild type PABP
cDNA with HindIII (1–237), MscI (1–358), or Tth111I (1– 432) (the
numbers indicate the amino acids retained from full-length PABP),
followed by transcription with T7 RNA polymerase in the presence of
the cap analogue and translation in the rabbit reticulocyte lysate. In
some cases, prior to their use in the pull-down assays, the reticulocyte
lysates were treated with either 100 units/ml cobra venom RNase
(RNase V1; Amersham Biosciences) for 30 min at 37 °C or with 250
units/ml micrococcal nuclease for 20 min at 20 °C in the presence of 1.5
mM CaCl2 followed by addition of EGTA to 6 mM. Both RNases efficiently degraded commercial poly(A) (data not shown). When specified,
the lysates were also supplemented with E. coli rRNA (250 ␮g/ml),
poly(A) (5 or 20 ␮g/ml), or poly(C) (20 ␮g/ml).
Pull-downs with the His-tagged proteins were carried out as described above but using the Ni-NTA resin (Qiagen). Labeled human
eIF4G was obtained by subcloning the XbaI/HindIII fragment from
plasmid pSK-HFC1 (41) into the same sites of the pBluescript KS vector
(Stratagene), followed by linearization with HindIII and in vitro transcription and translation as described above. His-tagged human eIF4A
from plasmid pET(His6-eIF4A) (42) and MS2/PABP-(372– 636) were
expressed and purified on Ni-NTA resin as recommended.
MS2 Tethering Constructs—The reporter plasmids MSC-15 and
MSA-15 were obtained by digestion of the MSC-GH and MSA-GH
plasmids (43) with XbaI/HindIII and ligation of the larger fragment to
the BamHI/HindIII fragment encoding the CAT gene from plasmid
pSV2-CAT. Both the vector and insert DNAs were in-filled with Klenow
DNA polymerase prior to ligation. The MSC-91 plasmid was constructed by inserting a BamHI/XbaI insert from the polylinker region of
plasmid pcDNA3 (Invitrogen) into the BamHI site of MSC-15. In this
case, the BamHI ligation was performed first, followed by the in-filling
of the protruding ends and a second round of ligation.
To produce the effector plasmids, the DNA fragments encoding the
various PABP deletions originally in the pGEX2T vector were first
cloned into the pET-MS2 plasmid (21) to generate MS2-PABP fusions.
Subcloning the in-filled BamHI PABP fragments from P372– 636 and
P237–584 into the BamHI site of pET-MS2, also in-filled, yielded MS2/
PABP-(372– 636) and MS2/PABP-(237–584), respectively. The resulting
plasmids were then digested with XbaI/EcoRV, in-filled, and the inserts
transferred to the EcoRV site of the eukaryotic expression vector
pcDNA3, under the control of the cytomegalovirus promoter. All cloning
steps and the final coding frames were confirmed by sequencing.
Transfection Assays—Approximately 4 ⫻ 105 human embryonic kidney cells (293-EBNA-BCRJ) were grown in each well of the 24-well
dishes with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, streptomycin (100 ␮g/ml), and penicillin (60 ␮g/ml). Subconfluent cultures were transfected in DMEM
without serum and antibiotics. Transfections were performed with 250
ng of reporter plasmid, 20 ng of pCMV␤ control plasmid (Clontech), and
2.5, 5, or 10 ng of effector plasmid (for the first two concentrations of the
effector plasmid DNA, the difference to 10 ng was supplemented with
DNA from the empty pcDNA3 vector). The plasmids were transfected
along with LipofectAMINE (Invitrogen) as recommended by the manufacturer, and the transfected cells were allowed to grow for ⬃36 h in
DMEM plus 10% fetal calf serum and antibiotics. Prior to harvesting,
the cells were washed twice with phosphate-buffered saline. Cells were
lysed with lysis buffer (100 mM Tris-HCl, pH 7.8, 0.5% Triton X-100) for
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Identification of a C-terminal PABP-PABP Interaction Domain
taining longer UTRs in the reticulocyte lysate. We conclude
that PABP-mediated repression in vitro requires more than 10
As in the leader region, and that it can operate even when the
A-tract is distal to the cap. This result is in agreement with
recently published data (38), as well as with the distance of 73
nucleotides from the cap to the A-tract in the human PABP
mRNA (23).
RRM 3/4 and the C Terminus of PABP Are Required for
Efficient Cap-distal Repression—To assess the contribution of
the different domains of PABP, including the C terminus, to its
activity as a repressor of translation, a series of different deletions in the PABP gene were prepared and the resulting proteins fused to GST were expressed in E. coli. Fig. 2A shows a
scheme describing the different deletions obtained during this
study, each named according to the number of the amino acids
retained from the wild type protein. The various proteins were
purified by affinity chromatography and quantified by SDSPAGE (see Fig. 2B for a selection of the various proteins used
in this study, as well as Fig. 5A (upper panel)).
