Vol. 268, No. 4, Issue of February 5, pp. 2937-2945, 1993 Printed in U. S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Mammalian Poly(A)-binding Protein I1 PHYSICAL PROPERTIES AND BINDING TO POLYNUCLEOTIDES*

(Received for publication, September 1, 1992)

Elmar WahleSg, Ariel Lustigl, Paul Jeno((, and Patrik Maurerl From the Departments of $Cell Biology, llBioph.ysica1 Chemistry, and )I Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 BaseiSwitzerland

The 49-kDa poly(A)-binding protein I1 (PAB 11) was purified to homogeneity from calf thymus. The 70-kDa poly(A)-binding protein I (PAB I) was obtained in dif- ferent fractions of the same preparation. Whereas PAB I1 stimulated poly(A) polymerase, PAB I was an inhib- itor. In analytical ultracentrifugation, the predomi- nant form of PAB I1 was a monomer of 50.3 kDa. A sedimentation constant of only 2.2 S indicated a dis- tinctly non-spherical shape. Binding was specific for single-stranded purine polyribonucleotides. The de- pendence of the dissociation constant on the length of oligoriboadenylate indicated a binding site size of 12 nucleotides. A single site was bound with a KO of 2 x lo”’ M, as determined by nitrocellulose filter binding assays. From fluorescence quenching and gel retarda- tion experiments, the packing ratio on poly(A) was estimated as 23 nucleotides/protein monomer.

Messenger RNAs in the cytoplasm and their nuclear pre- cursors are bound by two distinct sets of proteins (Dreyfuss, 1986; Dreyfuss et al., 1988). The most thoroughly character- ized of these RNA-binding proteins is a protein of approxi- mately 64-70 kDa that covers the poly(A) tails of mRNA (Blobel, 1973; Baer and Kornberg, 1983). In agreement with the genetic nomenclature in yeast (Sachs et al., 1986), we will call this protein PAB I.’ PAB I, very highly conserved from yeast to man (Sachs et al., 1986; Adam et aL, 1986; Grange et al., 1987; Zelus et al. 1989; Lefrere et al., 1990), contains at its N terminus four tandem repeats of the so-called RNP domain, a sequence of approximately 90 amino acids that is conserved between many RNA-binding proteins (reviewed by Kenan et al., 1991). This domain has been shown to mediate RNA binding in the poly(A)-binding protein (Sachs et al., 1987) as well as in other proteins (Kenan et al., 1991; Gorlach et al., 1992). In Saccharomyces cereuisiae, the poly(A)-binding pro- tein is encoded by a single essential gene (Sachs et al., 1986; Adam et al., 1986). The phenotype of a yeast strain that carries a temperature-sensitive pabl mutation as well as fur- ther genetic analysis provide evidence that the poly(A)-bind- ing protein plays an essential role in the initiation of trans-

* This work was supported by grants from the Kantons of Basel and the Schweizerischer Nationalfonds to Dr. Walter Keller. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2672071; Fax: 41-61-2672078. §To whom correspondence should be addressed. Tel.: 41-61-

The abbreviations used are: PAB I and PAB 11, poly(A)-binding protein I and 11; BSA, bovine serum albumin; CPSF, cleavage and polyadenylation specificity factor; FPLC, fast protein liquid chro- matography.

lation (Sachs and Davis, 1989, 1990; Sachs and Deardorff, 1992).

A second mammalian poly(A)-binding protein (PAB 11) has been purified that is distinct from PAB I as judged from its molecular mass of 49 kDa and partial amino acid sequences (Wahle, 1991b). In uitro, this protein functions in the poly- adenylation of mRNA precursors. Polyadenylation of mRNA (reviewed by Wahle, 1992; Wahle and Keller, 1992) is pre- ceded by a specific endonucleolytic cleavage of the precursor RNA. The addition of the poly(A) tail to the upstream cleav- age product is then initiated by the combined action of two factors: poly(A) polymerase (Wahle, 1991a) and the so-called cleavage and polyadenylation specificity factor (CPSF, pre- viously termed CPF (Christofori and Keller, 1988; Bienroth et al., 1991), SF (Takagaki et al., 1989) or PF2 (Gilmartin and Nevins, 1989)). CPSF binds the polyadenylation signal se- quence AAUAAA (Keller et al., 1991) and stimulates poly(A) polymerase to elongate specifically RNAs carrying this se- quence. After poly(A) polymerase, with the help of CPSF, has added 10-11 adenylate residues to the 3’-end of the mRNA precursor, PAB I1 binds to the growing poly(A) tail, further stimulating its elongation. Extension of a simple poly(A) primer by poly(A) polymerase, in the absence of CPSF, is stimulated 30-50-fold by PAB I1 (Wahle, 1991b).

PAB I1 activity was assigned to the 49-kDa protein based on the copurification of this protein with the activity and on the fact that partial amino acid sequences of this protein matched the RNP consensus. However, the 49-kDa protein was contaminated with other polypeptides that varied from preparation to preparation. Also, the “classical” poly(A)-bind- ing protein, PAB I, was not detected during the purification of PAB 11. We now report the purification of PAB I1 to homogeneity, confirming the assignment of poly(A)-binding and stimulatory activity to the 49-kDa polypeptide. Detection of PAB I in different fractions of the same preparation confirmed that the two proteins are distinct. We have also investigated the physical properties of PAB I1 and its inter- action with RNA.

EXPERIMENTAL PROCEDURES

Proteins-CPSF, purified as described (Bienroth et al., 1991), was a gift from S. Bienroth (Biozentrum, Basel). The poly(A) polymerase used here was the undegraded 82-kDa protein (Raabe et al., 1991; Wahle et al., 1991), prepared by a modification* of the published procedure (Wahle, 1991a). Due to a low yield of the undegraded protein and a slightly different behavior during column chromatog- raphy, the best fractions of poly(A) polymerase were only about 50% pure. A monoclonal antibody (10E10) directed against human PAB I was a gift from M. Gorlach and G. Dreyfuss (University of Pennsyl- vania, Philadelphia). T7 RNA polymerase was from Stehelin (Basel), SP6 RNA polymerase and sequencing grade trypsin from Boehringer Mannheim, and polynucleotide kinase from New England Biolabs.

