From the Plant Physiology/Biochemistry/Molecular Biology Program,
Department of Agronomy, University of Kentucky,
Lexington, Kentucky 40546
We have isolated cDNA clones encoding a novel
RNA-binding protein that is a component of a multisubunit poly(A)
polymerase from pea seedlings. The encoded protein bears a significant
resemblance to polynucleotide phosphorylases (PNPases) from bacteria
and chloroplasts. More significantly, this RNA-binding protein is able
to degrade RNAs with the resultant production of nucleotide
diphosphates, and it can add extended polyadenylate tracts to RNAs
using ADP as a donor for adenylate moieties. These activities are
characteristic of PNPase. Antibodies raised against the cloned
protein simultaneously immunoprecipitate both poly(A) polymerase and
PNPase activity. We conclude from these studies that PNPase is the
RNA-binding cofactor for this poly(A) polymerase and is an integral
player in the reaction catalyzed by this enzyme. The identification of this RNA-binding protein as PNPase, which is a chloroplast-localized enzyme known to be involved in mRNA 3'-end determination and
turnover (Hayes, R., Kudla, J., Schuster, G., Gabay, L., Maliga, P.,
and Gruissem, W. (1996) EMBO J. 15, 1132-1141), raises
interesting questions regarding the subcellular location of the poly(A)
polymerase under study. We have reexamined this issue, and we find that
this enzyme can be detected in chloroplast extracts. The involvement of
PNPase in polyadenylation in vitro provides a biochemical
rationale for the link between chloroplast RNA polyadenylation and RNA
turnover which has been noted by others (Lisitsky, I., Klaff, P., and
Schuster, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13398-13403).
 |
INTRODUCTION |
Poly(A) tails at the 3'-ends of RNAs are nearly ubiquitous in
biology; they are a hallmark of mature messenger RNAs in eukaryotes (1)
and have been identified as well in mitochondria (2), bacteria (3-7),
and chloroplasts (8, 9). In eukaryotes, poly(A) tails are important
cis-determinants of mRNA translatability and function in
concert with characteristic poly(A)-binding proteins to promote
translation initiation in vivo and in vitro (10). In contrast, in bacteria, poly(A) tails appear to be a determinant of
instability, serving to increase the rate with which RNAs are degraded
in vivo (5-7) and in vitro (11).
In our ongoing studies of mRNA polyadenylation in plants, we
identified a novel poly(A) polymerase that consisted of two distinct components (12), both of which were required for activity. One of these
(termed PAP-I in previous papers) copurified with a 43-kDa polypeptide
that could be recognized by monoclonal antibodies raised against the
yeast poly(A) polymerase (12). The other (PAP-III) included one or more
RNA-binding proteins of between 100 and 105 kDa in size; only RNAs
associated with these proteins could be polyadenylated by this enzyme
(13). Here, we describe the isolation of cDNAs encoding these
polypeptides. Analysis of these clones suggests that these RNA-binding
proteins are related and are in actuality polynucleotide phosphorylase.
In addition, we report that the poly(A) polymerase under study can be
found in chloroplasts. These findings raise interesting questions
regarding the interplay between RNA polyadenylation and turnover in
chloroplasts.
 |
MATERIALS AND METHODS |
Isolation of cDNA Clones--
PAP-III, purified through the
Mono Q stage (12, 13), was separated by
SDS-PAGE.1 Proteins were
transferred to Immobilon-P membranes (Millipore) by electroblotting and
the resolved polypeptides visualized by staining briefly with Coomassie
Brilliant Blue. The PAP-III polypeptides (13) were excised and
submitted to the University of Kentucky Macromolecular Structure
Analysis Facility for proteolytic digestion with endo-LysC and
NH2-terminal peptide sequencing. The resulting sequences
were used to design degenerate oligonucleotides intended to amplify the
coding segments for the corresponding peptides. These
oligonucleotides (5'-CGCTGCAGACIARIGCYTCIACYTC-3' and
5'-GCGGATCCGTIGARGTIGGICARGA-3', where I is inosine, R
is purine, and Y is pyrimidine) were used to amplify products from
a pea cDNA library (14). These products were cloned (after
digestion with BamHI and EcoRI) into
appropriately digested pBluescript (Stratagene) and the inserts of
different recombinants sequenced. Clones whose sequence matched those
predicted from the peptide sequences were used to probe the same
cDNA library, and several near full-length clones were identified.
