(Received for publication, July 14, 1995; and in revised form, September 9, 1995)
From the
We have constructed deletions in the nonconserved regions at the amino and carboxyl ends of the poly(A) polymerase (PAP) of Saccharomyces cerevisiae and examined the effects of these truncations on function of the enzyme. PAP synthesizes a poly(A) tail onto the 3`-end of RNA without any primer specificity but, in the presence of cellular factors, is directed specifically to the cleaved ends of mRNA precursors. The last 31 amino acids of PAP are dispensable for both nonspecific and specific activities. Removal of the next 36 amino acids affects an RNA binding domain, which is essential for the activity of the enzyme and for cell viability. This novel RNA binding site was further localized using additional deletions, cyanogen bromide cleavage of PAP cross-linked with RNA or 8-azido-ATP, and a monoclonal antibody against a COOH-terminal PAP epitope. A deletion that partially disrupts this domain has reduced nonspecific activity but functions in specific polyadenylation. In contrast, deletion of the first 18 amino acids of PAP has no effect on nonspecific polyadenylation but completely eliminates specific activity. This region is essential for enzyme function in vivo and is probably involved in the interaction of PAP with other protein(s) of the polyadenylation machinery.
The yeast Saccharomyces cerevisiae shares mechanisms of
mRNA maturation common to most eukaryotic species. One step in mRNA
biogenesis is the formation of a modified 3`-end, a complex and highly
regulated process that includes recognition of specific RNA sequence,
cleavage and polyadenylation, and termination of poly(A) addition once
a certain average tail length is reached(1, 2) . This
is accomplished by a multiprotein complex, which in yeast can be
separated into four factors (CF I, CF II, PF I, and PAP) ()essential for reconstitution of accurate 3`-end formation in vitro(3) . The yeast PAP is an important component
of the 3`-processing machinery. When mixed with partially purified CF I
and PF I, it is active only on RNA substrates containing specific
polyadenylation signal sequences (3) . However, replacement of
magnesium for manganese or separation of PAP from CF I and/or PF I (3) completely eliminates this specificity. Components in
mammalian cells, similar to CF I and PF I, provide analogous
functions(1, 2) . In this sense, PAP can be considered
as a catalytic subunit of a multicomponent enzyme, which requires at
least two other polypeptides to confer specificity for RNA substrate. A
similar situation has been described for vaccinia PAP, in which two
proteins comprise the holoenzyme(4, 5) .
The nature
of the interaction of PAP and other components, as well as the
interaction of PAP with RNA and ATP during catalysis, is mostly
unknown. Purified yeast PAP has been characterized(6) , and the
gene has been cloned and expressed in Escherichia
coli(7) . By using PAP as a bait in the two-hybrid system
for screening yeast DNA libraries, it has been shown that PAP interacts
with FIP1, a component of the PF I factor(8) , and with PIP2, a
protein homologous to ubiquitin-activating enzyme, and PIP3, a protein
of unknown function (9) . ()
The yeast PAP has strong homology in some regions with vertebrate PAPs (10) but also has unique nonconserved sequences located in the amino and carboxyl-terminal parts of the protein. There is a common RNA binding domain (RNP1) (7) whose role in PAP function has been confirmed by mutagenesis of the bovine gene(11) . Surprisingly, similar conservation does not exist when the eukaryotic PAP sequences are compared to that of PAP from E. coli(12, 13) or Vaccinia virus(5) . A knowledge of the mechanism of poly(A) polymerase action and a correlation of structure with function would provide useful insights into the regulation of polyadenylation and its interaction with other components of the polyadenylation complex. In this paper, we describe a previously uncharacterized region at the amino terminus of the yeast PAP, which is responsible for specific protein-protein interaction, and a new and unsuspected RNA binding site at the carboxyl end. We also discuss the importance of these regions to in vitro polyadenylation and cell viability.
