©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Poly(A) Tail Length Control Is Caused by Termination of Processive Synthesis (*)

(Received for publication, August 5, 1994; and in revised form, October 24, 1994)

Elmar Wahle (§)

From the Department of Cell Biology, Biozentrum, University of Basel, Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Poly(A) polymerase synthesizes poly(A) tails rapidly and processively only when the substrate RNA is bound simultaneously by two stimulatory proteins, the cleavage and polyadenylation specificity factor (CPSF) and poly(A)-binding protein II (PAB II). A burst of synthesis terminates after the addition of about 250 nucleotides, a length corresponding to that of newly synthesized poly(A) tails in vivo. Further elongation is slow. Length control can be reproduced with premade poly(A) tails of different lengths and is insensitive to large changes in the elongation rate. Thus, the control mechanism truly measures the length of the poly(A) tail. The stimulatory action of PAB II is similar on long and short tails. Coating of poly(A) with one PAB II molecule for approximately 30 nucleotides is required, such that the number of PAB II molecules in the polyadenylation complex is a direct measure of poly(A) tail length. CPSF also stimulates poly(A) polymerase on long and short tails. Long tails differ from short ones only in that they do not permit the simultaneous stimulation of poly(A) polymerase by CPSF and PAB II. Consequently, elongation of long tails is distributive. Thus, length control is brought about by an interruption of the interactions responsible for rapid and processive elongation of short tails. The 3`-end of the poly(A) tail is not sequestered in the protein-RNA complex when the correct length has been reached. Neither ATP hydrolysis nor turnover of the polymerized AMP is involved in length control.


INTRODUCTION

The poly(A) tails found at the 3`-ends of nearly all eukaryotic mRNAs are added during RNA processing in the cell nucleus and are later shortened after the RNA has been transported into the cytoplasm. The poly(A) tail has at least two different functions: the initiation of translation and the regulation of mRNA breakdown (reviewed by Sachs (1990), Jackson and Standart(1990), Bachvarova(1992), Wickens(1992), Sachs(1993), and Sachs and Wahle(1993)). A role in the export of mRNA from the cell nucleus is also possible (Wickens and Stephenson, 1984; Eckner et al., 1991).

Evidence for a role in translation initiation has been provided by genetic experiments in yeast (Sachs and Davis, 1989, 1990), studies on RNA introduced by electroporation into a number of different cell types (Gallie, 1991), in vitro translation experiments (Munroe and Jacobson, 1990), and microinjection of RNAs into oocytes (Galili et al., 1988; Vassalli et al., 1989; Paris and Richter, 1990; Sheets et al., 1994). The oocyte studies suggest that not only the presence or absence of a poly(A) tail is important but that its length can determine translational efficiency. Although this does not prove that tail length is also important for translation in other cells, regulated changes in poly(A) tail length have been described that are correlated with changes in translation in somatic cells (Robinson et al., 1988).

Evidence for a role of the poly(A) tail in the regulation of mRNA degradation comes from the observation that deadenylation is the first and rate-limiting step in the breakdown of at least some unstable mRNAs both in mammals and in yeast (Wilson and Treisman, 1988; Shyu et al., 1991; Muhlrad and Parker, 1992; Decker and Parker, 1993; Muhlrad et al., 1994). Sequences in the 3`-untranslated region and the coding sequence can control the half-life of the mRNA by influencing the rate of deadenylation (Shyu et al., 1991; Muhlrad and Parker, 1992; Lowell et al., 1992).

A control of the rate of deadenylation is useful only if the length of poly(A) initially added in the cell nucleus is also controlled. In mammalian cells, newly synthesized bulk poly(A) is approximately 200-250 nucleotides long (Brawerman, 1981). Individual mRNAs that have been examined, c-fos and several c-fos-beta-globin chimeras, have initial poly(A) tails of 150-300 nucleotides (Schiavi et al., 1994). A limitation of poly(A) tail synthesis to approximately 200 nucleotides has been observed during in vitro polyadenylation in crude nuclear extracts (Sheets and Wickens, 1989).

Nuclear polyadenylation is preceded by an endonucleolytic cleavage of the primary transcript at the site of poly(A) addition. In vitro, specific polyadenylation of a ``precleaved'' RNA already ending at the poly(A) site has been reconstituted from three proteins purified from mammalian tissue (reviewed by Wahle and Keller (1992) and Sachs and Wahle(1993)). Poly(A) polymerase, the enzyme that catalyzes polyadenylation, is devoid of any pronounced specificity with regard to the RNA substrate and is also almost inactive on its own (Wahle, 1991a). Two other proteins activate the enzyme. One of these is the cleavage and polyadenylation specificity factor (CPSF), (^1)which binds the polyadenylation signal AAUAAA that is present in almost every mRNA precursor (Bienroth et al., 1991; Keller et al., 1991; Murthy and Manley, 1992). The second stimulatory factor is a poly(A)-binding protein (PAB II), which binds the growing poly(A) tail (Wahle, 1991b; Wahle et al., 1993). Each of these two factors directs poly(A) polymerase specifically to RNAs carrying the respective protein binding site, either AAUAAA or an oligo(A) tail of at least 10 nucleotides. However, poly(A) polymerase works most efficiently when it is stimulated by both factors simultaneously (Wahle, 1991b). The enzyme on its own is entirely distributive. When held on the RNA by the combined efforts of CPSF and PAB II, the enzyme attains sufficient processivity to synthesize a complete poly(A) tail in one single binding event. Either stimulatory factor alone leads only to a very small increase in processivity (Bienroth et al., 1993).