These were sequenced using the dideoxy method (15).
Assays--
The assay for nonspecific plant poly(A) polymerase
activity has been described elsewhere (12, 13). In this study, bovine serum albumin was omitted from the polyadenylation reactions, and
poly(A) was added to a final concentration of 3.33 mg/ml except where
otherwise indicated. Unless noted otherwise PAP-I and PAP-III were
purified through the Mono Q steps (12). For the experiment in Fig. 4,
reactions were supplemented with the concentrations of unlabeled ADP
indicated.
To determine ADP-dependent adenylation, labeled RNA (RNA 3, described in Li et al. (13), in a total volume of 2 µl,
was mixed with 5 µl of PAP-III; the concentrations of labeled RNAs in
these reactions were about 40 nM. After 30 min at 30 °C,
5 µl of buffer I (12) and a revised poly(A) polymerase reaction mix
(167 mM Tris-HCl, pH 8.0, 267 mM KCl, 3.33 mM MgCl2, 0.33 mM EDTA, 3.33 mM dithiothreitol, 0.67% Nonidet P-40, and 3.33 mM ADP) were added. Reactions were incubated for 60 min at
30 °C, terminated by extraction with phenol and chloroform, and RNAs were recovered by precipitation with ethanol and separated on 6%
sequencing gels. Electrophoresis was carried out for a period of time
appropriate for the resolution of small RNAs; under these conditions,
larger RNAs failed to resolve well but instead were apparent as an
apparently homogeneous band. This property of our separations helps in
quantitating these reactions because the signals corresponding to
polyadenylated RNAs were localized in a small band rather than
dispersed (as would be the case if more extending runs were conducted).
Gels were visualized by autoradiography and quantitated using a
Molecular Dynamics PhosphorImaging System.
Phosphate-dependent RNA breakdown was assayed as above,
except ADP was replaced with 0.25 mM sodium phosphate, pH
7.4. 32P-Labeled RNA (rbcS-wt, derived from a
NsiI-HaeIII fragment of the pea
rbcS-E9 gene polyadenylation signal (16); 5,000-10,000 cpm,
corresponding to approximately 1 pmol, was used per reaction) was
preincubated with PAP-III for 15 min at 30 °C to permit binding of
RNA to PAP-III (for details of the labeling and purification of the
RNA, see Li et al. (13)). The PAP reaction mix, containing phosphate instead of ADP or ATP, was then added, and reactions were
incubated for 60 min at 30 °C (well within the period during which
the quantity of ADP increases in a linear manner). After halting
reactions with an equal volume of phenol/chloroform, 1-4-µl aliquots
of each reaction were applied to polyethyleneimine-cellulose thin layer
chromatography plates. The plates were developed in 0.3 M
LiCl and visualized by autoradiography. One µl each of 25 mM AMP, ADP, and ATP was applied as a standard (visualized
by irradiating plates directly with ultraviolet light). The
radioactivity in intact RNA (which remains at the origin) and ADP were
quantitated using a Molecular Dynamics PhosphorImaging System.
