Báo cáo khóa học: Human PABP binds AU-rich RNA via RNA-binding domains 3 and 4 pdf

Human PABP binds AU-rich RNA via RNA-binding domains 3 and 4
Rosemary T. Sladic
1
, Cathy A. Lagnado
1
, Christopher J. Bagley
1,2
and Gregory J. Goodall
1,2
1
Division of Human Immunology and Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, Australia;
2
Department of Medicine, The University of Adelaide, Australia
Poly(A) binding protein (PABP) binds mRNA poly(A) tails
and affects mRNA stability and translation. We show here
that there is little free PABP in NIH3T3 cells, with the vast
majority complexed with RNA. We found that PABP
in NIH3T3 cytoplasmic lysates and recombinant human
PABP can bind to AU-rich RNA with high affinity. Human
PABP bound an AU-rich RNA with K
d
in the n
M
range,
which was only sixfold weaker than the affinity for oligo(A)
RNA. Truncated PABP containing RNA recognition motif
domains 3 and 4 retained binding to both AU-rich and
oligo(A) RNA, whereas a truncated PABP containing RNA
recognition motif domains 1 and 2 was highly selective for
oligo(A) RNA. The inducible PABP, iPABP, was found to
be even less discriminating than PABP in RNA binding, with

affinities for AU-rich and oligo(A) RNAs differing by only
twofold. These data suggest that iPABP and PABP may in
some situations interact with other RNA regions in addition
to the poly(A) tail.
Keywords: PABP; iPABP; poly(A) binding protein; RNA-
binding protein; AU-rich element.
Poly(A) binding protein (PABP) occupies the poly(A) tails
of mRNA transcripts in eukaryotic cells and has important
influences on both the stability and the translation of
mRNA [1]. Studies with in vitro systems are consistent with
PABP acting as a physical impediment to limit the access of
exoribonuclease to the 3¢-end of polyadenylated RNA [2–4],
while more complex interactions also impact on mRNA
stability. Through interactions with proteins that assemble
on the 5¢-cap structure of mRNA, PABP participates in the
effective circularization of mRNA. One consequence of this
is that PABP helps limit the access of degradative enzymes
to the 5¢-end of the mRNA, by helping to stabilize the
binding of proteins such as the translation initiation factors
eIF4G and eIF4E [5–8], and possibly other proteins as well
[9].
In addition to its global effects on mRNA stability, PABP
participates in the action of certain cis-acting elements that
target particular mRNAs for rapid degradation. Some
vertebrate mRNAs contain AU-rich elements (AREs) that
act by a mechanism that includes acceleration of poly(A)
shortening [10,11]. As PABP is bound to the poly(A) tail,
it is likely to influence the poly(A) shortening rate, and
consistent with this, an in vitro system of AU-mediated
poly(A) shortening requires prior titration of PABP by

addition of poly(A) [2]. AU-rich elements can also target
mRNA in yeast for rapid degradation in a deadenylation
dependent manner [9,12]. In vertebrates, poly(A) shortening
is also the target of destabilizing elements that are structur-
ally distinct from the ARE [13,14].
The initiation of translation, and possibly translation
termination, are also influenced by PABP. The circulari-
zation of mRNA that results from the physical interaction
of PABP with the translation initiation factor eIF4G
[5–7,15] enhances the recruitment of both the 40S and 60S
ribosome subunits [16,17], resulting in an enhanced rate
of translation. PABP also interacts with the translation
termination factor eRF3, which may further enhance
translation by promoting the recycling of ribosomes on the
same mRNA [18].
Structurally, PABP consists of four RNA recognition
motif (RRM) domains that constitute the N-terminal half
of the molecule, and a C-terminal domain that has been
implicated in intermolecular interactions between PABP
molecules bound to a common poly(A) tail [19]. Studies of
the binding of yeast and Xenopus PABPs to homopolymeric
RNAs have shown that RRM domains 1 and 2 (RRM1,2)
are sufficient for high affinity binding to poly(A), while
RRM domains 3 and 4 can also bind poly(U) [19–21]. The
crystal structure of the RRM1,2 pair-complexed with
oligo(A) RNA has been determined [22], revealing a
continuous RNA binding trough in which lays an 8
nucleotide length of oligo(A) RNA.
We report here that PABP can bind with high affinity to
AU-rich RNA in vitro, in agreement with some other recent

