From the Institut für Biochemie,
Martin-Luther-Universität Halle, 06099 Halle and
§ Institut für Biochemie,
Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, 35392 Giessen, Germany
Received for publication, September 26, 2002, and in revised form, March 6, 2003
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ABSTRACT |
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The nuclear poly(A)-binding protein
(PABPN1) is involved in the synthesis of the mRNA poly(A) tails in
most eukaryotes. We report that the protein contains two RNA binding
domains, a ribonucleoprotein-type RNA binding domain (RNP domain)
located approximately in the middle of the protein sequence and an
arginine-rich C-terminal domain. The C-terminal domain also promotes
self-association of PABPN1 and moderately cooperative binding to RNA.
Whereas the isolated RNP domain binds specifically to poly(A), the
isolated C-terminal domain binds non-specifically to RNA and other
polyanions. Despite this nonspecific RNA binding by the C-terminal
domain, selection experiments show that adenosine residues throughout
the entire minimal binding site of ~11 nucleotides are recognized
specifically. UV-induced cross-links with oligo(A) carrying
photoactivatable nucleotides at different positions all map to the RNP
domain, suggesting that most or all of the base-specific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no
detectable influence on the interaction of the protein with RNA. The
N-terminal domain of PABPN1 is not required for RNA binding but is
essential for the stimulation of poly(A) polymerase.
In the cell, mRNA molecules and their precursors are
always bound by proteins. These proteins not only protect the
RNA from nucleases and undesirable interactions of its highly charged
surface but influence enzymes and other proteins that act upon the RNA at all stages of its maturation, function, and decay (1).
Characteristically, a single RNA-binding protein very often contains
more than one RNA binding domain. Different kinds of RNA binding
domains have been described (2). Among them, the RNA recognition motif
or RNP1-type RNA binding
domain is probably the best understood (1, 3, 4). The RNP domain
consists of ~90 amino acids forming a Another common RNA binding domain is the so-called RGG domain,
characterized by multiple copies of the amino acid sequence arginine-glycine-glycine, interspersed with phenylalanine and tyrosine
residues (9). The structure of the domain is not known, although a
spiral of The poly(A) tails at the 3'-ends of eukaryotic
mRNAs are bound by two different
proteins. Cytoplasmic poly(A)-binding protein2 (PABPC;
Pab1p in Saccharomyces cerevisiae) (26, 27) is found in all eukaryotes. Its main functions are in the initiation of translation (28) and in mRNA decay (29). PABPC contains four copies
of the RNP domain, the first and second being mainly responsible for
specific binding to poly(A) (30-32). In a co-crystal of these two
domains with oligoadenylate, A11, the Nuclear poly(A)-binding protein2 (PABPN1) (33, 34)
stimulates synthesis of the poly(A) tails of pre-mRNAs by
increasing the processivity of poly(A) polymerase (33, 35) and also
plays a role in poly(A) tail length control, i.e. in
limiting processive poly(A) tail synthesis to ~250 nucleotides (33,
35, 36). In addition, the protein may be involved in mRNA export
into the cytoplasm (37, 38). Although PABPN1 is conserved in most
organisms, it does not appear to exist in S. cerevisiae, as
its closest homolog in yeast is a cytoplasmic protein (Rbp29p) possibly
involved in translation (39). In vitro, PABPN1 binds with
high affinity and specificity to poly(A) and, almost equally well, to
poly(G) (33, 40). In vivo data are consistent with its
binding to poly(A) tails in nuclear RNA and support a role in mRNA
polyadenylation (37, 38, 41-43). Binding to poly(A) is moderately
cooperative (44). In binding to long poly(A), PABPN1 can form spherical particles of a defined size that accommodate ~250 nucleotides (45).
These particles appear to be in equilibrium with filamentous complexes.
The structure of PABPN1 is also of interest as short expansions of an
oligoalanine tract at the N terminus of the protein lead to the human
genetic disease oculopharyngeal muscular dystrophy, which is
characterized by the formation of insoluble PABPN1 aggregates in the
nuclei of muscle cells (43, 46).
Upon sequence inspection, an RNP-type RNA binding domain is evident
approximately in the middle of the PABPN1 amino acid sequence. Although
the C-terminal domain of the protein contains no RGG sequences, it is
arginine-rich, and all of its 13 arginines are asymmetrically
dimethylated (47). The contributions of different domains of the
protein to RNA binding have not been investigated so far. In this paper
we present evidence that both the RNP domain and the C-terminal
arginine-rich domain of PABPN1 contribute to RNA binding. The
N-terminal domain is essential for the stimulation of poly(A) polymerase.
DNA--
Sequences of DNA oligonucleotides used in the following
procedures are available upon request. All constructs were verified by
DNA cycle sequencing with a Prism 310 genetic analyzer (Applied Biosystems).
The bovine PABPN1 coding sequence (GenBankTM
accession number X89969) (34) inserted into the NdeI and
BamHI sites of pGM10 (48) was initially used for the
production of His6-tagged PABPN1 and variants in
Escherichia coli. Later, a modified pET19b expression vector
(Novagen) was used, in which the NcoI/NdeI
fragment encoding the tag was replaced by the corresponding fragment
from pGM10 encoding the peptide MAH6. The resulting vector,
which resulted in higher levels of recombinant proteins compared with
pGM10, will be referred to as pUK. Gel-purified PABPN1 cDNA
fragments were cloned into pUK according to standard procedures.
For silent mutagenesis of the PABPN1 coding sequence with the aim of
reducing the GC content, 18 overlapping and phosphorylated oligonucleotides spanning the first 360 bp were synthesized by TIB
Molbiol, Berlin, Germany. The oligonucleotides were designed such that
the desired ligation product contained overhanging
NdeI/XhoI ends. A mixture of all oligonucleotides
(20 nM each in 100 µl of ligation buffer without ATP)
were melted at 95 °C for 5 min. Annealing took place by slow cooling
of the reaction mix to room temperature in a water bath. After addition
of 1 mM ATP and 800 units of T4 DNA ligase, aliquots of
this mixture were incubated at six different temperatures between 12 and 40 °C for 15 min to 16 h, the incubation time depending on
the temperature. After DNA recovery by ethanol precipitation, one-half
of each ligation reaction was used for ligation into the
NdeI/XhoI-opened and dephosphorylated pGM-PABPN1 plasmid in 20-µl standard reactions. The presence of an
additional RsaI restriction site in the synthetic gene
fragment allowed for the initial identification of clones in which the synthetic sequence had replaced the beginning of the authentic open
reading frame. One such clone was then confirmed by DNA sequencing. After subcloning of the synthetic open reading frame into pUK, the
resulting plasmid pUK-synPABPN1 was used for the generation of
C-terminal deletion constructs, as well as for fusion protein constructs.