Wild type GST-PABP (P10 – 636) and the variant with the
whole of its C terminus deleted (P10 –370) were initially tested
in in vitro translation assays with the cyclin reporter mRNAs
described in Fig. 1. First, the RNAs with differing lengths of
A-tracts were tested with the full-length GST-PABP (P10 – 636)
and the P10 –370 variant. Both proteins were able to repress
the translation of RNAs containing either 61 or 20 A nt. As
expected, neither significantly affected the translation of the
reporter containing 10 A nt in the 5⬘-UTR (data not shown), in
agreement with the results in Fig. 1A. In contrast, when the
RNAs containing variations in the distance between the cap
and the 61-nt A-tract were translated in the presence of increasing amounts of PABP variants, a significant difference
could be seen between the two proteins (Fig. 3, A and B).
Full-length PABP efficiently repressed the translation of all
the distance constructs up to 126 nucleotides, but the PABP
variant lacking the C terminus (P10 –370) was a much less
effective repressor at A-tract distances greater than 41 nucleotides. This difference was not at the level of RNA binding, as
P10 – 636 and P10 –370 were equally able to bind 32P-labeled
probes consisting of the 5⬘-UTR of the different cyclin mRNAs
in a gel-shift assay (Fig. 3C). Strikingly, the PABP variant
P10 –190, which contains only the first two RRMs required and
sufficient for specific binding to poly(A) (12, 14), could only
effectively repress the translation of the reporter RNA containing the most cap-proximal A-tract, at the highest doses tested
(Fig. 3, D and E). We conclude that efficient repression of
cap-distal A-tract mRNA requires some or all of RRM3/4 and is
greatly enhanced by the C terminus.
Mapping the Domains Required for Cooperative Binding of
PABP to Poly(A)—The PABP C terminus confers homodimerization activity on PABP, bound to poly(A), as shown by bandshift assays in which labeled A23 probes (sufficient to bind only
one PABP molecule) formed multiple complexes at saturating
concentrations of PABP, but not of a variant only containing
the four RRMs (12). This band-shift strategy was used to assay
for homodimerization of various GST-PABP proteins in the
presence of a labeled poly(A) probe corresponding to the 61-ntlong PABP 5⬘-UTR A-rich sequence (Fig. 4A). From the work of
Kuhn and Pieler (12), we reasoned that this sequence is capable of binding three (possibly four) PABP molecules. Upon
incubation with increasing concentrations of GST-PABP,
(ranging from 4 to 260 nM), we observed multiple shifted complexes indicative of sequential filling of binding sites in a cooperative manner (Fig. 4B, P10 – 636). We estimate the Kd of
the PABP proteins bound to the labeled probe to vary between
5 and 15 nM, which are very similar values to those obtained
previously (12, 13, 46). To map the region involved in the
PABP/PABP interaction, GST fusion proteins containing Cterminal deletions (P10 –584, P10 –542, and P10 –370) were
used in comparison to GST-PABP. In full-length PABP and the
variants lacking part of or the complete PABC domain (P10 –
584 and P10 –542 respectively), binding of one molecule to the
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FIG. 1. Translational derepression by poly(A) of cyclin reporter mRNAs containing different poly(A) tracts in their 5ⴕ-UTR. Capped
cyclin mRNAs (0.5 ␮g/ml) containing A-tracts in their 5⬘-UTRs were translated in nuclease-treated rabbit reticulocyte lysate pre-incubated with
increasing concentrations of poly(A) (0, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 ␮g/ml). These mRNAs were translated alongside the ⌬(1–263) PABP mRNA
(0.25 ␮g/ml) as an internal control. The cyclin mRNAs did not contain a 3⬘ poly(A) tail, and the distance between the A tract and the cyclin initiator
AUG remained constant (70 nucleotides). A, translation profile of cyclin mRNAs with varied lengths of A-tracts (10, 20, or 61 A residues), at a fixed
distance of 41 nucleotides between the cap and the A-tract. B, results from A were quantitated by densitometry and the intensities of the cyclin
bands plotted as a function of the poly(A) concentration defining as 1 (dashed line) the value obtained in the absence of poly(A). C, translation of
mRNAs containing 61-nt A-tracts in their 5⬘-UTR, located at varying distances from the cap (10, 41, 86, and 126 nucleotides) in reticulocyte lysate
pre-incubated with increasing concentrations of poly(A) as in A and B. D, same as B but plotting the results shown in C.
Identification of a C-terminal PABP-PABP Interaction Domain
46361
probe was an intermediate stage followed at higher concentrations by binding of two, three, or even more molecules. In
contrast, P10 –370, although having an affinity to poly(A) similar to that of these proteins, differed significantly from them in
that multiple binding of the protein to the probe RNA was
substantially impaired. Thus, even at the highest concentration of P10 –370, a significant proportion of the probe was
bound by only one molecule and two molecules seemed to be the
most that could bind the probe (Fig. 4B). Similar results were
observed in the case of an A61 RNA probe, although here we
noted an increased efficiency of cooperative PABP/PABP interactions, which prevented detection of intermediate complex
formation (data not shown).
