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INTRODUCTION |
RNA editing is one of the most interesting and universal
RNA-processing mechanisms known to affect gene regulation. This process has been detected in a variety of organisms, such as trypanosomes and viruses (1, 2). In mammalian systems, both edited and unedited proteins are functional and have distinct properties (3-6).
For example, the edited and unedited subunits of the glutamate receptor
give rise to complex differing in calcium permeability (5). In
chloroplasts, RNA editing is a widespread processing event, creating
start and stop codons and most frequently altering coding sequences
(7-11). To date, a number of RNA editing sites have been identified in
chloroplasts with completely sequenced genomes, for example 26 sites in
maize (12), 26 sites in black pine (10), and 31 sites in tobacco (13).
The functional significance of the creation of an initiation codon and
a stop codon is easily understood (7, 8). Alteration of an internal
codon results in the conservation of functionally important amino
acids, suggesting that the conserved residues are critical for the
function of the protein (9-12, 14). However, we do not know whether
both unedited and edited proteins are functional in chloroplasts and
therefore whether chloroplast RNA editing is necessary.
An in vitro approach will give some answers to these
questions. Chloroplast-encoded polypeptides are mostly membrane
proteins forming large complexes, and it is difficult to reconstitute
such functional complexes using any in vitro approach.
Several soluble complexes containing chloroplast-encoded polypeptides,
such as ribulose-bisphosphate carboxylase, RNA polymerase, and
ATP-dependent protease, have not yet been reconstituted
in vitro. Here we present the first biochemical evidence for
the functional necessity of editing by using a reconstituted enzyme
involved in fatty acid synthesis.
Chloroplasts possess a key enzyme required for de novo fatty
acid synthesis. This enzyme is the prokaryotic form of acetyl-CoA carboxylase (ACCase).1 The
ACCase, different from the eukaryotic form of ACCase consisting of
multimers of a single multifunctional polypeptide, is multienzyme complex composed of the biotin carboxylase complex and the
carboxyltransferase (CT) enzyme (15). The latter enzyme contains the
nuclear-encoded
and the chloroplast-encoded
polypeptides
(15-17). We have recently reconstituted pea CT using a bicistronic
plasmid in which the accA and accD cDNAs
encoding the
and
polypeptides, respectively, were tandemly
ligated to a decahistidine tag (His tag). We have also shown that the
recombinant CT reconstituted in Escherichia coli has
properties similar to those of authentic CT (18). Because one
nucleotide of an internal codon of pea accD mRNA is
edited in chloroplasts (18) and a serine codon is converted to a
leucine codon, this reconstitution system provides a useful tool to
examine whether this RNA editing is necessary for CT.
Here we expressed in E. coli a construct containing the two
DNAs encoding the accA mRNA and either the unedited or
edited accD mRNA, and we measured the in
vitro activities of the resultant unedited or edited CT. Activity
was found in the edited CT, but not in the unedited CT, indicating that
editing is necessary for a functional enzyme, and the leucine residue
is critical in vitro. We found that editing at the same
position took place in four plants not having leucine residues at the
position, and we confirmed that editing is necessary for a functional
protein in vivo.
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EXPERIMENTAL PROCEDURES |
Materials--
Soybean (Glycine max, cv. Enrei),
Arabidopsis thaliana (ecotype Columbia), Brassica
napus, and black pine (Pinus thunbergii: a gift from
Dr. K. Shinohara) were used.
Plasmids--
The plasmid pHisADS, encoding unedited
accD mRNA, was constructed by inserting the
accD genomic DNA into the XhoI and
BlpI sites of pHisAD (18) instead of accD cDNA.
Expression and Purification--
The methods used have been
described previously (18) except that protease inhibitor tablets
(CompleteTM, EDTA-free, Roche Molecular Biochemicals) were
added to the buffer for the purification step. Purification of
recombinant His tag proteins followed the Novagen protocol. The column
was washed with 5 column volume of a washing buffer containing 60 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl (pH 7.9). A small portion of the recombinant
protein from pHisADS leaked out during washing. The eluate with a
solution containing 150 mM imidazole, 500 mM
NaCl, and 20 mM Tris-HCl (pH 7.9) was used for enzyme assay.
Immunoprecipitation--
Immnunoprecipitates were obtained by
anti-
polypeptide IgG and protein A-Sepharose CL-4B (Amersham
Pharmacia Biotech) (18).
Enzyme Assay--
CT activity and ACCase activity were measured
as described (18).
Sequencing--
Total RNA and DNA were prepared from seedlings
of soybean, Arabidopsis, Brassica, and black pine
described by Kozaki et al. (18). cDNA synthesis,
polymerase chain reaction, and direct DNA sequencing were
carried out (18).
Multiple Alignments--
The amino acid sequences of
accD genes were aligned using the CLUSTAL W program (19).
