(Received for publication, January 22, 1997, and in revised form, April 4, 1997)
From the Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel
Polyadenylation of mRNA in the chloroplast has recently been shown to target the RNA molecule for rapid exonucleolytic degradation. A model has been suggested in which the degradation of chloroplast mRNA is initiated by endonucleolytic cleavage(s) followed by the addition of poly(A)-rich sequences and rapid exonucleolytic degradation. When in vitro transcribed RNAs were incubated with chloroplast protein extract, competition between polyadenylated and non-polyadenylated RNAs for degradation resulted in the rapid degradation of the polyadenylated molecules and stabilization of their non-polyadenylated counterparts. To elucidate the molecular mechanism governing this effect, we determined whether the chloroplast exoribonuclease 100RNP/polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated 100RNP/PNPase degraded polyadenylated and non-polyadenylated RNAs at the same rate. However, when both molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. In RNA binding experiments, 100RNP/PNPase bound the poly(A) sequence with much higher affinity than other RNA molecules, thereby defining the poly(A)-rich RNA as a preferential substrate for the enzyme. 100RNP/PNPase may therefore be involved in a mechanism in which post-transcriptional addition of poly(A)-rich sequence targets the chloroplast RNA for rapid exonucleolytic degradation.
Photosynthesis and other essential biosynthetic plant cell
activities occur in the chloroplast. The chloroplast structural proteins and enzymes are encoded by both nuclear and chloroplast genomes. During its development, chloroplast gene expression is tightly
regulated at many levels, including that of mRNA accumulation (reviewed in Refs. 1-4). RNA metabolism involves a series of steps
that are dependent on RNA secondary structures, nucleases, and
regulatory RNA-binding proteins (5, 6). Similar to bacterial mRNAs,
most chloroplast mRNAs contain an inverted repeat sequence in their
3-untranslated region that can fold into a stable stem-loop structure
(7). Unlike nuclear encoded mRNA, most of the chloroplast mRNAs
are non-polyadenylated at the 3
-end in their steady-state condition.
Several RNA-binding proteins that may be involved in the processing,
maturation, and degradation of chloroplast RNAs have been characterized
over the past few years. For example, a family of proteins with an
RNA-binding recognition sequence motif has been described, of which
spinach 28RNP was most prominently characterized. Immunodepletion of
this protein from the chloroplast extract or the addition of
recombinant protein interfered with the in vitro 3-end
processing of chloroplast RNAs (8, 9). RNA binding properties of 28RNP
have been studied in detail and show that phosphorylation changes the
affinity of the protein for RNA (9-12). 28RNP is not itself a
ribonuclease. A search for such an enzyme, involved in the 3
-end
processing of chloroplast mRNAs, yielded a 100-kDa RNA-binding
protein (13). The purified 100-kDa protein had biochemical properties
very similar to one of the two exonucleases so far discovered in
bacterial cells, the polynucleotide phosphorylase (PNPase)1 (14). In addition, the deduced
amino acid sequence of the chloroplast 100RNP cDNA disclosed high
homology to the bacterial PNPase (13). The chloroplast RNA processing
and degradation system is therefore similar to recently discovered
mechanisms in Escherichia coli (15, 16). However, unlike
bacteria, plastid mRNA metabolism and associated enzymes are
controlled by the nucleus and may be regulated by both light and the
redox state of the chloroplast (13).
Post-transcriptional addition of poly(A) tail to the 3-end of mRNA
has been best characterized in eukaryotic cells for nuclear encoded and
viral mRNAs. The long poly(A) tail is an important determinant of
mRNA stability and maturation as well as initiation of translation
(17-19). Poly(A) tails have also been described for several bacterial
mRNA 3
-ends and for endonucleolytic and exonucleolytic sites of
the rpsO mRNA (20-26). In contrast to the nucleus and
cytoplasm of eukaryotic cells, where the poly(A) tail seems to
stabilize the nuclear encoded mRNA, the addition of poly(A) tails
to bacterial mRNAs promotes their degradation.