In the translation repression assay, the new variant P10 –
542, although lacking the PABC domain, was observed to be
nearly as active as the full-length protein in the repression of
reporter mRNAs with the A tract distal to the cap, with a
reduction in repression efficiency only at the 126-nt distance
(Fig. 4C). We conclude that the PABP C terminus, excluding
the PABC domain, is involved in cooperative PABP/PABP binding in the presence of poly(A) and enhances PABP activity as
repressor of translation.
PABP C Terminus, the Fourth RRM, and Poly(A) Are Required and Sufficient for Specific PABP/PABP Interaction—
The experiments above point to the region between amino acids
370 –542 as being involved in mediating PABP/PABP interactions and enhancing translational repression. To confirm these
results, we used pull-down assays in which PABP variants
were bound to glutathione-Sepharose beads and subsequently
incubated in the presence or absence of poly(A) with 35S-labeled
full-length PABP translated in rabbit reticulocyte lysate. A
second 35S-labeled protein, cyclin A, was included in the reaction as a control to monitor for nonspecific bead binding. Following incubation, and washing, the proteins bound to the
beads were analyzed by SDS-PAGE, and Coomassie Blue staining and autoradiography. The top panel in Fig. 5A shows that
approximately equal amounts of GST-PABP variants were
bound and eluted from the beads. As shown in the middle and
bottom panels of Fig. 5A, weak and equal cyclin protein binding
is observed in all lanes (this nonspecific binding was consistently enhanced following ribonuclease treatment, for unknown reasons). In the presence of poly(A), all N-terminal
GST-PABP variants containing at least RRM1/2 pull down
S-labeled PABP. Comparison with an aliquot of the initial
translation mix (Retic lane) allows us to estimate that approximately 20% of the 35S-labeled PABP is bound by GST-PABPs.
Variants P237–542 and P237– 636, which include a complete
RRM4 plus the 370 –542 part of the C terminus, identified as
necessary for cooperative binding to poly(A) (Fig. 4), or the
whole of the C terminus, respectively, also interact with the
translated PABP. In contrast, variant P372– 636, which only
consists of the PABP C terminus, interacts very weakly. This
level of binding was well above background binding; as shown
in the control lanes, labeled PABP did not bind immobilized
GST or glutathione-Sepharose beads. In the absence of poly(A),
and with the addition of cobra venom RNase to degrade any
remaining excess RNA, most of the fusion proteins, including
full-length GST-PABP, lose the ability to interact with 35SPABP. Surprisingly, the P237–542 and P237– 636 variants still
retain the capacity to bind to the labeled protein, suggesting
that the removal of the first three RRMs from at least one of the
binding molecules relieves an inhibitory mechanism, which
prevents PABP/PABP binding in the absence of poly(A). The
same results were obtained in several independent experiments, some using micrococcal nuclease in place of cobra
venom RNase (data not shown).
To rule out the possibility that the P237– 636 and P237–542
proteins bind full-length PABP through an RNA bridge (at
least when poly(A) is present), we tested their ability to bind
32
P-labeled A61 RNA probes using band-shift assays (performed as described in Fig. 4B). The P372– 636, P10 – 636, and
P10 –370 PABP variants were also included in this experiment
as controls (Fig. 5B). Only the proteins containing functional
RRMs 1 and 2 bound the A-tract probe, indicating that the
P237– 636 and P237–542 variants bound full-length PABP directly. We next tested the ability of GST-PABP variants to bind
truncated PABP proteins, labeled in vitro, to determine the
minimal optimal interaction region. Three different mRNAs,
coding for sequential C-terminal PABP deletions, which lack
the PABC domain, the whole of the C terminus, and the fourth
RRM plus the C terminus, respectively (see Fig. 2A for exact
positions of the deletions), were initially co-translated in the
rabbit reticulocyte lysate. Following translation, the reaction
was treated with micrococcal nuclease, to remove any remain35
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FIG. 2. Different GST-PABP fusion proteins used in this study. A, scheme of the GST-tagged PABP fusion proteins used in this study
(named according to the amino acids remaining from the full-length human PABP). The position of the GST tag as well as the four RRMs and the
PABC domain in PABP are indicated (see “Experimental Procedures” for details of the different proteins). Highlighted also are the positions of the
sites for the enzymes HindIII (H), MscI (M), and Tth111I (T), which cut after the codons for amino acids 237, 358, and 432, respectively. B,
Coomassie Blue-stained SDS-polyacrylamide gel of selected purified GST-PABP fusions used in this study. The various C-terminal and N-terminal
deletions are shown on the left and right panels, respectively, with P10 – 636 and P10 –370 in both for comparison. On the left are indicated sizes
in kDa of protein molecular mass markers. The dots indicate the position of the full-length, GST-PABP proteins.
