The RNA Binding Domains of the Nuclear poly(A)-binding Protein*,

Uwe KühnDagger , Anne Nemeth§, Sylke MeyerDagger , and Elmar WahleDagger ||

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta alpha beta beta alpha beta fold, in which a four-stranded beta -sheet is backed by two alpha -helices. The two central antiparallel beta -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 beta -sheet by hydrogen bonds and stacking interactions between bases and amino acid side chains (5-8).

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 beta -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).

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 beta -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).

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-Delta 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.

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-Delta 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).

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 [gamma -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).

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-Delta 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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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).

A PABPN1 variant with an N-terminal deletion including Ile160 (Delta 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. Delta 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.


                              
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Table I
Apparent dissociation constants of PABPN1 variants
Apparent affinities were determined by nitrocellulose filter binding assays as described under "Experimental Producedures." Numbers for the protein variants Delta N160 and C terminus were put in parentheses, because Delta N160 was prone to aggregation, and C terminus was purified under denaturing conditions and diluted into binding assays. ND, not determined.

Deletion of the arginine-rich C terminus from Asp258 (Delta 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 Delta 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).

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 (Delta 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 (Delta 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.

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 <=  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.

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 Delta 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 (Delta 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 Delta N113 mutation, and their effects on the affinity for immobilized wild-type protein was assayed. Whereas the Delta C8 mutant showed barely reduced binding, Delta 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-Delta N113 and variants in which progressive C-terminal deletions were combined with the Delta 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-Delta N113 and Delta N113-Delta C27; Mix2: Delta N113-Delta C8, Delta N113-Delta C20, and Delta N113-Delta C33; Mix3: Delta N113-Delta C27 and Delta N113-Delta C49), were loaded onto a 13% SDS-polyacrylamide gel, which was analyzed by phosphorimaging.

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).


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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.

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.


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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.


                              
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Table II
Enrichment of selected RNA fragments from cytidine interference assay
RNase A-dependent RNA fragments from the cytidine interference assay shown in Fig. 5 were quantified using the software supplied with the PhosphorImager. The amounts of individual fragments in each lane were normalized to the total radioactivity in that lane. The normalized radioactivity in each fragment in the input lane was then set to 100% and compared with the corresponding number from the binding reaction with 5 nM PABPN1. The selection against C-containing oligonucleotides was less severe at higher PABPN1 concentrations, but the numbers were qualitatively similar. The numbering of fragments is according to Fig. 5.

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 Delta 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 Delta 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 Delta 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 Delta 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 Delta N160 variant (500 nM).

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


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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.

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.


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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.

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 (Delta 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-Delta 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    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.