E. Wahle, unpublished data.

2937

2938 Mammalian Poly(A)-binding Protein II Calf intestinal phosphatase (grade I) was from Boehringer; the am- monium sulfate precipitate was pelleted in a microcentrifuge and dissolved in 30 mM Tris-HC1, pH 8.0, 3 M NaC1, 1 mM MgC12, 0.1 mM ZnC12. Methylated BSA was prepared as described (Wahle, 1991b).

Polynucleotides-Poly(A) (average chain length 80 nucleotides) was the preparation described previously (Wahle, 1991a). Poly(A) of defined size was isolated by preparative gel electrophoresis (Wahle, 1991b). Its length was determined by electrophoresis after 5’-end- labeling and partial hydrolysis in a manner similar to the one de- scribed below for oligo(A). Poly(U) (Boehringer Mannheim) was dissolved in HzO and used without further purification. Poly(U) of defined size was prepared in the same way as poly(A). Its length was estimated from comparison to defined poly(A) and DNA size stand- ards. Poly(G) (Pharmacia) was dissolved in 100 mM NaCl, extracted once with phenol-chloroform and precipitated with ethanol. The precipitate was washed twice with 70% ethanol, dried, and dissolved in H20. The concentrations of all polynucleotides were determined spectrophotometrically with extinction coefficients taken from the Pharmacia catalogue. The lengths of unfractionated poly(U) and poly(G) were comparable to that of poly(A) as judged from electro- phoresis of end-labeled material.

RNAs used as substrates for the specific polyadenylation reaction contained the L3 polyadenylation site of adenovirus-2. The RNAs were either L3pre, synthesized by SP6 RNA polymerase (Christofori and Keller, 1988), or a similar RNA slightly shortened at its 5’-end, synthesized by T7 RNA p~lymerase.~ A fraction of the RNA made by T 7 RNA polymerase was refractory to polyadenylation, even in the nonspecific Mn2+-dependent reaction, most likely due to some un- known modification of the 3”ends.

Oligo(A)-Poly(A) was partially hydrolyzed by incubation in 0.2 M NaOH for 30 min at 37 “C, followed by neutralization and desalting on a column of Bio-Gel-P6. Poly(A), partially hydrolyzed poly(A), and commercial oligo(A) (Pharmacia) were size fractionated in batches of up to 5 mg by chromatography on a 1-ml MonoQ FPLC column. The column was equilibrated in 25 mM Tris-HC1, pH 8.0, and developed with a 40-ml gradient from 0.2 to 1 M KC1 in 25 mM Tris-HC1, pH 8.0. Aliquots from representative fractions were 5’- end-labeled with polynucleotide kinase (Sambrook et al., 1989) and analyzed on a denaturing 12% polyacrylamide gel. Corresponding size classes from different MonoQ runs were combined, lyophilized, dis- solved in 500 pl of water, desalted on a Sephadex G-25 FPLC desalting column, lyophilized again, and dissolved in 100 pl of water. Their molar concentrations were estimated from the approximate length of oligo(A) and the UV absorption ( ~ ~ ~ 8 = 9, 800). These pools were expected to contain mixtures of 3’-OH ends, cyclic phosphate ends (from alkaline hydrolysis), and open 2’- and 3”phosphate ends. Cyclic phosphates were opened by an acid treatment (Bock, 1967): 10 p1 of 1 M HC1 was added and, after 20 min at room temperature, neutralized with 20 p1 of 1 M Tris base. The solution was then mixed with 150 pl of water and 30 p1 of 10 X phosphatase buffer (Sambrook et al., 1989). Approximately 1000 units of calf intestinal phosphatase were added for 100 nmol of oligo(A), and the mixture was incubated for 1 h at 37 “C. SDS (0.5%) and proteinase K (25 pg/ml) were added and, after 30 min at 37 “C, the mixture was extracted once with 1- butanol, then with phenol-chloroform, again with 1-butanol, and finally desalted as above. As a result of the phosphatase treatment, the oligo(A) obtained was expected to have uniform 5’-OH and 3’- OH ends so that its migration in gels or anion-exchange columns was only dependent on length, and that it could be directly labeled with polynucleotide kinase. All size classes were then repurified either by a second MonoQ chromatography or on a preparative denaturing 12% polyacrylamide gel. MonoQ column fractions were desalted as above. In the preparative gel, oligo(A) was visualized by UV shadowing. Bands were excised and oligo(A) was eluted overnight into 4 ml of 10 mM Tris-HC1, pH 8.0, 1 mM EDTA, 0.5% SDS. The solution was taken off the gel fragments, concentrated by extractions with 1- butanol, and desalted as above. Most of the size classes obtained in this way contained two major species of oligo(A). Their molar con- centrations were determined as above.

Aliquots from single size classes or mixtures were 5’-end-labeled with polynucleotide kinase and [T-~’P]ATP (Sambrook et al., 1989). Labeling reactions contained, in 50 pl, 20-40 pmol of oligo(A) and 200 pCi of [T-~*P]ATP. After 30 min at 37 “C, incorporation was determined in an aliquot by adsorption to DE81 paper as described (Stayton and Kornberg, 1983). The rest of the reaction mixture was

S. Bienroth, W. Keller, and E. Wahle, submitted for publication.

extracted once with an equal volume of phenol-chloroform and then dried by extraction with 1 ml of 1-butanol. After centrifugation for 15 min in a microcentrifuge, the pellet was washed with 1 ml of ethanol, dissolved in formamide loading buffer, and loaded on a denaturing 12% polyacrylamide gel. Single species of oligo(A) were cut out from the gel after brief autoradiography and eluted into 400 ~1 of 0.5% SDS a t 37 “C for 2-16 h. The solution was taken off the gel fragments and extracted successively with 1 ml of I-butanol, 200 pl of phenol-chloroform, and 1.2 ml of 1-butanol. After centrifugation for 15 min in a microcentrifuge, the pellet was washed with 1 ml of ethanol, dissolved in 50 or 100 pl of water and, after the addition of an equal volume of ethanol, stored at -20 “C.