Preparation of Antibodies and Immunoprecipitations--
The
complete p98 coding region was cloned into pGEX-2T or pGEX-3X (Amersham
Pharmacia Biotech) to produce protein for antibody production. The
appropriate fragment was produced by polymerase chain
reaction with suitable restriction sites and reading frame adjustments incorporated into the primers. Escherichia coli
carrying the recombinant plasmid was induced with isopropyl
1-thio-
-D-galactopyranoside, and protein was purified by
affinity chromatography using glutathione-Sepharose 4B, as recommended
by the manufacturer (Pharmacia). Affinity-purified fusion proteins were
eluted from the affinity matrix with SDS-PAGE sample buffer (0.1 M Tris-HCl, pH 6.8, 2.9 M
-mercaptoethanol, 4% SDS, 0.2% bromphenol blue, 20% glycerol), separated by SDS-PAGE, and transferred to nitrocellulose membranes by electroblotting. The
fusion proteins were visualized by staining briefly with Coomassie Brilliant Blue, excised, and the nitrocellulose was dissolved in
dimethyl sulfoxide. These preparations were used to inject rabbits (200 µg/injection); the schedule for injection and other details have been
described elsewhere (17). Monospecific antibodies were purified from
serum as described (15).
PAP-III was immunoprecipitated from heparin-Sepharose-purified poly(A)
polymerase preparations (12). 20 µl of antibody (in Tris-buffered
saline) was added to 20 µl of poly(A) polymerase and incubated at
room temperature for 4 h with shaking. 20 µl of a 50% (v/v)
suspension of protein A-Sepharose (Pharmacia), equilibrated in buffer I
(40 mM KCl, 25 mM Hepes-KOH, pH 7.9, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10% glycerol, and 5 µg/ml each of
leupeptin, chymostatin, and antipain), was then added and the
suspension incubated for 30 min on ice. The protein A-Sepharose was
then collected by centrifugation (30 s in a microcentrifuge) and washed
three times with 400 µl of buffer I. The pellet was suspended in 30 µl of buffer I, and 10 µl was assayed for PAP-III and PNPase
activity as described above.
Preparation of Extracts and Immunoblot Analysis--
Pea
chloroplasts were isolated as described by Orozco et al.
(18), and extracts were prepared by suspending chloroplasts in a lysis
buffer (62.5 mM Tris-HCl, pH 7.5, and 2 mM
MgCl2) and removing insoluble debris by centrifugation.
Chloroplast extracts typically had protein concentrations between 1 and
5 mg/ml. These extracts were fractionated and assayed for poly(A)
polymerase activity as described (12). Alternatively, chloroplast
extracts were evaluated by immunoblot analysis (15), using appropriate antisera.
Denatured extracts from pea leaves and roots, and extracts from
isolated nuclei, were prepared essentially as described by Yang and
Hunt (17). These extracts were examined by immunoblot analysis using
the antisera described in the text and figure legends.
For immunoblots, proteins were separated by SDS-PAGE and transferred to
a nitrocellulose membrane using a Trans-Blot Cell (Bio-Rad) following
the manufacturer's recommendations. Filters were then washed and
probed with antibodies (at dilutions of 1/500 to 1/1000) as described
elsewhere (15, 17).
 |
RESULTS |
PAP-III Is Similar to Bacterial and Chloroplast Polynucleotide
Phosphorylase--
Previously, we reported that preparations of
PAP-III contained a group of polypeptides between 100 and 105 kDa in
size, one or more of which could be cross-linked to exogenous RNAs and
to the products of polyadenylation (13). Moreover, an association of
substrate RNAs with these polypeptides was necessary for
polyadenylation to occur (13). Accordingly, we obtained
NH2-terminal sequences from proteolytic fragments derived
from these polypeptides. Because it was not possible to resolve the two
or three individual species noted earlier (13), we isolated these
polypeptides in a single sample for peptide sequencing. Five peptide
sequences were obtained, and this information was used to isolate
corresponding cDNA clones. The longest cDNA (pQL105a) contained
a single open reading frame capable of encoding a polypeptide of 897 amino acids (termed hereafter as p98), with a predicted size of about
98 kDa (Fig. 1). Because this open
reading frame contained all of the peptides identified by
NH2-terminal sequencing of endo-LysC fragments from the
purified protein sample, it would appear that the various species arise from post-translational modification of a single polypeptide.

