MOLECULAR AND CELLULAR BIOLOGY, Apr. 2006, p. 3085–3097
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 8
Evidence that Poly(A) Binding Protein C1 Binds Nuclear
Pre-mRNA Poly(A) Tails
Nao Hosoda, Fabrice Lejeune,† and Lynne E. Maquat*
Department of Biochemistry and Biophysics, University of Rochester, School of Medicine and Dentistry,
601 Elmwood Ave., Box 712, Rochester, New York 14642
Received 23 August 2005/Returned for modification 20 January 2006/Accepted 24 January 2006
In mammalian cells, poly(A) binding protein C1 (PABP C1) has well-known roles in mRNA translation and
decay in the cytoplasm. However, PABPC1 also shuttles in and out of the nucleus, and its nuclear function is
unknown. Here, we show that PABPC1, like the major nuclear poly(A) binding protein PABPN1, associates
with nuclear pre-mRNAs that are polyadenylated and intron containing. PABPC1 does not bind nonpolyade-
nylated histone mRNA, indicating that the interaction of PABPC1 with pre-mRNA requires a poly(A) tail.
Consistent with this conclusion, UV cross-linking results obtained using intact cells reveal that PABPC1 binds
directly to pre-mRNA poly(A) tails in vivo. We also show that PABPC1 immunopurifies with poly(A) polymer-
ase, suggesting that PABPC1 is acquired by polyadenylated transcripts during poly(A) tail synthesis. Our
findings demonstrate that PABPC1 associates with polyadenylated transcripts earlier in mammalian mRNA
biogenesis than previously thought and offer insights into the mechanism by which PABPC1 is recruited to
newly synthesized poly(A). Our results are discussed in the context of pre-mRNA processing and stability and
mRNA trafficking and the pioneer round of translation.
The 3? ends of almost all eukaryotic mRNAs and their
precursors consist of homopolymeric tails of adenosine, or
poly(A), that are added by poly(A) polymerase (PAP) during
the process of 3? end formation. There are two classes of
poly(A) binding proteins (PABPs) in mammalian cells (32, 40).
One class is exemplified by PABPN1, formerly called PABP2.
PABPN1 is primarily nuclear and plays a role in the synthesis
of poly(A) tails, but it also shuttles between the nucleus and
cytoplasm (5, 8, 9, 31, 53). The other class consists of the
primarily cytoplasmic PABPs, of which PABPC1, formerly
called PABI or PABP1, is the major form in mammalian so-
matic cells (40). In humans, at least four separate PABPC
genes and four pseudogenes have been identified (40).
PABPC1 influences mRNA translation and decay (18, 20, 23,
27, 29, 30, 50, 54–56), and it shuttles between the nucleus and
cytoplasm of at least some mammalian cells (1, 57, 58). Con-
sistent with the preferential compartmentalization of PABPN1
to the nucleus and PABPC1 to the cytoplasm, a physical inter-
action has been detected between PAP and PABPN1 but not
PABPC1 (28). Furthermore, PABPN1 associates with RNA
polymerase II during transcription and accompanies the re-
leased transcript to the nuclear pore (2). Given that PABPC1
can exist within nuclei, a key issue is whether PABPC1 binds to
transcripts inside the nucleus, and if it does, at which step in
The 5? ends of eukaryotic mRNAs and their precursors are
capped, and the cap is initially bound by the mostly nuclear cap
binding protein (CBP) heterodimer of CBP80 and CBP20 (25,
35, 38), which will be called CBP80/CBP20. CBP80/CBP20 is
detectably replaced by the mostly cytoplasmic eukaryotic trans-
lation initiation factor 4E (eIF4E) only after introns have been
removed by splicing (35). Evidence indicates that PABPC1 is a
component of CBP80/CBP20-bound mRNA. First, PABPC1,
like PABPN1, coimmunopurifies with CBP80 (11, 24). Second,
PABP-interacting protein 2, which inhibits the interaction of
PABPC1 with poly(A), inhibits nonsense-mediated mRNA de-
cay (NMD) (11). NMD occurs as a result of nonsense codon
recognition during a pioneer round of translation (11, 22, 24).
The pioneer round is defined as the translation of CBP80/
CBP20-bound mRNA, and it is distinct from steady-state
translation, which is defined as the translation of eIF4E-bound
mRNA. Therefore, PABPC1 is a functional component of
CBP80/CBP20-bound mRNA. Whether PABPC1 is acquired
prior to the completion of splicing and whether PABPN1 re-
mains associated with mRNA during the pioneer round of
translation have never been reported.
In this communication, we provide the first evidence that
PABPC1 can bind the poly(A) tails of unspliced pre-mRNA.
First, cell fractionation verifies the presence of PABPC1 within
the nuclei of both HeLa CCL2 and Cos 7 cells, which we use
in our studies. Second, not only PABPN1 but also PABPC1
coimmunopurifies with unspliced pre-mRNA. Since this find-
ing was unexpected, we provide evidence in control experi-
ments that the association of PABPC1 with unspliced pre-
mRNA occurs in vivo, rather than after cell lysis as an artifact
of immunopurification (IP). Furthermore, results of UV cross-
linking using intact cells reveal that PABPC1 interacts directly
with the poly(A) tail of unspliced pre-mRNA in vivo. Third,
PABPC1 coimmunopurifies with PAP. These findings indicate
that PABPC1 associates with transcripts earlier in mRNA bio-
genesis than previously thought. Therefore, it became impor-
tant to understand how long PABPN1 remains associated with
mRNA before it is completely replaced by PABPC1. We find
* Corresponding author. Mailing address: Department of Biochem-
istry and Biophysics, University of Rochester, School of Medicine and
Dentistry, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Phone:
(585) 273-5640. Fax: (585) 271-2683. E-mail: lynne_maquat@urmc
† Present address: Institut de Ge ´ne ´tique Mole ´culaire de Montpel-
lier, CNRS, Montpellier, France.
that NMD reduces the abundance of nonsense-containing
mRNA that is bound not only by PABPC1 but also by
PABPN1. This result provides the first direct evidence that
PABPN1 remains associated with mRNA during the CBP80/
CBP20-mediated pioneer round of translation.
In summary, our results extend current views by providing
evidence that PABPC1 is acquired by polyadenylated tran-
scripts during poly(A) tail synthesis. Roles for PABPC1 in
pre-mRNA processing and stability and mRNA trafficking and
the pioneer round of translation are discussed.
MATERIALS AND METHODS
Plasmid constructions. To construct pCMV-Myc-PABPC1, cDNA encoding
human PABPC1 was generated using PCR, pSHREKK-PABPC1 (47), and the
primer pair 5? GCCAGATCTCTATGAACCCCAGTGCCCCCAGG 3? (sense)
and 5? TGAGGTACCTTAAACAGTTGGAACACCGG 3? (antisense). The re-
sulting PCR product was cleaved with BglII and KpnI and inserted into the
corresponding sites of pCMV-Myc (Clontech).