The length of end-labeled oligo(A) was determined by band count- ing after partial hydrolysis and electrophoresis. An aliquot of the oligo(A) was mixed with 7.5 pg of carrier tRNA and lyophilized. It was dissolved in 27 p1 of water and divided into three portions. One portion received 1 pl of 0.5 M NaHC03 and was incubated for 5-20 min at 95 “C. (Shorter times were used for longer oligo(A).) The sample was then lyophilized and dissolved in water. A second sample was treated in the same way except that, after the heat treatment, 1 pl of 2 M HC1 was added. The third sample received only HCl immediately before lyophilization. Aliquots of all samples were sep- arated on a denaturing 12% polyacrylamide gel. The length of the undegraded oligo(A) was determined from the number of hydrolysis products under the following assumptions. The fastest migrating band was 5’-pAp-3’, whereas the most slowly migrating form was unde- graded 5’-(pA),-3’. Alkaline hydrolysis produced bands which dif- fered in length by units of one nucleotide and had mostly cyclic phosphate ends with some open phosphates. Acid treatment con- verted most but not all of the cyclic phosphates to open phosphates which migrated faster in the gel due to their additional negative charge. Acid treatment without prior alkali treatment resulted in a low degree of hydrolysis with open phosphates (Bock, 1967). The difference in migration between the undegraded species ((PA),) and the longest hydrolysis product (p(Ap)”.J was much larger than be- tween different hydrolysis products due to the lack of a 3’-phosphate in the undegraded oligo(A) and the interaction of the borate buffer with the cis-diol of the terminal ribose.

One preparation of end-labeled oligo(A) was also tested for sensi- tivity to aniline under conditions appropriate for cleavage of abasic sites (Peattie, 1983). No sensitivity was observed, indicating that the acid treatment used to open cyclic phosphates did not lead to any significant depurination.

Amounts of oligo(A) are given as moles of oligonucleotides, not of nucleotides.

Other Materials-Bio-Gel P-6 and Macroprep Q resins were from Bio-Rad, SP Trisacryl M from IBF (Villeneuve-la-Garenne, France), and DEAE-Sepharose FF, Sephadex G-150, and the FPLC columns and equipment from Pharmacia. Blue Sepharose was prepared as described (Bienroth et al., 1991). Centricon 30 filtration units were from Amicon, nitrocellulose filters BA 83 from Schleicher & Schuell, and DEAE paper DE81 from Whatman. [Y-~’P]ATP (3000 Ci/mmol) and other labeled nucleotides were from Amersham, phenylmethyl- sulfonyl fluoride from Boehringer, leupeptin and guanidinium hydro- chloride from Fluka, and pepstatin from Bachem. The pH of all buffers was adjusted at 1 M at room temperature.

Purification of Poly(A)-binding Proteins-The following buffers were used. Buffer 1 contained 50 mM Tris-HC1, pH 8 .0 , l mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, and salt as indicated. Buffer 2 was identical except that 20 mM Hepes-KOH, pH 7.9, replaced Tris. All operations were performed in the cold.

PAB I1 was prepared from 4 kg of calf thymus by a modification of the previously described procedure (Wahle, 1991b). Extract was prepared from 1 kg of tissue at a time, clarified by centrifugation, and applied to a 4-liter column of DEAE-Sepharose FF (Bienroth et al., 1991). The homogenization buffer, but not the buffers used in subsequent steps, contained the protease inhibitors phenylmethylsul- fonyl fluoride (0.5 mM), leupeptin (0.4 pg/ml), and pepstatin (0.7 pg/ ml). The column was eluted with a gradient up to 0.3 M KC1 as described (Bienroth et al., 1991) and then with a step of 1 M KC1 in the same buffer. The gradient fractions were assayed for CPSF (Bienroth et al., 1991), and active fractions were pooled. A second pool was made containing all fractions eluted later, including the salt step. CPSF and PAB I1 contained in the first pool were precipitated with ammonium sulfate, loaded onto a Blue Sepharose column, and CPSF was eluted with a KC1 gradient (Bienroth et al., 1991). The second DEAE pool was loaded directly onto the same Blue Sepharose column, which was then washed with 1.5-2 volumes each of 4 M NaCl

Mammalian Poly(A)-binding Protein II 2939

and 0.1 M KC1 in buffer 1 before PAB I1 from both DEAE pools was eluted as a single batch with 2 M guanidinium hydrochloride, 0.1 M KC1 in buffer 1. After overnight dialysis against 0.05 M KC1 in buffer 1, precipitated material was removed by centrifugation. The solution was diluted with buffer 1 lacking salt to the conductivity of 0.05 M KC1 in buffer 1 and applied to a Macroprep Q anion-exchange column (4 mg protein/ml column volume) equilibrated in the same buffer. The column was washed with equilibration buffer and eluted with a gradient (10 column volumes) from 0.05 to 0.4 M KCl. At this point, poly(A) binding activity was separated into several fractions: 15% of the activity did not hind to the column and also remained in the flow- through upon reloading. From this fraction, PAB I was purified as described below. 30-40% of the activity was eluted as a sharp peak around 0.18 M KCl. This peak corresponded to PAB I1 and was further purified. Some poly(A) binding activity was distributed throughout the fractions preceding PAB 11. Combined PAB I1 pools from 4 kg of thymus were dialyzed against buffer 2 containing 0.05 M KCl, and loaded onto an 8-ml MonoS FPLC column equilibrated in this buffer. The column was eluted a t a flow rate of 2 ml/min with a 320-ml gradient from 0.05 to 0.5 M KCl. PAB I1 was eluted a t 0.22 M KCI. The preparation is summarized in Table I. The purified protein was diluted in the buffer described (Wahle, 1991b).

PAB I was purified from the Macroprep Q flow-through fractions of the PAB I1 preparation. Poly(A) binding activity was recovered from the flow-through by batch adsorption to a quantity of Blue Sepharose approximately equal to the Macroprep Q column. The resin was packed into a column and directly eluted with 2 M guani- dinium hydrochloride, 0.1 M KC1 as described above. After dialysis against buffer 2 containing 0.05 M KCl, the solution was clarified by centrifugation and applied to a SP Trisacryl M cation-exchange column (4 mg protein/ml column volume) equilibrated in this buffer. After washing with 1 volume of the same buffer, the column was eluted with a gradient (10 column volumes) from 0.05 to 0.5 M KC1 in buffer 2. Activity was eluted in a very heterogeneous distribution with a peak around 0.3 M KC1. Only the peak material was pooled, dialyzed for 5 h against a 10-fold excess of buffer 2 lacking salt, and loaded onto a 1-ml MonoS FPLC column equilibrated in buffer 2 containing 0.05 M KCl. The column was washed with the same buffer and developed at a flow rate of 0.5 ml/min with a 40-ml gradient from 0.05 to 0.5 M KC1 in buffer 2. Most of the activity was eluted as a sharp peak at 0.2 M KC1. The pooled fractions were concentrated to 0.65 ml by centrifugation in a Centricon 30 device and applied to a Sephadex G-150 column (1.4 X 90 cm) equilibrated in buffer 1 containing 0.1 M KC1 and lacking dithiothreitol. The column was run at 8 ml/h and fractions of 2.8 ml were collected. The preparation is summarized in Table 11.