Post-transcriptional addition of poly(A)-rich sequences to chloroplast
psbA mRNA has recently been described (27, 28). Unlike
eukaryotic nuclear encoded and bacterial RNAs, the poly(A) moiety in
the chloroplast, which may be several hundred nucleotides long, was
found not to be a ribohomopolymer of adenosine residues, but rather
clusters of adenosines bounded mostly by guanosines and, on rare
occasion, by cytidines and uridines (27). When lysed chloroplasts were
incubated in the presence of yeast tRNA, thereby inhibiting exonuclease
activity, distinct endonucleolytic cleavage products accumulated (29).
Several of the endonuclease cleavage sites mapped by primer extension
perfectly matched the poly(A)-rich addition sites that were analyzed by
reverse transcription-polymerase chain reaction, suggesting a
degradation pathway for psbA mRNA in the chloroplast. In
this pathway, the degradation is initiated by endonucleolytic cleavage
followed by the addition of poly(A)-rich sequence to the 3-end of the
proximal cleavage product. Once the poly(A)-rich sequence is added,
this RNA molecule is rapidly degraded by exonuclease(s) (27). Indeed,
when synthetic polyadenylated RNA was incubated with a chloroplast
soluble protein extract together with its non-polyadenylated
counterpart, the polyadenylated RNA was rapidly degraded. In addition,
blocking mRNA polyadenylation with cordycepin inhibitor inhibited
exonucleolytic degradation, and endonucleolytic cleavage products
accumulated.2
In this study, we show that the chloroplast exoribonuclease 100RNP/PNPase exhibits preferential activity toward polyadenylated RNA. Purified 100RNP/PNPase degraded synthetic transcribed polyadenylated RNAs much faster than non-polyadenylated RNA. We also show this difference to be due to the high binding affinity of 100RNP/PNPase for the poly(A) sequence. These results suggest the preferential affinity of 100RNP/PNPase for poly(A)-rich sequences to be part of the mechanism in which post-transcriptional addition of poly(A)-rich sequence targets the chloroplast RNA for rapid exonucleolytic degradation.
Chloroplasts were isolated on Percoll gradients from
leaves of hydroponically grown spinach plants (Spinacia
oleracea cv. Viroflay) under 10.5 h of light and 13.5 h
of darkness as described previously (9). A soluble protein extract
capable of 3-end processing of chloroplast RNAs was prepared from
isolated intact chloroplasts as described (30).
Chloroplast soluble protein
extract was fractionated through a size-exclusion Superdex 200 column
(Pharmacia Biotech Inc.). Fractions containing protein complexes of
550-650 kDa were pooled and applied to a 1-ml heparin column (Hi-Trap,
Pharmacia) developed with a linear gradient of KCl in buffer E (20 mM HEPES, pH 7.9, 60 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 2 mM dithiothreitol, and 17% glycerol). Proteins eluted at
0.2 M KCl were dialyzed against buffer E and applied to a
Resource Q column (Pharmacia). The column was developed with a linear
gradient of KCl in buffer E, and 100RNP/PNPase was eluted at 0.3 M as a single silver-stained polypeptide (see Fig. 2).