To construct pCMV-Myc-PABPN1, pEGFP-C2-PABPN1 (16) was digested
with BglII and BamHI, and the resulting cDNA fragment encoding human
PABPN1 was inserted into the BglII site of pCMV-Myc.
To construct pGEX6P2-PABPN1 for PABN1 production in Escherichia coli,
pCMV-Myc-PABPN1 was digested with SalI and NotI, and the resulting cDNA
encoding human PABPN1 was inserted into the corresponding sites of
pGEX6P2 (Amersham Biosciences).
Cell transfections and protein and RNA purifications. Cos 7 cell transfections
using calcium (see Fig. 7) and HeLa CCL2 cell transfections using Lipofectamine
2000 (Invitrogen) (see Fig. 2 to 6) were as previously described (24, 36). Protein
and RNA were purified from total, nuclear, or cytoplasmic fractions also as
previously presented (3, 24, 35).
IPs. IPs were as previously reported (24) with antibodies described below.
When specified, extracts were mixed using end-over-end rotation for 2 h at 4°C.
UV cross-linking. HeLa CCL2 cells (107/150-mm dish) were exposed to UV
for 5 min in 4 ml of ice-cold Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal bovine serum with a Stratalinker UV Cross-Linker (Stratagene).
Nuclear extracts were subsequently prepared, and a fraction of each extract was
either incubated at 80°C for 10 min to dissociate noncovalent interactions or, as
a control, left on ice. IPs were as previously reported (24) except anti-PABPC1
(33) was used.
Western blotting. Proteins were electrophoresed in 10% polyacrylamide,
transferred to a nitrocellulose membrane (Amersham Biosciences), and probed
using rabbit polyclonal anti-PABPC1 (33), rabbit polyclonal anti-PABPN1
(raised against the peptide NH2-RGSGPGRRRHLVPGAGGEC-COOH; D.
Bear, M. Becher, T. Howard, and B. Reinert, unpublished data), rabbit poly-
clonal anti-phospholipase C gamma (anti-PLC-?) (Santa Cruz), mouse mono-
clonal antibody mAb414 (which recognizes the nucleoporin p62; BAbCO),
mouse monoclonal anti-Myc (BD Biosciences), goat polyclonal anti-glutathione
S-transferase (anti-GST; Amersham Biosciences), or rabbit polyclonal anti-
Reverse transcription-PCR (RT-PCR). ?-Globin (Gl) and major urinary pro-
tein (MUP) mRNAs were amplified as previously described (36). Intron-con-
taining Gl transcripts were amplified using the following primer pairs: (i) 5?
GCCTATTGGTCTATTTTCCC 3 ? (intron 1, sense) and 5? GAGGAGGGGA
AGCTGATATC 3? (intron 2, antisense), (ii) intron 1, sense (see above) and 5?
GGGTTTAGTGGTACTTGTGAGC 3? (exon 3, antisense), or (iii) 5? ACCAC
CGTAGACGCAGATCG 3? (exon 1, sense) and intron 2, antisense (see above).
Cellular histone H4 mRNA was amplified using 5? CCTGCGGTCATGTCCG
GCCTGTG 3? (sense) and 5? GCGCTTGAGCGCGTTAACCACATCCATGG
CTGTGACGG 3? (antisense). Cellular triosephosphate isomerase (TPI) pre-
mRNA was amplified using 5? ACCTTGGCTTCATCTCTTCC 3? (intron 2,
sense) and 5? GTGTCTGTCCAAACCTATTG 3? (intron 5, antisense), MUP
pre-mRNA was amplified using 5? AGATAGAAGATAATGGCAAC 3? (intron
1,sense) and 5? AGGCGTGAGACCATACCAGG 3? (intron 2, antisense), cel-
lular ribosomal protein L36 (RPL36) pre-mRNA was amplified using 5? GAG
AGAAGCTGCTTAACTAG 3? (intron 1, sense) and 5? GGCCCTGACTCCC
ATCCCAC 3? (intron 2, antisense), and cellular RPL36 mRNA was amplified
using 5? AAAGTGACCAAGAACGTGAG 3? (exon 1, sense) and 5? TCTTGG
CGCGGATGTGCGTC 3? (exon 3, antisense).
PABPC1 is detected in the nuclear fraction of HeLa CCL2
cells. Although PABPC1 has been characterized as exclusively
cytoplasmic in selected HeLa and Cos cell lines (48), more
recent studies demonstrate that PABPC1 shuttles between the
nucleus and cytoplasm in at least some HeLa and NIH 3T3 cell
lines (1, 57, 58). We utilized cell fractionation and rabbit poly-
clonal anti-PABPC1 (33) to determine if PABPC1 was present
in the nuclear fraction of HeLa CCL2 cells, since we used these
cells in subsequent studies (see below). As a control, we also
used rabbit polyclonal anti-PABPN1 (33) to assess the cellular
distribution of PABPN1. Importantly, anti-PABPC1 specifi-
cally reacts with PABPC1, and anti-PABPN1 specifically reacts
with PABPN1 based on two criteria. First, Western blotting of
E. coli-produced and purified PABPC1, which is ?71 kDa (19),
reacted only with anti-PABPC1, and E. coli-produced and pu-
rified PABPN1, which is ?33 kDa but migrates in sodium
dodecyl sulfate-polyacrylamide as ?49 kDa (46), reacted only
with anti-PABPN1 (data not shown). Second, anti-PABPC1
and anti-PABPN1 reacted with appropriately sized HeLa
CCL2 cell or Cos 7 cell proteins by Western blotting (data not
shown). Cos 7 cells were also used in subsequent studies (see
Anti-PABPC1 may react not only with PABPC1 but also
with other members of the PABPC class. However, we con-
cluded that PABPC1 is the primary if not sole PABPC that was
detected in our experiments for the following three reasons.
First, PABPC1 is the most abundant of the PABPCs in somatic
cells (17, 40). Second, RT-PCR analysis of PABPC1, PABPC3,
PABPC4 (also called inducible PABP), and PABPC5 mRNAs
(40) in HeLa CCL2 and Cos 7 cells revealed that, in addition
to PABPC1 transcripts, only PABPC4 transcripts were de-
tected (data not shown). Furthermore, the ratio of PABPC4
and PABPC1 transcripts in the two cell types was only ?1:8
and ?1:20, respectively (data not shown). Third, and most
important, the anti-PABPC1 used in this study was raised
against a peptide that is specific to PABPC1 (33).