The polyadenylation apparatus reconstituted as described above also reproduces the length control observed either in vivo or in extracts. Once a poly(A) tail length of close to 250 nucleotides has been reached, further elongation becomes very slow (Wahle, 1991b; Bienroth et al., 1993). This paper describes the reproduction of true length control in vitro and presents evidence that it is due to a switch from processive to distributive synthesis.


EXPERIMENTAL PROCEDURES

Polyadenylation Proteins

PAB II was taken from the peak fraction of the preparation described earlier (Wahle et al., 1993). Protein concentration was 0.47 mg/ml as determined by a Bradford assay (Bradford, 1976) and 0.54 mg/ml as determined by quantitative amino acid analysis of a dialyzed aliquot (Wahle et al., 1993). Calculations were based on a concentration of 0.5 mg/ml. CPSF was purified by a modification of the procedure of Bienroth et al.(1991). Spermine-agarose chromatography was left out. The pool of the poly(A)-Sepharose column was concentrated by centrifugation through Centricon-30 and further purified by gel filtration on a Superose 6 prep grade fast protein liquid chromatography column. Active fractions were pooled and concentrated on Centricon-30. Purity and activity were comparable with those of published preparations (Bienroth et al., 1991). Total protein concentration as determined by a Lowry assay (Scopes, 1987) was 0.52 mg/ml. Concentration measurements by densitometric scanning of the 73-kDa subunit in a SDS-polyacrylamide gel stained with Coomassie Brilliant Blue and comparison to a BSA standard gave 0.13 mg/ml, assuming equimolar amounts of all subunits. Active CPSF as determined in a gel retardation assay (Bienroth et al., 1991) with excess L3pre RNA was at least 200 pmol/ml, corresponding to 0.07 mg/ml. The latter concentration was the basis of calculations. Poly(A) polymerase was a gift from Georges Martin. The protein was expressed in Escherichia coli from the bovine cDNA (Wahle et al., 1991; Raabe et al., 1991) under the control of a T7 promoter with 6 N-terminal histidine residues. It was purified on a Ni-nitrilotriacetic acid column (Hochuli et al., 1987) followed by chromatography on a MonoQ-fast protein liquid chromatography column. (^2)The protein was nearly homogeneous on a SDS-polyacrylamide gel. The specific activity in the presence of Mn and saturating amounts of oligo(A) primer was 19 times 10^6 units/mg, very similar to the activity of the 60-kDa proteolytic fragment purified from calf thymus (Wahle, 1991a). (^3)The concentration of poly(A) polymerase (0.15 mg/ml) was based on a comparison with a BSA standard on a SDS-polyacrylamide gel stained with Coomassie Brilliant Blue. Yeast poly(A) polymerase purified to homogeneity from recombinant E. coli (Lingner et al., 1991b) was a gift from Pascal Preker. Dilution buffer for all proteins was 5 mM Tris-HCl, pH 8.0, 10% (v/v) glycerol, 50 mM NaCl, 0.5 mM EDTA, 0.2 mg/ml methylated BSA, 0.01% (v/v) Nonidet P-40, 1 mM dithiothreitol.