Because the sequence similarities between accD protein and
eukaryotic forms of ACCase are low, we performed the sequence
similarity search using the UCSC SAM-T98 program with default
parameters (20). This method uses an iterative hidden-Markov model and
uncovers subtle relationships missed by single-pass data base search
methods (21, 22). The amino acid sequence of E. coli accD
gene was used as a query. The amino acid sequences of eukaryotic forms
of ACCase, ACC1, are aligned with those of accD genes based
on the result of this search.
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RESULTS |
Comparison of Unedited and Edited Enzymes--
Pea accD
consists of 1,770 base pairs and encodes the
polypeptide of 590 amino acid residues (23). Only one editing site, where a cytosine is
changed into a uracil, has been found at nucleotide position 800 (18).
This corresponds to the second position (underlined) of the 267th
codon, a UCG (serine) codon that is converted into a
UUG codon (leucine) in the mRNA. The cDNAs
encoding accA mRNA and either the unedited or edited
accD mRNA were bicistronically inserted into the pET-19b
vector, and we obtained two expression plasmids, pHisADS (Fig.
1A) and pHisAD. The difference
between the two plasmids is a base at position 800.

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Fig. 1.
Expression of pHisAD and pHisADS.
A, the plasmid pHisADS, encoding unedited accD
mRNA, was constructed by inserting the accD genomic DNA
into the XhoI and BlpI sites of pHisAD (18)
instead of accD cDNA. Into the pET19b vector,
accA cDNA, a ribosomal binding site (rbs),
and accD genomic DNA were inserted. The asterisk
indicates the only difference between pHisADS and pHisAD. The base in
pHisADS at this position was cytosine and that in pHisAD was uracil.
B, SDS-PAGE of the crude extract from E. coli
(55-µl culture) containing pHisAD or pHisADS and the fraction (6 µg
of protein) purified on a nickel column. The gel was stained with
Coomassie Brilliant Blue. C, immunoblot. The fraction (0.6 µg of protein) purified on a nickel column was separated by SDS-PAGE
and probed with an anti- or anti- polypeptide serum.
D, immunoprecipitates. Anti- polypeptide IgG was added to
the fraction (50 µg of protein) purified on a nickel column, and the
precipitates separated by SDS-PAGE were stained with Coomassie
Brilliant Blue.
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The pHisADS plasmid was expressed in E. coli. The resultant
CT complex was partially purified using a nickel column and compared with that resulting from the expression of pHisAD (Fig. 1, B
and C). Upon SDS-PAGE, both fractions gave a major band with
a molecular mass of 102 kDa and two minor bands of 72 and 87 kDa. The
102-kDa band reacted with the anti-
polypeptide serum, and the
87-kDa band reacted with anti-
polypeptide serum. The 72-kDa band
was a nonspecifically bound protein from E. coli. In
pHisADS, the His tag was fused to the
polypeptide, and the
polypeptide copurified on a nickel column. In addition, when anti-
polypeptide IgG was added to the eluate from the nickel column, the
polypeptide coprecipitated with the
polypeptide (Fig.
1D). These results indicate that the
polypeptide is
associated with the
polypeptide. The abundance of the
polypeptide exceeds that of the
polypeptide (Fig. 1B),
indicating a difference in expression of the two polypeptides. A
certain amount of the
polypeptide was not associated with the
polypeptide, as shown previously (18), and a small portion of the
polypeptide associated with the
polypeptide. There was almost no
difference of antigenicity between the eluates from pHisAD and pHisADS.
Thus, the level of expression of the unedited complex is similar to
that of the edited complex.
Both the unedited and edited complexes were mainly eluted by a buffer
containing 150 mM imidazole and 500 mM NaCl
from a nickel column. Freezing and thawing of the eluate did not affect
the solubility of the edited complex, but affected the unedited
complex, resulting in an insoluble complex. Dialysis of the eluate of
the unedited complex against a buffer containing 50 mM
Tricine-KOH (pH 8) and 150 mM NaCl resulted in
precipitates. In contrast to this, the edited complex was soluble, even
in 50 mM Tricine-KOH (pH 8). Thus, the solubility of the
unedited complex was different from that of the edited complex. A
serine residue at position 267 is probably not compatible with the
formation of a soluble complex with the
polypeptide, whereas a
leucine residue is required.
The eluate from the nickel column obtained from the expression of
pHisAD had CT activity as reported previously (18) and had ACCase
activity when a complex of biotin carboxylase with the biotin carboxyl
carrier protein was added (Table I).
However, the fraction from pHisADS had neither CT nor ACCase activity
upon addition of the biotin carboxylase complex, although the unedited complex was soluble and did not precipitate under the assay conditions used. To find any CT activity of the unedited complex under different conditions, we also assayed under an altered malonyl-CoA (0.1-1 mM) and salt concentration (0-100 mM NaCl) but
did not detect any activity. The protein degradation in both eluates
was not observed during incubation. These results indicate that the
amino acid at position 267 is important and that RNA editing in
accD mRNA is required for CT activity and therefore for
ACCase activity in vitro. Probably the
polypeptide,
having a serine residue instead of leucine at position 267, formed a
complex of inappropriate conformation for catalytic activity.