Nuclear and cytoplasmic fractions of HeLa CCL2 cells were
prepared (35) and analyzed by Western blotting using anti-
PABPC1, anti-PABPN1, anti-PLC-? (which controls for the
purity of the nuclear fraction), and mAb414 (which recognizes
the p62 component of the nuclear basket and controls for the
purity of the cytoplasmic fraction). The nuclear fraction proved
to be devoid of detectable PLC-?, which is essentially exclu-
sively a cytoplasmic protein (Fig. 1) (12). The cytoplasmic
fraction contained at most 7% ? 5% of total-cell p62, which is
largely nuclear (Fig. 1) (14). While PABPC1 was primarily in
the cytoplasmic fraction, 25% ? 3% of total-cell PABPC1 was
in the nuclear fraction (Fig. 1). In contrast, PABPN1 was
largely in the nuclear fraction, although 18% ? 2% of total-cell
PABPN1 was in the cytoplasmic fraction (Fig. 1). We conclude
that PABPC1 exists within the nuclear fraction of HeLa CCL2
cells. PABPC1 also exists within the nuclear fraction of Cos 7
cells (data not shown; D.G. Bear, personal communication).
PABPC1, like PABPN1, copurifies with unspliced pre-mRNA,
partially spliced pre-mRNA, and fully spliced mRNA but not
nonadenylated RNA. Since PABPC1 is present in nuclear frac-
tions and is a functional component of the pioneer translation
initiation complex, it was of interest to determine if PABPC1
3086HOSODA ET AL.MOL. CELL. BIOL.
binds the intron-containing precursors of the pioneer transla-
tion initiation complex. HeLa CCL2 cells were transiently
transfected with two plasmids: (i) a pmCMV-Gl test plasmid
that encodes Gl RNA (61) and (ii) a phCMV-MUP reference
plasmid that encodes MUP RNA and controls for variations in
transfection efficiency and RNA recovery (4). Neither RNA
was expressed from the HeLa cell genome. Protein and RNA
were isolated before and after IP of nuclear extract using
anti-PABPC1, anti-PABPN1, or, to control for nonspecific IP,
normal rabbit serum (NRS).
RT-PCR was used to amplify the region that extends from
the first intron into the last intron of Gl pre-mRNA or the
region that extends from the first intron into the second intron
of MUP pre-mRNA. Results demonstrated that each intron-
PABPC1 or anti-PABPN1 but not NRS (Fig. 2A and B, com-
pare lanes 2 and 3 to lane 1 in each). The identity of each
RT-PCR product was verified by PCR analysis of pmCMV-Gl or
phCMV-MUP using the same primer pair that was used to am-
plify the corresponding transcript (Fig. 2A and B, lanes 4).
RT-PCR was also used to amplify sequences that extend from
the first intron into the last intron of RPL36 pre-mRNA or se-
quences that extend from the second intron into the penultimate
intron of triosephosphate isomerase (TPI) pre-mRNA, each of
which was derived from the HeLa cell genome. Results dem-
onstrated that each intron-containing pre-mRNA was likewise
immunopurified with anti-PABPC1 or anti-PABPN1 but not
NRS (Fig. 2C and D, compare lanes 2 and 3 to lane 1 in each).
The identity of the TPI RT-PCR product was verified by PCR
analysis of pmCMV-TPI (60) using the same primer pair that
was used to amplify the corresponding HeLa cell TPI tran-
script (Fig. 2D, lane 4).
To assess if IP of each pre-mRNA using either anti-PABPC1
or anti-PABPN1 depends on the presence of a poly(A) tail,
histone H4 mRNA that derived from the HeLa cell genome
was also assessed. Histone H4 transcripts undergo neither
splicing nor polyadenylation (15). RT-PCR was used to am-
plify almost the full length of histone H4 mRNA. Results
demonstrated that this nonadenylated mRNA was not detect-
ably immunopurified with either anti-PABPC1 or anti-PABPN1
(Fig. 2A, lanes 2 and 3). Here again, the identity of the RT-PCR
product was corroborated by PCR analysis of pmCMV-H4 (44)
using the same primer pair that was used to amplify HeLa cell
histone H4 mRNA (Fig. 2A, lane 5).
We conclude that PABPC1, like PABPN1, can bind intron-
containing pre-mRNA. We also conclude that binding depends
on a poly(A) tail, since nonadenylated RNA fails to bind either
PABP. Consistent with this view, while splicing can occur co-
transcriptionally, unspliced but polyadenylated pre-mRNA
does exist in cells (6, 26, 42, 43). Furthermore, the presence of
unspliced but polyadenylated Gl pre-mRNA in samples ana-
lyzed in Fig. 2 was corroborated by RT-PCR using primers that
amplified sequences extending from Gl intron 2 into the
poly(A) tail (data not shown).
An association of PABPC1 with intron-containing pre-
mRNA has never been reported. Therefore, it was particularly
important to determine if the association occurred in vivo or
only after cell lysis as an artifact of the experimental procedure
(45). If the association occurred in vivo, then it should be
detected after IP of nuclear extract from cells that coexpressed
Myc-PABPC1 and Gl pre-mRNA. However, the association
should not be detected after IP of a mixture of (i) nuclear
extract from cells that expressed Myc-PABPC1 but not Gl
pre-mRNA and (ii) nuclear extract from cells that expressed
Gl pre-mRNA but not Myc-PABPC1.
To determine if PABPC1 and Gl pre-mRNA interact in vivo,
HeLa CCL2 cells were transiently transfected so that the total
amount of introduced DNA was constant (25 ?g) per trans-
fection (see the legend to Fig. 3). In these transfections,
pCMV-Myc (12 ?g) controlled for the absence of pmCMV-
PABPC1 (12 ?g), pCMV-Myc (3 ?g) controlled for the ab-
sence of phCMV-MUP (3 ?g), and pCMV-Myc (10 ?g) con-
trolled for the absence of pmCMV-Gl (10 ?g). Nuclear
extracts were prepared, and protein and RNA were isolated
prior to and after IP using anti-Myc. IPs were performed be-
fore or after mixing specific nuclear extracts.
Western blotting using anti-Myc revealed that Myc-PABPC1
was expressed at a similar level in cells that had been cotrans-
fected with pmCMV-Gl, phCMV-MUP, and pCMV-Myc-PABPC1
and in cells that had been cotransfected with pCMV-Myc,
phCMV-MUP, and pCMV-Myc-PABPC1 (Fig. 3A, compare
Myc-PABPC1 was immunopurified with similar efficiency from
the corresponding nuclear extracts (Fig. 3A, compare lanes 7
Using RT-PCR, the amount of Gl pre-mRNA that was im-
munopurified using anti-Myc and extracts of cells cotrans-
fected with pmCMV-Gl, phCMV-MUP, and pCMV-Myc-
PABPC1 was defined as 100% (Fig. 3B, lane 7). Only 9% ?