    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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Varani, G., and Nagai, L. (1998) Annu. Rev. Biophys. Biomol. Struct. 27, 407-445[CrossRef][Medline] [Order article via Infotrieve]
2. Burd, C. G., and Dreyfuss, G. (1994) Science 265, 615-621[Medline] [Order article via Infotrieve]
3. Nagai, K., Oubridge, C., Ito, N., Avis, J., and Evans, P. (1995) Trends Biochem. Sci. 20, 235-240[CrossRef][Medline] [Order article via Infotrieve]
4. Nagai, K. (1996) Curr. Opin. Struct. Biol. 6, 53-61[CrossRef][Medline] [Order article via Infotrieve]
5. Oubridge, C., Ito, N., Evans, P. R., Teo, C.-H., and Nagai, K. (1994) Nature 372, 432-438[CrossRef][Medline] [Order article via Infotrieve]
6. Price, S. R., Evans, P. R., and Nagai, K. (1998) Nature 394, 645-650[CrossRef][Medline] [Order article via Infotrieve]
7. Handa, N., Nureki, O., Kurimoto, K., Im, I., Sakamoto, H., Shimura, Y., Muto, Y., and Yokoyama, S. (1999) Nature 398, 579-585[CrossRef][Medline] [Order article via Infotrieve]
8. Deo, R. C., Bonanno, J. B., Sonenberg, N., and Burley, S. K. (1999) Cell 98, 835-845[Medline] [Order article via Infotrieve]
9. Kiledjian, M., and Dreyfuss, G. (1992) EMBO J. 11, 2655-2664[Abstract]
10. Ghisolfi, L., Joseph, G., Amalric, F., and Erard, M. (1992) J. Biol. Chem. 267, 2955-2959[Abstract/Free Full Text]
11. Kambach, C., Walke, S., Young, R., Avis, J. M., de la Fortelle, E., Raker, V. A., Lührmann, R., Li, J., and Nagai, K. (1999) Cell 96, 375-387[Medline] [Order article via Infotrieve]
12. Ghisolfi, L., Kharrat, A., Joseph, G., Amalric, F., and Erard, M. (1992) Eur. J. Biochem. 209, 541-548[Abstract]
13. Bagni, C., and Lapeyre, B. (1998) J. Biol. Chem. 273, 10868-10873[Abstract/Free Full Text]
14. Adinolfi, S., Bagni, C., Musco, G., Gibson, T., Mazzarella, L., and Pastore, A. (1999) RNA 5, 1248-1258[Abstract/Free Full Text]
15. Vanhamme, L., Perez-Morga, D., Marchal, C., Speijer, D., Lambert, L., Geusken, M., Alexandre, S., Ismaili, N., Göringer, H. U., Benne, R., and Pays, E. (1998) J. Biol. Chem. 273, 21825-21833[Abstract/Free Full Text]
16. Abdul-Manan, N., O'Malley, S. M., and Williams, K. R. (1996) Biochemistry 35, 3545-3554[CrossRef][Medline] [Order article via Infotrieve]
17. Darnell, J. C., Jensen, K. B., Jin, P., Brown, V., Warren, S. T., and Darnell, R. B. (2001) Cell 107, 489-499[Medline] [Order article via Infotrieve]
18. Gary, J. D., and Clarke, S. (1998) Prog. Nucleic Acid Res. Mol. Biol. 61, 65-131[Medline] [Order article via Infotrieve]
19. Valentini, S. R., Weiss, V. H., and Silver, P. A. (1999) RNA 5, 272-280[Abstract/Free Full Text]
20. Raman, B., Guarnaccia, C., Nadassy, K., Zakhariev, S., Pintar, A., Zanuttin, F., Frigyes, D., Acatrinei, C., Vindigni, A., Pongor, G., and Pongor, S. (2001) Nucleic Acids Res. 29, 3377-3384[Abstract/Free Full Text]
21. Cobianchi, F., Karpel, R. L., Williams, K. R., Notario, V., and Wilson, S. H. (1988) J. Biol. Chem. 263, 1063-1071[Abstract/Free Full Text]
22. Casas-Finet, J. R., Smith, J. D., Kumar, A., Kim, J. G., Wilson, S. H., and Karpel, R. L. (1993) J. Mol. Biol. 229, 873-889[CrossRef][Medline] [Order article via Infotrieve]