RNA

The standard polyadenylation substrate L3pre, derived from the L3 polyadenylation site of adenovirus-2, and conditions for its synthesis by SP6 RNA polymerase have been described (Christofori and Keller, 1988; Bienroth et al., 1993). RNA was made without a cap. Products were run on 6% polyacrylamide gels in 1 times TBE, 8.3 M urea (Sambrook et al., 1989), and the RNA was eluted overnight at 37 °C in 750 mM ammonium acetate, 10 mM magnesium acetate, 0.1 mM EDTA, 0.5% SDS, mixed with 0.1 volume of buffer-saturated phenol. The RNA was extracted once with phenol/chloroform and precipitated with ethanol. No carrier tRNA was added. The amount of RNA obtained was calculated from the specific activity of [alpha-P]UTP and the number of U residues in the RNA. Transcription reactions also produced a small amount of a slightly shorter transcript. Because it was polyadenylated with the same efficiency as the correct RNA, no attempt was made to separate the two. Polyadenylated RNAs were made from gel-purified L3pre by the nonspecific Mn-dependent polyadenylation reaction essentially as described (Wahle, 1991b; Bienroth et al., 1993). RNAs with poly(A) tails of the desired lengths were gel-purified as above. Poly(A) tail lengths were determined on analytical polyacrylamide gels with end-labeled DNA fragments as markers. L3pre lacking poly(A) (expected size, 65 nucleotides) had an apparent length of approximately 70 nucleotides. tRNA (Boehringer Mannheim) was dissolved, extracted with phenol/chloroform, precipitated with ethanol, washed with 70% ethanol, and dissolved in water. Concentration measurements assumed that A = 1 corresponded to 40 µg/ml. Poly(A) (Boehringer Mannheim) was dissolved in water, extracted with phenol/chloroform, and dialyzed exhaustively against water. The concentration was determined spectrophotometrically with =9800. Measurements after complete alkaline hydrolysis with = 15,400 (3`-AMP, pH 13) were in excellent agreement (less than 4% deviation). As analyzed by 5`-end labeling with polynucleotide kinase and gel electrophoresis under denaturing conditions, the poly(A) had a very heterogeneous size distribution. Most chains were longer than 200 nucleotides with a large fraction longer than the longest marker (622 nucleotides).

Other Materials

SP6 RNA polymerase, yeast hexokinase, proteinase K, ATPS, and AMPPNP were from Boehringer Mannheim. RNAguard, Superose 6 prep grade, and other fast protein liquid chromatography columns and equipment were from Pharmacia Biotech Inc., radiolabeled nucleotides were from Amersham, Centricon-30 filtration devices were from Amicon, Inc., and polyethyleneimine plates for thin-layer chromatography were from Merck. Polyvinyl alcohol (cold water soluble) was from Sigma, and Ni-nitrilotriacetic acid resin was from Diagen (Hilden, FRG). Methylated BSA was made as described (Wahle, 1991b).

Polyadenylation Reactions

Reactions contained, in 25 µl, 25 mM Tris-HCl, pH 7.9 (measured at 1 M and room temperature), 10% (v/v) glycerol, 2.6% (w/v) polyvinyl alcohol, 50 mM KCl, 2 mM MgCl(2), 0.05 mM EDTA, 0.01% (v/v) Nonidet P-40, 0.4 mg/ml methylated BSA, 0.5 mM ATP, 1 mM dithiothreitol, 4 units of RNAguard, 80 fmol of radiolabeled substrate RNA, 9 fmol of poly(A) polymerase, 100 fmol of CPSF, and 200 fmol of PAB II. For some experiments, tRNA was added at 2.5 µg/reaction, and 18 fmol of poly(A) polymerase was used. For longer poly(A) tails, the amount of PAB II was increased as indicated for individual experiments. Incubations were at 37 °C for the time indicated. For kinetic experiments, larger reaction mixtures (e.g. 10times = 250 µl) were assembled on ice in the absence of ATP and then prewarmed at 37 °C. Polyadenylation was started by the addition of ATP. Reactions were terminated by the addition of 2-fold concentrated proteinase K digestion buffer (Wahle, 1991a) and water to a final volume of 100 µl. Digestion was carried out with 10 µg of proteinase K and 2.5 µg of tRNA for 30 min at 37 °C. RNA was precipitated with 2.5 volumes of ethanol, washed with 70% ethanol, dried, dissolved in formamide loading buffer, and analyzed on 6% polyacrylamide gels (40 cm long) in 1 times TBE, 8.3 M urea (Sambrook et al., 1989). Rates of polyadenylation were calculated from estimates of average poly(A) tail length after different times of reaction. Incorporation of [P]AMP into unlabeled RNA was measured by adsorption to DE81 paper (Stayton and Kornberg, 1983). In experiments in which the production of ADP and AMP in polyadenylation reactions was measured by polyethyleneimine thin-layer chromatography, incorporated nucleotides were taken to be those remaining at the origin.

Other Procedures

SDS-polyacrylamide gels were run according to Laemmli(1970). Nucleotides were analyzed by thin-layer chromatography on polyethyleneimine plates with 1 M formic acid, 0.5 M LiCl as a solvent.