In Vitro Transcription of RNA
The plasmids used for
in vitro transcription of parts of the mRNA from the
spinach chloroplast genes psbA (encoding the D1 protein of
photosystem II) and petD24 (encoding subunit IV of the
cytochrome b6f complex) have been
described previously (9, 31). To generate RNA containing the
psbA amino acid coding region (nucleotides 946-1028
according to Zurawski et al. (32)) with an additional 14 adenosines at the 3
-end, the reverse transcription-polymerase chain reaction fragment described by Lisitsky et al. (see
clone 5 in Fig. 1 of Ref. 27) was used. The reverse
transcription-polymerase chain reaction fragment was cloned into the
pUC57-dT vector (MBI Inc.) and subcloned into pBluescript SK
(Stratagene) using BamHI and KpnI restriction
sites. To generate the RNA with 14 adenosines at the 3
-end, the
plasmid was linearized with BamHI and transcribed using T7
RNA polymerase. The transcribed RNA was then hybridized to an adapter
oligonucleotide (27) and digested with RNase H as described previously
(9). To generate RNA without the additional 14 adenosines, the
above-described RNA molecule was hybridized to an oligo(dT) adapter
oligonucleotide (27) and then digested with RNase H. To generate RNA
with poly(A) sequence located within the molecule, the RNA transcribed
from the plasmid linearized with BamHI was used. Therefore,
this RNA contained 18 nucleotides derived from the oligo(dT) adapter
oligonucleotide (27) and 5 nucleotides derived from the pUC57 vector,
3
to the 14 adenosines. RNAs were transcribed using T7 RNA polymerase
and radioactively labeled with [
-32P]UTP to a specific
activity of 8-10 × 103 and 8-10 × 104 cpm/fmol for RNA degradation and UV cross-linking
experiments, respectively (9). The full-length transcription products
were purified on 5% denaturing polyacrylamide gels. In
vitro polyadenylation of RNA was performed by incubating the
in vitro transcribed RNA with a chloroplast protein extract
(1 mg/ml) in buffer E containing 0.5 mM ATP for 30 min
(27). The polyadenylated RNA was purified by denaturing PAGE.
In vitro RNA degradation experiments were carried out as described previously (27). Briefly, in vitro synthesized RNA (2 fmol) was incubated with the chloroplast soluble protein extract (1 mg/ml) or with isolated 100RNP/PNPase (0.4 µg/ml) for the times indicated in the figure legends. Following incubation, the RNA was isolated and analyzed by gel electrophoresis and autoradiography (9).
UV Cross-linking AssayUV cross-linking of proteins to
[-32P]UTP-labeled RNA was carried out as described
previously (8-10). Briefly, 3 fmol of RNA (240,000 cpm) were incubated
with 20 µg of chloroplast proteins in 15 µl of buffer E. To avoid
the poly(A) RNA degradation by 100RNP/PNPase observed in this work, the
RNA/protein mixture was immediately exposed to UV light at 4 °C
without preincubation at room temperature. Indeed, when UV
cross-linking was performed with preincubation at room temperature, no
differences were observed in the binding of 100RNP/PNPase to the
different RNA molecules (data not shown) (33). Following 1.8 J of
UV irradiation in a UV cross-linking apparatus (Hoefer Scientific
Instruments), the RNA was digested with 5 µg of RNase A at 37 °C
for 1 h, and the proteins were fractionated by SDS-PAGE. The label
transferred from the RNA to the proteins was detected by
autoradiography and quantified with a Fuji imaging analyzer.
The 100RNP/PNPase cDNA (13) was subcloned into a pMal-cR1 vector (Bio-Lab Inc.) using the BamHI/HindIII sites. The recombinant fusion protein was expressed and purified on amylose resin according to the manufacturer's protocol, with an additional purification step using a heparin column. Polyclonal antibodies were generated in rabbit as described previously (8). Unlike the mature protein, the maltose-binding 100RNP/PNPase-fused recombinant protein exhibited neither RNA binding nor exonuclease activity (data not shown).
SDS-PAGE fractionation and detection of 100RNP/PNPase on immunoblots probed with specific antibodies were performed as described previously (8). Protein concentration was determined using the Bio-Rad protein assay kit.
Binding of 100RNP/PNPase to Immobilized RibohomopolymersChloroplast protein extract (1 mg) was applied to immobilized ribohomopolymer columns (0.1 ml; Sigma). Following extensive washing of the columns with buffer E, the bound proteins were successively eluted with 0.5, 1, and 2 M KCl in buffer E. Proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with specific antibodies to 100RNP/PNPase.