The pJPAP1 expression plasmid was
a kind gift of J. Lingner and W. Keller(7) . Serial deletions
of the 5`- and 3`-ends of the PAP gene were made using polymerase chain
reaction and natural restriction sites. To delete the last 67 amino
acids of PAP, pJ6PAP was created by digesting pJPAP1 with BstEII and PvuII, and to delete the last 20 amino
acids (pJ
7PAP), pJPAP1 was digested with BglII and PvuII. The digestion products were blunted with DNA polymerase
I (Klenow) fragment (New England Biolabs), and the large fragments were
circularized using DNA ligase (New England Biolabs). pJ
8PAP
(deleting the last 55 amino acids), pJ
9PAP (deleting the last 43
amino acids), pJ
10PAP (deleting the last 31 amino acids),
pJ
14PAP (deleting amino-terminal amino acids 3-18), and
pJ
15PAP (deleting amino-terminal amino acids 3-44) were made
by replacement of the PAP gene sequence between the SacI and PvuII sites in pJPAP1 with appropriate polymerase chain
reaction fragments (Fig. 1). For testing function of the
truncated PAPs in yeast, the same fragments were inserted into pHCp50
plasmid, a derivative of YCp50(18) , with the HindIII
fragment of yeast chromosomal DNA containing PAP1 sequence.
pHCp50 was a kind gift of Dr. W. Keller. All restriction and modifying
enzymes were from New England Biolabs.
Figure 1: Schematic diagram of yeast poly(A) polymerase showing known functional and structural domains and sites of deletion used in this study. The scale represents the number of amino acids, and the relative positions of restriction sites used for truncations and subcloning are indicated. The positions of epitopes recognized by two different monoclonal antibodies (mAb) against the yeast PAP (21) are also marked. The table (inset) represents the reference number of the deletion, the number of amino acids (-a.a.) deleted from the amino (N)- or carboxyl (C)-terminal parts of the protein, and the nonspecific activity of each truncated recombinant PAP in E. coli cell lysate expressed as a percentage of that of the wild type enzyme.
Yeast whole extract was prepared as described (3) except that the spheroplasting step was omitted. Fractions containing CF II/PF I were concentrated to 4 mg/ml, and CF I was further purified about 100-fold and used at a concentration of 0.4 mg/ml. All extracts and factors were frozen in liquid nitrogen and stored at -70 °C.
Protein concentrations were determined by the procedure of Bradford (20) using the Bio-Rad kit and bovine serum albumin as a standard. Protein markers were from New England Biolabs.
To assay specific polyadenylation, the reactions (16 µl) were assembled on ice and contained 1 mM magnesium acetate, 75 mM potassium acetate, 2% polyethylene glycol(8000), 2 mM ATP, 20 mM creatine phosphate, 10 nM (16 ng) labeled pre-cleaved mRNA, and 1.5 µM (0.6 µg) tRNA. The reactions contained either 1 µl of yeast whole cell extract (20 µg of protein) or 2 µl of CF I fraction (0.8 µg of protein), 2 µl of CF II/PF I (8 µg of protein), and 1 µl of diluted PAP (13 ng of protein) and were incubated at 30 °C for 20 min and stopped with proteinase K and SDS as described previously(3, 21) . The reaction products were fractionated on 5% polyacrylamide, 8.3 M urea gels and visualized by autoradiography.
For cyanogen bromide (CNBr) cleavage(22) , UV-cross-linked samples were precipitated with 10% trichloroacetic acid, rinsed with ethyl ether, and resuspended in 100 µl of 70% formic acid containing 10-100 µg/ml freshly sublimated CNBr. After an overnight reaction at room temperature, samples were diluted with water and lyophilized twice. The resulting peptides were separated on a 16% polyacrylamide gel using Tris-tricine-SDS buffer(23, 24) , transferred to PVDF membrane, and detected by autoradiography. The membrane was treated for immunoblot analysis using PAP-specific monoclonal antibody as described previously (21) .
Truncated forms of PAP were purified using the
protocol described under ``Materials and Methods.'' It is
interesting that some of the COOH-terminal truncated proteins exhibited
chromatographic behavior different from that of the full-length enzyme.
For example, on a DEAE-Sephacel column, wild type PAP comes out in the
first half of the flow through fractions at a KCl concentration of 100
mM, but 8PAP eluted from DEAE-Sephacel with 250 mM KCl. On a phosphocellulose column, this effect is even more
dramatic. Wild type PAP elutes at 400 mM KCl,
10PAP at
300 mM, and
9PAP at 50 mM KCl;
8PAP does
not bind phosphocellulose at all and appears in the flow-through
fractions at 0 mM KCl. This altered behavior of
9PAP and
8PAP correlates with the large drop-off in nonspecific activity
observed with these truncations and may indicate that we have deleted a
phosphate binding site.