Protein Analysis-Routine protein estimations were done by the method of Bradford (1976). The concentration of PAB I1 in the MonoS peak fraction was determined by quantitative amino acid analysis. A sample of the protein was dialyzed overnight against 10% formic acid. Amino acid analysis was carried out in duplicate with aliquots of the dialyzed material according to Knecht and Chang (1986). The two analyses differed by less than 1%. The concentration of the dialyzed material was compared to the original sample by SDS- polyacrylamide gel electrophoresis, followed by Coomassie staining and densitometry. Four different gels, each with 4-6 aliquots of each sample, gave concentrations of the original sample of 0.507, 0.524, 0.473, and 0.671 mg/ml, average 0.544 mg/ml. The concentration of the same sample determined with the Bradford assay was 0.47 mg/ ml.

For trypsin digestion of PAB I, an aliquot from the Sephadex G- 150 column containing 50 pg of protein was mixed with 0.1% SDS and 10 mM dithiothreitol and incubated for 1 h at 37 "C. 50 mM iodoacetamide was added and the incubation was continued for 30 min at room temperature in the dark. Then 50 mM dithiothreitol was added, and the sample was dialyzed overnight against 25 mM NH,HCO,, 0.1% SDS. The protein was lyophilized, dissolved in 100 pl of HzO, and precipitated by t.he addition of 900 pl of ethanol. The precipitate was collected by centrifugation, washed with ethanol, dried under vacuum, and dissolved in 50 p1 of 8 M urea, 50 mM Tris-HC1, p H 8.0, 2 mM CaC12. It was diluted with 150 pl of the same buffer lacking urea and digested with 1.25 pg of trypsin for 5 h a t 37 "C. Another 1.25 pg of trypsin was added, and incubation was continued overnight. Peptides were separated by reversed-phase HPLC on a Hewlett Packard 1090 liquid chromatograph. A Vydac 218 TP 52 column (2.1 X 250 mm) was used and eluted at 150 pl/min with the gradient conditions described by Stone and Williams (1988). One peptide was sequenced on an Applied Biosystems 477A pulse-liquid

phase sequencer. Phenylthiohydantoin-amino acids were detected online on a model 120A analyzer (Applied Biosystems).

SDS-polyacrylamide gels were run according to Laemmli (1970). Analytical Ultracentrifugation-Sedimentation velocity and sedi-

mentation equilibrium studies were performed in a Beckman model E analytical ultracentrifuge equipped with UV absorption optics and a photoelectrical scanning device. 1 ml of the MonoS peak fraction of PAB I1 (Fig. 1) was dialyzed for 13 h in ice against 1 liter of 50 mM Tris-HC1, pH 8.0, 100 mM KC1, 1 mM EDTA, 0.5 mM dithiothre- itol. Aliquots of this sample were loaded into a 12-mm double-sector Epon cell in an An-D rotor. The sedimentation velocity runs were carried out at 56,000 rpm at 2 1 "C. Sedimentation constants were corrected to 20 "C and water. Sedimentation equilibrium runs were performed a t 12,000 and 18,000 rpm. In this case, the cell was filled only to about 3-mm column height with 0.12 ml solvent or solution. The run took 24 h a t 18 "C. Molecular weights were determined by a linear regression computer program that adjusts the base line to obtain the best linear fit of 1nA (A = absorption) uersus ? ( r = distance from rotor center). A partial specific volume of 0.73 ml/g was used.

Fluorescence Measurements-Fluorescence measurements were taken at 20 "C on a Jasco FP-777 spectrofluorometer a t an excitation wave length of 280 nm and an emission wavelength of 335 nm. 555 pmol of PAB I1 was used in 500 ~l of filter-binding buffer. High molecular weight poly(A) was added in small aliquots. The decrease in fluorescence was corrected for dilution by the poly(A) solution.

Filter Binding Assays-The activity of poly(A)-binding proteins was measured a t room temperature with the nitrocellulose filter binding assay described previously (Wahle, 1991b), except that the sample was applied directly to the filter without dilution. For com- petition assays, the rRNA used as a competitor in the standard assay was omitted, and other polynucleotides were added as indicated. For KD determinations, no competitor RNA was used. 5'-End-labeled oligo(A) of the length indicated was used a t 5 fmol/reaction (total volume 100 pl), and varying amounts of PAB I1 were added. Dilution buffer was added such that the combined volume of PAB I1 sample and dilution buffer was 5 pl. This improved the reproducibility of the assay, possibly due to BSA and Nonidet P-40 introduced with the dilution buffer. Since the omission of competitor RNA caused an increased background, 1 ml of rRNA solution (5 pg/ml in wash buffer) was passed through the filter before the sample was applied.

The equation KD = ( [ R ] X [P])/[RP] where R is RNA and P is protein can be converted to [RP]/[R,] = [PI/(& X [PI) with [R,] = [R] + [RP]. This equation, equivalent to the Michaelis-Menten equation for enzyme reactions, can be further converted to 1/ [RP] = (&/[Rt]) X (l /[P]) + (l/[Rt]). A plot of l/[RP] against 1/ [PI, equivalent to a Lineweaver-Burk plot, has a y intercept of l/[R,]. R, is the maximum number of complexes that can be formed. The x intercept is - (1/&).

Gel Retardation Assays-The binding reaction was the same as the one used for filter binding assays except that the volume was 25 pl, and 0.01% Nonidet P-40 and 0.2 mg/ml methylated BSA were in- cluded. After incubation at room temperature for approximately 10 min, the samples were loaded onto a native polyacrylamide gel run- ning a t 250 V. The gel (20 cm long, 1 mm thick) contained 6% acrylamide (acrylamide/bisacrylamide = 80:l) in 0.5 X TBE buffer (Sambrook et al., 1989) and was prerun for 90 min a t 250 V with 0.5 X TBE as running buffer. A sample of bromphenol blue was loaded into a separate lane, and electrophoresis was carried out until the dye had migrated to approximately 2 cm from the bottom.

Polyadenylation Assays-AAUAAA- and CPSF-dependent poly- adenylation was carried out as described (Wahle, 1991b) except that tRNA, creatine phosphate, and creatine kinase were omitted.