2% of this pre-mRNA was immunopurified using anti-Myc and
FIG. 1. Not only PABPN1 but also PABPC1 is present in the nu-
clear fraction of HeLa CCL2 cells. Nuclear (lane 1) and cytoplasmic
(lane 2) fractions were analyzed by Western blotting using anti-PLC-?,
mAb414 (which recognizes the nucleoporin p62), rabbit polyclonal
anti-PABPC1, or rabbit polyclonal anti-PABPN1. The four leftmost
lanes, which analyzed threefold dilutions of total-cell protein, indicate
that the analysis is semiquantitative. The amount of total-cell PLC-?,
p62, PABPC1, or PABPN1 that was nuclear was 5% ? 1%, 93% ?
5%, 25% ? 3%, or 82% ? 2%, respectively. Results are representative
of two independently performed experiments.
VOL. 26, 2006 PABPC1 BINDS UNSPLICED PRE-mRNA3087
FIG. 2. Both PABPC1 and PABPN1 bind to intron-containing transcripts that derive from either transiently introduced plasmids or the HeLa
cell genome. HeLa CCL2 cells (107/150-mm dish) were transiently transfected with pmCMV-Gl (10 ?g) and phCMV-MUP (3 ?g). Protein and
RNA were isolated before (?) and after IP of the nuclear extract as described in the legend to Fig. 1 and analyzed by Western blotting (data not
shown; IP efficiencies were 9% ? 2% when anti-PABPC1 was used and 38% ? 4% when anti-PABPN1 was used) and RT-PCR. To the right of
each panel, sense (1) and antisense (2) PCR primers are specified by arrows above the introns to which they correspond within each transcript
3088 HOSODA ET AL.MOL. CELL. BIOL.
a mixture of (i) extract from cells cotransfected with pCMV-
Myc, phCMV-MUP, and pCMV-Myc-PABPC1 and (ii) extract
from cells cotransfected with pmCMV-Gl and pCMV-Myc
(Fig. 3B, lane 9). As expected, anti-Myc immunopurified nei-
ther Gl pre-mRNA nor MUP mRNA with extract from cells
that did not express Myc-PABPC1 (Fig. 3B, lanes 6 and 8).
These data indicate that only 9% ? 2% of Gl pre-mRNA that
was bound by Myc-PABPC1 was due to binding in vitro. In
other words, the bulk (i.e., 89 to 93%) of PABPC1 that copuri-
fied with Gl pre-mRNA reflected binding in vivo rather than
binding after cell lysis. Notably, the level of Myc-PABPC1 was
threefold the level of endogenous PABPC1 when cells were
harvested (data not shown). Considering that pre-mRNA is
compete with endogenous PABPC1 for binding to pre-mRNA.
If the association of PABPC1 with pre-mRNA typifies
pre-mRNA that is productively processed to translationally
active mRNA, then the partially and fully spliced products of
pre-mRNA should also be bound by PABPC1. To test this
hypothesis, the association of PABPN1 and PABPC1 with un-
spliced, partially spliced, and fully spliced Gl transcripts was
assessed using the same nuclear IPs that were analyzed in Fig.
2. RT-PCR was used to amplify from the first to the last exon
of Gl RNA. Results demonstrated that fully spliced (Gl
mRNA), partially spliced (intron 1 only) pre-mRNA, and an
electrophoretically inseparable mixture of unspliced (i.e., con-
taining introns 1 and 2) and partially spliced (containing intron
2 only) pre-mRNAs were immunopurified using anti-PABPC1
or anti-PABPN1 but not NRS (Fig. 4A, left; compare lanes 2
and 3 to lane 1). The same RT-PCR products were subse-
quently analyzed using a lower-percentage acrylamide gel that
separated pre-mRNA containing introns 1 and 2 from pre-
mRNA containing intron 2 only. Results revealed that un-
spliced pre-mRNA and all three possible spliced variants were
immunopurified using anti-PABPC1 or anti-PABPN1 but not
NRS (Fig. 4A, right, compare lanes 2 and 3 to lane 1).
RT-PCR was also used to amplify sequences from the first
exon to the last intron of Gl transcripts. Results showed that
partially spliced (intron 2 only) pre-mRNA and unspliced (in-
trons 1 and 2) pre-mRNA were immunopurified using anti-
PABPC1 or anti-PABPN1 but not NRS (Fig. 4B, compare
lanes 2 and 3 to lane 1). Notably, the identity of RT-PCR
products that derived from Gl transcripts that were unspliced
or contained only intron 1 was verified by PCR analysis of,
respectively, pmCMV-Gl or pmCMV-Gl ?(intron 2) (61) us-
ing the same primer pair that was used to amplify the corre-
sponding RNA (Fig. 4A, lanes 4 and 5, and B, lane 4). These
findings indicate that PABPC1 binds unspliced Gl pre-mRNA
and remains associated during splicing.
PABPC1 can be UV cross-linked in vivo to form a heat-
resistant complex with Gl pre-mRNA and Gl mRNA but not
with histone mRNA. To determine if the association of PABPC1
with Gl pre-mRNA is direct, the ability of PABPC1 to form
covalent UV cross-links with Gl pre-mRNA was examined.
HeLa CCL2 cells that had been transiently transfected with
pmCMV-Gl were either exposed to UV at 0°C, which induces
covalent RNA-protein cross-links in vivo, or not exposed to
UV. Nuclear extracts were subsequently generated, and a frac-
tion of each extract was either incubated at 80°C for 10 min,
which dissociates noncovalent interactions, or left at 0°C. Pro-
tein and RNA were then isolated from the variously treated
extracts before or after IP using anti-PABPC1 or, to control for
nonspecific IP, NRS.
Western blotting of samples prior to IP using anti-PABPC1
revealed that exposure to UV, heat, or both reduced the re-
covery of PABPC1 (Fig. 5A, compare lane 1 to lanes 2 through
4). However, IP efficiencies for all extracts were comparable
(Fig. 5A, compare lane 6 to lanes 8, 10, and 12; Fig. 5, legend).
RT-PCR of samples prior to IP revealed that exposure to UV,
heat, or both also reduced the recovery of Gl mRNA, Gl
pre-mRNA, and histone H4 mRNA (Fig. 5B, compare lane 1
to lanes 2 through 4). Additionally, anti-PABPC1 immunopu-
rified more Gl mRNA than Gl pre-mRNA in the absence of
UV or heat, consistent with the relative steady-state levels of
the two RNAs (Fig. 5B, lane 6).