reports [23,24]. We identified the region within PABP that is
responsible for binding to an AU-rich RNA and also found
that the closely related protein, iPABP [25], binds to
AU-rich RNA with almost as high an affinity as it binds to
oligo(A) RNA. This suggests that PABP in vivo could bind
not only to poly(A), but also to other sites on some
mRNAs,ifpresentatalevelinexcessofthatrequiredto
occupy all the available sites on poly(A) tails.
Correspondence to G. J. Goodall, Hanson Institute, IMVS,
Frome Road, Adelaide, S.A., 5000, Australia.
Fax: + 61 88232 4092, Tel.: + 61 88222 3430,
E-mail:
Abbreviations: ARE, AU-rich element; PABP, poly(A) binding
protein; REMSA, RNA electrophoretic mobility shift assay;
RRM, RNA recognition motif.
(Received 31 October 2003, accepted 28 November 2003)
Eur. J. Biochem. 271, 450–457 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03945.x
Materials and methods
Preparation of cell extracts
NIH3T3 fibroblasts were grown to near-confluence in
Dulbecco’s modified Eagles’ medium with 7.5% (v/v) fetal
bovine serum (Gibco). For preparation of cytoplasmic
extracts (essentially according to [26]), cells were harvested
by scraping, washed in cold NaCl/P
i
and lysed on ice in
buffer containing 40 m
M
KCl, 10 m
M

Hepes, pH 7.9, 3 m
M
MgCl
2
,1m
M
dithiothreitol, 0.2% (v/v) Nonidet P-40
(NP-40), 5% (v/v) glycerol, 8 lgÆmL
)1
aprotinin, 2 lgÆmL
)1
leupeptin and 0.5 m
M
phenylmethanesulfonyl fluoride.
Nuclei were removed by centrifugation at 14 000 g for
2min.CaCl
2
was added to the supernatant, followed by
micrococcal nuclease (Worthington Biochemical Corpora-
tion, Lakewood, NJ, USA)
1
to give final concentrations of
1m
M
and 12.5 lgÆmL
)1
, respectively. Following incubation
at 25 °C for 10 min, the nuclease was inactivated by
addition of EGTA to a final concentration of 4 m
M

.
UV crosslinking assay
NIH3T3 cell extracts (0.3–0.5 lg of protein) were incubated
for 10 min at room temperature in 40 m
M
KCl, 10 m
M
Hepes, pH 7.6, 3 m
M
MgCl
2
,1m
M
dithiothreitol and 5%
(v/v) glycerol with 25 ng yeast RNA (Boehringer) prior
to the addition of probe RNA (26 fmol), various molar
excesses of cold competitor RNA and 270 ng tRNA, then
further incubated for 30 min at room temperature. The
reaction mixes (final volume 12 lL) were irradiated in
96-well polypropylene microtitre plates on ice with 100 mJ
of 254 nm UV light ( 35 s) using a Stratalinker 1800
apparatus (Stratagene), then electrophoresed on 11% (w/v)
SDS/PAGE under reducing conditions. Gels were fixed,
dried and analysed using a Molecular Dynamics Phos-
phorImager and
IMAGEQUANT
version 3.3 (Molecular
Dynamics, Sunnyvale, CA, USA).
Immunoprecipitation
For immunoprecipitation with the anti-human PABP

monoclonal Ig 10E10 [27] or control monoclonal anti-
bodies, 10 lg of cytoplasmic extract from HEL 299
human embryonic lung fibroblasts (ATCC, Manassus,
VA, USA)
2
was digested with micrococcal nuclease and
UV crosslinked as described above but with a UV dose of
750 mJ, in the presence of 78 fmol of probe. Samples were
then made to 100 lL with buffer A containing 0.2% (v/v)
NP-40 and 0.6 UÆmL
)1
RNasin, after which 50 lLofa
25% slurry of Protein A-Sepharose (Pharmacia) equili-
brated in the same buffer was added. The tubes were then
placed on a rotating wheel at 4 °C for 1 h. The resulting
precleared supernatant was incubated on ice for 1 h in the
presence of 0.5–5 lL (as shown) of ascites fluid prior to
the addition of a further 50 lL of Protein A-Sepharose
slurry and incubated on a rotating wheel at 4 °Cfor1h.
The beads were washed three times in buffer A containing
0.2% (v/v) NP-40 and twice in buffer A containing 0.5%
(v/v) NP-40 and 100 m
M
NaCl. SDS/PAGE loading dye
was added and the samples heated at 98 °Cfor5min
before SDS/PAGE on an 11% (w/v) gel.
Preparation of RNA oligonucleotides
RNA was prepared by in vitro transcription performed
using partial duplex oligonucleotide templates encoding the
T7 RNA polymerase promoter, essentially according to the