RESULTS

Purification of PAB ZZ-PAB I1 was purified from calf thymus by a procedure that was modified from the one de- scribed earlier (Wahle, 1991b) (Table I). The poly(A) binding activity of the protein was measured by a nitrocellulose filter binding assay (see "Experimental Procedures"). The activity was quantitated only after endogenous nucleic acids had been removed by Blue Sepharose chromatography. The subsequent anion-exchange chromatography separated PAB I1 from other poly(A)-binding proteins: approximately 15% of the activity applied to the column was present in the flow-through and

2940 Mammalian Poly(A)-binding Protein I1 TABLE I

Purification of P A B I1 PAB I1 was purified from 4 kg of calf thymus as described under

“Experimental Procedures.” The purification was monitored by meas- urements of the poly(A) binding activity. For technical reasons, protein and activity were not determined in Fraction I1 (DEAE- Sepharose).

Fraction Protein Activity Specific activitv

mg unit X 1oJ unit f mg I (extract) 155,700 111 (Blue Sepharose) 1,060 1,243 IV (Macroprep Q)

6,833 32

V (MonoS) 7.2 2,060 2,246

286,100 70,200

identified as PAB I (see below). PAB 11 bound to the column and was eluted as a sharp peak around 180 mM KCl. A low level of activity was distributed throughout the fractions preceding the PAB I1 peak, forming two distinct peaks in one column but not in another (data not shown). Thus, the total yield of poly(A) binding activity was higher than indicated in Table I, which refers only to PAB 11. After the final MonoS column, the protein was electrophoretically homogeneous (Fig. 1, A and B ) . Its specific activity in the filter binding assay was higher than reported before, reflecting the improved purification but also some variability in the assay depending on the particular batch of labeled poly(A) used.

Specific AAUAAA-dependent polyadenylation of a radio- labeled RNA ending at the L3 polyadenylation site of adeno- virus-2 was carried out by purified poly(A) polymerase and CPSF. In this reaction, PAB I1 had the stimulatory activity that has been described in detail (Wahle, 1991b) (Fig. IC).

Purification of PAB I-The poly(A) binding activity that failed to bind to the anion-exchange column was further purified as summarized in Table 11. The activity was distrib- uted quite heterogeneously in cation-exchange chromatogra- phy. Since only the major peak was used for further purifi- cation, the yield was low (Table 11). The final gel filtration chromatography resolved two peaks of activity, the first of which corresponded to a 73-kDa protein (Fig. 2, A and B ) . Human PAB I also has an apparent molecular mass of 73 kDa in SDS-polyacrylamide gel electrophoresis; its molecular mass calculated from the cDNA sequence is 70 kDa (Grange et al., 1987). Tryptic peptides were prepared from the 73-kDa protein. The amino acid sequence of the only peptide se- quenced was completely identical with amino acids 331-345 of human PAB I (Grange et al., 1987). In a Western blot, the 73-kDa protein also reacted with a monoclonal antibody (10E10) directed against human PAB I (data not shown). Thus, by the criteria of activity, molecular mass, primary structure, and antigenic properties, the 73-kDa protein is bovine PAB I. The heterogeneous behavior during the puri- fication, including the second activity peak in the gel filtration column (Fig. 2) is possibly explained by a sensitivity of PAB I to proteolytic degradation in the absence of protease inhib- itors. However, the existence of additional poly(A)-binding proteins (Setyono and Greenberg, 1981; Tian et al., 1991) in these fractions is also possible. While only small quantities of PAB I were obtained compared to PAB 11, this does not permit conclusions regarding their abundance in the cell: the yield of PAB I during purification was much lower, unknown quantities may have been lost in the first DEAE column, and the efficiencies of extraction from tissue may not be the same.

In contrast to PAB 11, PAB I inhibited the specific poly- adenylation reaction (Fig. 2C). The amount of PAB I required for inhibition was slightly higher than the amount of PAB I1 required for stimulation. The stimulatory effect of PAB I1 was maximal around 2 units/25 p1. PAB I had a barely

detectable effect at 1 unit/25 pl and inhibition was not com- plete at 3 units125 pl (Fig. 2C and data not shown). Inhibitory activity was associated with both peaks of poly(A) binding activity in the final gel filtration column, although the effect of the second peak was significantly stronger. RNA molecules partially elongated in the presence of PAB I (Fig. 2C, lane 7) differed in length by units of approximately 25 nucleotides, the stretch of poly(A) covered by this protein (Baer and Kornberg, 1980; Sachs et al., 1987).

Physical Properties of PAB II-The molecular weight of denatured PAB 11, as determined by SDS-polyacrylamide gel electrophoresis, is 49,000 (Wahle, 1991b, Fig. 1). Upon glyc- erol gradient centrifugation, PAB I1 sedimented as a single sharp peak with a recovery of 88%. Comparison to marker proteins gave a sedimentation constant of 2.2 S, unusually low for a protein of this size (Fig. 3). Gel filtration of PAB I1 on Sephadex G-150 also resulted in a single sharp peak with 55% recovery. The apparent Stokes radius, determined by comparisqn with marker proteins (Siegel and Monty, 1966), was 37.6 A (data not shown).

From the sedimentation coefficient, the apparent Stokes radius, and an assumed partial specific volume of 0.73 ml/g, a native molecular weight of 33,000 was calculated (Siegel and Monty, 1966). Since this was significantly smaller than the molecular weight determined by SDS-polyacrylamide gel elec- trophoresis, the native molecular weight was also determined by analytical ultracentrifugation. A sample of PAB I1 was dialyzed against buffer lacking glycerol, and a homogeneous solution was placed into the rotor cell. In a sedimentation velocity run, two components were resolved. The sedimenta- tion coefficient was calculated from the rate at which the solution was cleared from the protein, as monitored by scan- ning at 280 nm (Schachman, 1959). One component, approx- imately 75% of the material, sedimented at 2.2 S, in good agreement with glycerol gradient centrifugation. The other component sedimented at 5.5 S (data not shown). In a second run, the native molecular weight was determined by sedimen- tation equilibrium. In this method, the molecular weight is determined from the gradient of protein concentration when sedimentation is balanced by diffusion (Schachman, 1959). At a rotor speed of 18,000 rpm, again two components were resolved. For the smaller one, a molecular mass of 50.3 kDa was calculated at an assumed partial specific volume of 0.73 ml/g. Since the molecular mass of the larger species could not be determined accurately, the run was continued at a lower speed, 12,000 rpm. When the protein distribution had come to a new equilibrium, a molecular mass of 96 kDa was deter- mined for the larger species, and an even larger species with a molecular mass of approximately 214 kDa was detected (data not shown). In order to determine whether the larger species were generated by a time-dependent association or aggregation, another sedimentation velocity run was carried out. Indeed, the component of 2.2 S now represented only 50% of the sample, the other 50% sedimenting at approxi- mately 20 S (data not shown). At the end of the experiment, poly(A) binding activity of the protein sample was deter- mined. A sample that had been stored on ice during the entire experiment had retained 61% of its specific activity compared to the protein before dialysis. The samples from the first velocity run, the equilibrium run, and the second velocity run had retained 46, 53, and 31% of their specific activities, respectively.