As described above (Fig. 2A), anti-PABPC1 failed to immu-
nopurify histone H4 mRNA because it lacked a poly(A) tail
(Fig. 5B, lane 6). Anti-PABPC1 immunopurified Gl mRNA
and Gl pre-mRNA after UV treatment without subsequent
heat treatment (Fig. 5B, lane 8), as well as with subsequent
heat treatment (Fig. 5B, lane 12), but not with heat treatment
in the absence of UV treatment (Fig. 5B, lane 10). When we
compensated for the reduced recovery of Gl mRNA and Gl
pre-mRNA that occurred after UV and heat treatments, we
found that the efficiency with which Gl mRNA coimmuno-
purified with PABPC1 after treatment was essentially the same
as the efficiency with which Gl pre-mRNA coimmunopurified
with PABPC1 after treatment (compare 73% ? 9% to 75% ?
13%) (Fig. 5C).
We conclude that nuclear PABPC1 can be UV cross-linked
to Gl mRNA and Gl pre-mRNA in a way that is resistant to
heat that dissociates noncovalent bonds. The simplest inter-
pretation of these results is that nuclear PABPC1 interacts
directly with the poly(A) tail of not only Gl mRNA but also Gl
pre-mRNA in intact cells.
PABPC1 coimmunopurifies with PAP-?. To gain a better
understanding of when in pre-mRNA metabolism PABPC1
associates with newly synthesized poly(A) tails, we tested if
HeLa cell PABPC1 coimmunopurifies with HeLa cell PAP.
Bovine PABPN1 that was synthesized in vitro using rabbit
reticulocyte lysates has been shown to copurify with bovine
PAP that was synthesized in E. coli as a GST-tagged protein
(28). This and other findings indicate that PABPN1 stimulates
PAP activity, and thus poly(A) tail elongation, by recruiting
PAP to substrate RNAs (28). In theory, PABPC1 could also
coimmunopurify with PAP.
diagram. Transcript exons are indicated as boxes, and transcript introns are specified as lines. (A) RT-PCR of Gl pre-mRNA produced from
pmCMV-Gl and cellular histone H4 mRNA. PCR of pmCMV-Gl or pmCMV-H4 provided a control for transcript size. (B) RT-PCR of MUP
pre-mRNA produced from phCMV-MUP and, as a control, PCR of phCMV-MUP. (C) RT-PCR of cellular RPL36 pre-mRNA. (D) RT-PCR of
cellular TPI pre-mRNA and, as a control, PCR of pmCMV-TPI. Results are representative of two independently performed experiments.
VOL. 26, 2006 PABPC1 BINDS UNSPLICED PRE-mRNA3089
The ability of PABPC1 to coimmunopurify with PAP-?, one
of several HeLa cell PAP isoforms, was tested for two reasons.
First, this isoform is known to function in a mechanism that
depends on both cleavage-polyadenylation specificity factor
and the AAUAAA polyadenylation signal (34). Second, high-
quality anti-PAP-? is available (34). Protein was isolated be-
fore or after IP using nuclear extracts from untransfected
HeLa CCL2 cells and rabbit polyclonal anti-PAP-? or, as a
negative control, NRS.
but failed to immunopurify the nucleoporin p62 (Fig. 6A, lane 2).
Anti-PAP-? also immunopurified PABPC1 (Fig. 6A, lane 2),
demonstrating that PAP-? and PABPC1 do, indeed, interact.
Even though the interaction may not be direct, these data
FIG. 3. IP of PABPC1 with Gl pre-mRNA is attributable to binding in vivo rather than to binding during extract preparation. HeLa CCL2 cells
(107/150-mm dish) were transiently transfected with the following plasmids so that the total amount of introduced DNA was constant: lanes 1 and
6, pmCMV-Gl (10 ?g), phCMV-MUP (3 ?g), and pCMV-Myc (12 ?g); lanes 2 and 7, pmCMV-Gl (10 ?g), phCMV-MUP (3 ?g), and
pCMV-Myc-PABPC1 (12 ?g); lane 3, pmCMV-Gl (10 ?g), pCMV-Myc (3 ?g), and pCMV-Myc (12 ?g); lane 4, pCMV-Myc (10 ?g), phCMV-
MUP (3 ?g), and pCMV-Myc (12 ?g); lane 5, pCMV-Myc (10 ?g), phCMV-MUP (3 ?g), and pCMV-Myc-PABPC1 (12 ?g). Protein and RNA
were isolated from nuclear extracts before (?) or after IP using anti-Myc. IPs were performed prior to (lanes 6 and 7) or after the specified extracts
were mixed (lane 8 analyzed a mixture of the extracts that were analyzed separately in lanes 3 and 4; lane 9 analyzed a mixture of the extracts that
were analyzed separately in lanes 3 and 5). (A) Western blotting using anti-Myc. (B) RT-PCR of Gl pre-mRNA from intron 1 into intron 2 (introns
1 and 2) and MUP mRNA to determine the extent of PABPC1 binding to Gl pre-mRNA that occurs after cell lysis. This extent was calculated
to be 9% ? 2% (lane 9) of PABPC1 binding to Gl pre-mRNA that occurred in cells that had been cotransfected with pmCMV-Gl, phCMV-MUP,
and pCMV-Myc-PABPC1, which was defined as 100% (lane 7). The asterisk indicates an uncharacterized RT-PCR product that did not interfere
with experimental interpretations. Results are representative of two independently performed experiments.
3090 HOSODA ET AL.MOL. CELL. BIOL.
suggest that PABPC1 may be acquired by newly synthesized,
polyadenylated transcripts during poly(A) tail synthesis. Anti-
PAP-? reproducibly immunopurified a larger percentage of
nuclear PABPN1 (13% ? 3%) than nuclear PABPC1 (5% ?
1%). These percentages indicate that there is likely to be more
of each PABP associated with poly(A) tails after polyadenyla-
tion than with PAP-? during polyadenylation, which makes
sense, given that most poly(A) tails in a cell are not associated
The co-IP of both PABPN1 and PABPC1 with PAP-? im-
plies that antibody to either PABP should immunopurify the
other PABP. Using nuclear fractions from untransfected HeLa
CCL2 cells, anti-PABPN1 immunopurified not only PABPN1
but also PABPC1 and, as expected, failed to immunopurify p62
(Fig. 6B, lane 3). Unexpectedly, however, while anti-PABPC1
immunopurified PABPC1, it failed to immunopurify PABPN1
(Fig. 6B, lane 2). Failure may be experimentally induced, e.g.,
because anti-PABPC1 binding to PABPC1 interferes with
PABPC1 binding to PABPN1. Alternatively, the fraction of
cellular PABPC1 that associates with PABPN1 may be suffi-
ciently low to preclude easy detection.