Deletions mutants of PABPN1 were generated by PCR using Pwo
DNA polymerase (Hybaid AGS, Heidelberg, Germany) and primers
introducing a new start codon as part of an NdeI site or a
stop codon followed by a new BamHI site, respectively.
Phosphorylated and purified PCR fragments were subcloned into
SmaI-cut pGEM3z (Promega). After double digestion with
NdeI or XhoI combined with BamHI, the
shortened fragments of the PABPN1 coding region were cloned into the
pUK vector or the pUK-synPABPN1 construct opened at the same
restriction sites. For GST pull down experiments, the
XhoI/BamHI fragments coding for C-terminal
truncations were subcloned into the pUK-PABPN1-
Single amino acid substitutions were made with the use of a PCR-based
method (49). Positive clones were identified with the help of newly
created restriction sites and verified by DNA sequencing.
For the generation of GST and protein A fusion proteins, the sequences
encoding the respective tags were PCR-amplified with Pwo DNA
polymerase and primer pairs containing additional 5'-NdeI sites. The plasmid pGEX5 × 1, bp 258-945 (Amersham
Biosciences) was used as a template for the GST gene, and the
plasmid pBS1761, bp 785-1225 (50) was used for the protein A tag. PCR
products were inserted into the NdeI site of
pUK-synPABPN1.
Proteins--
Protein concentrations were determined with
Bradford reagent (Bio-Rad) and/or by SDS-polyacrylamide gel
electrophoresis followed by Coomassie staining and gel imaging with
bovine serum albumin as a standard. Calf thymus PABPN1 was isolated as
described (40). The untagged wild-type PABPN1 used in the experiment of
Fig. 9 and related assays was produced in E. coli from the
authentic cDNA sequence cloned into pT7-7 (34) and purified
essentially as described (34, 40). For expression of His-tagged PABPN1 variants, pUK or pGM10 constructs were transformed into
electrocompetent BL21 (DE3) pUBS520. The plasmid pUBS520 facilitates
the translation of genes containing rare arginine codons by
co-expression of the corresponding tRNAArg (51). Growth
conditions, further treatment of cells, and protein purification were
according to Benoit et al. (52) with the following variations: the Ni-NTA column was washed with 50 mM sodium
phosphate, 10 mM Tris-HCl, pH 8.0, and an additional step
with 6 column volumes of buffer containing 75 mM imidazole.
Proteins were eluted with 5 ml of buffer containing 500 mM
imidazole and further purified on a 1-ml MonoQ fast protein liquid
chromatography column (Amersham Biosciences) to remove nucleic acids.
The variant PABPN1-
Bovine poly(A) polymerase was a kind gift of Georges Martin
(Biozentrum, Basel, Switzerland). DNA modifying enzymes were from New
England Biolabs.
RNA--
Homopolymers were from Sigma, and E. coli
ribosomal RNA was from Roche Diagnostics. Chemically synthesized RNA
oligonucleotides were from IBA (Göttingen, Germany).
Size-fractionated homopolymers were prepared by gel purification (45)
or by ion exchange chromatography (40). RNA concentrations were
determined as described (36, 40). 5'-Labeling of RNA was performed
using [ RNA Binding Assays--
Filter binding assays were carried out
essentially as described (40). For determination of the binding
constants, 112 fmol (as mononucleotides) of radioactively labeled RNA
was incubated with increasing amounts of PABPN1 variants in 40 µl of
RNA binding buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol,
0.2 mg/ml methylated bovine serum albumin, 0.01% Nonidet P-50, 1 mM EDTA, 1 mM dithiothreitol, 100 mM KCl). After 30 min of incubation at room temperature, 35 µl of each reaction were applied to nitrocellulose filters
(Schleicher & Schuell) pre-treated with 1 ml of wash buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl) containing 5 µg/ml rRNA. After rinsing with 5 ml of ice-cold wash buffer, the
filter-bound radioactivity was measured by scintillation counting.
Apparent KD or K50 values were
determined both from direct and double-reciprocal plots.
For the electrophoretic mobility shift assay, 5'-labeled gel-purified
RNA was incubated with increasing amounts of PABPN1 variants in 20 µl
of RNA binding buffer (see above). After incubation for 30 min at room
temperature, 15-µl aliquots of the reactions were loaded onto a
native agarose/polyacrylamide composite gel (55). Gels were dried and
analyzed with a PhosphorImager (Amersham Biosciences).
Cytidine Substitution Interference Assay--
C-spiked
A12 was made by IBA (Göttingen) using 95% A- and 5%
C-precursors for each step of synthesis. As the adenosine at the 3'-end
was covalently coupled to the support during synthesis, there was no C
substitution at this position. Gel-purified C-spiked A12
and homogeneous A12 used as a control were 5'-labeled, and about 2.5 nM labeled oligonucleotides (as 5'-ends) were
incubated at ambient temperature for 15 min in 100 µl of RNA binding
buffer containing different concentrations of calf thymus PABPN1. The reaction mixtures were applied to a pre-treated nitrocellulose filter
and washed once with 2 ml of ice-cold wash buffer (see above). Each
filter was then treated for 30 min at 37 °C with 20 µg of
proteinase K (Merck) in 300 µl of elution buffer containing 100 mM Tris-HCl, pH 7.5, 12.5 mM EDTA, 150 mM NaCl, 1% SDS, and 2 µg of rRNA. The eluted RNA was
precipitated with 3 volumes of ethanol and digested for 30 min at
37 °C with 1 ng of RNase A in 10 µl of 5 mM Tris-HCl,
pH 8.0, 1 mM EDTA. The recovered radioactivity was
determined by scintillation counting of 1-µl aliquots. Equal amounts
of radioactivity were analyzed on a 20% polyacrylamide gel
(40-cm-long) containing 8.3 M urea. Autoradiography and
quantification of RNA fragments was done with the help of a
PhosphorImager (Amersham Biosciences). Digestion of the C-spiked
A12 was complete, and unsubstituted A12 was
found to be resistant to RNase A under the conditions used.