Two different
RNA substrates, both of which terminate at the GAL7 poly(A)
site, were used to assay the ability of the enzyme to participate with
other protein factors in specific polyadenylation. The first substrate
contains 161 nucleotides of the wild type GAL7 sequence
upstream of the poly(A) site, and the second is identical except for a
deletion of a 12-nucleotide UA repeat, an element essential for
specific activity (3) (Fig. 2A, lanes 1 and 2). Purified recombinant PAP was mixed with either of
these substrates in the presence of CF I and PF I. As expected, in the
absence of PAP, no poly(A) addition activity is observed (Fig. 2A, lane 3). The 10PAP exhibits the
same specific activity as the wild type enzyme (Fig. 2A, lanes 4 and 5). As might be
expected, the amount of polyadenylated product in reactions containing
9PAP is less (Fig. 2A, lane 6), but all
of this is the result of specific polyadenylation, since no product is
observed using the mutated substrate (Fig. 2A, lanes 7-9).
Figure 2:
Specific polyadenylation assay of
truncated PAPs. Panel A, the activity of COOH-terminal
deletion mutants. Precleaved radiolabeled GAL7 wild type (lanes 1, 3-6) or mutant transcripts (lanes
2, 7-9) were assayed with yeast extract (lanes
1 and 2) or with partially purified CF I and PF I (lanes 3-9) supplemented with PAPs (lane 3, no
PAP; lanes 4 and 7, wild type PAP; lanes 5 and 8, 10PAP; lanes 6 and 9,
9PAP). Panel B, the activity of the amino-terminal
deletion mutant. Precleaved radiolabeled GAL7 transcript was
assayed with yeast extract (lane 1) or partially purified CF I
and PF I (lanes 2-4) supplemented with wild type PAP (lane 3),
14PAP (lane 4), or without PAP (lane 2). Lanes 5 (wtPAP) and 6 (
14PAP)
show a nonspecific assay using GAL7 transcript in presence of
manganese and without specificity factors.
Elimination of amino acids 3 through 18
(14PAP) from the amino-terminal end of PAP has no effect on
nonspecific activity (Fig. 1). However, this truncation
completely knocks out specific polyadenylation activity (Fig. 2B, lanes 3 and 4). The
14PAP used in this experiment is active for nonspecific
polyadenylation, since it exhibits wild type activity when magnesium is
replaced with manganese (Fig. 2B, lanes 5 and 6).
Figure 3: Panel A, UV cross-linking of PAP to RNA. Randomly radiolabeled GAL7 precleaved RNA, with (lane 2) and without (lane 1) competitor tRNA, was UV cross-linked to PAP, digested with RNase A, separated on a 10% polyacrylamide gel in Tris glycine SDS buffer, and visualized by autoradiography. Panel B, PAP was cross-linked to GAL7 precleaved RNA labeled in different ways, and the complexes were digested with RNase A and partially cleaved with CNBr. Protein fragments were separated on a 16% polyacrylamide gel in Tris-tricine-SDS buffer and blotted onto PVDF membrane. Peptides were visualized by immunostaining with antibody specific for the PAP carboxyl end (lanes 4-6) and radioactive peptides by autoradiography (lanes 1-3); lanes 1 and 4, 3`-end-labeled RNA; lanes 2 and 5, 5`-end-labeled RNA, lanes 3 and 6, randomly labeled RNA. The size and position of CNBr-generated peptides detected with the PAP COOH-terminal specific antibody is indicated on the right. This immunostaining reveals a ladder of partial digestion products containing C9. Panel C, diagram of the nine peptides (C1-C9) generated by CNBr cleavage of purified recombinant PAP. The peptides are arranged in order from the amino terminus (left) to carboxyl terminus (right). The molecular masses of the fragments are calculated from the number of amino acids.