As shown by a different experimental approach, the second
interpretation appears to be correct. In this approach, HeLa
CCL2 cells were transiently transfected with pCMV-Myc-
PABPC1, pCMV-Myc-PABPN1, or pCMV-Myc. These plas-
mids produced Myc-tagged PABPC1, Myc-tagged-PABPN1,
FIG. 4. PABPC1, like PABPN1, binds to unspliced, partially spliced, and fully spliced Gl transcripts. Gl transcripts were analyzed before (?)
and after IP using the same nuclear extract that was analyzed in Fig. 2 and the specified antibody. To the right of each panel, sense (1) and antisense
(2) PCR primer pairs are indicated by arrows above each uppermost transcript diagram, where exons are shown as boxes and introns are shown
as lines. (A) RT-PCR of Gl exon 1 through exon 3 and, as a control, PCR of the same region of pmCMV-Gl or pmCMV-Gl ?(intron 2). Samples
were electrophoresed in either 5% (left) or 3.5% (right) polyacrylamide, the latter of which separated unspliced pre-mRNA (introns 1 and 2) from
partially spliced pre-mRNA (intron 2 only). Dots indicate intron 1-only RNA, and triangles indicate an inseparable mixture of Gl RNA containing
introns 1 and 2 and Gl RNA containing intron 2 only. (B) RT-PCR of Gl exon 1 through intron 2 and, as a control, PCR of pmCMV-Gl. Results
are representative of two independently performed experiments.
VOL. 26, 2006 PABPC1 BINDS UNSPLICED PRE-mRNA 3091
or Myc tag, respectively. Protein was purified from nuclear
fractions before and after IP using anti-Myc. Results of West-
ern blotting using anti-Myc indicated that although Myc-
PABPN1 was expressed at a higher level than Myc-PABPC1,
both Myc-PABPN1 and Myc-PABPC1 were readily detectable
both before IP (Fig. 6C, lanes 2 and 3) and after IP (Fig. 6C,
lanes 5 and 6). Western blotting using anti-PABPN1 confirmed
that Myc-PABPC1 coimmunopurifies with cellular PABPN1
(Fig. 6C, lane 6). Western blotting using anti-PABPC1 re-
vealed that Myc-PABPN1 indeed coimmunopurifies with cel-
FIG. 5. PABPC1 can be UV cross-linked in vivo to form heat-resistant complexes with Gl pre-mRNA and Gl mRNA but not histone H4
mRNA. HeLa CCL2 cells (107/150-mm dish) that had been transiently transfected with pmCMV-Gl (10 ?g) were either exposed to UV for 5 min
to induce covalent RNA-protein cross-links in vivo (?UV) or not exposed to UV (?UV). Nuclear extracts were subsequently prepared, and a
fraction of each extract was either incubated at 80°C for 10 min (?Heat) to dissociate noncovalent interactions or left on ice (?Heat). Protein and
RNA were isolated from the variously treated extracts before IP (?IP) or after IP using anti-PABPC1 or, as a control for nonspecific IP, NRS.
(A) Western blotting using anti-PABPC1 demonstrated that IP efficiencies were 25% ? 6%. The four leftmost lanes, which analyzed threefold
dilutions of nuclear protein prior to IP, indicate that the analysis is semiquantitative. (B) RT-PCR of Gl mRNA, Gl pre-mRNA, or histone H4
mRNA. Samples before IP (lanes 1 to 4) consist of half the cell equivalents of samples after IP. The four leftmost lanes, which analyzed twofold
dilutions of nuclear RNA prior to IP, demonstrate that the analysis is quantitative. (C) Quantitation of Gl mRNA and pre-mRNA levels that were
immunopurified using anti-PABPC1. After UV and heat treatments, the level of immunopurified Gl mRNA was normalized to the level of Gl
mRNA prior to IP. The normalized level was subsequently expressed as a percentage of the normalized level of the Gl mRNA that had not been
exposed to UV or heat treatment, which was defined as 100. The same analysis was performed for Gl pre-mRNA. Values derive from two
independently performed experiments.
3092 HOSODA ET AL.MOL. CELL. BIOL.
lular PABPC1, although a relatively dark exposure was re-
quired for detection (Fig. 6C, lane 5, darker exposure).
Additionally, Western blotting using anti-PAP-? showed that
both Myc-PABPC1 and Myc-PABPN1 coimmunopurified with
PAP-? (Fig. 6C, lanes 5 and 6).
PABPN1, like PABPC1, is present on mRNA that is a sub-
strate for NMD. Given our finding that PABPC1 binds poly-
adenylated transcripts earlier than was previously appreciated,
it became important to gain insight into how long PABPN1
remains associated with these transcripts. CBP80 coimmuno-
FIG. 6. PABPC1, like PABPN1, coimmunopurifies with PAP-?. (A) Protein from nuclear extracts of untransfected HeLa CCL2 cells (107/
150-mm dish) was immunopurified using anti-PAP-? or, as a control for nonspecific IP, NRS. Western blotting using anti-PAP-? or mAb414, which
recognizes nucleoporin p62, demonstrated that the IP efficiency for anti-PAP-? was 53% ? 7% and that the nucleoporin p62 was not detectable
in the IP. (B) Protein from nuclear extracts of untransfected HeLa CCL2 cells (107/150-mm dish) was immunopurified using anti-PABPC1,
anti-PABPN1, or, as a control for nonspecific IP, NRS. Western blotting revealed that the IP efficiency for anti-PABPC1 or anti-PABPN1 was
11% ? 6% or 35% ? 4% and that the nucleoporin p62 was not detectable in either IP. (C) HeLa CCL2 cells (107/150-mm dish) were transiently
transfected with 30 ?g of pCMV-Myc-PABPC1 (lanes 2 and 5), 3 ?g of pCMV-Myc-PABPN1 plus 27 ?g of pCMV-Myc (lanes 3 and 6) or, as a
control, 30 ?g of pCMV-Myc (lanes 1 and 4). Nuclear extracts were generated and analyzed either before (?) IP (lanes 1 through 3) or after IP
using anti-Myc (lanes 4 through 6). Western blotting using anti-Myc demonstrated that the IP efficiency for Myc-PABPC1 or Myc-PABPN1 was
45% ? 5% or 56% ? 8%, respectively. The specificity of each IP was evident from the absence of detectable p62. Samples before IP (lanes 1
through 3) consist of half the cell equivalents of samples after IP. The three leftmost lanes (A and B) or four leftmost lanes (C), which analyzed
threefold dilutions of protein, indicate that the analysis is semiquantitative. Results are representative of two independently performed experi-
VOL. 26, 2006 PABPC1 BINDS UNSPLICED PRE-mRNA3093
purifies with the C-terminal domain of RNA polymerase II
(35) and, in the insect Chironomus tentans, binds cotranscrip-
tionally to RNA caps (52). Additionally, Chironomus tentans
PABPN1 can be visualized on elongating transcription com-
plexes (2), and mammalian PABPN1 binds to RNA during
poly(A) tail synthesis (53). However, though PABPN1 copuri-
fies with CBP80, it may be removed by the time CBP80/
CBP20-bound mRNA undergoes the pioneer round of trans-
lation (11, 24, 37).