RESULTS

Reconstitution of Length Control with Premade Poly(A) Tails

Derivatives of the precleaved polyadenylation substrate RNA L3pre carrying poly(A) tails of different lengths were prepared (see ``Experimental Procedures'') and used in polyadenylation assays with purified poly(A) polymerase, CPSF, and PAB II. These reactions with premade poly(A) tails faithfully reproduced the length control seen during de novo poly(A) synthesis. An L3pre derivative carrying 30 adenylate residues at its 3`-end was elongated to an average tail length of approximately 250 nucleotides within 1 min or less. Further elongation was slow. This corresponds to the kinetics described previously (Wahle, 1991b; Bienroth et al., 1993). A derivative of the same RNA already carrying 250 adenylate residues did not undergo the initial burst of rapid elongation; it was only elongated in the slow mode (Fig. 1). RNAs with intermediate tail lengths were elongated rapidly to a tail length of 250 nucleotides before the elongation rate decreased (see below). Thus, the length control mechanism measures the length of the poly(A) tail present, rather than the time that poly(A) polymerase has spent elongating the substrate RNA. Further evidence for this conclusion will be presented below. From the same experiment one may also conclude that, whereas the burst of elongation of short tails is processive, the slow elongation of long tails is distributive. In the reaction, substrate RNA was present in excess over poly(A) polymerase. Elongation of a small fraction of the short tails to a length of 250 nucleotides with no extension of the majority of the substrate at early times is indicative of the processive nature of the reaction as described before (Bienroth et al., 1993). Under the same conditions, the entire population of RNA molecules carrying long tails was elongated in a synchronous manner, demonstrating a distributive behavior of the polymerase. Some shortening of the substrate, evident in Fig. 1, was due to an apparently nonspecific 3`-exonuclease activity that contaminates CPSF preparations. Exonucleolytic degradation of the labeled substrate RNA could be suppressed by the addition of tRNA (see below). However, for the sake of simplicity, this was not done in most experiments.


Figure 1: Poly(A) tail length control with premade tails. A 25times reaction mixture lacking RNA and ATP was set up on ice and divided into three equal portions. Two of these were mixed with the substrate RNAs indicated at the bottom, and a 25 µl aliquot was withdrawn from each (0 min time point). The rest of each mixture was prewarmed for 3 min at 37 °C. Reactions were started by the addition of ATP, and additional aliquots were taken at various times after ATP addition as indicated. The size of DNA markers (in nucleotides) is indicated on the left.



Protein Requirements

Length control was only observed in reactions containing poly(A) polymerase with both stimulatory factors, CPSF and PAB II. When the reaction was carried out with either factor alone, elongation was slow even though a 10-fold higher concentration of poly(A) polymerase was used compared with the reaction containing both stimulatory factors (Fig. 2). The low rate of polyadenylation is due to the distributive nature of the reaction under these conditions (Bienroth et al., 1993). Most importantly, when poly(A) tail length was plotted versus time (Fig. 2B), no sudden drop in chain growth rate was observed at any particular tail length. A progressive decrease in the chain growth rate in the CPSF-dependent reaction is probably caused by the increasing distance between the CPSF binding site and the 3`-end to be elongated. The decrease in rate in the PAB II-dependent reaction is likely to be due to the decreasing ratio of PAB II to poly(A) (see below).


Figure 2: Length control requires CPSF and PAB II. A, a 33times standard reaction mixture containing L3pre-A was set up in the absence of all polyadenylation factors and ATP and divided into four equal portions. Polyadenylation factors were added to three of these as indicated. The two tubes containing either only PAB II or only CPSF received a 10-fold higher amount of poly(A) polymerase (90 fmol/25 µl). A 25-µl aliquot was withdrawn from each tube (0 min time point), and the rest was prewarmed for 3 min at 37 °C. The reactions were started by the addition of ATP, and additional aliquots were taken as indicated. The size of DNA markers (in nucleotides) is indicated on the left. B, poly(A) tail lengths were estimated from A and plotted against reaction time. Open circles, reactions containing CPSF and PAB II; filled circles, reactions containing CPSF; squares, reactions containing PAB II.



Standard reaction conditions included 80 fmol of RNA, 100 fmol of CPSF, 200 fmol of PAB II, and 9 fmol of poly(A) polymerase. When the concentration of CPSF in the reaction was increased, length control was unchanged. Likewise, a 2-fold or 10-fold increase in PAB II concentration did not relieve length control (data not shown, but see Fig. 4). Measurements of the initial rate of poly(A) chain growth showed that the standard amount of PAB II was optimal with 9 or 180 fmol of poly(A) polymerase. Whereas at the higher concentration of poly(A) polymerase a significantly larger fraction of precursor RNA was elongated immediately after the start of the reaction, the initial rate of chain growth was not affected; it was 20-25 nt/s at either concentration of poly(A) polymerase ( Fig. 3and data not shown). This confirms the processive nature of the reaction. Other experiments showed that, in contrast, the elongation of long tails was proportional to the concentration of poly(A) polymerase, as expected for a distributive reaction (see below).


Figure 4: PAB II dependence of burst elongation with different initial poly(A) tail lengths. A 26times reaction mixture was assembled in the absence of RNA, ATP, and PAB II. Poly(A) polymerase was used at 90 fmol/25 µl. The mixture was split into three equal portions, which were mixed with RNAs as indicated at the bottom. 25 µl aliquots were then dispensed, and PAB II was added as indicated. One aliquot from each mixture was mixed with proteinase K digestion buffer without prior incubation to show the unreacted substrate (first lane in each set of eight). The others were prewarmed to 37 °C for 2 min. Reactions were started by the addition of ATP and stopped after 30 s by the addition of proteinase K digestion buffer. The size of DNA markers (in nucleotides) is indicated on the left.