Chloroplast soluble protein extract has been used to
analyze transcription, 3-end processing, and polyadenylation of
in vitro transcribed RNA corresponding to chloroplast genes
(7, 9, 13, 27, 28, 31, 34, 35). In vitro transcribed RNA without a stem-loop structure at the 3
-end is rapidly degraded in this
extract (31). In a previous work, we showed that when polyadenylated
and non-polyadenylated RNAs were incubated in this extract, the
polyadenylated RNA was rapidly degraded, whereas its non-polyadenylated
counterpart was stabilized (27). This may have been because the
polyadenylated RNA had a higher affinity for the degradation enzyme(s),
thereby excluding it from the non-polyadenylated RNA. To verify this
hypothesis, the same experiment was performed with increasing amounts
of chloroplast extract (and consequently, increasing amounts of RNA
ribonucleases). When an RNA molecule corresponding to part of the
petD mRNA was incubated in the chloroplast extract at a
concentration of 0.4 mg/ml, it was rapidly degraded (Fig.
1A). The same RNA was then mixed with its
polyadenylated counterpart in such a way that an identical number of
molecules were incubated in each reaction mixture. As compared with the system including only non-polyadenylated RNA (Fig. 1A), the
polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was
stabilized (Fig. 1B). Increasing protein concentrations
resulted in accelerated degradation of the polyadenylated RNA, but no
degradation of the non-polyadenylated RNA. Finally, at a concentration
of 8 mg/ml, 20 times greater than that originally used, the
polyadenylated RNA disappeared within 5 min, and the non-polyadenylated
RNA was also degraded, albeit more slowly than when incubated alone at 0.4 mg/ml (Fig. 1, A and D). Variance in the
stability of the RNAs was not due to the difference in their lengths
since the addition of other nucleotides has no such effect (27, 36). Since the two RNAs differed only in the presence or absence of a
poly(A) tail, this result suggested that the poly(A)-tail has a higher
affinity than non-polyadenylated RNA for the ribonucleases in the
chloroplast protein extract. Because we had previously shown the
poly(A) tail to be a target for exonucleolytic degradation of RNA
(27),2 we questioned whether a purified exoribonuclease
would exhibit such a preference.
100RNP/PNPase Exonuclease
A nuclear encoded chloroplast 100-kDa RNA-binding protein exhibiting exoribonuclease activity and amino acid sequence homologous to the bacterial polynucleotide phosphorylase (100RNP/PNPase) has recently been described (13). To analyze the activity of 100RNP/PNPase in degrading polyadenylated RNA and since we were unsuccessful in expressing a recombinant active 100RNP/PNPase in E. coli, the enzyme was purified to homogeneity from chloroplast protein extract (Fig. 2A). Purified 100RNP/PNPase consisted of one polypeptide (which sometimes appeared as a doublet) that reacted with specific antibodies generated against the recombinant maltose-binding protein fused to 100RNP/PNPase (Fig. 2B). When the purified protein was incubated with in vitro transcribed RNA, the RNA was rapidly degraded (Fig. 2C) (13).
In addition to exoribonuclease activity, the E. coli PNPase disclosed in vitro RNA polymerase activity when incubated in the presence of nucleosides (14). The polymerase activity is activated when there are sufficient diphosphates and a very low concentration of inorganic phosphate (14). Chloroplast 100RNP/PNPase also showed RNA polymerase activity when incubated with RNA in the presence of ADP (data not shown). This result suggested an interesting possibility: 100RNP/PNPase might also be involved in post-transcriptional addition of poly(A)-rich sequences to RNA in the chloroplast. However, we had previously shown that depletion of 100RNP/PNPase from the chloroplast protein extract did not interfere with in vitro polyadenylation activity (27). In addition, a poly(A) polymerase had been purified from spinach chloroplast extract (37).3 If the chloroplast 100RNP/PNPase activity is modulated similar to the E. coli PNPase, then the concentrations of inorganic phosphate and nucleotide diphosphate in the chloroplast would promote the degradation rather than the polymerization activity.2 Combined, these results show that additional experiments are required to clarify the in vivo mode of action of 100RNP/PNPase.