View larger version (70K):
[in this window]
[in a new window]
|
Fig. 1.
p98 is similar to chloroplast and bacterial
PNPases. The deduced amino acid sequence of p98 (noted here as
PAP-III; GenBank accession number AF010578) is shown on the top
line. The similarity with the spinach 100 ribonucleoprotein
(cpPNP; 23) and E. coli PNPase (ecoPNP; 19) is shown as
well. Homology was determined with ClustalW 1.6. Positions of amino
acid identity or chemical similarity (with respect to a consensus of
any two of the three sequences) are shown as capital letters
and dissimilar positions as lowercase letters. The peptides
derived from purified PAP-III from which NH2-terminal
sequences were obtained are underlined. The domain of the
E. coli PNPase which is homologous to the RNA binding domain
of ribosomal protein S1 (19, 20) is double underlined.
|
|
p98 displayed significant homology to a number of proteins in various
data bases. Most prominent among these were several polynucleotide
phosphorylases (or PNPases; Fig. 1); for example, the amino acid
identity of p98 and the E. coli PNPase was 40% (with
respect to p98) in the region of homology (amino acids 83-842 of p98).
The similarity with PNPases extended through the COOH-terminal domain
that contains the ribosomal protein S1-related RNA binding domain (Fig.
1; 19, 20), consistent with our finding that p98 is an RNA-binding
protein (13). p98 was also highly homologous to a chloroplast protein
described previously, a 100-kDa ribonucleoprotein from spinach which is
also related to the E. coli PNPase and has PNPase activities
(23; Fig. 1); in this case, the amino acid identity was 76%. The
similarity of p98 to the E. coli and spinach PNPases was
lowest at the NH2 terminus. This is most likely because of the presence of a chloroplast-targeting sequence in one or both polypeptides; the NH2-terminal domain of p98 is rich in
hydroxylated amino acids and is free of acidic residues,
characteristics commonly found in transit peptides (21).
The sequence similarity of p98 to PNPase suggested a biochemical
similarity as well. Consequently, we assayed purified PAP-III for
phosphate-dependent breakdown of RNA (with a subsequent
production of NDPs) and for the extension of RNA substrates using NDPs
as donors for nucleotide units, two activities associated with PNPase enzymes (22). After incubation of PAP-III with inorganic phosphate and
RNA that had been labeled with [
-32P]ATP, labeled ADP
could be detected (Fig. 2A);
this ADP formation was dependent on the presence of phosphate. In
addition, incubation of labeled RNA with PAP-III in the presence of ADP
resulted in a substantial elongation of the labeled RNA (Fig.
2B). These results indicate that PAP-III has biochemical
activities similar to those of a PNPase.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2.
PAP-III has PNPase activities.
Panel A, phosphate-dependent formation of ADP.
RNA that had been prepared using labeled ATP was prebound with PAP-III,
and ADP formation following the addition of phosphate was monitored by
thin layer chromatography. 5 (lane 1), 10 (lane
2), or 20 (lane 3) µl of Mono Q-purified PAP-III was
added in each 59-µl reaction. In the sample for lane 4,
the conditions were identical to those of the sample for lane
3, but inorganic phosphate was omitted from the reaction. The
sample for lane 5 was prepared as those for lanes
1-3, but PAP-III was replaced with buffer I. Panel B,
ADP-dependent adenylation. Labeled RNA was prebound with
PAP-III and ADP-dependent adenylation monitored following
the addition of ADP-containing reaction mix. Adenylation reactions were
incubated for 60 min (lane 1) and analyzed as described
under "Materials and Methods." In the sample for lane 2,
the conditions were identical to those of the sample for lane
1, but ADP was omitted from the reaction. In the sample for
lane 3, the conditions were identical to those of the sample
for lane 1, but PAP-III was replaced with buffer I.