UV-induced RNA/Protein Cross-links--
Desalted
5-iodo-UMP-modified oligonucleotides
(A2-5iU-A10 and
A10-5iU-A2) were purchased from IBA
(Göttingen) and used without additional purification for
5'-labeling with minimal exposure to light. Binding reactions were done
in 100 µl of RNA binding buffer (see above) that contained 2 nM (as 5'-ends) of either of the two modified oligonucleotides in the presence of either 50 nM His-tagged
wild-type PABPN1 or 500 nM of the deletion variants,
respectively. After 10 min of incubation at room temperature, each
binding reaction was evenly distributed to five wells of a 96-well
microtiter plate. The plate was placed on top of an ice-cooled aluminum
block at a distance of 4-4.5 cm to an inverted UV table (Fluolink;
Renner GmbH). After 30 min of irradiation at 312 nm, the aliquots from each binding reaction were recombined, and 20 µl of each irradiated RNA/protein mix were digested with 200 ng of protease Lys-C (sequencing grade; Roche Diagnostic) at ambient temperature. Aliquots were taken as
indicated and analyzed on a 10% Tricine-SDS-polyacrylamide gel (56).
The gel was dried, and radiolabeled proteolytic fragments were analyzed
by phosphorimaging.
Polyadenylation Assays--
Gel-purified, 5'-labeled
A80 was used in polyadenylation assays in the presence of
Mg2+ (specific polyadenylation reaction) as described (36).
25-µl reactions contained 80 fmol (as 5'-ends) of A80, 40 fmol of poly(A) polymerase, and the indicated amounts of PABPN1.
Protein Interaction Assays--
GST-PABPN1 fusion proteins were
expressed and purified by Ni-NTA chromatography as described above
without any additional purification step. pUK-PABPN1-
375 µl of glutathione-Sepharose suspension (30 µl/binding reaction;
Amersham Biosciences) was washed five times with 0.5 ml of NET4 buffer
(20 mM Tris-HCl, pH 8.0, 100 mM
NaCl2, 0.5 mM EDTA, 0.5% Nonidet P-40). The
beads were resuspended in 625 µl of NET4 buffer containing 25 µg of
bovine serum albumin and ~40 µg of GST-PABPN1 fusion protein. After
15 min of incubation at room temperature with thorough mixing, 5 mM CaCl2, 2250 units of micrococcal nuclease,
and 12.5 µg RNase A were added. After 30 min at ambient temperature
with mixing, the beads were washed three times with 1.2 ml of NET4
buffer and resuspended in 1.2 ml of NET4 buffer. 100-µl aliquots were
mixed with 3 µl of Xenopus PABPC1 translation mix (as a
negative control), which had been diluted to 50 µl with NET4 buffer,
and 3 µl of PABPN1 translation mix and incubated for 30 min at room
temperature with vigorous mixing. Free proteins were removed by five
washes with 0.5 ml of ice-cold NET4 buffer each. Bound proteins were
eluted by addition of 40 µl of SDS sample buffer and incubation for 5 min at 90 °C. One-half of the bound proteins, as well as 10% of the
protein input, were resolved on a 13% SDS-polyacrylamide gel. The gel
was dried, and radioactive proteins were identified by phosphorimaging.
Variants of bovine PABPN1 were expressed in E. coli. A
significantly improved expression was achieved by use of a partially synthetic cDNA in which the GC content of the first 300 nucleotides was reduced to 47%, compared with 80% in the wild-type sequence, without a change in the encoded amino acid sequence (see
"Experimental Procedures"). Proteins contained an N-terminal His
tag and were purified by metal affinity chromatography. One or, for
some variants, two additional chromatographic purification steps were
required to achieve electrophoretic homogeneity and to remove nucleic
acids as judged from the UV spectrum.
Both RNP Domain and C-terminal Domain Contribute to RNA
Binding--
Sequence analysis suggests that PABPN1 consists of three
distinct domains; an RNP-type RNA binding domain (approximately from Met161 to Thr257) separates a mostly acidic
N-terminal domain from a basic C-terminal domain. In a phylogenetic
comparison, the RNP domain is highly conserved whereas only the
arginine-rich character with a high frequency of RXR or RG
motifs is conserved in the C terminus. Within the N-terminal domain, a
potential amphipathic
A PABPN1 variant with an N-terminal deletion including
Ile160 (
Deletion of the arginine-rich C terminus from Asp258
(
As a further test of the role of the RNP domain in RNA binding in the
context of full-length PABPN1, three point mutations were generated.
One of these, K213Q, had no significant influence on the affinity of
the protein for oligo(A). In contrast, the Y175A and the F215A
mutations, singly or in combination, strongly reduced binding to
oligo(A) or poly(A). Amino acid side chains corresponding to
Tyr175 and Phe215 are solvent-exposed in those
RNP domains whose structures have been solved. This makes a structural
distortion by the mutations unlikely. The data thus confirm the
importance of the RNP domain for RNA binding (Table I).