The centrifugation data show that, even at the rather high protein concentration employed, PAB I1 exists largely as a monomer of 50 kDa. However, under the centrifugation con- ditions, PAB I1 has a tendency to form dimers and higher

FIG. 1. Purification of PAB 11. A , profile of the final Monos column of the preparation summarized in Table I. For details, see “Experimental Procedures.” H, SDS-polyacrylamide gel electropho- resis of Monos fractions. Aliquots of 2 pl of the fractions indicated at the bot- tom were loaded on a 10% gel, which was stained with Coomassie Brilliant Blue. The molecular weights of size standards (lane M) are indicated on the left. C, stimulation of specific polyadenylation by PAB 11. The assay was carried out with purified poly(A) polymerase, CPSF, and an L3pre substrate RNA made by T7 RNA polymerase as described in “Ex- perimental Procedures.” Lane I con- tained no additional proteins. Lane 2 contained 20 ng of an earlier preparation of PAB I1 as a positive control. Lanes 3- 13 contained 0.5 pl of a 1:lOO dilution of the Monos fractions indicated at the top. RNA was analyzed on a denaturing 12% polyacrylamide gel. Sizes (in nucleo- tides) of DNA standards (lane M) are indicated on the left.

Mammalian Poly(A)-binding Protein II A

2941

Fraction No

B 205. 116. 97. 66.

L5.

29.

Frochon 28 29 30 31 32 33 3 L 35 36 37 38

C

Fraclion 28 29 30 31 32 33 34 35 36 37 38

404-

160- 147-

122- 110-

90-

76 -

67- - Subslrale-

h I L 3 4 3 b

TABLE I1 Purification of PAB I

PAB I was purified from the Macroprep Q flow-through of the PAB I1 preparation described in Table I (see “Experimental Proce- dures”). The purification was monitored by poly(A) binding activity. Data for the Sephadex G-150 column refer only to the 70-kDa protein.

Fraction Protein Activity Specific activity

rng unitx lo? unitlrng IV (Macroprep Q) 366 1,033 2,800 V (SP-Trisacryl) 23.1 164 7,100 VI (Monos) 4.2 108 25,700

0.27 31.5 116,700 VI1 (G-150)

oligomers. From the sedimentation coefficient, the native molecular weight, and again an assumed partial specific vol- ume of 0.73 ml/g, a frictional coefficient of 1 X lom7 g/s can be calculated (Cantor and Schimmel, 1980). A spherical pro- tein of the same molecular weight, the same partial specific

/ 8 Y 10 I 1 12 13

volume, and a hydration of 0.3-0.4 would have a frictional coefficient of 5.215.3 x lo-’ g/s (Cantor and Schimmel, 1980). Thus, the frictional ratio (Perrin shape factor) for PAB I1 is 1.9-2.0. For an ellipsoid, this would correspond to an axial ratio near 20 (Cantor and Schimmel, 1980). While the shape of PAB I1 is unknown, this number does serve to illustrate the extent to which PAB I1 deviates from the behavior of a spherical protein.

Specificity of RNA Binding-PAB I1 binds specifically to poly(A) as compared to poly(C) and ribosomal RNA (Wahle, 1991b). In a previous experiment, poly(G) bound only 10-fold weaker than poly(A) (Wahle, 1991b). Since the preparation of poly(G) used in that experiment had a very low average chain length, the experiment was repeated with high molec- ular weight poly(G). This new batch of poly(G) competed with radiolabeled poly(A) for binding to PAB I1 with the same efficiency as unlabeled poly(A) (data not shown). Thus, PAB I1 has the same affinity for poly(A) and poly(G). All other

2942 Mammalian Poly(A)-binding Protein 11

A

,d.” 15 / 20 25 30 35 LO

Frochon No

B 205-.- - . 116

66 97

L 5

29

-.-._ ”. . - ..

Fractlon 20 21 22 23 2L 25 26 27 28 29 30 31 32 33 34 35

C Fracllon 20 22 24 26 28 x) 32 3L 36

21 23 25 27 29 31 33 35

LOL- 309-

2LO- *ol 2 1 7 z 180 190=

160- 147-

122-

110-

90-

76-

M 1 2 3 5 6 7 8 9 IO I 1 12 I3 IL 15 16 17 18 19 20

FIG. 2. Purification of PAB I. A, profile of the final Sephadex G-150 gel filtration column of the preparation summarized in Table 11. For details, see “Experimental Procedures.” B, SDS-polyacryl- amide gel electrophoresis of the G-150 fractions. Aliquots of 10 pl were loaded on a 10% polyacrylamide gel, which was stained with Coomassie Brilliant Blue. C, inhibition of specific polyadenylation by PAB I. Assays were carried out with an L3pre substrate RNA made by SP6 RNA polymerase (see “Experimental Procedures”). All reac- tions contained purified poly(A) polymerase and CPSF, except lane I, which contained no poly(A) polymerase. Lane 2 received no addi- tional protein. Lane 3 received 2.5 ng of purified PAB 11. Lanes 4-20 contained 1 pl of the G-150 column fractions indicated at the top. RNA was analyzed on a denaturing 12% polyacrylamide gel. Sizes (in nucleotides) of DNA size standards (lane M) are indicated on the left.

polynucleotides used previously and in this paper had chain lengths comparable to that of the labeled poly(A).