Polyadenylated RNA Is Specifically Degraded by Purified 100RNP/PNPaseThere are two possible ways of accounting for the
observed competition of degradation enzymes for polyadenylated RNA
in the presence of chloroplast protein extract (Fig. 1). The
preferential activity toward polyadenylated RNA could be a phenomenon
intrinsic to the exonuclease polypeptide(s) itself. Alternatively,
additional auxiliary proteins that confer this preferential activity
may be required. To determine which of these scenarios was valid, competition experiments were performed using the isolated enzyme. In vitro transcribed RNA corresponding to part of the
petD mRNA and the same RNA with the addition of 100 adenosines were incubated with purified 100RNP/PNPase, separately or in
combination. As shown in Fig. 3, the RNAs were degraded
at similar rates when incubated alone. However, when they were
combined, the non-polyadenylated RNA was stabilized (Fig.
3B). Moreover, under our experimental conditions, the
polyadenylated RNA was degraded at the same rate when incubated
separately or with non-polyadenylated RNA (Fig. 3). Increasing the
100RNP/ PNPase concentration led to accelerated degradation of the
RNA molecules, similar to the situation shown in Fig. 1 for the protein
extract (data not shown) (compare with Fig. 4, where the
protein/RNA ratio was 2.5 times higher than that in Fig. 3). These
results demonstrated that the purified exonuclease 100RNP/PNPase
preferentially degrades polyadenylated RNA and suggested that
competition for degradation of polyadenylated RNA is due to the
enzyme's intrinsic properties, rather than to auxiliary proteins.
RNA Is Not Rapidly Degraded When the Poly(A) Stretch Is Not at the 3
100RNP/PNPase, like the bacterial
PNPase, is an exonuclease that processively degrades the RNA from the
3-end (13, 14). Since the experiments described above suggested that
chloroplast 100RNP/PNPase has a higher affinity for polyadenylated RNA,
we questioned whether or not the poly(A) stretch needs to be located at
the 3
-end of the RNA molecule. To this end, RNA molecules representing
part of the psbA amino acid coding region with or without
the addition of 14 adenosines as well as with the addition of 14 adenosines and 23 bases, including all four nucleotides, at the 3
-end
were synthesized (see "Materials and Methods") (marked A, B, and C in Fig. 4, respectively).
These molecules were mixed and incubated with purified 100RNP/PNPase.
The results presented in Fig. 4 show that the non-polyadenylated RNA
and the RNA with the internal poly(A) stretch were degraded at a
similar rate, whereas the RNA with the poly(A) at the 3
-end was much
more rapidly degraded. Therefore, to compete for 100RNP/PNPase
degradation activity, the poly(A) tail needs to be located at the
3
-end of the RNA molecule.
Two possible
mechanisms could explain the preferential degradation of polyadenylated
RNA by 100RNP/PNPase. In the first, the enzyme discriminates between
substrates because, once bound to the RNA molecule, it degrades
polyadenylated RNA much faster than non-polyadenylated RNA. The second
mechanism implies similar degradation rates, but higher binding
affinity of 100RNP/PNPase for polyadenylated RNA. Therefore, a higher
affinity for poly(A) sequence would result in preferential activity
toward the polyadenylated molecules when the enzyme concentration is
limited. The first potential mechanism can be excluded by the
observation that polyadenylated and non-polyadenylated RNAs were
degraded at a similar rate when incubated separately with isolated
100RNP/PNPase (Fig. 3). Nevertheless, we intended to determine whether
or not this protein displays a higher affinity for poly(A). To measure
the binding affinities of 100RNP/PNPase for different RNA molecules, it
was fractionated through immobilized ribohomopolymer columns.