Figure 3: The initial rate of burst elongation. An 8times reaction mixture was assembled with L3pre-A in the absence of ATP. Compared with the standard conditions, a 20-fold higher concentration of poly(A) polymerase was used (180 fmol/25 µl). A 25-µl aliquot was withdrawn (0 min time point). The rest of the mixture was prewarmed for 3 min at 37 °C and then kept at this temperature. 25-µl aliquots were drawn up into a pipette tip, expelled into prewarmed tubes containing ATP, drawn up into the same pipette tip, and transferred to proteinase K digestion buffer. The size of DNA markers (in nucleotides) is indicated on the left.



The requirement for only a 2-3-fold molar excess of PAB II over RNA was surprising. An estimate of a packing density of 1 PAB II molecule/23 adenylate residues (Wahle et al., 1993) suggests that the amount of PAB II used in these experiments is sufficient to coat the short oligo(A) tails present at the beginning of the reaction but not the long tails generated during elongation. Therefore, the PAB II requirement was tested during burst elongation of different initial poly(A) tail lengths (Fig. 4). As mentioned above, burst elongation at optimal PAB II concentrations always generated the same final poly(A) tail length, irrespective of the initial tail length. Higher concentrations of PAB II did not overcome the length limitation; in fact, they were inhibitory with respect both to the tail length generated and the number of molecules elongated. Suboptimal concentrations of PAB II also led to shorter average poly(A) tails. With 80 fmol of an A tail, 200 fmol of PAB II were optimal (8 adenylates/PAB II based on initial tail length), with an A tail, 300 fmol of PAB II were optimal (20 adenylates/PAB II), and with an A tail, 400 fmol of PAB II were optimal (30 adenylates/PAB II). Although it is apparent that optimal elongation requires more than a single molecule of PAB II for each poly(A) tail, the effect of different PAB II concentrations was not very strong, and the interpretation of PAB II titrations in this type of reaction is not straightforward (see ``Discussion''). Thus, the PAB II requirement was also tested in the elongation of poly(A) tails in the absence of CPSF. This simpler reaction showed a much more pronounced dependence on the exact PAB II concentration. The elongation of 80 fmol of an RNA with an A tail was most efficient with 1200 fmol of PAB II. This corresponds to a ratio of between 1 PAB II/23 adenylate residues, based on the initial tail length, and 1 PAB II/30 adenylate residues, based on the final tail length of 450 nucleotides. Higher concentrations of PAB II were inhibitory. Additional titrations, some of them with shorter poly(A) tails, gave comparable ratios (Table 1), their accuracy being limited by the number of nucleotides added during the reaction. Saturation of the same molar quantities of shorter tails at lower concentrations of PAB II is not only further evidence that coating of the tail is required but also demonstrates that the experiments measured the stoichiometry of the PAB II requirement rather than the affinity of PAB II for poly(A). In the elongation of a simple unlabeled poly(A) primer with radiolabeled ATP, the amount of PAB II required for maximum incorporation was also proportional to the amount of poly(A) present. Saturation was achieved near 35 nucleotides/PAB II monomer (Fig. 5). Inhibition became apparent with amounts of PAB II exceeding 1/30 adenylate residues. In these experiments, AMP incorporation at the optimum levels of PAB II was between 20 and 38% of the poly(A) added as a primer. The optimal ratio of protein to poly(A) based on the initial poly(A) concentration may thus be slightly overestimated.




Figure 5: PAB II dependence of poly(A) elongation in the absence of CPSF. A 34times reaction was assembled lacking CPSF, PAB II, RNA, and ATP. Poly(A) polymerase was included at 360 fmol/25 µl. The mixture was split into three equal aliquots, which received unlabeled high molecular weight poly(A) at 273, 137, and 55 pmol of AMP/25 µl, respectively. 25-µl aliquots of each mixture were mixed with PAB II as indicated. Reactions were started by the addition of ATP and transfer to 37 °C. After 30 min, they were stopped by application to DE81 paper. Background was determined by application of ice-cold reaction mixtures lacking PAB II to DE81 paper immediately after the addition of ATP. The value obtained (0.8 pmol of AMP) was subtracted from the incorporation measured in all other reactions. Open circles, 273 pmol of poly(A)/25 µl; filled circles, 137 pmol of poly(A)/25 µl; triangles, 55 pmol of poly(A)/25 µl.