46362
Identification of a C-terminal PABP-PABP Interaction Domain
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FIG. 3. Translation repression by GST-PABP variants of the cyclin mRNAs containing 5ⴕ-UTR A-tracts. The cyclin reporter mRNAs
described in Fig. 1C were translated along with the ⌬(1–263) PABP mRNA (control) in the presence of increasing concentrations of GST-PABP
proteins. A, comparison of the translation of the different mRNAs in the presence of increasing concentrations of full-length GST-PABP P10 – 636
(0, 1.6, 3.3, 6.5, 13, and 26 ␮g/ml) or the P10 –370 variant (0, 1.4, 2.8, 5.6, 11.3, and 22.5 ␮g/ml). B, the autoradiograms shown in A were quantitated
and analyzed as in Fig. 1, but here for each experiment the ratio between the intensities of the cyclin and the control PABP bands was plotted as
a function of the GST-PABP concentration, normalizing as 1 (dashed line) the value obtained in the absence of GST-PABP (non-normalized
cyclin/control ratios of 3.05, 2.95, 1.2, and 0.55 for the 10-, 41-, 86-, and 126-nt distance mRNAs, respectively). C, band-shift analysis with the
5⬘-UTR of the cyclin mRNAs used in the translation assays. The various cyclin DNA constructs tested in A and B, linearized after the cyclin ATG
(StyI site), were transcribed in the presence of [␣-32P]ATP. The resulting labeled RNAs (10,000 cpm or ⬃0.7 ng) were incubated with either wild
type GST-PABP or the P10 –367 variant (14 ␮g/ml final concentration) and the protein-RNA complexes analyzed on 4% nondenaturing polyacrylamide gels. ORF, open reading frame. D, translation repression assay of PABP variant P10 –190 (0, 2.6, 5.3, and 10.5 ␮g/ml) with the 10-, 41-, and
86-nt distance cyclin mRNAs tested in A. E, the results from D were quantitated and plotted as in B (non-normalized cyclin/control ratios of 3.2,
2.8, and 2.15 for the 10-, 41-, and 86-nt distance mRNAs, respectively). The stimulation observed for the 86-nt distance in the last concentration
(10.5 ␮g/ml) is not reproducible and is only observed in cyclin/control ratio because of a small decrease in the control PABP band in the last lane
of the experiment. In all translation assays, the concentration of the control mRNA was 0.25 ␮g/ml. Cyclin mRNA concentrations: 10 and 41 at 2.5
␮g/ml; 86 and 126 at 3.75 ␮g/ml.
ing RNA, and excess ribosomal RNA was added as nonspecific
competitor, after the addition of EGTA to quench micrococcal
nuclease activity. The reaction was then divided into two aliquots, and either poly(A) or poly(C) was added prior to incubation with various bead-bound GST-PABPs (Fig. 5C). In the
presence of poly(A), the three GST-PABPs bring down the three
labeled proteins at equivalent levels, compared with their ini-
tial reticulocyte levels. Indeed, as observed previously (Fig.
5A), P237– 636, although not able to bind poly(A) (Fig. 5B), is as
efficient as the P10 –370 and P10 – 636 proteins that do bind
poly(A) (Fig. 5C). Even the 1–237 labeled protein that lacks the
entire C terminus and the fourth RRM is brought down in
similar proportions to the longer proteins. (We also note that
the minor labeled proteins, truncated versions of PABP arising
Identification of a C-terminal PABP-PABP Interaction Domain
46363
from the use of internal AUGs or from premature termination,
interact with immobilized PABP.) We cannot rule out a direct
interaction between the various proteins but it is more likely
that the added poly(A), which allows multiple molecules of
PABP to associate, and the endogenous rabbit reticulocyte
PABP, act as bridges for any poly(A)-binding protein, resulting
in their association with the GST-PABP variants. In the presence of poly(C), as observed above after RNase treatment,
neither the wild type nor the C terminus lacking variant (P10 –
370) can bind any of the labeled protein, whereas P237– 636
could relatively efficiently bring down the largest of these proteins (1– 432), which retains the first 50 amino acids of the C
terminus. With further deletions (protein 1–358, which loses
the remaining C terminus), this binding is severely impaired
(3-fold reduction in binding), although some minor binding can
still be seen even with the labeled protein 1–237 (⬃15% of that
observed for protein 1– 432). Identical results were obtained
with the GST variant P237–542 (data not shown).
Together, these pull-down experiments confirm that the region of the C terminus immediately after the fourth RRM, but
excluding the PABC domain, plays a significant role in mediating the interaction between PABP molecules. A role for
RRM4 in this self-association is also implied by our findings,
because it is required in the GST fusion for maximal interaction (compare P372– 636 and P237–542/636; Fig. 5A) and its
removal from the labeled PABP deletion abolishes most of the
remaining binding activity to the P237– 636 variant in the
presence of poly(C) (Fig. 5C). Alternatively, the residual activity observed between P237– 636 and the labeled protein 1–237
may indicate minor binding to the other RRMs, even in the
absence of RRM4. This latter observation is consistent with the
overall sequence similarity between RRMs 2, 3, and 4 of ⬃45–
50% in human PABP, with RRM1 being more divergent. In
summary, gel-shift assays and pull-down experiments indicate
that RRM4 and the part of the C terminus, up to but not
including PABC, mediates PABP self-association. This interaction is dramatically enhanced in the presence of poly(A); in the
absence of poly(A), only truncated versions of PABP, lacking
RRM1/2, retain the ability to bind other PABP molecules.