Figure 4: Specificity of UV cross-linking of 8-azido-ATP to PAP was determined by competition with different nucleic acids. Competitors were preincubated with PAP and 15 µM 8-azido-ATP prior to irradiation, and complexes were separated on a 10% SDS-polyacrylamide gel and visualized by autoradiography. Lanes 1 and 11, no competitor; lane 2, 250 µM ATP; lane 3, 250 µM GTP; lane 4, 250 µM CTP; lane 5, 250 µM UTP; lane 6, 250 µM 2`-dATP; lane 7, 250 µM 3`-dATP; lane 8, 0.3 µM poly(A); lane 9, 100 µM tRNA; lane 10, 10 µM single-stranded DNA, a 24-base oligonucleotide.
To investigate the specificity of the azido-ATP cross-linking, we
performed a competition assay by mixing different nucleic acids or
nucleotides with PAP and azido-ATP before UV irradiation. RNA
molecules, and even single-stranded DNA, competed with azido-ATP for
PAP binding much more effectively than ATP and other nucleotide
triphosphates (Fig. 4). These results suggest that azido-ATP is
most likely interacting with an RNA binding site. To determine if this
site corresponded to the one at the PAP carboxyl end, azido-ATP was
cross-linked to wild type PAP, 10PAP, and
9PAP (Fig. 5).
9PAP cross-links to 8-azido ATP much less
efficiently than wild type PAP, and
10PAP cross-links more
efficiently, consistent with the results obtained for RNA binding by
enzyme kinetic analysis.
Figure 5:
The efficiency of 8-azido-ATP binding of
different truncated PAPs was determined by UV cross-linking followed by
separation on a 10% SDS-polyacrylamide gel, which was stained with
silver (lanes 1-3), and radioactive proteins were
visualized by autoradiography (lanes 5-7). Lanes 1 and 5, wild type PAP; lanes 2 and 6,
10PAP; and lanes 3 and 7,
9PAP. Lane
4, broad range marker proteins (New England
Biolabs).
To our surprise, 8-azido-ATP labeled the
same peptide as did the randomly labeled RNA (Fig. 6, lanes
1 and 3). To further confirm that the carboxyl-terminal
peptide was being labeled, we cross-linked azido-ATP to 10PAP,
which lacks the last 31 amino acids and would give a correspondingly
shorter terminal peptide after CNBr cleavage. The labeled peptides
after partial CNBr cleavage of
10PAP shifted down the predicted
distance (Fig. 6, lane 2), and this truncated PAP lost
the epitope recognized by the COOH-terminal specific antibody (Fig. 6, lane 4)(21) .
Figure 6:
CNBr digest of wild type PAP (lane 1 and 3) and 10PAP (lanes 2 and 4)
UV-cross-linked with radiolabeled 8-azido-ATP. Samples were separated
on 16% polyacrylamide gel in Tris-tricine-SDS buffer and blotted onto
PVDF membrane. Peptides were visualized by immunostaining with antibody
specific for the PAP carboxyl end (lanes 3 and 4) and
radioactive peptides by autoradiography (lanes 1 and 2). The size and position of CNBr-generated peptides is
indicated on the right (see Fig. 3, panels B and C). The carboxyl-terminal peptides (C9) of wild type (a) and
10PAP (b) are indicated with arrows.
The results described in this report present new features of the yeast poly(A) polymerase. A novel RNA binding site has been found and characterized in the carboxyl end of PAP, and a sequence involved in specific protein-protein interaction has been identified in the first 18 amino acids.
From amino acids 80-390, the yeast PAP
has a strong, 70% homology to mammalian PAPs (Fig. 1)(7) . The RNP1 sequence, a conserved motif found
in many RNA binding proteins(27) , is located in this region.
The first 79 amino acids and the last 178 amino acids of yeast PAP have
very loose or very little homology to the mammalian counterparts.
However, elimination of the first 44 amino acids (15PAP) or the
last 67 amino acids (
6PAP) completely inactivates the yeast
enzyme. On the other hand, elimination of 16 amino acids from the
amino-terminal end (
14PAP) or a COOH-terminal deletion of 31 amino
acids (
10PAP) has no effect on the nonspecific activity of the
yeast PAP.