Since NMD in mammalian cells targets CBP80/CBP20-
bound mRNA but not detectably eIF4E-bound mRNA, one
way to determine if PABPN1 remains associated with poly(A)
during the pioneer round of translation is to assess if anti-
PABPN1 immunopurifies nonsense-containing mRNA that
has been reduced in abundance by NMD. If it does, then
PABPN1 must be a component of the pioneer translation
initiation complex. To this end, Cos 7 cells were transiently
transfected with two plasmids: (i) a pmCMV-Gl test plasmid
that encodes either nonsense-free (Norm) or nonsense-con-
taining (Ter) ?-Gl mRNA (Fig. 7A) (61) and (ii) the phCMV-
MUP reference plasmid. Protein and RNA were purified from
nuclear fractions before and after IP using anti-PABPC1, anti-
PABPN1, or, as a control for nonspecific IP, NRS.
Western blotting of proteins from the nuclear fraction dem-
onstrated that anti-PABPC1 immunopurified PABPC1 (Fig.
7B, top, lanes 3 and 4) and anti-PABPN1 immunopurified
PABPN1 (Fig. 7B, bottom, lanes 5 and 6), whereas NRS im-
munopurified neither PABP (Fig. 7B, lanes 1 and 2). Further-
more, anti-PABPN1 immunopurified PABPC1 (Fig. 7B, top,
lanes 5 and 6). Therefore, the two PABPs coimmunopurify has
expected (Fig. 6). As in Fig. 6B, anti-PABPC1 failed to immu-
nopurify PABPN1 (Fig. 7B, bottom, lanes 3 and 4), although
an interaction between Myc-PABPC1 and cellular PABPN1
was detectable, which is consistent with data indicating that the
two proteins do copurify (Fig. 6C).
RT-PCR of RNA from the nuclear fraction, where the
NMD of nonsense-containing Gl mRNA occurs (36, 61), dem-
onstrated that, prior to IP, the level of Gl Ter mRNA was 22%
? 5% the level of Gl Norm mRNA (Fig. 7C, compare lane 2
to lane 1). The level of Gl Ter mRNA that was immunopuri-
fied using either anti-PABPC1 or anti-PABPN1 was similarly
reduced (Fig. 7C, compare lane 6 to lane 5 or lane 8 to lane 7,
respectively). As expected, NRS immunopurified neither Gl
mRNA nor MUP mRNA (Fig. 7C, lanes 3 and 4). These data
indicate that NMD targets mRNA that is bound by PABPC1
and PABPN1 and that PABPN1 remains bound during the
pioneer round of translation.
Our results provide the first evidence that PABPC1, like
PABPN1, can associate in vivo with intron-containing, poly-
adenylated transcripts that go on to be productively spliced.
Furthermore, PABPC1, like PABPN1, binds directly to the
poly(A) tail of these transcripts. First, PABPC1 is present
within the nuclei of both cell types that we examined (Fig. 1;
data not shown). Second, anti-PABPC1, like anti-PABPN1,
immunopurifies every unspliced pre-mRNA, partially spliced
pre-mRNA, and fully spliced mRNA that we tested (Fig. 2 and
4). Third, cellular PABPC1 binding to these transcripts is de-
FIG. 7. Both PABPN1 and PABPC1 are present on nonsense-con-
taining mRNA that was reduced in abundance by NMD. Cos 7 cells
(4 ? 107/150-mm dish) were transfected with a pmCMV-Gl test plas-
mid (10 ?g), either nonsense-free (Norm) or containing a TAG non-
sense codon at position 39 (Ter), and the phCMV-MUP reference
plasmid (3 ?g). Protein and RNA from nuclear fractions were purified
either before (?) or after IP using anti-PABPC1, anti-PABPN1, or, as
a control for nonspecific IP, NRS. (A) Diagram of pmCMV-Gl.
ATG(0) indicates the translation initiation codon, TAG(39Ter) indi-
cates the nonsense codon that typifies pmCMV-Gl Ter, and TAA(147)
indicates the normal translation termination codon. The thick gray bar
represents the CMV promoter, boxes represent exons, and lines be-
tween boxes represent introns. (B) Western blotting of nuclear frac-
tions using anti-PABPC1 or anti-PABPN1. IP efficiencies using anti-
PABPC1 or anti-PABPN1 were 24% ? 9% or 38% ? 6%,
respectively. The four leftmost lanes, which analyzed twofold protein
dilutions, indicate that the analysis is semiquantitative. (C) RT-PCR of
nucleus-associated Gl and MUP mRNAs. The three leftmost lanes,
which analyzed twofold dilutions of RNA, demonstrate that the anal-
ysis is quantitative. Numbers immediately below the figure represent
the level of Gl mRNA normalized to the level of MUP mRNA, where
the normalized level of Gl Norm mRNA prior to or after IP using
anti-PABPC1 or anti-PABPN1 was defined as 100%. Results are rep-
resentative of three independently performed experiments.
3094 HOSODA ET AL.MOL. CELL. BIOL.
pendent on a poly(A) tail, as evidenced by the failure of anti-
PABPC1 to immunopurify nonadenylated histone H4 mRNA
(Fig. 2 and 5). Fourth, anti-PABPC1 immunopurifies pre-
mRNA, largely as a consequence of binding in vivo and not
because of an experimental artifact (Fig. 3). Fifth, cellular
PABPC1 remains UV cross-linked to pre-mRNA under con-
ditions that dissociate noncovalent bonds, and cross-linking
depends on the presence of a poly(A) tail (Fig. 5). Sixth,
cellular PAP-? coimmunopurifies with cellular PABPC1, and
Myc-PABPC1 coimmunopurifies with cellular PAP-? (Fig. 6).
Our ability to detect an interaction between PAP-? and
PABPC1 contrasts with data demonstrating that purified bo-
vine PAP-? and Xenopus laevis PABPC1 do not detectably
interact (28). However, Xenopus PABPC1 may be unable to
interact with human PAP because of incompatibility between
species, because mammalian PABPC1 functions in ways that
Xenopus PABPC1 does not, or because in vitro binding con-
ditions were insufficient to support the interaction of PABPC1
We also find that E. coli-produced and purified PABPC1
copurifies with E. coli-produced and purified GST-PABPN1
but not GST alone (data not shown). However, considering
there are several examples of proteins that interact in vitro but
not apparently in cells (e.g., Staufen1 and Staufen2) (49; H. A.