procedure of Milligan et al. [28]. For example, the oligo-
nucleotides 5¢-TAATACGACTCACTATAGG-3¢ (univer-
sal upper strand) and 5¢-CT
(25)
CCTATAGTGAGTCGT
ATTA-3¢ (sequence specific lower strand for A25) direct
transcription of the A25 sequence when annealed and used
as a template in the in vitro transcription reaction. The other
RNA oligonucleotides were similarly encoded using appro-
priate lower strands. In vitro transcription was performed
for 4 h at 37 °C in a reaction volume of 30 lL containing
annealed template at 2.5 pmolÆmL
)1
,40mgÆmL
)1
PEG
8000, 40 m
M
MgCl
2
,1m
M
spermidine, 50 m
M
dithio-
threitol, 0.01% (v/v) Triton X-100, 40 m
M
TrisCl, pH 8.1,
5mgÆmL
)1

BSA, 40 m
M
each of CTP, ATP, GTP and
UTP, [
32
P]GTP[cP] to a final specific activity of 0.17 CiÆ
mmol
)1
,4.5UÆlL
)1
T7 RNA polymerase (Promega) and
8UÆmL
)1
pyrophosphatase. Following template removal
by digestion with RQ1 DNase (Promega), the transcripts
were purified on 16% (w/v) polyacrylamide/8
M
urea gels.
All transcripts were treated with calf intestinal phosphatase
(Promega) under manufacturers’ recommended conditions
before the incorporation of c
32
P into 5 pmol of transcripts
from [
32
P]ATP[cP] by T4 polynucleotide kinase (Promega)
under manufacturer’s recommended conditions. Transcripts
were purified on 16% (w/v) polyacryamide/8
M
urea gels

andstoredin1m
M
EDTA.
PABP constructs
Sequence encoding amino acids 10–636 of the major
cytoplasmic PABP, PABPC1 (NCBI RefSeq NM_002568)
was subcloned from plasmid containing PABP cDNA
[27] into the end-filled NdeI site of the histidine tagged
vector pET28a+ (Novagen) to produce the construct
pEThPABP. Two stop codons were introduced by site
directed mutagenesis at amino acids 385 and 386 (end of
RRM domain 4) into pBSPABP (BamHI cloned fragment
containing PABP cDNA in pBlueScript vector) from which
sequence encoding PABP amino acids 10–636 was sub-
cloned into the end-filled NdeI site of the histidine tagged
vector pET28a+ (Novagen) to produce the construct
pEThPABP1234. Sequence encoding PABP RRM1 and
RRM2 (amino acids 10–179) was PCR amplified from
pBSPABP using oligonucleotides incorporating an NdeI
restriction site at the 5¢-end and a stop codon followed by a
SacI site at the 3¢-end. The resultant NdeI–SacI digested
PCR product was cloned into similarly digested pET28a+
vector to produce the construct pEThPABP12. Sequence
encoding PABP RRM3 and RRM4 (amino acids 190–374)
was PCR amplified from pBSPABP using oligonucleotides
incorporating an NdeI at the 5¢-end and a stop codon and a
BamHI site at the 3¢-end. The resultant NdeI–SacI digested
PCR product was cloned into similarly digested pET28a+
vector to produce the construct pEThPABP34. Two
alternate changes were introduced at PABP amino acids