Elongation of Long Poly(A) Tails

Although poly(A) tails of more than 250 adenylate residues fail to undergo the processive elongation typical of short tails, they are nevertheless good primers for the PAB II-stimulated activity of poly(A) polymerase. In the experiments described in Table 1, excessively long tails were elongated by 100 or more nucleotides in the presence of the optimal PAB II concentration, whereas PAB II-independent elongation was barely detectable. In fact, more precise measurements showed that the elongation rate of long tails was, if anything, slightly higher than the elongation rate of short tails at their respective PAB II optima (Table 2). Similarly, poly(A) polymerase assisted by CPSF was only 5-fold slower on long compared with short tails. This may reflect the distance between the 3`-end being elongated and the CPSF binding site. A serious deficiency of long tails was apparent only in the presence of both PAB II and CPSF; long tails were extended 300-fold more slowly than short tails. Whereas the simultaneous presence of both factors increased the elongation rate for short tails by a factor of 300-1000 compared with either factor alone, the effect was only 2-5-fold for long tails (Table 2). When, in the presence of both stimulatory factors, the concentration of poly(A) polymerase was increased up to 10-fold, the elongation rate of long tails increased proportionally (data not shown). This is further evidence for a distributive mechanism as discussed above. The rate of extension of long tails in the presence of both CPSF and PAB II was at least as high as with either factor alone and about 50-fold higher than in the absence of any stimulatory factor (near 0.1 nt/min under these conditions). Thus, length control is not due to an inhibition of the elongation of long poly(A) tails. Neither is it due to a lack of interaction between poly(A) polymerase and CPSF or PAB II. Rather, it is caused by the inability of long tails to mediate the simultaneous effects of PAB II and CPSF on poly(A) polymerase that are the basis for the burst of processive elongation of short tails.



Accessibility of 3`-Ends

A simple possibility for length control would be the length-dependent formation of an RNA-protein complex that prevents further elongation by sequestering the 3`-end of the RNA. Although the lack of inhibition evident from Table 2does not suggest this model, it can be more rigorously tested by the use of an enzyme that elongates the RNA nonspecifically. One such enzyme is poly(A) polymerase of yeast. Because the yeast enzyme does not respond to mammalian CPSF and PAB II (Lingner et al., 1991a, 1991b) (additional data not shown), a 90-fold larger amount of yeast poly(A) polymerase compared with the mammalian enzyme was used for reasonable chain growth rates. No control of poly(A) tail length was found. With an RNA carrying 30 A residues, the initial rate of elongation was 14 nt/min until a total tail length of 70 As had been reached. The elongation rate was 8 nt/min in the interval between 125 and 300 adenylates and 6 nt/min between 300 and 500 adenylates. In a control reaction with a second aliquot of the same reaction mixture, mammalian poly(A) polymerase showed the expected kinetics. When a mixture of short and long premade tails was used, yeast poly(A) polymerase in the presence of CPSF and PAB II elongated both substrates at similar rates (25 nt/min for short tails and 10 nt/min for long tails), whereas the mammalian enzyme first elongated the short tails to nearly 250 adenylates before it touched the long tails (Fig. 6). In conclusion, these experiments suggest that there is no sequestration of the 3`-end of the poly(A) tail to terminate elongation. When in an experiment as shown in Fig. 6both yeast and mammalian poly(A) polymerase were present simultaneously, no sequestration was observed either. In more general terms, CPSF and PAB II are not sufficient for length control. The mammalian poly(A) polymerase is needed, because, in the presence of the two stimulatory factors, this enzyme but not the yeast enzyme is capable of processive burst elongation.


Figure 6: Competition of polyadenylated and oligoadenylated RNA. A 25times reaction mixture containing 40 fmol/25 µl each of L3pre-A and L3pre-A was assembled in the absence of poly(A) polymerase and ATP. PAB II was used at 1200 fmol/25 µl. A 4times aliquot was mixed with 36 fmol of bovine poly(A) polymerase (PAP), and a 5times aliquot was mixed with 3900 fmol of yeast poly(A) polymerase. 25-µl aliquots were withdrawn from each tube (0 min time point). The rest was prewarmed for 2 min at 37 °C before the reactions were started by the addition of ATP. Additional aliquots were withdrawn as indicated. The size of DNA markers (in nucleotides) is indicated on the left.