|
|
Several attempts were made to isolate and characterize a GST-p98 fusion
protein in a soluble form from extracts prepared from E. coli. However, the fusion protein (as well as thrombin-treated protein) produced could not be eluted from the glutathione-Sepharose affinity matrix under nondenaturing conditions, thereby precluding a
direct assay of the fusion protein. Thus, as an alternative, we raised
antibodies against the gel-purified fusion protein and examined the
ability of these antibodies to immunoprecipitate PAP-III and PNPase
activities. After affinity purification, these antibodies specifically
recognized a group of polypeptides that copurified with PAP-III
activity (Fig. 3). The mobilities of this group of polypeptides were similar to those present in purified PAP-III
(13), and this was consistent in these gels with the predicted size (98 kDa; Fig. 3).

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 3.
p98 copurifies with PAP-III activity.
Samples of the pea poly(A) polymerase or PAP-III from different stages
of purification were analyzed by immunoblotting, using
affinity-purified antibody against p98. Lane 1, 24 µg of
purified GST protein (not GST-p98). Lane 2, 1 µg of Mono
Q-purified PAP-III. Lane 3, 4 µg of
heparin-Sepharose-purified poly(A) polymerase. Lane 4, 7 µg of DEAE-purified poly(A) polymerase. Lane 5, 85 µg of
whole leaf extract. The positions of size standards are shown to the
right of the blot.
|
|
Subsequently, a partially purified preparation of this poly(A)
polymerase (through the heparin-Sepharose step; this preparation contains PAP-I and PAP-III (see Ref. 12)) was treated sequentially with
affinity-purified anti-p98 antibodies and protein A-Sepharose. After
pelleting and washing the protein A-Sepharose, proteins that were bound
to the pellets were assayed for poly(A) polymerase and PNPase activity.
As shown in Table I, substantial PAP-III activity (defined as PAP-I-dependent poly(A) polymerase
activity) could be detected in the immunoprecipitate. Importantly, the
dependence on PAP-I (compare the +PAP-I and
PAP-I columns in Table I)
indicates that PAP-III was efficiently immunoprecipitated but that the
PAP-I that is present in the partially purified PAP preparation was not. The immunoprecipitate also possessed significant PNPase activity (Table I). In contrast, no significant activity could be seen in
samples treated with protein A-Sepharose but not with antibody.
View this table:
[in this window]
[in a new window]
|
Table I
Anti-PAP-III antibodies immunoprecipitate PAP-III and PNPase
activity
Partially purified PAP (after the heparin-Sepharose step (12)) was
treated with antibody raised against a PAP-III-GST fusion protein and
affinity purified using the same protein. Complexes containing this
antibody were adsorbed to protein A-Sepharose, collected by
centrifugation, washed extensively, and assayed for PAP-III activity
(defined as PAP-I-stimulated poly(A) polymerase activity) and PNPase
activity (here, phosphate-dependent ADP formation from RNAs
prepared with labeled ATP). The immunoprecipitate was compared with
purified PAP-III (after the Mono Q step (12)) and with a mock
immunoprecipitation in which antibody was omitted. PAP activity is
represented as cpm incorporated from [ -32P]ATP into
polynucleotide in 120 min, and PNPase activity as the percent of label
in RNA converted to ADP in the same time.
|
|
The Combination of PAP-I + PAP-III Is a Poly(A)
Polymerase--
The realization that p98 is a PNPase raises some
important questions regarding the polyadenylation activity observed
with the combination of PAP-I + PAP-III. In particular, it is possible that the observed activity (12, 13), which was measured as incorporation of label from [
-32P]ATP into long
molecules, might be the result of the action of a PNPase using trace
amounts of [
-32P]ADP. This ADP might be present as a
contaminant in commercial [
-32P]ATP preparations or
might be produced by the action of unanticipated ATPases, nucleases,
and nucleoside diphosphate kinases, or other contaminants. To evaluate
this, the effect of added unlabeled ADP on the incorporation of label
from [
-32P]ATP was examined. As seen in Fig.