In several proteins, short C-terminal extensions of the RNP domain
contribute significantly to RNA binding (57, 58). To determine whether
this is the case in PABPN1 or whether the entire C-terminal domain is
involved in poly(A) binding, we prepared and assayed a set of
progressive C-terminal deletions. The shortest of these deletions,
removing the last eight amino acids ( The C-terminal Domain Promotes Self-interaction and Cooperative RNA
Binding--
The data presented in the preceding section show that
C-terminal deletion mutants of PABPN1 have a smaller preference than the wild-type for poly(A) as opposed to oligo(A). Thus, they should exhibit lower cooperativity, or, in other words, the C-terminal domain
should be responsible for the cooperativity of RNA binding. Cooperativity of the wild-type protein is low (cooperativity
parameter
In the absence of poly(A) and at elevated concentrations, full-length
PABPN1 forms irregular multimers as shown by analytical ultracentrifugation (34),3
chemical cross-linking (34), electron microscopy (45), and UV
spectroscopy. In contrast to the wild-type protein, the RNA Binding Specificity--
The RNA binding specificity of the
PABPN1 variants was determined both by direct binding assays and by
competition assays. The full-length protein bound tightly to poly(A)
and almost as tightly to poly(G) but not to poly(U) or poly(C) (data
not shown) as reported earlier (33, 40). Neither a deletion of the N terminus nor of the C terminus had a major influence on the specificity of binding, and the isolated RNP domain also bound specifically to
poly(A), binding at least 100-fold more weakly to poly(U) and ignoring
poly(C) (Fig. 4 and data not shown). The
C-terminal domain, in contrast, bound with roughly similar affinity to
all four homopolymers, and binding to poly(A) was very sensitive to
competition by either rRNA or heparin (Fig. 4 and data not shown).
The minimum length of oligo(A) required for high affinity binding of
PABPN1 is 10 to 11 nucleotides (34, 44). poly(A)-specific binding of
the RNP domain and indiscriminate binding of the C-terminal domain to
different polynucleotides suggest that one block of nucleotides within
this sequence might be bound specifically by the RNP domain and a
second block non-specifically by the C-terminal domain. However, in an
initial test of this hypothesis, neither synthetic
A5C5 nor C5A5 was bound
by PABPN1 to any detectable extent. Thus, the following selection
experiment was designed to identify those bases within the
oligonucleotide A12 that are specifically recognized by
PABPN1; chemical synthesis of A12 was carried out such that
there was a small percentage of cytidine substitution at positions 1 through 11. This mixture of cytidine-spiked oligo(A) was incubated with
increasing amounts of PABPN1, and bound oligonucleotides were separated
from free RNA by filtration over nitrocellulose. Bound RNA was
recovered from the filters and digested with RNase A under conditions
permitting cleavage 3' of C but not of A. The digestion products were
analyzed on a denaturing polyacrylamide gel (Fig.
5). Surprisingly, all cleavage products
were underrepresented in the protein-bound RNA, whereas the RNase
A-resistant A12, lacking all C substitutions, was enriched (Table II). This result shows that PABPN1
discriminates against C substitutions at all positions. In other words,
most if not all adenosine residues in the minimal binding site seem to
be recognized in a base-specific manner. The resolution of the
experiment is limited by the fact that the oligonucleotide used is one
or two nucleotides longer than the minimal binding site of the protein. Thus, PABPN1 can probably bind these molecules in two or three registers, and, consequently, one cannot be certain that every single
nucleotide is indeed bound in a specific manner. However, the
experiment shows that there is no block of consecutive nucleotides bound in a nonspecific manner. The use of an oligonucleotide slightly longer than the minimal binding site probably also contributes to the
less stringent selection for A at either end of the molecule.
The selection result can be explained in two ways; either the
C-terminal domain contributes to base discrimination, or the RNP domain
interacts with most of the bases in A12 in a specific manner, with the C-terminal domain providing additional nonspecific contacts. To investigate contacts between the protein and the bases in
oligo(A), UV cross-linking experiments were carried out with two
synthetic 13-mers, each carrying a single photoactivable nucleotide,
5-iodo-UMP, within an oligo(A) context. One oligonucleotide had this
substitution at position 3 (rA2-5iU-A10) and the other one at
position 11 (rA10-5iU-A2). As these
oligonucleotides are only two to three nucleotides longer than the
minimal interaction site of PABPN1, the cross-link(s) induced upon UV
irradiation should be characteristic for the position of each activable
nucleotide and its interaction with a particular protein domain. The
oligonucleotides were bound by PABPN1 with an apparent
KD of 15-30 nM, slightly less tightly
than unsubstituted oligo(A) (data not shown). Cross-linking of the 32P-labeled oligonucleotides, detected by SDS gel
electrophoresis as the transfer of label to PABPN1, was linearly
dependent on the time of irradiation at 312 nm up to 30 min. Under
standard conditions, 5-8% of either oligonucleotide was cross-linked
to wild-type PABPN1. As the cross-linking efficiency was about 10-fold lower with unsubstituted A14, 90% of the cross-links must
have been to the substituted position. Cross-linking efficiency with the PABPN1 Binds RNA as a Monomer--
For the interpretation of the
preceding experiments, knowledge of the binding stoichiometry is
essential. Previous quantitative binding experiments have all led to
the conclusion that PABPN1 binds oligo(A) with a 1:1 stoichiometry and
thus as a monomer (34, 44).5
As an additional test, the following experiment was carried out. A
chimeric protein was prepared in which the IgG binding domain of
protein A was fused to the N terminus of PABPN1. This protein was
compared with wild-type PABPN1 in a gel shift experiment (Fig. 7). Both proteins bound labeled
A70 with similar affinity and formed well defined ladders
of retarded bands with increasing occupancy of the RNA. The lowest band
in the ladder of the fusion protein migrated much more slowly than the
lowest band in the ladder of the wild-type protein. When the two
proteins were mixed, no additional band of intermediate mobility
appeared between these two bands, strongly suggesting that the
respective lowest bands contain a single protein bound to the RNA. As a
positive control, a retarded band not observed with either protein
alone and thus representing a mixed occupancy was seen at a higher
position in lanes 10 through 13 (labeled with an
arrow in Fig. 7). The experiment supports previous
conclusions that the RNA binding unit of PABN1 is a monomer. Binding as
a preformed stable dimer, which would also be consistent with the data,
can be excluded, because PABPN1 is a monomer in analytical
ultracentrifugation at up to 2 µM.3
Arginine Methylation Has No Influence on RNA Binding--
In
PABPN1 purified from calf thymus, the last thirteen arginine residues
are all asymmetrically dimethylated. Analysis by mass spectrometry and
sequencing excluded other modifications of the protein except a likely
acetylation of the N terminus (47). PABPN1 made in E. coli
is not methylated. Thus, a direct comparison of RNA binding by these
two preparations of the protein should reveal a possible influence of
arginine methylation on RNA binding. Protein expressed in E. coli without a His tag and purified by conventional chromatography
was used in these assays to exclude any influence of the tag. The two
proteins were compared for binding to oligo(A) and poly(A) by
nitrocellulose filter binding experiments. Their specificities were
examined by competition experiments, and binding to poly(A) was also
checked by protein titrations in gel shift assays (Fig.