Stability of Poly(A) Tails

DNA polymerases as well as RNA polymerases have exonucleolytic activities (Brutlag and Kornberg, 1972; Kornberg and Baker, 1992; Surratt et al., 1991; Kassavetis and Geiduschek, 1993), and the same has been claimed for poly(A) polymerase (Abraham and Jacob, 1978; Tarui and Minamikawa, 1989). Therefore, the possibility was considered that exonucleolytic shortening of the poly(A) tail might play a role in length control. For these experiments, the exonuclease activity contaminating the CPSF preparation (see above) had to be suppressed by tRNA as a nonspecific competitor. This did not cause any change in length control. When under these conditions poly(A) synthesis was abruptly stopped at a late stage by ATP depletion with hexokinase and glucose, the poly(A) tails that had been synthesized were completely stable, even though they exceeded a length of 250 nucleotides (data not shown). When a mixture of short and excessively long tails was exposed to the polyadenylation factors in the absence of ATP, both were completely stable during extended incubations. Upon addition of ATP, short tails were preferentially extended in a processive manner (Fig. 7). Finally, when under similar conditions [alpha-P]ATP was used to elongate unlabeled L3pre RNA, AMP production (as a result of exonucleolytic excision of incorporated nucleotides) was at the limit of detection, at most 5% of the net incorporation. Analysis of additional aliquots of the same reaction by gel electrophoresis demonstrated that length control was normal, and both the gel pattern and calculations showed that incorporation was almost exclusively into the polyadenylation substrate and not the tRNA used to suppress the exonuclease (data not shown). In conclusion, exonuclease activity is not involved in poly(A) tail length control. Tests under stringent conditions have also not detected any poly(A)-degrading activity in poly(A) polymerase itself.^3


Figure 7: Stability of poly(A) tails. A 10times reaction mixture containing 40 fmol/25 µl each of L3pre-A and L3pre-A was assembled in the absence of poly(A) polymerase and ATP. Reactions contained tRNA and PAB II at 2000 fmol/25 µl. A 25-µl aliquot was withdrawn (0 min time point). The rest was mixed with poly(A) polymerase (18 fmol/25 µl) and transferred to 37 °C. Aliquots were taken after 5, 10, and 20 min. After 21 min, ATP was added, and additional aliquots were withdrawn as indicated. The size of DNA markers (in nucleotides) is indicated on the left.



Role of ATP

We have previously reported that poly(A) polymerase can use ATP analogs with nonhydrolyzable beta- bonds (Bienroth et al., 1993). Length control is also functional with these analogs; in a kinetic experiment, elongation with AMPPNP proceeded approximately 50-fold more slowly than with ATP and slowed down at a tail length of about 200 nucleotides. After 90 min, when elongation had become very slow, an aliquot of the reaction mixture was added to fresh oligoadenylated precursor RNA. This fresh substrate was still elongated, effectively competing out the previously made tails (data not shown). Thus, in spite of the long incubation time, inactivation of proteins was not the reason for the decrease in the elongation rate. Experiments with ATPS also showed normal length control with an elongation rate intermediate between ATP and AMPPNP (data not shown). In a competition between long and short tails, the polyadenylation machinery almost exclusively selected the short RNA in the presence of ATP, ATPS, or AMPPNP (Fig. 8). Thus, the hydrolysis of the beta- bond of ATP is not required for length control. (All experiments involving AMPPNP were done in the presence of hexokinase and glucose to exclude possible ATP contaminations. In control experiments with ATP, hexokinase completely abolished polyadenylation within seconds.) From the same experiments, one may also conclude that length control is essentially insensitive to large changes in the rate of chain elongation. This confirms that, as discussed above, the length control mechanism truly measures the length of the poly(A) tail present.


Figure 8: Competition between short and long tails in the presence of ATP analogs. An 18times reaction mixture was set up with 40 fmol/µl each of L3pre-A and L3pre-A in the absence of ATP. PAB II was used at 2000 fmol/25 µl. Three tubes each received a 5times aliquot of the mixture, one of which also received hexokinase (1.25 units) and glucose (10 mM). 25-µl aliquots were withdrawn from each tube (0 min time points), and the rest was prewarmed for 3 min at 37 °C. Reactions were started by the addition of ATP, ATPS, or AMPPNP, respectively. AMPPNP was added to the mixture containing hexokinase and glucose. Additional aliquots were taken as indicated. The rest of the original 18times mixture was used to demonstrate that hexokinase and glucose prevented polyadenylation (not shown). The size of DNA markers (in nucleotides) is indicated on the left.




DISCUSSION

The cell controls the length of poly(A) tails in three ways: during synthesis in the nucleus, during shortening in the cytoplasm, and, at least in special cases, during readenylation in the cytoplasm. The first type of control, limitation of poly(A) tail synthesis to a length of approximately 250 nucleotides, can be reconstituted from three purified proteins, poly(A) polymerase, CPSF, and PAB II. The two stimulatory factors, which are also responsible for the primer specificity of the polyadenylation reaction, act by forming a complex that includes poly(A) polymerase and the substrate RNA. This complex is able to synthesize a full-length poly(A) tail processively without disintegration (Bienroth et al., 1993). The length of poly(A) tails generated during this processive burst of synthesis was higher in this study than in previous experiments (Wahle, 1991b). The reason seems to be that length control depends on certain properties of poly(A) polymerase.^3 Whereas previously the proteolytically shortened poly(A) polymerase from calf thymus (Wahle, 1991a) was used, the experiments reported here were carried out with a full-length protein purified from recombinant E. coli. This observation suggests that length control is not rigid and may be subject to regulation.