4, relatively low concentrations of
unlabeled ADP had a modest stimulatory effect on the incorporation of
label from ATP into long molecules. Because the lowest concentration of
ADP added in this experiment (0.02 mM) was at least 10-fold greater than possible trace contamination of [
-32P]ADP
in the preparations of [
-32P]ATP we use (labeled ADP
was not detectable by thin layer chromatography), we conclude that ADP
present in the substrate mixture is not responsible for the
ATP-dependent polyadenylation observed here or previously. In addition, because unlabeled ADP concentrations comparable to the ATP
concentrations (0.25 mM) used in our experiments had no effect on activity (Fig. 4), we conclude that conversion of ATP to ADP
is not responsible for the observed ATP-dependent
polyadenylation. Thus, the combination of PAP-I + PAP-III in fact can
use ATP to extend poly(A) tracts on substrate RNAs.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
ADP does not inhibit
ATP-dependent adenylation by PAP-I + PAP-III.
Increasing concentrations of ADP were added to standard polyadenylation
reactions containing 10 µl of Mono Q-purified PAP-I and 0.4 (squares), 1 (diamonds), or 2.5 (circles) µl of PAP-III. Reactions were terminated after
120 min and incorporation of label into product determined as described
under "Materials and Methods."
|
|
The Poly(A) Polymerase under Study Is a Chloroplast
Enzyme--
The above results suggest that the poly(A) polymerase we
have characterized (12, 13) may be located in the chloroplast (from
which other plant PNPases have been isolated (23)). To test this, we
prepared extracts from purified pea chloroplasts and assayed these for
poly(A) polymerase activity. Using our standard assay, we could detect
activity that, like the enzyme isolated whole cell extracts, did not
require the addition of exogenous RNAs (Table
II). This lack of dependence on
exogenously added RNA is one of the hallmarks of PAP-III isolated from
whole leaf extracts (13) and is the result of the association of RNA
with PAP-III during purification. The specific activity in crude
chloroplast extracts was comparable to that after fractionation of
whole cell extracts on DEAE-Sepharose (Table II), which suggests that
poly(A) polymerase activity is enriched through the purification of
chloroplasts. Fractionation of the chloroplast extracts on
DEAE-Sepharose, heparin-Sepharose, and Mono Q columns yielded results
identical to those obtained with whole cell extracts: fractions
analogous to PAP-I and PAP-III could be obtained (not shown). These
observations support the hypothesis that the novel poly(A) polymerase
we have studied is a chloroplast-localized enzyme.
This possibility was tested further by examining chloroplast
extracts for the presence of p98 by immunoblotting. Because poly(A) polymerase is typically presumed to be a nuclear enzyme, pea nuclear extracts were also examined. As seen in Fig.
5, p98 is readily detectable in
chloroplast extracts. In contrast, this protein was absent from nuclear
extracts (Fig. 5). Probing identical blots with antibodies against U1
small nuclear ribonucleoprotein (snRNP; a nuclear marker) and the large
subunit of ribulose-1,5-bisphosphate carboxylase (a chloroplast marker)
indicated that the chloroplast and nuclear extracts were free from
appreciable cross-contamination (Fig. 5). In addition to these results,
p98 could not be detected in roots (not shown). These observations are
all consistent with the hypothesis that PAP-III is a chloroplast
enzyme.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
p98 is present in chloroplast extracts.