8 and data not shown). These assays should have been able to detect differences in affinity, cooperativity, or specificity, but none of them revealed any significant difference between the methylated and the unmethylated form of the protein. MgCl2 reduces the affinity of PABPN1 for poly(A) about
20-fold (45) and might be expected to affect arginine-phosphate
contacts. However, binding assays in the presence of 2 mM
MgCl2 did not show any differences between the two proteins
either. We conclude that asymmetric dimethylation of arginines does not
influence the RNA binding properties of PABPN1.
The N-terminal Domain of PABP2 Is Required for the Stimulation of
poly(A) Polymerase--
The stimulation of poly(A) polymerase by
PABPN1 was assayed by the extension of a radiolabeled poly(A) primer.
Whereas the wild-type protein stimulated the elongation ~50-fold, the
N-terminally truncated protein ( The primary structure of the nuclear poly(A)-binding protein
suggests a separation into three domains, which is supported by the
experiments reported here. The acidic N-terminal domain is clearly
dispensable for RNA binding but essential for the stimulation of
poly(A) polymerase. The RNP domain is essential but not sufficient for
poly(A) binding. Full affinity is seen only in the presence of the
arginine-rich C- terminal domain.
The designation of the C terminus, starting at Asp258, as a
separate domain is based on (i) the high degree of sequence
conservation up to but not beyond this residue (see Supplemental
Material) and (ii) the dimethylation of all arginine residues
C-terminal of Asp258 in contrast to the almost complete
absence of this modification N-terminal of position 258 (47). A domain
junction at this point is further supported by a preferred trypsin
cleavage site in the immediate vicinity in limited proteolysis
experiments.4 Although both the RNP domain and the
C-terminal domain interact with RNA, separate functions can be
distinguished; the RNP domain binds specifically to poly(A), as shown
both by the ability of the isolated domain to discriminate against
other polynucleotides and by its ability, in the context of the entire
protein, to form specific cross-links to oligo(A) substituted with
photoactivable bases. In contrast, the C-terminal domain appears to
bind non-specifically to RNA; when tested as an isolated peptide, it
cannot distinguish between different polynucleotides, and no
cross-links of this domain to photoactivatable bases were detectable in
the context of wild-type PABPN1.
In addition to its interaction with RNA, the C-terminal domain is
responsible for the self-association of PABPN1, as also reported
recently by others (59). Probably as a consequence of self-association,
the C-terminal domain mediates the moderate cooperativity of RNA
binding. This was shown by a reduced preference for longer poly(A) with
C-terminally truncated proteins and by an increased cooperativity when
the relative contribution of the C-terminal domain to the RNA
interaction was increased by point mutations in the RNP domain. The
distal part of the C-terminal domain is essential for the homotypic
interaction of PABPN1, whereas the proximal part of this domain is more
important for RNA binding. It is unlikely that the effects of the
C-terminal domain on RNA binding are merely indirect and a reflection
of protein-protein interactions. First, the cooperativity of RNA
binding is low, whereas the effect of a C-terminal deletion on RNA
binding is severe. Thus, loss of cooperativity cannot account for the
reduction in RNA affinity. Second, a C-terminal deletion also strongly
reduces the affinity for oligo(A), which cannot be bound in a
cooperative manner. Contacts between the abundant arginine residues of
the C-terminal domain and the phosphate backbone would be the simplest explanation for the contribution of this domain to RNA binding. In
agreement with such a possibility, binding of wild-type PABPN1 to RNA
is sensitive to increasing salt concentration, whereas binding of the
isolated RNP domain is completely resistant up to 500 mM
NaCl.6
Even though the C-terminal domain of PABPN1 contains no RGG sequences,
all of its other properties are very similar to those of RGG domains,
as summarized in the Introduction and as follows: an apparently
sequence-independent interaction with RNA, a role in self-association
of the protein and cooperative RNA binding, interactions with other
proteins,6 a high content of arginine, tyrosine, and
phenylalanine, and, most specifically, the asymmetric dimethylation of
arginine residues (47). In agreement with other studies (19, 20), we
found no influence of arginine methylation on RNA binding. Possibly, the modification affects interactions with other proteins, as has been
reported both for symmetric and asymmetric arginine dimethylation (60-63). The C-terminal domain has been suggested to be involved in
nucleocytoplasmic transport of the protein (38), and asymmetric arginine dimethylation is known to affect nucleocytoplasmic protein distribution (25, 64-68).
The selection experiment (Fig. 5) suggests that PABPN1 recognizes
adenine bases in a specific fashion throughout its binding site. Both
the properties of the isolated PABPN1 domains and the cross-linking
data suggest that base recognition is mediated exclusively by the RNP
domain. Quantitative evaluation of the change in PABPN1 affinity with
the lengths of a series of ribooligoadenylates led to an estimate of
the minimal binding site size of PABPN1 of 10 to 11 nucleotides (34,
44). Because PABPN1 binds RNA as a monomer and contains a single RNP
domain, this seems to indicate that the RNP domain may interact with as
many as 10 nucleotides. However, this would be very unusual in light of
the known structures of RNP domain-RNA complexes; in a complex between
the first two RNP domains of the cytoplasmic poly(A)-binding protein
and A11, each RNP domain interacts with no more than four
nucleotides (8). Interaction with a small number of nucleotides (up to
six) is frequent for RNP domains, in particular those binding
unstructured single-stranded nucleic acids (7, 69), but also in at
least one case of a structured RNA target (70). A larger number of nucleotides is bound only in structured RNAs; in stem-loop II of U1
snRNA, seven nucleotides in the single-stranded loop plus the first
base pair of the stem form direct contacts with the RNP domain of U1A
protein (5), and 12 nucleotides are contacted by the RNP domain of the
U2B" protein, again in a stem-loop structure (6).