In competition experiments, poly(U) also inhibited binding t o labeled poly(A) with the same efficiency as unlabeled poly(A) (data not shown). This could either be due to true competition or to hybrid formation and an inability of PAB I1 to bind to double-stranded poly(A)-poly(U). Binding to poly(U) was measured directly. Radiolabeled poly(A) of a defined length of approximately 70 nucleotides was bound with 50% efficiency by 5 X lo-” M PAB I1 in a nitrocellulose filter binding experiment. A 400-fold higher concentration of PAB I1 was required for 50% binding of poly(U) of the same

Fracllon No

FIG. 3. Glycerol gradient centrifugation of PAB 11. 50 p1 of the peak fraction of the Monos column of Fig. 1 was applied to a 12- ml 20-40s glycerol gradient in 50 mM Tris-HC1, pH 8.0, 100 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol in tubes of 14 X 95 mm. The following marker proteins were applied to parallel gradients alone or in pairwise combinations: cytochrome c (1.9 S), RNase A (2 S), chymotrypsinogen (2.5 S), ovalbumin (3.5 S), bovine serum albu- min (4.5 S), aldolase (7.35 S), and yeast alcohol dehydrogenase (7.6 S). Gradients were run for 64 h a t 4 “C and 39,000 rpm (= 275,000 gmaX) in a Kontron TST 41.14 rotor and unloaded from the top (39 fractions/tube). Marker proteins were located by their UV absorb- ance. Their peak positions are indicated by arrowheads. PAB I1 was identified by filter binding assays.

0.0 ‘ I 1 2 3 4

IIrPAB Ill (1InM)

FIG. 4. Determination of the dissociation constant for PAB I1 and A14. Filter binding experiments were carried out as described in “Experimental Procedures” with 5 fmol of Al,/assay.

chain length. Similar results were obtained in gel retardation assays (data not shown). Poly(U) thus probably inhibits bind- ing of PAB I1 to poly(A) by hybrid formation. This was confirmed by the following experiment. Labeled poly(A) was annealed to a 2-fold weight excess of unlabeled poly(U), and binding by increasing amounts of PAB I1 was measured. The PAB I1 concentration required for the binding of a certain quantity of poly(A)-poly(U) was 20-fold higher than for bind- ing of the same amount of poly(A). Thus, PAB I1 binds inefficiently to poly(A)-poly(U). Poly(C), incubated with poly(A) under the same conditions, had no effect (data not shown).

Poly(dA) was a poor competitor for labeled poly(A) in binding experiments with PAB 11: an 800-fold weight excess was required to reduce binding by 50% (data not shown).

In summary, PAB I1 binds specifically to single-stranded purine polyribonucleotides.

Dissociation Constant and Minimal Binding Site-Oligo(A) of defined length was prepared and used in nitrocellulose filter

Mammalian Poly(A)-binding Protein 11 2943

binding experiments to determine the KO for binding of PAB 11. Binding at room temperature had no time dependence between 1 and 30 min. On the filter, the complexes were reasonably stable. Doubling of the volume of wash buffer did not reduce the amount of complex, and a 10-fold larger volume reduced it by only 25% (data not shown). Results of a typical experiment with A14 are shown in Fig. 4. The x intercept indicates a KO of 2 X lo-’ M. The y intercept of the plot is a measure of the maximum number of complexes formed (see “Experimental Procedures”). The average in 16 experiments with All to A14 was 3.54 fmol. The discrepancy between this number and the 5 fmol of oligo(A) that were used for the reactions could be explained by the assumption that the complexes were retained on the filter with an efficiency of 71%. However, other explanations are also possible (Carey et al., 1983). The half-life of the PAB II.oligo(A) complexes was too short to be reliably measured by dilution below the KO followed by filtration after different intervals. A crude esti- mate is 2 s (data not shown). From this half-life and the equilibrium constant, an association rate constant near 2 X 10’ s” M-’ can be estimated. This is similar to comparable protein-RNA associations (Pingoud et al., 1975; Werner, 1991).

In Fig. 5 , the data from titration experiments with oligo(A) of different lengths are combined. A pronounced length de- pendence of the KO between A7 and A12 and little length dependence with longer oligonucleotides indicates that A12 is the minimal site for high affinity binding.

Binding of PAB I1 to oligo(A) was salt-sensitive. At 100 mM NaCl, the concentration at which all measurements were done, binding to A2,, was reduced to approximately 15% of the binding observed in the absence of salt. A further increase to 200 mM NaCl reduced binding again by a factor of 10. Binding to Alz was reduced by a factor of 3 by an increase from 0 to 100 mM NaCl and further reduced by a factor of 10 upon an increase to 200 mM NaCl (data not shown).

Packing Ratio of PAB ZZ-Upon excitation at 280 nm, PAB I1 showed fluorescence with a broad maximum around 340 nm. This fluorescence was quenched by binding of poly(A) (Fig. 6). Titration of a fixed amount of protein with increasing amounts of poly(A) revealed an initial steep linear increase in quenching, followed by a much shallower linear increase at high concentrations. The latter increase was assumed to be a nonspecific effect. Saturation of the protein, determined as the intercept of the two linear portions of the curve, occurred at 23 adenylate residues/protein monomer (Fig. 7).

The validity of this stoichiometry rests on the accuracy with which the concentration of active protein was deter- mined. Total protein concentration was determined by quan- titative amino acid analysis (see “Experimental Procedures”). Protein activity was determined in a filter binding experiment by a stoichiometric titration with All-14 a t a fixed concentra- tion of 4 X M, high above the KO. In this experiment, one protein monomer bound 1.4-1.5 molecules of oligo(A) (Fig. 8). If one assumes an efficiency of retention of 71% (see above), the stoichiometry was 2.0-2.1 molecules of oligo(A)/ molecule PAB 11. The most straightforward interpretation of this titration is that the protein preparation is 100% active with possibly two RNA-binding sites/protein. In view of the obvious uncertainties that influence this titration, the pres- ence of more than one binding site has to be verified by independent experiments.

Gel retardation assays were in apparent agreement with a packing density of 23 adenylate residues/protein. In native gels, As5 formed three different retarded complexes whereas Ass formed four such complexes (Fig. 9). However, the inter-

pretation of these experiments is not straightforward. In native gels, we have never been able to observe any complex formation with a single site (A1*). Thus, there is no proof that the fastest complex represents the binding of a single molecule of PAB 11. Also, additional complexes were observed. Minor complexes can be seen near the top of the gel in Fig. 9. These had the same specificity as the major complexes, being com- peted by poly(A) but not by poly(U) (data not shown). When gels were run at 10 “C rather than at room temperature, other complexes appeared. These were of irregular abundance, weak bands being interspersed with strong ones, and their number was higher than the length of the polynucleotide divided by the binding site size, A12. Thus, these complexes probably represent either some kind of stable conformational hetero- geneity or the formation of protein-protein complexes.