Extracts from purified nuclei (ne) and chloroplasts
(cp), containing 15 µg of protein, were separated by
SDS-PAGE, transferred to nitrocellulose, and the filters probed with
antibodies against p98 (anti-PAP-III), U1 snRNP
(anti-U1), and the large subunit of
ribulose-1,5-bisphosphate carboxylase (anti-rbcL) as
described under "Materials and Methods."
|
|
 |
DISCUSSION |
Several results, from this and previous studies, support our
conclusion that PAP-III consists at least in part of PNPase. Before, we
had noted the presence, in highly purified preparations of PAP-III, of
a group of approximately 100-105-kDa polypeptides (13). One or more of
these polypeptides could be cross-linked to the labeled products of
polyadenylation, indicative of a close association with the poly(A)
polymerase reaction. Moreover, there was a requirement that substrate
RNAs for the poly(A) polymerase be bound to one or more of these
proteins (13). In the present study, we have isolated cDNA clones
based on peptide sequences obtained from these RNA-binding
polypeptides. Antibodies raised against the product of the polypeptide
encoded by the p98 clone recognize polypeptides of about 100 kDa (Fig.
3), and these antibodies immunoprecipitate PAP-III activity efficiently
(Table I). These observations indicate that the encoded protein (p98)
is a component of PAP-III. The identity of p98 with PNPase is indicated
by the extensive amino acid sequence similarity with known PNPases
(Fig. 1) and by the copurification (Fig. 2) and coimmunoprecipitation (Table I) of PNPase and PAP-III activities.
PNPase has been identified previously in chloroplasts (23), thus
suggesting a chloroplast localization for the poly(A) polymerase we
have characterized. Two lines of evidence presented here support this
hypothesis. First, poly(A) polymerase activity is present in
chloroplast extracts (Table
II),2 and the activity that
can be purified from such extracts is biochemically indistinguishable
from the activity that has been purified from whole leaf extracts
(Table II). Second, p98 is present in chloroplast extracts (Fig. 5). We
have not been able to detect this polypeptide in root extracts
(data not shown), an observation that suggests that p98 is not present
in mitochondria or other subcellular compartments that are present in
root cells. However, we cannot rule out the presence of multiple
locations for p98 in photosynthetic tissues, or in highly specialized
cell types in roots.
p98 is very similar to that of a previously described spinach PNPase
(23), but these sequences diverge at their NH2 and COOH
termini. The NH2-terminal divergence might be expected in a
chloroplast transit peptide. The COOH terminus of p98 retains the
ribosomal protein S1-related RNA binding domain found in bacterial PNPases, whereas the spinach PNPase lacks most of this domain (Fig. 1).
Because p98 serves as an RNA-binding cofactor in the reaction catalyzed
by the chloroplast poly(A) polymerase (13), the COOH-terminal sequence
differences raise the interesting possibility that plants may possess
more than one form of PNPase, only some of which are suitable as
partners for polyadenylation.
PNPase is an important part of the RNA turnover machinery in bacteria
(11, 24, 25), acting to degrade RNAs in a 3'
5' direction via its
phosphorolytic activity. In chloroplasts, polynucleotide phosphorylase
is associated with a complex that helps to determine mRNA 3'-ends
and can also degrade RNAs in vitro (23). Poly(A) tracts can
destabilize RNAs in chloroplast extracts, and chloroplast RNAs of a
transient (and presumably unstable) nature in vivo also may
have polyadenylate tracts at their 3'-ends (8, 9). Our results provide
a possible link between polyadenylation and increased RNA turnover in
chloroplasts. This follows from the observation that only those RNAs
that are associated with p98 (which the present study indicates is
identical to PNPase) can be polyadenylated by the poly(A) polymerase we
have characterized (13); because p98 is also PNPase, our results
indicate that, in vitro, the products of polyadenylation by
a chloroplast poly(A) polymerase are necessarily associated with an
enzyme known to be involved in RNA degradation.
We thank Carol Von Lanken for excellent
technical assistance and Martha Peterson for helpful suggestions with
the manuscript. We are grateful to Robert Houtz and A.S.N. Reddy for
the gifts of anti-RBCL and anti-U1 antisera, respectively.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF010578.