Tethered PABP C Terminus Enhances Translation Repression by MS2 of mRNA Containing MS2 Binding Sites in Their
5⬘-UTR—To investigate the role of the C terminus in enhancing translation repression by PABP in vivo, we undertook a
tethered strategy whereby MS2 binding sites were inserted in
the 5⬘-UTR of CAT reporter genes and, in a second plasmid, the
MS2 protein was fused to either the PABP C terminus (amino
acids 372– 636) or its fourth RRM plus the proximal part of the
C terminus (amino acids 237–542 missing the PABC domain),
or used alone (Fig. 6A). The MS2 protein is known to repress
translation/expression of reporter genes containing MS2 binding sites in their 5⬘-UTR, as long as it is placed within the first
40 or so nucleotides after the cap. It is not very efficient at
preventing translation if the distance between the cap and the
MS2 binding site is increased further (43, 47). Fig. 6 (B and C)
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FIG. 4. Cooperative binding to poly(A) by PABP does not require the PABC domain. A, A-rich tract from the 5⬘-UTR of the human PABP
mRNA used as probe in the assays. B, band-shifts, performed by incubating the 32P-labeled A-rich probe with increasing concentrations of the
PABP proteins P10 – 636, P10 –584, P10 –542, and P10 –370 and analysis on a 4% nondenaturing polyacrylamide gel. Protein concentrations used
were 0, 0.38, 1.5, 6, and 24 ␮g/ml. The arrows indicate the different protein-RNA complexes. C, translation repression assay with the P10 –542
variant. The 10-, 41-, 86-, and 126-nt distance cyclin mRNAs (Fig. 1C), as well as the control ⌬(1–263) PABP mRNA, were translated as described
in Fig. 3 in the presence of increasing concentrations of P10 –542 (0, 7.5, and 30 ␮g/ml). D, results from C were quantitated and plotted as a function
of the recombinant protein concentration.
46364
Identification of a C-terminal PABP-PABP Interaction Domain
describes the scheme of the CAT reporters and the MS2 fusion
proteins. Two different CAT constructs were made with the
high affinity MS2 binding site MSC placed at 15 and 91 nucleotides from the transcription start site, MSC-15 and MSC-91
respectively, and a third construct contained the low affinity
MS2 binding site MSA placed at the 15-nucleotide distance,
MSA-15 (47).
Plasmids encoding the reporter genes and the protein fusions
were co-transfected in 293 cells along with a third plasmid
encoding ␤-galactosidase to serve as transfection control. After
30 –35 h of transfection, the cells were harvested and assayed
for CAT and ␤-galactosidase activities. To compensate for variations in transfection efficiency, the ratios between the CAT
and ␤-galactosidase activities were determined and for each set
of transfections, this ratio was compared with a control transfection where no MS2 expression plasmid was included (Ctrl).
In some experiments, RNA was extracted as well and used for
Northern blots to confirm approximately equal expression and
stability of the different CAT and MS2 genes (Fig. 7D). First,
the cells were transfected with the MSC-15 CAT reporter plasmid along with increasing concentrations of the plasmids expressing the different MS2 fusion proteins (Fig. 7A). All three
proteins led to a dose-dependent decrease in CAT activity, with
a maximum reduction of ⬃40%. A minor enhancement of repression was observed by the fusion proteins containing the
PABP C terminus. In cells transfected with the reporters con-
taining the mutated MS2 binding site (MSA-15), no reduction
in CAT activity was observed for MS2 on its own and less than
20% reduction for the MS2-PABP C terminus fusions, showing
that expression of the MS2 fusions is not causing a general
nonspecific inhibition of CAT expression (Fig. 7B). We then
analyzed the effect of the fusion proteins on the reporter CAT
mRNA bearing a cap-distal MS2 binding site (MSC-91, Fig.
7C). Although less than 10% reduction of CAT activity was
observed even with the highest dose of the MS2 plasmid tested,
both fusions containing the PABP fragments were still capable
of efficiently (⬃40% reduction) repressing CAT expression.
These effects were largely at the level of translation, as we did
not observe corresponding changes in RNA levels (Fig. 7D). The
tethered strategy has also been used by us to specifically investigate reduction in CAT mRNA abundance as well as translation in 293 cells mediated by PABP binding to its 5⬘-UTR
(48). Full-length PABP fused to MS2, like the MS2/C terminus
fusions described above, despite reducing the expression of the
CAT construct MSC-91, does not affect the reporter mRNA
levels.