Eleven amino acid side chains in the first RNP domain of PAPBC
form specific hydrogen bonds to the adenine bases (8). Only four of
those amino acids are conserved in PABPN1. In the second RNP domain,
nine amino acid side chains are involved in specific hydrogen bonds to adenine bases. Only one of those amino acid residues
is conserved in PABPN1. Although the question of the number of bases
contacted by the RNP domain of PABPN1 will be resolved only by the
structure determination of a PABPN1-oligo(A) complex, this comparison
suggests that the mode of oligo(A) binding may indeed be different
between the two types of poly(A)-binding proteins.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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fold, in
which a four-stranded
-sheet is backed by two
-helices. The two
central antiparallel
-strands carry the highly conserved amino acids
of the RNP1 and RNP2 motifs. Different members of the RNP protein
family can bind structured or extended RNA molecules in a
sequence-specific manner. As seen in several co-crystals, the RNA is
bound on the surface of the
-sheet by hydrogen bonds and stacking
interactions between bases and amino acid side chains (5-8).
-turns has been proposed based on spectroscopic data (10).
A possibly related arginine-rich domain found at the C termini of
several of the spliceosomal Sm core proteins was not ordered in
a crystal structure in the absence of RNA (11). The RGG domain usually
occurs in proteins in conjunction with one or more other RNA binding
domains, e.g. of the RNP or K homology type (2) and
is often considered unable to discriminate between different RNA
sequences. Instead, it is thought to increase the RNA binding affinity
of a protein in a nonspecific manner, the specificity being determined
by the other RNA binding domain(s) (12-14). However, sequence- or
structure-specific binding by means of an RGG domain has been proposed
for several proteins (9, 15-17). A characteristic feature of the RGG
domain is the asymmetric dimethylation of the arginine side chains
within RGG sequences (18). A possible modulation of RNA binding by
arginine methylation has frequently been discussed, but binding of the
yeast protein Hrp1p to a specific RNA sequence was not affected by
arginine methylation (19). The affinity of a synthetic RGG domain
peptide for nonspecific RNA was also independent of arginine
methylation, although CD spectroscopy suggested that the methylated
peptide had a different structural effect on the RNA compared with the unmethylated peptide (20). Several RGG domains are involved in
protein-protein interactions; RGG domain-dependent
self-association of the hnRNP A1 protein leads to moderate
cooperativity of RNA binding (21, 22), but the same domain can also
interact with other proteins (23). Similarly, RGG domains of other
proteins serve in protein-protein interactions (24, 25).
-sheet surfaces of the two RNP domains form an almost continuous platform that binds an
extended conformation of the oligonucleotide. The 3'-half of the
oligonucleotide is associated with the N-terminal RNP domain (8).
EXPERIMENTAL PROCEDURES
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N113 construct opened
at the same sites. The clone expressing the RNP domain of PABPN1
encodes the amino acids 161-257, and the C terminus consists of amino
acids 258-306.
N160 was additionally loaded onto a Superdex-200
fast protein liquid chromatography gel filtration column to separate
monomeric protein from aggregates. The His-tagged C terminus of PABPN1
was insoluble under the conditions described above. Therefore, Ni-NTA
purification was performed in the presence of 8 M urea
following the protocol supplied by Qiagen. Elution was done with 2 ml
of buffer containing 250 mM imidazole, 8 M
urea, 100 mM sodium phosphate, 10 mM Tris-HCl, adjusted to pH 8.0. The protein concentration of the His-tagged C
terminus was measured photometrically (1 A280 = 530 µg/ml).
-32P]ATP (Amersham Biosciences) and T4
polynucleotide kinase according to standard procedures (53).
Incorporated radioactivity and RNA yields were measured using DE81
filter binding (54).
N113 constructs
with C-terminal truncations (see above), as well as a pRSET-construct
coding for a Xenopus laevis protein PABPC (31),
were used for in vitro synthesis of radioactively labeled
poly(A)-binding proteins. In vitro translations were done in
the presence of [35S]methionine (PerkinElmer Life
Sciences) with the T7 TNT-coupled reticulocyte lysate system
(Promega) according to the manufacturer's instructions. At the end of
the reaction, RNA was removed by addition of 10 mM
CaCl2, 300 units of micrococcal nuclease (MBI Fermentas), and 1 µg RNase A and incubation for 30 min at room temperature. After
addition of 20 mM EGTA, the translation mixes were frozen in liquid nitrogen and stored at
80 °C for further use.
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-helix (residues 119 to 146 in the bovine
sequence) is present in all known orthologues. The remaining part of
the N-terminal domain is variable (see Supplemental Material).
N160) tended to aggregate; only a
fraction of the protein present in the cell lysate was soluble, and gel
filtration as the final purification step showed a large proportion of
the protein eluting with the excluded volume whereas the rest was
distributed throughout the column.
N160 taken from the included
fractions of the gel filtration column was assayed for binding to
oligo(A) or poly(A) by nitrocellulose filter binding assays. Binding
was relatively more efficient at lower protein concentration, again
suggesting aggregation. However, the apparent K50,
extrapolated from the assays carried out at low protein concentrations,
was not much higher than that of the wild-type protein (Table
I). Thus, the N-terminal domain does not
appear to make a significant contribution to RNA binding. This
conclusion is supported by data discussed below.
Apparent dissociation constants of PABPN1 variants
N160 and C terminus were put in parentheses,
because
N160 was prone to aggregation, and C terminus was purified
under denaturing conditions and diluted into binding assays. ND, not
determined.
C49) reduced the apparent affinity for A14 ~200-fold.
The apparent affinity for A70 was reduced even more
strongly, 900-fold (Table I). This suggests that both the RNP domain
and the arginine-rich domain contribute to RNA binding. To confirm this
result, both of these domains were expressed separately as His-tagged
proteins. The RNP domain (Met161 to Thr257) was
obtained in large quantities in soluble form. Its affinity for oligo(A)
or poly(A) was similar to that of the
C49 variant. This confirms
that the RNP domain indeed binds RNA and that the N terminus has little
or no effect on RNA binding. The C-terminal domain (from
Asp258) was purified under denaturing conditions. Upon
dilution into binding assays, this protein bound oligo(A) at least as
tightly as the RNP domain (Table I).
C8), had little effect on the
binding of oligo(A) or poly(A). Further deletions progressively reduced
the affinity for poly(A) (Fig. 1).