337 in pBSPABP by site directed mutagenesis. Sequence
encoding PABP amino acids 10–636, including the change
F337V, was subcloned from pBSPABP into the end-filled
Ó FEBS 2003 Human PABP Binds AU-rich RNA via RRM 3 and 4 (Eur. J. Biochem. 271) 451
NdeI site of pET28a+ to produce the construct
pEThPABP-F337V. Sequence encoding PABP amino acids
10–636, including the change F337D was subcloned from
pBSPABP into the end-filled NdeI site of pET28a+ to
produce the construct pEThPABP–F337D.
Sequence encoding iPABP (PABPC4, NCBI RefSeq
NM_003819) amino acids 1–644 was subcloned as an
NcoI–XbaI fragment from plasmid piPABP [25] into the
NcoI–NheI site of pET28a+ to produce the construct
pEThiPABP.
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays were performed in a
total volume of 15 lL. Protein (1.5 lL) diluted to an
appropriate concentration in protein buffer (50 m
M
NaPO
4
,
pH 7.8, 300 m
M
NaCl, 10% glycerol, 2 m
M
dithiothreitol
and 0.05% NP-40) was added to 10 fmol of radioactively
labeled RNA in 13.5 lL binding buffer [10 m
M

Tris/HCl
3
,
pH 8.0, 10% (v/v) glycerol, 0.05% (v/v) NP-40, 1 m
M
dithiotheitol, 70 m
M
KCl, 100 ngÆmL
)1
BSA] and incuba-
ted on ice for 15 min. Binding reactions were electrophore-
sed in nondenaturing 7% (w/v) polyacrylamide gels [60 : 1,
1· Tris/borate/EDTA
4
and 0.1% (v/v) Triton X-100] at 4 °C
at 220 V for 120 min. Gels were visualized with the use of
a Molecular Dynamics PhosphorImager. Free RNA bands
were quantitated using
IMAGEQUANT
image analysis soft-
ware (Molecular Dynamics). The amount of free probe
vs. log His-PABP concentration was plotted using
PRISM
version 1.03 (Graph Pad Software). Apparent K
d
was
determined as the EC
50
, which is the protein concentration
at which 50% of RNA was bound.

Purification of recombinant protein
Transformed strains of BL21(DE3) were grown at 37 °Cin
superbroth (3.2% bactotryptone, 2.0% yeast extract, 0.5%
NaCl, pH 7.2) and 30 lgÆmL
)1
kanamycin to an A
600
value
of 0.5. His-tagged protein expression from the transformed
plasmid was induced by the addition of isopropyl thio-b-
D
-galactoside (0.5 m
M
final concentration). After 2 h of
growth at 37 °C, cells were harvested by centrifugation,
frozen and resuspended in 30 mL lysis buffer (50 m
M
NaPO
4
, 300 m
M
NaCl, 10 m
M
imidazole, pH 8.0). Thawed
cells were sonicated and cell debris was removed by
centrifugation. For Ni-nitriloacetic acid purification, the
cleared supernatant was incubated with 1 mL of
Ni-nitrilotriacetic acid agarose (Qiagen) for 2 h at 4 °C
with constant mixing on a rotating wheel. The Ni-nitrilo-
triacetic acid resin was transferred into a column and

washed twice with 10 mL wash buffer (50 m
M
NaPO
4
,
300 m
M
NaCl, 20 m
M
imidazole, pH 8.0) before the His-
tagged protein was eluted from the column with elution
buffer (50 m
M
NaPO
4
,300m
M
NaCl, 250 m
M
imidazole,
pH 8.0). Ni-nitrilotriacetic acid purified proteins were
buffer-exchanged into poly(U) binding buffer (10 m
M
Tris/HCl, pH 8.0, 1 m
M
dithiothreitol, 200 m
M
NaCl) with
Centripreps (Amicon) to reduce the imidazole concentration
to below 10 m

M
and bound to 1 mL of equilibrated poly(U)
Sepharose (Pharmacia) at room temperature. The protein
bound Sepharose was transferred to a column, washed twice
with 10 mL poly(U) binding buffer and eluted with poly(U)
elution buffer (10 m
M
Tris pH 8.0, 1 m
M
dithiothreitol,
800 m
M
NaCl). Protein concentrations were determined
by comparison with dilutions of BSA on Sypro Orange
(Molecular Probes) stained SDS/polyacrylamide gels using
Molecular Dynamics
IMAGEQUANT
software.
Results
PABP binds to AU-rich RNA
We employed a UV cross-linking assay to detect proteins
in cytoplasmic extracts from NIH3T3 cells that bind to
AU-rich RNAs. To ensure that proteins in the extract that
are tightly bound to endogenous mRNA and consequently
not readily available to bind the probe in the UV cross-
linking assay would also be detected, we treated the
cytoplasmic extract with micrococcal nuclease and then
inactivated the nuclease with EGTA before performing the
UV cross-linking assay. The nuclease treatment released a
protein that, when UV cross-linked to the probe, migrated