DISCUSSION

The purification of PAB I1 reported here confirms the earlier assignment of activity to the 49-kDa polypeptide. Partial peptide sequences of PAB I1 showed that the protein is distinct from PAB I (Wahle, 1991b). Both proteins have now been obtained from the same tissue, and both can be detected in HeLa cell lysates by Western blotting with specific an t ib~d ies .~ Immunofluorescence microscopy shows a nuclear localization of PAB 114 whereas an antibody to yeast PAB I showed a cytoplasmic localization (Adam et al., 1986). Thus, both proteins can occur in the same cell in apparently differ- ent locations and presumably have different roles in poly(A) metabolism. This is also suggested by their opposite effects on poly(A) polymerase. The inhibitory effect of bovine PAB I on bovine poly(A) polymerase is in agreement with the inhibitory effect of yeast PAB I on yeast poly(A) polymerase (Lingner et al., 1991). It is not known why yeast PAB I stimulates bovine poly(A) polymerase (Wahle, 1991b).

Analytical ultracentrifugation established that the native molecular weight of the predominant form of PAB I1 is similar to its denatured molecular weight and hence that the protein is monomeric. The dimers and oligomers observed may not be significant. They were only seen in the analytical ultracen- trifuge runs near 20 “C in which a high initial protein concen- tration increased even more during the experiment. Also, the content of oligomers appeared to increase over a period of days in which the protein was kept at a high concentration and in the absence of glycerol. Glycerol gradient centrifuga- tion and gel filtration at low temperatures showed only single activity peaks. Since both the drop in specific activity in the analytical ultracentrifugation experiment and the amount of oligomeric material were near 50%, it cannot be excluded that the oligomerization was associated with an inactivation.

The native molecular weight of a protein is frequently determined from its sedimentation coefficient and the Stoke’s radius according to the method of Siege1 and Monty (1966). In experiments with several proteins involved in pre-mRNA processing (Wahle, 1991a; Lingner et al., 1991; Kramer, 1992; this paper), the native molecular weights obtained by this method were significantly lower than the molecular weights determined by electrophoresis under denaturing conditions, and thus obviously too low. The most likely explanation is that interactions of the proteins with the gel filtration matrix led to an underestimation of the Stoke’s radius. This is suggested by the observation that both bovine poly(A) polym- erase and PAB I1 were not recovered a t all from a Superose FPLC gel filtration column, and poly(A) polymerase was clearly retarded on a Sephacryl column.’ A slight retardation

S. Krause and E. Wahle, unpublished data.

2944 Mammalian Poly(A)-binding Protein 11

Lenglh of Oligo(A) (Nucleolldes)

FIG. 5. Length dependence of the dissociation constant. Data from a number of titration experiments as in Fig. 7 were combined. Each point represents the average of two to seven titration experi- ments.

0 2 0 4 0 6 0

Poly(A) Added ( m o l Nucleolldes)

FIG. 7. Titration of PAB I1 with poly(A). Fluorescence of 555 pmol of PAB I1 in 500 p1 of buffer was measured a t an excitation wave length of 280 nm and an emission wave length of 335 nm. Small aliquots of poly(A) were added in volumes of 1-5 pl. After each addition, fluorescence was recorded with time until equilibrium was reached. Protein fluorescence was corrected for the buffer background and dilution by the poly(A) solution.

PAB I1 (pmol)

FIG. 8. Titration of oligo(A) with PAB 11. Filter binding assays were carried out with 41 pmol of A,,-lc and the amounts of PAB I1 indicated.

A65

0.27 136 27.2 I # r “ -

PABn(na) 0 0.14 0.54 2.72 0

A85

0.27 1.36 27.2 0.14 0.54 232

c I

300 320 3L0 360 380 400 Wavelength (nm)

FIG. 6. Fluorescence spectrum of PAB I1 and quenching by poly(A). Fluorescence of 555 pmol of PAB I1 in 500 pl of buffer was measured at an excitation wave length of 280 nm (top curve). The next five curves toward the bottom represent the fluorescence spectra after repeated additions of a constant amount of poly(A) (10.6 nmol as nucleotides). The bottom curve shows the spectrum of buffer.

1 2 3 4 5 6 7 8 9 IO 11 12 13 14

FIG. 9. Gel retardation assays with poly(A) and PAB 11. The assay was carried out as described under “Experimental Procedures” with 2.5 fmol of poly(A)/reaction. Lanes 1-7 contained A, and lanes 8-14 contained A,. Amounts of PAB I1 added are indicated at the top.

may not be immediately obvious, and the resulting error in the Stoke’s radius will lead to a low estimate of the native molecular weight. Whereas the error is evident if the molec- ular weight obtained is lower than in SDS-polyacrylamide gel electrophoresis, it may not be noticed in the case of dimeric or oligomeric proteins. Our observations suggest that the problem might be recognized by differences in the behavior of the protein on different gel filtration resins.

The binding site size of PAB 11, 12 adenylate residues, is very similar to that of yeast PAB I (Sachs et al., 1987). The apparent packing ratio of PAB I1 and poly(A), 23 adenylate residues/protein monomer, is also similar to that of PAB I (Sachs et al., 1987). The simplest interpretation of this pack- ing ratio is that a PAB I1 monomer covers 23 nucleotides on a single molecule of poly(A). This model is supported by the results of gel retardation assays (Fig. 9). However, the gel retardation experiments cannot be interpreted with certainty (see above), and a stoichiometric titration (Fig. 8) suggests the possibility that a single molecule of PAB I1 might bind more than one polynucleotide. Therefore, other interpreta-

Mammalian Poly(A)-binding Protein 11 2945

tions of the data cannot be excluded at present. Nuclease protection experiments, which first showed a packing density near 25 nucleotides for PAB I (Baer and Kornberg, 1980), did not result in any specific degradation pattern in the case of PAB 11.'

Acknowledgments-We are grateful to Walter Kel ler for generous suppor t and he lpfu l d i scuss ions . We thank Mat th ias Gor lach and Gideon Dreyfuss for the monoclonal antibody to h u m a n P A B I, Silke Bienroth for puri f ied CPSF, and Walter Keller and Angel Kramer for cri t ically reading the manuscript .

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