However, the affinity for A14 was reduced merely 10-fold by a deletion of the last 33 amino acids, in contrast to a 100-fold effect
on poly(A) binding. Only further deletions had a stronger effect on
oligo(A) binding (Fig. 1). Moreover, as pointed out above, a complete
deletion of the C terminus (
C49) reduced binding to poly(A) more
strongly than binding to oligo(A). These data suggest that binding to
an isolated site (A14) differs from binding to several
contiguous sites as in poly(A); the RNP domain and the proximal part of
the arginine-rich domain play a dominant role in binding to oligo(A)
whereas the distal part of the arginine-rich domain is important for
binding to poly(A).
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Fig. 1.
Progressive C-terminal deletions of PABPN1
affect RNA binding. His-tagged PABPN1 and C-terminal deletion
mutants were purified from E. coli and used in filter
binding assays with radioactively labeled gel-purified A70
or fast protein liquid chromatography-purified A14 as
described under "Experimental Procedures." The apparent
KD for A14 (squares) and the
K50 for A70 (circles) were plotted against the
extent of the C-terminal deletion.
50) (44), so that a further reduction of
cooperativity by C-terminal deletions cannot be measured easily.
However, the Y175A/F215A double mutant in the RNP domain displayed a
clearly sigmoidal binding curve, i.e. increased
cooperativity (Fig. 2). As a control, binding of this mutant to A14 showed a normal hyperbolic
dependence on protein concentration (data not shown). Presumably, a
weakened RNA interaction of the RNP domain leads to a higher relative
contribution of the C-terminal domain to the total binding energy.
Enhanced cooperativity of such RNP domain mutants is consistent with a role of the C-terminal domain in cooperative binding and also suggests
that binding energies of the two domains are not additive.
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Fig. 2.
Substitution of Tyr175 and
Phe215 in PABPN1 leads to an increased cooperativity in
poly(A) binding. Filter binding experiments were carried out with
His-tagged PABPN1 (circles) and the double mutant
Y175A/F215A (triangles) and radioactively labeled
A70 as described under "Experimental Procedures." The
inset shows the data points for low protein concentrations
on a different scale.
C49 variant
shows no evidence of scattering in its UV
spectrum,4 suggesting that
the C-terminal domain mediates self-association of the protein. This
was directly demonstrated by interaction assays in which a fusion of
PABPN1 with GST was immobilized on glutathione beads and used to
bind radiolabeled variants of PABPN1 prepared by in vitro
translation. Whereas the wild-type protein bound but weakly, an
N-terminal deletion mutant (
N113) bound strongly to the affinity
resin (Fig. 3). Binding to the
immobilized GST-PABPN1 fusion protein was ~6-fold stronger than to
GST alone (data not shown). The association was resistant to treatment
with micrococcal nuclease and thus independent of RNA. PABPC, used as a
specificity control, did not bind to immobilized PABPN1. C-terminal
deletions were combined with the
N113 mutation, and their effects on
the affinity for immobilized wild-type protein was assayed. Whereas the
C8 mutant showed barely reduced binding,
C20 and all larger
C-terminal deletions prevented binding (Fig. 3). Thus, in agreement
with a requirement for the C-terminal domain for cooperative RNA
binding, this domain also promotes the self-association of PABPN1.
Addition of 0.5 µM A10 to the binding
reactions after inactivation of micrococcal nuclease did not prevent
the self-association of PABPN1 (data not shown). Because
A10 is too short to bind two molecules of PABPN1,
self-association must be direct even under these conditions. The result
shows that the self-association is compatible with RNA binding of
PABPN1, i.e. it is not a consequence of the protein being
detached from RNA.
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Fig. 3.
The C terminus of PABPN1 is necessary for
self-interaction. GST-PABPN1 was immobilized on
glutathione-Sepharose and incubated with 35S-labeled
PABPN1- N113 and variants in which progressive C-terminal deletions
were combined with the
N113 mutation. Labeled cytoplasmic PABPC from
X. laevis was included as a negative control.
After washing, one-half of the bound proteins from each reaction, as
well as 10% of the protein input (Mix1: PABPN1-
N113 and
N113-
C27; Mix2:
N113-
C8,
N113-
C20, and
N113-
C33; Mix3:
N113-
C27 and
N113-
C49),
were loaded onto a 13% SDS-polyacrylamide gel, which was analyzed by
phosphorimaging.
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Fig. 4.
Binding specificity of RNP domain and C
terminus. Filter binding assays were performed with radioactively
labeled RNA homopolymers (circles, poly(A);
triangles, poly(U); squares, poly(C)) and
increasing amounts of purified His-tagged proteins as indicated.
Non-fractionated RNA was used for binding reactions with the RNP domain
under standard conditions (see "Experimental Procedures"). The C
terminus, purified in the presence of 8 M urea, was
directly diluted into standard binding reactions containing 850 fmol of
homopolymers (as mononucleotides) with an average length of 34 nucleotides.
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Fig. 5.
Cytidine interference assay.
5'-32P-Labeled, cytidine-doped A12 was
incubated with 5, 10, and 25 nM PABPN1 purified from calf
thymus, and protein-bound RNA was selected by nitrocellulose filter
binding and recovered from the filters. Recovery was ~13, 20, and
30% of the input RNA at the three protein concentrations. One-half of
each sample was treated with RNase A. An aliquot of the unselected
starting pool was also digested. An internally labeled transcript
(L3pre) was used as a control for the RNase A digestion.
RNase A-digested and non-digested RNA was loaded on a 20% PAGE
containing 8.3 M urea and analyzed by phosphorimaging.
Approximately equal amounts of radioactivity were loaded to facilitate
comparison. RNA fragments are numbered from the 5'-end. The faint
unnumbered bands below A12 are most likely
A11 and A10 resulting from chain termination
during synthesis. These oligonucleotides are expected to migrate much
more slowly than oligonucleotides of the same length resulting from
RNase A digestion because of the absence of a 3'-phosphate and complex
formation of the borate buffer with the 3'-terminal cis-diol.
Enrichment of selected RNA fragments from cytidine interference assay
C33 mutant (used at 500 nM) was similar to that of
the wild-type protein (used at 50 nM). Cross-linking was
lower with the
C49 mutant, probably because of the fact that this
protein could only be used at 500 nM even though it binds
oligo(A) with a KD of ~1500 nM.