at approximately 90 kDa in SDS/PAGE (Fig. 1A). When
the time of UV cross-linking was extended, a second, faster
migrating band from micrococcal nuclease-treated extracts
was also seen. Digestion of the crosslinked complexes with
RNase A converted both bands to a single species migrating
at  79 kDa, suggesting both bands are derived from the
same protein (data not shown). Using competitor RNAs of
various sequence composition and with a range of effect-
iveness in destabilizing mRNA in vivo [11], we found that
this protein preferentially bound to AU-rich RNA
(Fig. 1C), although the selectivity of the protein for
destabilizing AU-rich sequences in comparison to non-
destabilizing AU-rich sequences was not sufficient to
strongly suggest that it is involved directly in the destabil-
izing function of AREs in vivo (data not shown). The
protein also bound with high affinity to poly(A) RNA
(Fig. 1C). Furthermore, the UV crosslinked complex could
be immunoprecipitated with a monoclonal antibody to
PABP (Fig. 1D), suggesting that that the crosslinked
protein is most likely PABP.
Identification of the domains responsible for binding
to AU-rich RNA
To further verify that PABP can bind AU-rich RNA, we
prepared a recombinant His
6
-tagged form of the major
cytoplasmic PABP (PABPC1), and measured its affinity
for poly(A) and AU-rich RNA in an RNA electrophoretic
mobility shift assay (REMSA). The AU-rich RNA used
is similar in sequence to an ARE from the GM-CSF

3¢-UTR. To estimate the relative affinity for each RNA, a
constant low concentration of radiolabelled RNA was
incubated with a range of protein concentrations, after
which the bound and unbound RNAs were separated by
native PAGE. Under these conditions, the apparent K
d
can be determined from the concentration of protein at
which 50% of the RNA probe is bound [29]. We
compared the binding of PABP to a 26 nucleotides
AU-rich probe, AU4, an oligo(A) RNA (A25) and a
mixed sequence RNA, M8 (Fig. 2). The recombinant
PABP bound the A25, AU4 and M8 RNAs with apparent
affinity constants of 0.7, 3.9 and 7.7 n
M
, respectively
452 R. T. Sladic et al. (Eur. J. Biochem. 271) Ó FEBS 2003
(Fig. 2). Thus, purified recombinant PABP binds prefer-
entially to poly(A) as expected but can also bind other
RNAs with considerable affinity.
To determine which domains of PABP contribute to
binding the AU-rich RNA, we prepared truncated forms
of PABP as His6-tagged proteins. PABP1234 contains the
four RRM domains but not the large C-terminal domain,
while PABP12 and PABP34 contain RRM domains 1 and
2, and RRM domains 3 and 4, respectively (Fig. 3). The
Fig. 1. Detection of an AU-binding protein by UV crosslinking assay
and identification as PABP. (A) UV crosslinking assays were per-
formed with 0.5 lg of NIH3T3 cytoplasmic extract, with and without
prior digestion of the extract with micrococcal nuclease, using UV
doses of 100 mJ or 750 mJ as shown. Lane 1 extract was preincubated

without addition of micrococcal nuclease. Lane 2 extract was pre-
incubated with micrococcal nuclease but in the presence of an inhibi-
tory level (4 m
M
)ofEGTA.Lane3extractwaspreincubatedwith
micrococcal nuclease for 10 min before the nuclease was inactivated by
addition of EGTA. (B) Sequences of the probe and competitor RNAs
used in UV crosslinking assays. The M8 sequence has UUU motifs
converted to CUC motifs. (C) UV crosslinking assays using micro-
coccal nuclease treated extract in the presence of 0, 2.6, 6.4, 16, 40 and
100–fold molar excess of competitor RNA as indicated. (D) Immu-
noprecipitation of the UV cross-linked complex with anti-PABP mAb
10E10, or irrelevant control mAb.
Fig. 2. Affinity of recombinant PABP for oligo(A), AU-rich and mixed
sequence RNAs measured by REMSA. (A) Sequences of RNA probes
used in REMSAs. (B) Representative REMSAs in which each probe
was incubated with a twofold dilution series from 1.25 to 80 n
M
of
recombinant PABP. (C) Binding curves of free probe vs. protein
concentration. The data shown are from an experiment in which the
PABP concentrations were adjusted to give a high density of points in
the vicinity of the EC
50
. K
d
values calculated from three replicate
experiments are shown in Table 1.
Fig. 3. Full length and truncated forms of recombinant PABP. (A)
Schematic of the His