Kuzmiak and L. E. Maquat, unpublished data), it would be
premature to conclude that PABPN1 and PABPC1 interact
directly in mammalian cells. We favor the interpretation that
PABPC1 associates with unspliced pre-mRNA directly via the
poly(A) tail because PABPC1 can be UV cross-linked to pre-
mRNA in intact cells. Nevertheless, we cannot exclude the
possibility that PABPC1 also associates via PABPN1.
In view of the unexpectedly early step at which PABPC1 is
acquired during mRNA biogenesis, it became important to
determine how long PABPN1 remains associated with mRNA,
given that PABPN1 is ultimately replaced by PABPC1. We
have found that PABPN1 remains associated with mRNA dur-
ing the pioneer round of translation, for which CBP80/CBP20-
bound mRNA serves as a template. This was evidenced by the
ability of anti-PABPN1 to immunopurify nonsense-containing
mRNA that had already been reduced in abundance by NMD
(Fig. 7). Even though anti-CBP80 immunopurifies PABPN1
and PABPC1 (11, 24) and anti-eIF4E immunopurifies only
PABPC1 (11, 24), we cannot be certain that PABPN1 is no
longer present after CBP80 and CBP20 are replaced by eIF4E,
since the fraction of eIF4E-bound mRNA that is bound by
PABPN1 may be too small to detect.
We conclude that both PABPN1 and PABPC1 bind to the
poly(A) tail of newly synthesized transcripts in the nucleus, at
least in some cases prior to splicing. Consistent with this, not
all splicing occurs cotranscriptionally in mammalian cells and,
consequently, unspliced or partially spliced polyadenylated
transcripts do exist (6, 26, 42, 43; data not shown).
PABPN1 has been shown to function in nuclear polyadenyl-
ation by recruiting PAP to transcripts and, by so doing, increas-
ing the processivity of PAP and controlling the length of the
newly synthesized poly(A) tail (5, 28, 53). PABPN1 has also
been functionally implicated in mRNA transport, since it shut-
tles between the nucleus and the cytoplasm (9, 10), and immu-
noelectron microscopy demonstrates that it is present on nu-
clear messenger ribonucleoprotein particles (mRNPs) that
transit the nuclear envelope (2). A functional homolog to
mammalian PABPN1 in Saccharomyces cerevisiae has not been
found. The S. cerevisiae homolog to mammalian PABPC1,
Pab1p, is known to play a role in both cytoplasmic mRNA
metabolism, including mRNA translation and decay, as well as
in nuclear RNA metabolism, including polyadenylation (32,
40) and mRNA export (7). Recent studies have shown that
PABPC1, like yeast Pab1p (7), shuttles between the nucleus
and the cytoplasm (1, 58).
Our data indicate PABPC1 associates with nuclear pre-
mRNP prior to intranuclear transport by directly binding
poly(A), most likely simultaneously with PABPN1. PABPC1
bound to newly synthesized poly(A) tails may function in pre-
mRNA metabolism. For example, it could protect nuclear pre-
mRNA from decapping, which is known to occur within nuclei,
much as it protects cytoplasmic mRNA from decapping (30,
55, 56). As another example, PABPC1 could promote nuclear
poly(A) nuclease 2 (Pan2)-mediated pre-mRNA poly(A) trim-
ming, as it does in the cytoplasm (51), since both Pan2 and
Pan3, the latter of which tethers Pan2 to PABPC1, are known
to shuttle between the nucleus and the cytoplasm (59). In fact,
Pan2p and Pan3p, the S. cerevisiae orthologs of mammalian
Pan2 and Pan3, are known to regulate nuclear poly(A) tail
length (39, 41).
PABPC1 may also influence the metabolism of nuclear
mRNP. For example, as polyadenylated mRNP approaches the
nuclear pore and is unfolded for transit through the nuclear
pore complex (13), poly(A)-bound PABPC1 may play a role
during transit by further nucleating PABPC1 assembly and
concomitantly removing PABPN1, a process that would be
completed after the pioneer round of translation. Another
possibility is that PABPC1 primarily assembles with RNP and
functions in the nucleus at a step that is much earlier than
transit across the pore. In either scenario, PABPC1 may also
be involved in the localization of particular mRNAs to specific
regions of the cytoplasm. For example, PABPC1 was recently
shown to bind directly to paxillin, which is an abundant protein
of focal complexes at the leading edges of migrating cells (57,
58). The PABPC1-paxillin complex localizes to the perinuclear
endoplasmic reticulum and the leading edge of the migrating
cell plasma membrane in mouse fibroblasts. Thus, nuclear
PABPC1 could play a role in assembling proteins at the 3?
untranslated region of an mRNP that are subsequently re-
quired for site-specific cytoplasmic localization of that mRNP.
PABPC1 function within nuclei would occur prior to its
earliest certified role, which is during the pioneer round of
translation. This round of translation involves CBP80/CBP20-
bound mRNA, supports the decay of nonsense-containing
mRNAs, precedes the translation of eIF4E-bound mRNA, and
most often occurs in association with nuclei, probably during
mRNA export to the cytoplasm (11, 24, 35, 37, 58). It is likely
that PABPC1 augments the translation of CBP80/CBP20-
bound mRNA in a manner similar to its augmentation of the
translation of eIF4E-bound mRNA. PABPC1 binding to
eIF4G increases the efficiency of translation initiation (27, 32),
and eIF4G is a functional component of the pioneer transla-
tion initiation complex (37). Furthermore, PABPC1 binding to
eukaryotic translation release factor 3 (21) may increase the
efficiency of translation termination.
Future studies aim to determine at what step in RNA me-
VOL. 26, 2006PABPC1 BINDS UNSPLICED PRE-mRNA 3095
tabolism the bulk of PABPC1 binding to poly(A) tails occurs
and specifically how PABPC1 functions prior to its role in the
pioneer round of translation.
We thank Rick Lloyd for anti-PABPC1, Luiz Penalva and Jack
Keene for pSHREKK-PABPC1, Dave Bear for anti-PABPN1, Guy
Rouleau for pEGFP-C2-PABPN1, Anders Virtanen for anti-PAP-?,
Yi-Tao Yu and Allan Jacobson for useful information, and Dave Bear
and Holly Kuzmiak for helpful conversations and comments on the
This work was supported by NIH R01 GM59614 to L.E.M. N.H. was
supported in part by a fellowship from the Japan Society for the
Promotion of Science.
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