Cross-linked proteins were digested with the lysine-specific protease
Lys-C, and labeled peptides were analyzed by SDS-polyacrylamide gel
electrophoresis. The patterns of cross-linked peptides were similar
with both oligonucleotides, and one major radiolabeled peptide was
observed for each of the three proteins (Fig.
6A). As the size of this
peptide varied with the extent of the C-terminal deletion, the peptide
derived from the wild-type protein must have contained the C-terminal
domain (which is devoid of lysine residues), but, as a comparable
cross-link was obtained with the
C49 mutant, cross-linking must have
been outside the C-terminal domain. Digestion with AspN also produced
very similar patterns of radioactive peptides with the wild-type and
both C-terminal deletions (data not shown). The
N160 mutant could
also be cross-linked with either oligonucleotide. The labeled
polypeptide was resistant to Lys-C digestion and barely smaller than
the digestion product obtained with the wild-type protein (Fig.
6B). Together with the results of the C-terminal deletion
mutants, this demonstrates unequivocally that the cross-links are in
the RNP domain. Under all conditions, the two different
oligonucleotides produced similar patterns of cross-linked peptides.
Thus, nucleotides at both position 3 and position 10 were cross-linked
to the RNP domain.
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Fig. 6.
RNA/Protein cross-linking reveals direct
contacts in the RNP domain of PABPN1. A, binding
reactions containing either radioactively labeled
A2-5iU-A10 or
A10-5iU-A2 and recombinant PABPN1
(50 nM) or C-terminally truncated PABPN1 versions (500 nM) were irradiated with 312 nm UV light. Cross-linked
PABP2/RNA complexes were digested for 0, 1, or 16 h with the
protease Lys-C. Radioactive proteolytic fragments were resolved on a
10% Tricine-SDS-polyacrylamide gel, which was dried and analyzed by
phosphorimaging. The fat band at the bottom of
the gel is free RNA. B, the same experiment was carried out
with the wild-type protein (50 nM) and the N160 variant
(500 nM).
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Fig. 7.
PABPN1 binds poly(A) as a monomer.
Standard binding reactions for electrophoretic mobility shift assays
were performed with radioactively labeled A70 and
increasing amounts of purified calf thymus PABPN1, recombinant protein
A-PABPN1 fusion protein, or mixtures of both as indicated. Complexes
were separated on a native polyacrylamide gel, which was dried and
analyzed by phosphorimaging. The arrow points to RNA/protein
complexes observed exclusively in reactions containing both PABPN1
variants.
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Fig. 8.
Asymmetric dimethylation of arginines does
not influence RNA binding. An electrophoretic mobility shift assay
was carried out with PABPN1 from calf thymus (CT) and
recombinant non-tagged PABPN1 (E. coli). Calf thymus PABPN1
was the same preparation used in the analysis of post-translational
modification (47). Radioactively labeled, gel-purified A100
was used for binding reactions containing the given concentrations of
PABPN1 in binding buffer. RNA/protein complexes were subjected to
non-denaturing polyacrylamide gel electrophoresis. The dried gel was
autoradiographed.
N160) was entirely inactive (Fig.
9). Gel shift experiments confirmed that
the protein bound the poly(A) primer under the conditions used for the
extension assay (data not shown). Thus, the N-terminal domain is
essential for the stimulation of polyadenylation.
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Fig. 9.
The N terminus of PABPN1 is necessary for PAP
stimulation. Specific polyadenylation reactions containing
radioactively labeled substrate A80, poly(A) polymerase,
and increasing amounts of either recombinant wild-type PABPN1 or the
N-terminal deletion variant PABN1- N160 were incubated for 15 min at
37 °C as indicated. Another reaction with a 10-fold higher PAP
concentration was done as a control. poly(A) was recovered and analyzed
on a 10% polyacrylamide gel with 8.3 M urea. The sizes (in
nucleotides) of labeled DNA fragments serving as size markers are
indicated on the right.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Gudrun Scholz for skillful technical assistance, to Till Scheuermann and Elisabeth Schwarz for sharing unpublished data and reading the manuscript, to Olfert Landt for help with oligonucleotide design for the synthetic PABPN1 gene, to Georges Martin for poly(A) polymerase, and to Christopher Böhm, Kathrin Brunk, Henning Friedrich, and Anne Knoth for help with some experiments.
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FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to E. W. and U. K.) and from the Fonds der Chemischen Industrie (to E. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a supplemental figure.
¶ Present address: GeneScan Analytics GmbH, Engesserstr. 4, 79108 Freiburg, Germany.
To whom correspondence should be addressed. Tel.:
49-345-5524920; Fax: 49-345-5527014; E-mail:
ewahle@biochemtech.uni-halle.de.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M209886200
2 In the designation of poly(A)-binding proteins, we follow the recommendations of the HUGO Gene Nomenclature Committee (www.gene.ucl.ac.uk/nomenclature/). The cytoplasmic poly(A)-binding protein (PABPC) exists in several variants (PABPC1 through PABPC4 in humans) and is usually called PAB in the literature. The nuclear poly(A)-binding protein (now called PABPN1) was initially described as PAB II (33,34) and later renamed PABP2 (46).
3 H. Lilie and S. Meyer, unpublished data.
4 T. Scheuermann and E. Schwarz, personal communication.
5 Note that initial calculations concerning the interaction between PABPN1 and oligo(A) (40) were based on the misleadingly high apparent molecular weight of the protein in SDS gels and were later corrected (34).
6 Y. Kerwitz, U. Kühn, H. Lilie, A. Knoth, T. Scheuermann, H. Friedrich, E. Schwarz, and E. Wahle, submitted.
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ABBREVIATIONS |
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The abbreviations used are: RNP, ribonucleoprotein; GST, glutathione S-transferase; PABPN1, poly(A)-binding protein, nuclear 1; PABPC, poly(A)-binding protein, cytoplasmic; 5iU, 5-iodouridine; Ni-NTA, nickel-nitrilotriacetic acid; Tricine, N-[2-hydroxy-1,1-bis(hydro xymethyl)ethyl]glycine.
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