Deletion of an additional 12 amino acids (9PAP) from
the COOH-terminal end of PAP causes a dramatic loss of nonspecific
activity in crude extract, decreases the protein's ability to
bind phosphocellulose, and increases the constant of RNA binding (K
) by 50-fold. The sequence removed in
9PAP (Fig. 7A) is highly basic; seven of the amino acids are
lysine and arginine. Our data are consistent with a model in which this
deleted sequence is part of an RNA binding site. After deletion of the
next 12 amino acids (
8PAP), only a low level of activity remains,
and deletion of the next 12 amino acids (
6PAP) completely
inactivates the enzyme. A sequence related to that deleted in the yeast
8PAP and
9PAP is located in the amino-terminal region of
mammalian PAPs (28, 29) and Xenopus PAP (10) (Fig. 7A). Similarity can also be found in
parts of the Vaccinia (5) and E. coli enzymes(12) . However, this stretch does not resemble any
of the previously described RNA binding motifs(27) .
Figure 7: Comparison of the amino acid sequences of the novel RNA binding site (A) and specificity domain (B) of yeast PAP with other poly(A) polymerases.
Because
recombinant yeast PAP can use ATP instead of RNA as a primer ()and 8-azido-ATP binds to the carboxyl RNA binding site, we
suspected that this site might bind the 3`-end of the RNA primer in
preparation to receive an adenosyl residue. However, by using RNAs
labeled randomly or at either end, we could show that, even though this
site is essential for activity, it is not specific for any portion of
the RNA strand. This conclusion was supported by the binding
competition assay where even single-stranded DNA competed effectively.
These results also imply that the authentic ATP binding site is very
specific and that the bulky azido group in this position of the analog
interferes with binding.
A good candidate for a 3`-RNA binding site may be the RNP1 motif, which is very conserved among the eukaryotic, nonviral PAPs (10) and is adjacent to a potential catalytic domain(11) . Mutations in the RNP1 of bovine PAP inhibit both specific and nonspecific activities, and this defect could be rescued by increasing the concentration of RNA(11) , suggesting it is indeed involved in RNA binding, perhaps of the 3`-end. The fact that the major yeast PAP peptide cross-linked to 3`-end-labeled RNA after CNBr cleavage is similar in size to the yeast RNP1-containing peptide lends support to this hypothesis.
Deletion of 16 amino
acids(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) from
the amino terminus of the yeast PAP (14PAP) completely knocked out
the specific activity of the enzyme, leaving the nonspecific activity
untouched. Regions with strong similarity (56%) to this yeast sequence
are found in the amino-terminal ends of the higher eukaryotic enzymes (Fig. 7B), but this region has not been correlated with
any particular function. The discovery of a specificity determinant in
this part of the yeast PAP was surprising because previous mutagenesis
of the bovine PAP indicated that a domain with similar function
coincided with a nuclear localization signal (NLS1) in the
COOH-terminal part of the protein(11) . This region has no
homology to the motif we have identified, and it is possible that two
different domains contribute to the interactions of PAP with other
polyadenylation factors.
Such a model invoking multiple contacts would not be implausible, since it has been shown that the processivity of the mammalian PAP is stimulated by either CPSF, the mammalian specificity factor, or PABII, a nuclear poly(A) binding protein, but is maximal when both factors are present simultaneously(30, 31) . Interestingly, the U1A protein can down-regulate PAP activity, apparently through a domain which is further toward the carboxyl end of PAP than the NLS1 site, yet this interaction does not affect the ability of PAP to stimulate the cleavage reaction(32) , again suggesting that two factors may contact PAP at the same time.
Recent studies are beginning to clarify the association of the yeast PAP with equivalent factors. For example, application of the two-hybrid system identified FIP1 as a protein that interacts with the yeast PAP (8) . FIP1 is a component of PF I(8) , a factor that works in conjunction with CF I, the yeast analog of CPSF, to catalyze specific poly(A) addition(3) . It appears that FIP1 links PAP to RNA14, a subunit of CF I. Depletion of PAP from yeast-processing extracts with PAP-specific antibody leads to a loss of both cleavage and poly(A) addition activity(21, 33) . Both activities can be restored if only CF I is added to the depleted extracts, suggesting that a direct interaction of CF I with PAP may also exist. It will be interesting to determine which protein(s) interact with PAP through the amino-terminal domain identified in this study.