23. Cartegni, L., Maconi, M., Morandi, E., Cobianchi, F., Riva, S., and Biamonti, G. (1996) J. Mol. Biol. 259, 337-348[CrossRef][Medline] [Order article via Infotrieve]
24. Bouvet, P., Diaz, J.-J., Kindbeiter, K., Madjar, J.-J., and Amalric, F. (1998) J. Biol. Chem. 273, 19025-19029[Abstract/Free Full Text]
25. Yun, C. Y., and Fu, X.-D. (2000) J. Cell Biol. 150, 707-717[Abstract/Free Full Text]
26. Sachs, A. B., Bond, M. W., and Kornberg, R. D. (1986) Cell 45, 827-835[Medline] [Order article via Infotrieve]
27. Adam, S. A., Nakagawa, T., Swanson, M. S., Woodruff, T. K., and Dreyfuss, G. (1986) Mol. Cell. Biol. 6, 2932-2943[Medline] [Order article via Infotrieve]
28. Sachs, A. (2000) in Translational Control of Gene Expression (Sonenberg, N. , Hershey, J. W. B. , and Mathews, M. B., eds) , pp. 447-465, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
29. Schwartz, D. C., and Parker, R. (2000) in Translational Control of Gene Expression (Sonenberg, N. , Hershey, J. W. B. , and Mathews, M. B., eds) , pp. 807-825, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
30. Burd, C. G., Matunis, E. L., and Dreyfuss, G. (1991) Mol. Cell. Biol. 11, 3419-3424[Medline] [Order article via Infotrieve]
31. Kühn, U., and Pieler, T. (1996) J. Mol. Biol. 256, 20-30[CrossRef][Medline] [Order article via Infotrieve]
32. Deardorff, J. A., and Sachs, A. B. (1997) J. Mol. Biol. 269, 67-81[CrossRef][Medline] [Order article via Infotrieve]
33. Wahle, E. (1991) Cell 66, 759-768[Medline] [Order article via Infotrieve]
34. Nemeth, A., Krause, S., Blank, D., Jenny, A., Jenö, P., Lustig, A., and Wahle, E. (1995) Nucleic Acids Res. 23, 4034-4041[Abstract]
35. Bienroth, S., Keller, W., and Wahle, E. (1993) EMBO J. 12, 585-594[Abstract]
36. Wahle, E. (1995) J. Biol. Chem. 270, 2800-2808[Abstract/Free Full Text]
37. Chen, Z., Li, Y., and Krug, R. M. (1999) EMBO J. 18, 2273-2283[Abstract/Free Full Text]
38. Calado, A., Kutay, U., Kühn, U., Wahle, E., and Carmo-Fonseca, M. (2000) RNA 6, 245-256[Abstract/Free Full Text]
39. Winstall, E., Sadowski, M., Kühn, U., Wahle, E., and Sachs, A. B. (2000) J. Biol. Chem. 275, 21817-21826[Abstract/Free Full Text]
40. Wahle, E., Lustig, A., Jenö, P., and Maurer, P. (1993) J. Biol. Chem. 268, 2937-2945[Abstract/Free Full Text]
41. Krause, S., Fakan, S., Weis, K., and Wahle, E. (1994) Exp. Cell Res. 214, 75-82[CrossRef][Medline] [Order article via Infotrieve]
42. Calado, A., and Carmo-Fonseca, M. (2000) J. Cell Sci. 113, 2309-2318[Abstract/Free Full Text]
43. Calado, A., Tomé, F. M. S., Brais, B., Rouleau, G. A., Kühn, U., Wahle, E., and Carmo-Fonseca, M. (2000) Hum. Mol. Genet. 9, 2321-2328[Abstract/Free Full Text]
44. Meyer, S., Urbanke, C., and Wahle, E. (2002) Biochemistry 41, 6082-6089[CrossRef][Medline] [Order article via Infotrieve]
45. Keller, R. W., Kühn, U., Aragon, M., Bornikova, L., Wahle, E., and Bear, D. G. (2000) J. Mol. Biol. 297, 569-583[CrossRef][Medline] [Order article via Infotrieve]
46. Brais, B., Bouchard, J. P., Xie, Y. G., Rochefort, D. L., Chretien, N., Tome, F. M., Lafreniere, R. G., Rommens, J. M., Uyama, E., Nohira, O., Blumen, S., Korcyn, A. D., Heutink, P., Mathieu, J., Duranceau, A., Codere, F., Fardeau, M., and Rouleau, G. A. (1998) Nat. Genet. 18, 164-167[Medline] [Order article via Infotrieve]