6
-tagged proteins. (B) Coomassie stained SDS/
polyacrylamide gel of recombinant proteins purified by nickel chro-
matography and poly(U) affinity column. Lane 1, PABP; lane 2,
PABP1234; lane 3, PABP12; lane 4, PABP34.
Ó FEBS 2003 Human PABP Binds AU-rich RNA via RRM 3 and 4 (Eur. J. Biochem. 271) 453
His
6
-tagged proteins were purified by chromatography on
Ni-nitrilotriacetic acid columns, and subsequently by
affinity chromatography on poly(U) sepharose to ensure
that the proteins were correctly folded and competent to
bind RNA.
Removing the C-terminal domain from PABP made little
change to the affinity for A25, and resulted in a small
increase in the affinity for AU4 and M8 (PABP1234, Fig. 4,
Table 1). Thus, the primary determinants for RNA binding
in human PABP are within the four RRM domains, as they
are in the Xenopus protein [19]. Further truncation to
remove domains 3 and 4 (producing PABP12) caused a
large decrease in the binding of the AU-rich and mixed
sequence RNAs. The apparent K
d
of PABP12 for AU4 was
113 n
M
(87-fold change compared to PABP1234). The
affinity for M8 was also severely reduced, with apparent
K
d

of 308 n
M
(128-fold change compared to PABP1234).
However the apparent K
d
forA25was1.8n
M
, which is only
slightly decreased compared to PABP1234. As PABP12 is
less than half the size of PABP1234, two molecules of
Fig. 4. Measurement of apparent K
d
values of truncated forms of PABP for various RNA sequences. REMSAs were performed using a fixed probe
concentration (0.67 p
M
) and with a range of protein concentrations, the limits of which are indicated in n
M
units. Binding experiments with
PABP1234, PABP12 and PABP34, are shown in the left, middle and right columns, respectively. The top row shows REMSAs using A25 probe, the
second row shows REMSAs with AU4 probe and the third row shows REMSAs with M8 probe. Above each gel a vertical arrow indicates the point
at which 50% of probe has been bound by protein. Free probe in each lane was quantitated by phosphorimage analysis. The bottom panel in each
column shows the plots of free probe vs. protein concentration, from which the apparent K
d
values were determined. At high protein concentration,
the PABP12 and PABP34 proteins can bind probe with 2 : 1 stoichiometry, which perturbs the binding profile at the higher concentrations. The K
d
determination is slightly affected by this, but only to a very small degree, because at the protein concentration that leaves 50% free probe, very little
trimeric complex is formed.
Table 1. Apparent K
d

values for PABP proteins. Apparent K
d
values
were determined by REMSA as described in Materials and methods.
The data for each K
d
determination are pooled from three experiments.
95% Confidence limits are shown in brackets.
Protein
K
d
for A25
(n
M
)
K
d
for AU4
(n
M
)
K
d
for M8
(n
M
)
PABP 0.67 (0.61–0.74) 3.9 (3.7–4.1) 7.7 (6.5–9.1)
PABP1234 0.74 (0.61–0.81) 1.3 (1.2–1.4) 2.4 (2.2–2.6)
PABP12 1.8 (1.6–1.9) 113 (82–157) 308 (88–1074)

PABP34 1.5 (1.3–1.6) 2.9 (2.8–3.1) 12 (11–13)
454 R. T. Sladic et al. (Eur. J. Biochem. 271) Ó FEBS 2003
PABP12 can be accommodated per probe molecule,
resulting in a second, lower mobility band at higher
concentrations of PABP12. This has only a slight effect on
the apparent K
d
, which is calculated from measurement of
free probe, because at the protein concentration that leaves
50%freeprobe,verylittleofthe2:1complexisformed.
The results suggest that much of the binding affinity for
poly(A) results from binding to domains 1 and 2, but that
these domains do not contribute much to the binding of
AU-rich RNA. In contrast, PABP34, containing just
domains 3 and 4, bound the AU4 RNA almost as well as
it bound the A25 RNA. The apparent K
d
of PABP34 for
A25 was 1.5 n
M
and for AU4 was 2.9 n
M
, while the
apparent K
d
for M8 was 11.7 n
M
. Thus, the major
contribution to the binding of PABP to AU-rich RNA
comes from domains 3 and 4.

