(Received for publication, August 5, 1994; and in revised form, October 24, 1994)
From the
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.
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--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), ()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.
Figure 1:
Poly(A) tail length control with
premade tails. A 25 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.
Figure 2:
Length control requires CPSF and PAB II. A, a 33 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 26 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 8 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 34 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.
Figure 6:
Competition of polyadenylated and
oligoadenylated RNA. A 25 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 4
aliquot was mixed with 36 fmol of
bovine poly(A) polymerase (PAP), and a 5
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.
Figure 7:
Stability of poly(A) tails. A 10
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.
Figure 8:
Competition between short and long tails
in the presence of ATP analogs. An 18 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 5
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, ATP
S, or AMPPNP,
respectively. AMPPNP was added to the mixture containing hexokinase and
glucose. Additional aliquots were taken as indicated. The rest of the
original 18
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.
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. 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.