Once a full-length tail has been made, polyadenylation essentially terminates by a transition from processive to distributive elongation. Direct evidence for a distributive elongation of long poly(A) tails is a synchronous elongation of all RNA molecules under conditions of limiting poly(A) polymerase and a dependence of the elongation rate on the concentration of poly(A) polymerase. By both criteria, elongation of short tails is processive. The processivity of the latter reaction has also been demonstrated by a primer challenge experiment (Bienroth et al., 1993). Also, under conditions that only allow distributive elongation of short tails (presence of only one stimulatory factor), there is no significant difference between the elongation rates of short and long tails. Thus, a switch from processive to distributive elongation as the mechanism for length control also explains the protein requirements of the reaction; only those conditions that are appropriate for processive elongation of short tails show length control. This includes the presence of both CPSF and PAB II and the use of a homologous poly(A) polymerase.

Under all other conditions, long and short tails behave quite similarly. Also, under conditions appropriate for length control, long tails still grow at a rate far exceeding the unstimulated rate observed with poly(A) polymerase alone. Thus, there is no lack of stimulation by individual factors and no inhibition of the elongation of long tails. In particular, the two RNA binding factors do not sequester the 3`-end of the RNA in a nonaccessible complex once the correct length has been reached. There is only a specific inability of long tails to mediate the interaction of all three proteins that is responsible for processive elongation.

The length control mechanism involves a true measurement of poly(A) tail length, as evidenced by the function of length control with premade poly(A) tails of different lengths, the lack of sensitivity to large changes in the rate of elongation, and the ability of the polyadenylation machinery to discriminate between short and long tails when challenged with a mixture of the two.

What is the yard stick that the polyadenylation complex uses to measure poly(A) tail length? The most obvious possibility is that the complex senses the number of PAB II molecules it contains. PAB II can coat high molecular weight poly(A) at a stoichiometry of nearly 25 nt/protein (Wahle et al., 1993), and there is no reason why it should not do so during poly(A) tail elongation. In the absence of CPSF, elongation of a poly(A) tail clearly requires its binding of multiple molecules of PAB II. The optimal amount of PAB II tends to be slightly less than expected from measurements of binding stoichiometry; thus elongation may be most efficient when tails are not completely coated. The requirement for a similar amount of PAB II in the presence of CPSF is more apparent when the poly(A) tails present at the start of the reaction are longer (Fig. 4). This reveals that the experiment is probably unable to measure the true PAB II optimum. The higher concentrations that are optimal for the long tails generated during the reaction are inhibitory for the short ones present initially ( Fig. 4and Fig. 5).

If the measurement of poly(A) tail length involves coating the tail with PAB II, one might predict that, at the low PAB II concentrations sufficient to induce processive elongation of short tails, length control should not be functional. Evidently, this is not the case. This might mean that the termination of processive elongation depends on poly(A) tail length in a manner not involving the number of PAB II molecules. However, detailed kinetics always showed a lack of synchrony during burst elongation (Fig. 3). Thus, the number of PAB II molecules bound to an individual poly(A) tail may differ from what is calculated from the molar ratio in the entire reaction, because the population of RNA molecules is probably heterogeneous. Some molecules may recruit a sufficient amount of PAB II to terminate processive synthesis, whereas others may be devoid of PAB II and thus also fail to be elongated efficiently.

Regardless of the exact mechanism, a disruption of the polyadenylation complex must take place once 250 As have been polymerized. The nature of this event remains to be determined.

From careful in vivo pulse-labeling studies of HeLa cells, Sawicki et al.(1977) concluded that there were two distinct modes of adenosine incorporation into nuclear poly(A) tails. Synthesis of a full-length poly(A) tail of about 230 nucleotides was found to take less than a minute. In addition, poly(A) tails that were already full-length at the time of pulse labeling were subject to a slower end addition with 5-10 nucleotides added in a 2-min pulse. It is reassuring that the kinetics described here for the purified in vitro system can fully account for the in vivo incorporation kinetics.


FOOTNOTES

(^1)
The abbreviations used: CPSF, cleavage and polyadenylation specificity factor; AMPPNP, adenylyl beta,-imidodiphosphate; ATPS, adenosine 5`-O-(3-thiotriphosphate); BSA, bovine serum albumin; PAB II, poly(A) binding protein II; nt, nucleotide(s).

(^2)
G. Martin and W. Keller, personal communication.

(^3)
T. Wittmann and E. Wahle, unpublished results.

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

§
Recipient of a Heisenberg fellowship from the Deutsche Forschungsgemeinschaft, Federal Republic of Germany. To whom correspondence should be addressed: Dept. of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.: 41-61-2672071; Fax: 41-61-2672078.


ACKNOWLEDGEMENTS

I thank Georges Martin and Pascal Preker for their gifts of purified poly(A) polymerases, Walter Keller for his support, and Witek Filipowicz, Andreas Jenny, Walter Keller, Lionel Minvielle, and Helen Rothnie for reading the manuscript.


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