Chloroplast protein extract was applied to a poly(A)- or
poly(U)-agarose column, and the bound proteins were eluted with
increasing salt concentrations. The amount of 100RNP/PNPase in both the
unbound and bound fractions was detected by probing an immunoblot with
specific antibodies. Fig. 5 shows that most of the
100RNP/PNPase was depleted from the extract by the poly(A) column,
whereas a much smaller amount was depleted by the poly(U) column. In
addition, 100RNP/PNPase was eluted from the poly(A) column with 1 M KCl, whereas a concentration of only 0.5 M
eluted the protein from the poly(U) column. No binding activity of
100RNP/PNPase on the poly(G) or poly(C) column was observed under these
conditions (data not shown). To define the binding activities of
100RNP/PNPase for different RNA molecules in an additional system, we
carried out UV cross-linking competition experiments (9, 10).
Chloroplast protein extract was incubated with
[32P]UTP-labeled RNA corresponding to the psbA
3-end. 100RNP/PNPase is strongly labeled in this UV cross-linking
binding assay (13). Increasing amounts of a competitor were used to
define a quantitative I50 number, defined as the
concentration of competitor required to reduce the UV cross-linking
signal to 50% (9, 10). Due to the preferential activity of
100RNP/PNPase toward polyadenylated RNA, these experiments were
performed as rapidly as possible and at a low temperature.
Nevertheless, the results obtained with poly(A) should be taken as a
minimum value because the concentration of poly(A) in the mixture was
lower than that added to the system due to degradation. The results
presented in Fig. 6 concurred with the column binding
studies, showing the higher binding affinity of 100RNP/PNPase for
poly(A). The affinity for poly(U) was about three to four times lower,
and that for poly(G) and for RNA corresponding to the 3
-end of
psbA was 15-20 times lower compared with the values
obtained with poly(A). Taking the poly(A) binding affinity as a minimum
value and because maximal competition had already been obtained with a
4-fold excess of poly(A) (Fig. 6), the actual differences in binding
affinities are assumed to be even greater. Taken together, these
results demonstrated that 100RNP/ PNPase binds poly(A) with much
higher affinity than any other RNA sequence. Therefore, it competes for
the degradation of polyadenylated RNA molecules by binding to poly(A)
and by being excluded from the non-polyadenylated RNA molecules.
A possible pathway for the degradation of mRNA in the
chloroplast has recently been suggested (27, 28).2 The
first event is endonucleolytic cleavage(s), which produces RNA with no
stem-loop structure at the 3-end. Following these cleavages, the
proximal fragments are polyadenylated at their 3
-end by the addition
of poly(A)-rich sequences. The polyadenylated RNAs are then rapidly
digested by the exonuclease(s), possibly due to the higher enzyme
affinity for the poly(A)-rich sequence. Indeed, blocking the
polyadenylation of RNA in an RNA degradation assay based on lysed
chloroplasts had the same effect as the addition of yeast tRNA, an
exonuclease inhibitor (29): RNA degradation was inhibited, and
endonucleolytic cleavage products accumulated.2 Moreover,
polyadenylated RNA was specifically degraded in a chloroplast protein
extract and competed with non-polyadenylated RNA for the degradation
machinery (Fig. 1) (27). In this work, we provide a more profound
understanding of the last step of this degradation mechanism. We show
that an exonuclease isolated from spinach chloroplasts, 100RNP/PNPase,
uses RNA with a poly(A) tail as the preferred substrate, suggesting
that specific degradation of polyadenylated RNA is a phenomenon
intrinsic to the exonuclease and that other proteins are not required.
Thus, polyadenylation of RNA degradation products at their 3
-end
increases their binding affinity for the exonuclease 100RNP/PNPase,
rendering these RNAs more susceptible to the enzyme's activity.