47. Smith, J. J., Rücknagel, K. P., Schierhorn, A., Tang, J., Nemeth, A., Linder, M., Herschman, H. R., and Wahle, E. (1999) J. Biol. Chem. 274, 13229-13234[Abstract/Free Full Text]
48. Martin, G., and Keller, W. (1996) EMBO J. 15, 2593-2603[Abstract]
49. Picard, V., Ersdal-Bardju, E., Lu, A., and Bock, S. C. (1994) Nucleic Acids Res. 22, 2587-2591[Abstract]
50. Puig, O., Caspary, F., Rigault, G., Rutz, B., Bouveret, E., Bragado-Nilsson, W., Wilm, M., and Seraphin, B. (2001) Methods 24, 218-229[CrossRef][Medline] [Order article via Infotrieve]
51. Brinkmann, U., Mattes, R. E., and Buckel, P. (1989) Gene 85, 109-114[CrossRef][Medline] [Order article via Infotrieve]
52. Benoit, B., Nemeth, A., Aulner, N., Kühn, U., Simonelig, M., Wahle, E., and Bourbon, H.-M. (1999) Nucleic Acids Res. 27, 3771-3778[Abstract/Free Full Text]
53. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
54. Stayton, M., and Kornberg, A. (1983) J. Biol. Chem. 258, 13205-13212[Abstract/Free Full Text]
55. Rüegsegger, U., Beyer, K., and Keller, W. (1996) J. Biol. Chem. 271, 6107-6113[Abstract/Free Full Text]
56. Schägger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-379[Medline] [Order article via Infotrieve]
57. Görlach, M., Burd, C. G., and Dreyfuss, G. (1994) J. Biol. Chem. 269, 23074-23078[Abstract/Free Full Text]
58. Avis, J. M., Allain, F. H.-T., Howe, P. W. A., Varani, G., Nagai, K., and Neuhaus, D. (1996) J. Mol. Biol. 257, 398-411[CrossRef][Medline] [Order article via Infotrieve]
59. Fan, X., Dion, P., Laganiere, J., Brais, B., and Rouleau, G. A. (2001) Hum. Mol. Genet. 10, 2341-2351[Abstract/Free Full Text]
60. Friesen, W. J., Massenet, S., Paushkin, S., Wyce, A., and Dreyfuss, G. (2001) Mol. Cell 7, 1111-1117[CrossRef][Medline] [Order article via Infotrieve]
61. Bedford, M. T., Frankel, A., Yaffe, M. B., Clarke, S., Leder, P., and Richard, S. (2000) J. Biol. Chem. 275, 16030-16036[Abstract/Free Full Text]
62. Xu, W., Chen, H., Du, K., Asahara, H., Tini, M., Emerson, B. M., Montminy, M., and Evans, R. M. (2001) Science 294, 2507-2511[Abstract/Free Full Text]
63. Mowen, K. A., Tang, J., Zhu, W., Schurter, B. T., Shuai, K., Herschman, H. R., and David, M. (2001) Cell 104, 731-741[Medline] [Order article via Infotrieve]
64. Shen, E. C., Henry, M. F., Weiss, V. H., Valentini, S. R., Silver, P. A., and Lee, M. S. (1998) Genes Dev. 12, 679-691[Abstract/Free Full Text]
65. Green, D. M., Marfatia, K. A., Crafton, E. B., Zhang, X., Cheng, X., and Corbett, A. H. (2002) J. Biol. Chem. 277, 7752-7760[Abstract/Free Full Text]
66. Côté, J., Boisvert, F.-M., Boulanger, M.-C., Bedford, M. T., and Richard, S. (2003) Mol. Biol. Cell 14, 274-287[Abstract/Free Full Text]
67. Nichols, R. C., Wang, X. W., Tang, J., Hamilton, J., High, F. A., Herschman, H. R., and Rigby, W. F. C. (2000) Exp. Cell Res. 256, 522-532[CrossRef][Medline] [Order article via Infotrieve]
68. Pintucci, G., Quarto, N., and Rifkin, D. B. (1996) Mol. Biol. Cell 7, 1249-1258[Abstract]
69. Ding, J., Hayashi, M. K., Zhang, Y., Manche, L., Krainer, A. R., and Xu, R.-M. (1999) Genes Dev. 13, 1102-1115[Abstract/Free Full Text]
70. Allain, F. H.-T., Bouvet, P., Dieckmann, T., and Feigon, J. (2000) EMBO J. 19, 6870-6881[Abstract/Free Full Text]


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