Initially the transfection results were quite surprising, as we
previously showed that the GST-PABP C terminus (P372– 636)
seemed to have only a limited ability for PABP-PABP binding
in the absence of poly(A) (Fig. 5A). Although we expected the
intracellular system to be comparable to a poly(A)-rich envi-
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FIG. 5. Delineation of the region required for the interaction between GST-PABP variants and 35S-labeled PABP. A, GST-PABP
fusion proteins were immobilized on glutathione-Sepharose beads in the presence of BSA and incubated with 35S-labeled wild type PABP and cyclin
A translated in the rabbit reticulocyte lysate. The proteins bound to the beads were eluted in SDS sample buffer and analyzed in SDSpolyacrylamide gels by Coomassie Blue staining and autoradiography. The dots indicate the position of the full-length, undegraded, GST-PABP
proteins. The assay was performed in the presence of 5 ␮g/ml poly(A) or after treatment of the reticulocyte lysate with RNase V1 to eliminate any
residual RNA. The lane marked Beads represents the sample where no protein was added to the beads. B, GST-PABP variants were tested for their
ability to bind poly(A) in band-shift assays, carried out as described in Fig. 4A but using a 32P-labeled 63-A RNA probe and 5% nondenaturing
polyacrylamide gels. As positive controls, PABP variants P10 – 636 and P10 –370 were also included. C, 35S-labeled PABP deletions were obtained
by linearizing PABP cDNA with various restriction enzymes (HindIII for 1–237, MscI for 1–358, and Tth111I for 1– 432; the numbers indicate the
amino acids remaining in the labeled proteins) followed by transcription with T7 RNA polymerase and translation in the rabbit reticulocyte lysate.
The translation reaction was then treated with micrococcal nuclease and incubated with E. coli rRNA and poly(C) or poly(A) (both at 20 ␮g/ml) prior
to incubation with the GST-PABP variants indicated. In A and C, the lane marked Retic indicates an equivalent amount of the translation products
used in the assay.
Identification of a C-terminal PABP-PABP Interaction Domain
46365
N-terminal His tag and tested whether MS2-PABP-(372– 636)
immobilized on Ni-NTA beads interacts with 35S-labeled
PABP. As shown in Fig. 7E, this protein does indeed bind
full-length PABP, even in the absence of poly(A). Control assays were also performed using as the labeled protein a human
eIF4G variant (41), which lacks the motif required for binding
PABP while retaining those required to bind eIF4A (16). No
interaction between MS2-PABP-(372– 636) and labeled eIF4G
is observed (Fig. 7E). In contrast, recombinant His-tagged human eIF4A can only recover traces of the labeled PABP, while
binding very efficiently to labeled eIF4G. These experiments
then confirmed that the C terminus on its own, or at least when
positioned after an adequate fusion protein, mediates PABP/
PABP interaction and can enhance the limited activity of the
RNA-binding protein MS2 in blocking the translation of
mRNAs by the small ribosomal subunit at cap-distal positions.
DISCUSSION
ronment, we examined whether the N-terminal MS2 portion
stabilized the fusion protein (MS2-PABP-(372– 636)) and enhanced its ability to bind full-length PABP. Therefore, we took
advantage of the fact that the MS2 half of the protein has an
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FIG. 6. Tethering approach to investigate the role in vivo of the
PABP C terminus in enhancing translational repression. A, experimental strategy used to test the effect of MS2-PABP fusions on the
translation of CAT reporter mRNAs containing a MS2 binding site in
their 5⬘-UTR. B, scheme of the different plasmids encoding the reporter
mRNAs. Three different reporter plasmids were used, two coding for a
high affinity MS2 binding site positioned at 15 and 91 nt from the cap
(MSC-15 and MSC-91, respectively) and a third coding for a MS2
binding site containing a C to A mutation (MSA-15), which drastically
reduces the binding of the MS2 protein to the mRNA (47). C, MS2PABP fusions tested in the assay.
The results presented in this report contribute in multiple
aspects to the study of the poly(A)-binding protein, with implications both in PABP function/synthesis as well as control of
mRNA translation in general. By examining the requirements
for PABP to act as a translational repressor, we confirm that
PABP can be effective in preventing translation by binding
A-tracts at locations both proximal and distal to the cap. In the
latter case, PABP very likely blocks the scanning of the 40 S
ribosomal subunit along the mRNA, as has been recently
shown (38). However, the ability to bind poly(A) is not sufficient
for efficient translational repression at distal sequences, because removal of the PABP C terminus somewhat reduces its
ability to inhibit translation (Figs. 3 and 4). A second significant contribution from this work is the identification, within
the PABP C terminus, of the region required for PABP/PABP
interaction and cooperative binding to poly(A) (Figs. 4, 5, and
7). Major implications from this observation will be discussed
below.
Translation of PABP mRNA is controlled by at least two
independent mechanisms: autoregulation mediated by the
PABP protein (25–27), and coupling to the cellular growth
stage mediated by the polypyrimidine stretch in its 5⬘ end (23).