Effect of mutation of a residue predicted to contact
AU-RNA
In an investigation of the RNA binding properties of yeast
PABP, Deardorff and Sachs [30] found that mutation of a
single Phe residue in RRM4 to Val caused a large reduction
in binding to a U-rich RNA (UUUUGUUGUUUU
UUUUCUAG), without having a drastic effect on binding
to oligo(A). This result is consistent with our findings with
human PABP that the RRM3,4 pair is important for
binding a U-rich RNA (AU4) and furthermore suggests
that an equivalent mutation in the human PABP may affect
binding to AU-rich RNA without a major effect on binding
to poly(A). We aligned the sequences of the human and
yeast PABPs and identified a Phe residue in human PABP
(Phe337) that is equivalent to the yeast Phe366 (Fig. 5). We
also modeled the structures of human and yeast RRM3,4
on the published crystal structure of human RRM1,2 [22],
which indicated that the RNA binding pocket in RRM4 is
similar in the yeast and human PABPs (data not shown). To
test whether Phe337 in human PABP is crucial for binding
to AU-rich RNA, we made mutant forms of the protein,
PABP-F337V and PABP-F337D, in which Phe337 was
mutated to Val and Asp, respectively. The recombinant
His
6
-tagged mutant proteins were purified and their affinity
for oligo(A), AU-rich and mixed sequence RNA was
measured by REMSA and compared to that of the wild-
type protein (Fig. 5). In contrast to the effect of this
mutation in the yeast protein, mutation of this residue in the

human protein did not reduce the affinity for any of the
RNAs tested. Thus, yeast and human PABP may differ in
their interaction with U-rich RNAs, despite their similarity
in sequence and predicted structure, although the possibility
also remains that differences in the sequences of the U-rich
probes used in the two studies were responsible for the
different outcomes.
Binding AU-rich RNA is also a property of iPABP
The inducible poly(A) binding protein, iPABP, is closely
relatedtoPABPinsequence(79%identity).RRM
domains 1 and 2 are especially conserved between the
two proteins, sharing 95% sequence identity but RRM
domains 3 and 4 are slightly less conserved (78% and 88%,
respectively), raising the possibility that the iPABP may
have similar affinity to PABP for poly(A) but have
different affinity for AU-rich RNA. iPABP is expressed
at low levels in resting human T cells, but its mRNA is
induced rapidly following T cell activation [25], although
the function of iPABP is unknown. To assess whether
iPABP binds AU-rich RNA we prepared His
6
-tagged
iPABP and measured its binding to oligo(A) and AU-rich
RNA by REMSA (Fig. 6). The apparent K
d
of iPABP for
the oligo(A) RNA was 1.1 n
M
, which is a slightly lower
affinity than that of PABP for oligo(A). However, the

apparent K
d
of iPABP for AU-rich RNA was 2.4 n
M
,
which is a slightly higher affinity than PABP has for the
AU-rich RNA. Thus the affinity of iPABP for AU-rich
RNA is almost as high as its affinity for oligo(A) (2.4 n
M
and 1.1 n
M
, respectively).
Fig. 5. Measurement of apparent K
d
values of PABP Phe337 mutants
for different RNA sequences. (A)Alignmentofthesequencesofyeast
and human PABPs in the vicinity of yeast Phe366, showing the high
degree of sequence conservation in this region of the proteins. The
yeast Phe336 and the equivalent residue in human PABP that was
chosen for mutation are indicated by asterisks. (B) REMSAs were
performed and binding curves constructed as in Fig. 4. The binding
curves for PABP-F337V and PABP-F337D are shown in the upper
and lower graphs, respectively. (C) Apparent K
d
values, with 95%
confidence limits shown in brackets, for the mutant PABPs binding to
each RNA. The data are pooled from three experiments. For com-
parison, the apparent K
d
for PABP binding to each RNA, determined