Analysis of the proteins involved in the processing of the 3-end of
the RNA in the chloroplast revealed a high molecular mass complex
harboring 100RNP/PNPase and an endonuclease that cross-reacted with
antibodies prepared against the E. coli endonuclease RNase E
(13). RNA maturation and degradation in the chloroplast may therefore
resemble the prokaryotic process, preserving its ancestral origin.
Indeed, recent studies have confirmed the important role played by
polyadenylation in the decay of E. coli transcripts. These
poly(A) tails are added to at least some transcripts at multiple sites,
rendering them extremely susceptible to degradation by an as yet
undefined mechanism (20-26). A significant proportion of this
polyadenylation occurs at endonucleolytic cleavages sites within the
coding and 3
-ends of the transcripts (26). Taken together, these
results suggest that the mechanism of
polyadenylation-dependent exonucleolytic RNA degradation
may be similar in bacteria and chloroplasts.
To date, two exonucleases have been identified in bacteria, both
working in the 3 to 5
direction (5, 38). The isolation of a
chloroplast exonuclease homologous to the bacterial PNPase and the
similarity of their RNA degradation systems suggest that the situation
in the chloroplast is similar to that in the bacterial cell. In this
work, we show that purified 100RNP/PNPase preferably degrades
polyadenylated RNA, similar to the situation in the chloroplast extract. However, the identification and characterization of other chloroplast exonucleases are required to determine whether the preferred poly(A) RNA degradation phenomenon is also shared by the
other exonucleases. Indeed, higher in vitro degradation
activity of bacterial RNase II for polyadenylated RNA was recently
detected (39). In that direction, a potential RNase II homologue from spinach chloroplast is under
purification.4
Isolated 100RNP/PNPase was active as a poly(A) polymerase (data not shown). However, depletion of 100RNP/PNPase from the chloroplast extract by heparin or single-stranded DNA columns did not interfere with in vitro polyadenylation activity (27). Moreover, a poly(A) polymerase has been isolated from chloroplast extract (37).3 A similar situation of in vitro polymerase activity of the PNPase and the existence of poly(A) polymerases was reported for bacteria (14). RNA polyadenylation was even enhanced in bacteria cells where the PNPase was inactivated (26). Combined, these results suggest that in vivo, 100RNP/PNPase might be active only as an exonuclease, probably by inhibition of its polyadenylation activity by an as yet unknown mechanism. Another possibility is that the degradation as opposed to the polymerization activity is modulated by the concentration of phosphate ions. The polymerization activity of the bacterial PNPase and chloroplast 100RNP/PNPase is highly inhibited by a relatively low concentration of inorganic phosphate (13-15). Taking into account the concentrations of inorganic phosphate and nucleoside diphosphates in the chloroplast, the degradative reaction is highly favored. Therefore, detailed biochemical characterization of the 100RNP/PNPase action in the high molecular mass complex and the factors that modulate its activity will probably help to resolve this issue.
100RNP/PNPase, like the bacterial PNPase, is a processive
exoribonuclease that binds to the 3-end and digests the RNA nucleotide by nucleotide, without disconnecting from the molecule (13). Competition experiments showed high binding affinity of 100RNP/PNPase for poly(A) (Figs. 5 and 6). The experiment presented in Fig. 4 shows
that in order for the non-polyadenylated RNA to compete for
100RNP/PNPase, the poly(A) stretch must be located at the 3
-end. Taken
together, these results suggest that the high affinity of 100RNP/PNPase
for the poly(A) stretch directs the enzyme to the poly(A) part of the
RNA, and only if this part is at the 3
-end of the molecule can the
enzyme start digesting the RNA. Since 100RNP/ PNPase works
processively along the RNA, it remains bound to it and is thereby
excluded from the non-polyadenylated molecules.
We thank Ruth Haddar for excellent technical assistance.