In this study, by using cyclin reporter mRNAs with the A-tracts
in their 5⬘-UTR, we were able to concentrate on one aspect of
PABP mRNA translation, avoiding the influence of other sequences present in PABP mRNA, which may affect its translation or stability (28). The A-rich sequence required for PABP
mRNA translation repression differs from other sequences that
have a similar function such as the IRE, in that it does not
easily assume a secondary or tertiary structure, which may
prevent ribosomes from scanning along the mRNA. The A-rich
sequence also does not need to be positioned proximally to the
cap structure (this work and Ref. 38). We reasoned that specific
properties of PABP might be required for it to be an effective
repressor and decided to use this activity as a tool to study the
contribution of individual domains within PABP to its function.
Full-length PABP represses the translation of cyclin mRNAs
bearing A-tracts in their 5⬘-UTR, whether the A-tracts are
proximal or distal to the cap, although with somewhat higher
efficiency in the former case. On the other hand, repression by
PABP variants lacking the C terminus is much more sensitive
to the distance of the A-tract from the cap (Fig. 3). Thus, the
C-terminal region of the PABP, although not interfering with
binding to poly(A), is involved in the repression mechanism. Its
role may be to increase the size of the complex bound to the
RNA, not only by recruiting other PABP molecules but perhaps
also by allowing the association of other proteins to create a
multiprotein complex that blocks ribosomal scanning. Previous
reports also hint at the contribution of the C terminus to PABP
46366
Identification of a C-terminal PABP-PABP Interaction Domain
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FIG. 7. Repression by MS2 of the CAT reporter mRNA bearing a cap-distal MS2 binding site is enhanced by tethering the C
terminus of PABP. Plasmids encoding the different reporter mRNAs as well as the MS2-PABP fusions were co-transfected in 293 cells, and 30 –35
h later the cells were harvested and assayed for CAT and ␤-galactosidase activity. Three different transfection reactions were performed for each
experimental point, and the mean values and standard deviations were determined. The ratio of CAT to ␤-galactosidase activity was set to 1 for
the control cells transfected without any MS2 fusions. Results shown are representative of at least three independent transfection experiments.
A, effect of the different MS2-fusions on expression of MSC-15 mRNA. Three different concentrations of the effector plasmids were used (2.5, 5, and
10 ng). B and C, same as A but using the plasmids encoding the MSA-15 and MSC-91 CAT mRNAs, respectively. D, Northern blots analysis
comparing the mRNA levels of the CAT, ␤-galactosidase (b-gal), and MS2 fusions in 293 cells transfected as in C with the CAT construct MSC-91.
Probes used for the Northern blots are indicated. On the right panel are the results from the CAT/␤-galactosidase activity for the experiment used
for the RNA analysis. This result is representative of several different experiments. E, pull-down assay to confirm binding of MS2-PABP-(372– 636)
to 35S-labeled PABP. His-tagged MS2-PABP-(372– 636) was immobilized on Ni-NTA beads and incubated with 35S-labeled PABP or eIF4G (lanes
1 and 2). As a control, His-tagged human eIF4A was immobilized on Ni-NTA beads and incubated with 35S-labeled PABP or eIF4G (lanes 3 and
4). Ctrl, control.
function. In Xenopus oocytes, a minimal PABP consisting of
RRMs 1 and 2 did not seem to be able to bind efficiently to
mRNAs or prevent their deadenylation when overexpressed in
oocytes, in contrast to wild type PABP (49). In a HeLa-derived
cell line, a similar PABP variant (as well as one containing the
four RRMs but no C terminus) was still able to bind efficiently
Identification of a C-terminal PABP-PABP Interaction Domain
46367
to poly(A) and shuttle to the cellular nucleus although its
export back to the cytoplasm was impaired (22).
The C-terminal region of PABP was previously reported to be
involved in mediating protein/protein interactions, and more
specifically PABP multimerization in the presence of poly(A)
(12). The recent description of the PABC domain very much
filled a gap in the knowledge about PABP function by mapping
the motif involved in interactions with eRF3, PAIP1 and PAIP2
(19, 50, 51). Our results build upon these discoveries by confirming the participation of the C terminus in PABP/PABP
interaction, while ruling out any participation of the PABC
domain, and suggest a significant additional contribution of the
fourth RRM. These results may relate to those obtained in
yeast, where neither the first two RRMs nor the last 50 amino
acids, which includes most of the PABC domain, are involved in
mRNA stabilization by PABP (21). The fourth RRM is one of
the least characterized of the PABP RRMs. An analysis of
sequence conservation of RRMs from PABPs of distantly related organisms shows that RRM 4 is as conserved as RRM 2,
which is known to be involved in such important functions as
specific poly(A) binding and interaction with eIF4G. In contrast, RRM 3 is much less conserved. RRM 4 has been ascribed
roles in cellular viability and in translation (see Introduction),
but no interacting factors have yet been identified. Although its
involvement in the protein/protein interactions necessary for
PABP multimerization may in part explain its conservation
(this work), it is also possible that it interacts with still unknown protein or RNA factors (18).