in Fig. 2, is also shown in the table.
Ó FEBS 2003 Human PABP Binds AU-rich RNA via RRM 3 and 4 (Eur. J. Biochem. 271) 455
Discussion
We found that both the poly(A) binding protein, PABP1,
and the inducible poly(A) binding protein, iPABP, bind to
an AU-rich RNA that is predominantly U-rich, and thus
does not resemble the major in vivo RNA substrate for
PABP, which is poly(A). The affinity of the interaction of
these proteins with the AU-rich RNA is comparable to the
affinities that some other RNA-binding proteins have for
their in vivo RNA substrates. For example, the AU-binding
protein AUF1/hnRNPD, which is involved in the rapid
degradation of some mRNAs [31–33], binds to
AU-containing RNAs with K
d
values of 10–20 n
M
[34].
Thus, although PABP binds primarily to mRNA poly(A)
tails, PABP or iPABP could potentially bind additional
RNA sites if the amount of cytoplasmic PABP exceeded
that required to occupy the available poly(A) tails. When
carrying out the UV cross-linking assay with cytoplasmic
extract from NIH3T3 cells we did not detect cross-linking of
the RNA probe to PABP if the extract was not first treated
with micrococcal nuclease. This suggests that the majority
of PABP in growing but unstimulated NIH3T3 cells is
tightly complexed to RNA [most likely to poly(A)] and that
there is very little PABP available for binding to RNA
sequences of lower affinity than poly(A). This is consistent

with the finding that a negative feedback regulation circuit,
involving an A-rich region in the PABP mRNA 5¢-UTR,
normally limits the expression of PABP [35,36]. However it
is possible that higher levels of PABP are present in some
situations. For example, iPABP mRNA is markedly
induced in activated T cells [25], although it is not known
whether this results in an increase in the availability of either
PABP or iPABP to bind RNA sequences other than
poly(A).
We found that iPABP shares the property of binding
AU-rich RNA with PABP1. iPABP barely distinguishes
between an oligo(A) RNA, A25, and an AU-rich RNA,
while the affinity of recombinant PABP1 for an oligo(A)
RNA was only sixfold greater than for the AU-rich RNA.
The binding of PABP to poly(A) in vivo may be further
stabilized by interactions with translation initiation factors
bound at the 5¢-cap [6] and also by cooperative interactions
between multiple PABP molecules bound to long poly(A)
tails [19]. Whether such protein–protein interactions might
affect binding to other RNA sequences is not known. In
addition, it is possible that post-translational modifications
such as phosphorylation [24,37] or methylation [38], might
differentially affect the affinity of PABP for different RNA
sequences, thereby changing the degree of selectivity of
PABP for the different RNA sequences.
It is interesting that the selectivity of RNA binding by
PABP is markedly partitioned between the RRM1,2 and
RRM3,4 domain pairs. The RRM1,2 pair is highly
selective for poly(A) over the AU-rich RNA, whereas the
RRM3,4 pair barely distinguishes between the two RNA

sequences. Our results are consistent with studies of PABP
from Xenopus [19], which showed the domain 1,2 pair to
have greater selectivity than the domain 3,4 pair for
poly(A) over other homopolymeric sequences, although in
this case affinity constants were not determined. That the
selectivity has been maintained during evolution suggests it
may play a role in the function of PABP, although we have
no indication to date what that function may be. One
possible role for PABP binding to an AU-rich RNA region
of a viral mRNA has been proposed recently. Human
papillomavirus type 1 late mRNA contains an AU-rich
region in its 3¢-UTR that negates enhancement of trans-
lation by the poly(A) tail. This AU-rich element was found
to bind PABP, leading to the suggestion that the element
may interfere with the interaction of PABP with eIF4G,
thereby preventing circularization of the mRNA and
reducing its rate of translation [23]. However, it remains
to be determined whether this is the mechanism through
which the papillomavirus AU-rich element functions, and
whether such an effect might occur with any cellular
mRNAs.
Acknowledgements
We thank Jeff Ross for initially suggesting PABP as the identity of the
major band in the UV cross-links, Matthias Goerlach and Gideon
Dreyfuss for providing PABP cDNA, Tullia Lindsten for iPABP
cDNA and Gideon Dreyfuss for PABP mAb. This work was supported
by a program grant from the National Health and Medical Research
Council of Australia.
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