The region of the C terminus bordered by RRM 4 and the
PABC domain first came to notice when PABP sequences from
yeast and human were compared, because of its high content of
the amino acids proline and glutamine (52–54) (Fig. 8). Strikingly, in human PABP, roughly 1 in every 5 or 6 amino acids in
this region is proline and 1 in every 3 is either glutamine,
asparagine, or arginine, and there are no negatively charged
amino acids in the entire 150-amino acid region. Alignments of
the C terminus plus the fourth RRMs from PABPs of distantly
related organisms shows no consistent sequence homology
within this “linker” region, although the bias for the amino
acids proline, glutamine, and, to a lesser extent, asparagine
and arginine is maintained, as is the lack of acidic residues
(Fig. 8).
Until now, the strongest evidence pointing to the proline
glutamine-rich linker region being involved in protein-protein
interaction arose from a study of yeast PABP (Pab1p). Pbp1p
(for Pab1p-binding protein) was obtained in a screen for factors
that interact with the PABP C terminus. The specific interaction between Pab1p and Pbp1p does not require the last 24
amino acids of the PABP C terminus, which includes part of
PABC (55). A likely candidate for mediating the Pab1p/Pbp1p
interaction is a segment within the Pab1p linker region (italicized in Fig. 8) found to be partially homologous to two segments in the Pbp1p C terminus. Pab1p mutations that prevent
the interaction map within this segment, which was proposed
to be involved in homomultimerization (for both Pab1p/Pab1p
or Pbp1p/Pbp1p multimers) or heteromultimerization (for
Pab1p/Pbp1p multimers). Interestingly, removal of the RRMs
from Pab1p actually enhances its binding to Pbp1p (55). This
observation is reminiscent of the PABP pull-down assays described here, where removal of RRMs 1–3 appears to relieve an
inhibitory constraint that prevents PABP/PABP interaction in
the absence of poly(A).
Downloaded from www.jbc.org at CAPES/MEC - UFPI on October 6, 2007
FIG. 8. Sequence alignment of PABP regions containing RRM 4 and the C terminus from homologues of representative organisms.
Protein homologues to the constitutive human PABP sequence (accession no. P11940) from selected organisms were chosen, and the regions
encompassing RRM4 plus the C terminus (as defined in the human sequence) were aligned with the help of Blast and DNAstar software. Accession
numbers: frog (Xenopus laevis), CAA40721; fly (Drosophila melanogaster), P21187; worm (Caenorhabditis elegans), CAA21572; plant (Arabidopsis
thaliana), AAA61780; yeast (Saccharomyces cerevisiae), AAA34787. Residues identical to the human sequence are shaded. Human RRM 4 is boxed,
and the PABC domain is overlined (19). The yeast Pab1p region involved in the interaction with Pbp1p (55) is in italics. The region in human PABP
shown to be required for arginine methylation by CARM1 is underlined with double arrows indicating the two principal arginine targets (58).
Single arrows indicate the last amino acids present in the truncated proteins shown in this study. Asterisks (*) indicate the prolines present in the
human PABP linker region.
46368
Identification of a C-terminal PABP-PABP Interaction Domain
Acknowledgments—Plasmids pSK-HFC1 and pET(His6-eIF4A) were
a generous gift from I. Ali and R. Jackson. We thank A. Kornblight for
the pSV2-CAT vector, M. Hentze for the MSC-GH and MSA-GH plasmids, and M. Wickens for the pET-MS2. We acknowledge members of
the de Melo Neto, Standart, and Martins de Sa groups for helpful
assistance and general discussion.
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Evidence from various sources indicates that PABP activity
can be regulated by different means. An example of such modulation has been observed in mammals, where binding of human eRF3 to PABP abolishes its ability to multimerize when
binding to poly(A) (56). Phosphorylation of plant PABP has also
been shown to play a role in regulating its binding to poly(A),
because only the modified form binds poly(A) cooperatively
(57). Theoretically, phosphorylation of the proline linker region
could reduce its net positive charge and enhance PABP-PABP
interaction. Recently, human PABP was identified as a substrate of the coactivator-associated arginine methyltransferase
or CARM1 (58). Modification of target proteins by arginine
methyltransferases has been shown to interfere with proteinprotein interactions, and, in one example, the arginine residues
flank proline-rich motifs (59). Intriguingly, the region in PABP
methylated by CARM1 has been mapped to the amino acids
384 – 478 (58), within the linker region that we show to be
involved in PABP-PABP interaction (see Fig. 8). However, because the recombinant protein binds cooperatively to poly(A),
at least for human PABP, neither methylation nor phosphorylation is probably required for the PABP-PABP interaction.
Nevertheless in vivo, these modifications might play an important role in regulating poly(A)-binding and PABP-PABP interactions. Further work is still needed to elucidate these possibilities and to define precisely how the multiple PABP domains
interact.