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Table I
Enzyme activities
CT activity of the eluate from a nickel column was measured as
described previously (18). ACCase activity was measured after the
addition of a biotin carboxylase complex with a biotin carboxyl carrier
protein prepared by anion exchange chromatography of a pea chloroplast
extract (18). This specific activity was expressed on the basis of the
amount of the recombinant CT protein. Results are the means of two
independent determinations. Almost the same results were obtained for
two independent enzyme preparations.
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Occurrence of RNA Editing in Other Plants--
The in
vitro evidence described above suggests that RNA editing is
required for functional enzyme in vivo. To examine this proposition, we looked at whether RNA editing occurs in plants not
having the leucine codon necessary for a functional enzyme at the
position in question of the accD gene. If RNA editing occurs to create a leucine codon at the same position in such plants, we can
conclude that editing is needed to form in vivo functional CT.
First, we compared the amino acid sequences deduced from the
accD gene of 15 land plants, 6 algae, and 2 bacteria. Pea CT
polypeptide has a putative zinc finger motif
(CX2CX15CX2C), which is conserved among all the
polypeptides reported. A
substituted site at position 267 in the pea is located at position 15 downstream from the last cysteine residue of this motif. Multiple
alignments surrounding the substituted site showed conservation of
about 30 amino acid residues among land plants (Fig.
2). In particular, 10 amino acid residues
from positions 260 to 269, except for 267, were conserved. At position
267, leucine was found in 15 organisms, not only in land plants but
also in algae and bacteria. Serine was found in five angiosperms, and
proline was found only in black pine. Methionine (plastid 5) and
isoleucine (plastid 11) are a conservative substitution for leucine.
Leucine and its similar amino acids were also conserved in the
eukaryotic form of ACCase composed of a single multifunctional
polypeptide, ACC1. Thus, we found six plants not having leucine or its
similar residues at the position in question.

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Fig. 2.
Similarities among the accD
and ACC1 sequences around the RNA editing
site. The arrow indicates the RNA editing site of pea
accD. Amino acid residues identical in half or more of the
sequences are shaded in black, and conservative
substitutions are shaded in gray. The left column
indicates species abbreviations as follows: Pea, Pisum
sativum; Soybean, G. max;
Arabidopsis, A. thaliana; Brassica,
B. napus; Dodder, Cuscuta reflexa;
Pine, P. thunbergii; Plastid 1,
Picea abies; Plastid 2, Nicotiana
tabacum; Plastid 3, Solanum tuberosum;
Plastid 4, Spinacia oleracea; Plastid
5, Oenothera elata subsp. Hookeri; Plastid
6, Epifagus virginiana; Plastid 7,
Angiopteris lygodiifolia; Plastid 8,
Marchantia polymorpha; Plastid 9,
Physcomitrella patens; Plastid 10,
Chlorella vulgaris; Plastid 11, Cyanidium
caldarium; Plastid 12, Nephroselmis
olivacea; Plastid 13, Porphyra purpurea;
Cyano 1, Synechocystis PCC6803; Cyano
2, Synechococcus PCC7942; Bacterium 1,
E. coli; Bacterium 2, Bacillus
subtilis; Eukaryote 1, Saccharomyces
cerevisiae; Eukaryote 2, A. thaliana
(cytosolic ACCase); Eukaryote 3, Homo
sapiens.
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Next, to survey possible occurrence of editing, we compared each codon
at the position 267. The data showed that the serine codon of the
Southern Asian dodder, soybean, Arabidopsis, and Brassica accD gene is UCG, as in peas, and the proline codon
of the black pine accD gene is CCA. RNA editing in
chloroplasts is mostly a cytosine-to-uracil change at the second
nucleotide position of the triplet (14). If a cytosine of the second
nucleotide of UCG (serine) and CCA (proline) is
converted to a uracil, the resultant UUG and
CUA triplets encode a leucine, and there is a possibility
for such editing to occur.
To examine whether such editing does occur, we compared the nucleotide
sequences of the accD gene and its cDNA from soybeans, Arabidopsis, Brassica, and black pine by direct
DNA sequencing (Fig. 3). The observed
gene sequence of the soybeans, Arabidopsis, and
Brassica accD at the position in question were all
TCG, which agreed with the reported data, but the cDNA
sequences were all converted to TTG. The observed sequence
of the black pine gene is CCA, which agreed with the
reported data, but the sequence of its cDNA was converted to
CTA. Thus, cytosine residue at the second position of the
triplet was converted to a uracil at mRNA. These results indicate
that the predicted editing occurred and that a leucine codon was
created by RNA editing. Thus, editing at the same position was also
shown to take place in the
polypeptide mRNA in all the plants
tested, indicating that editing is needed to synthesize a functional
enzyme in vivo and is necessary for ACCase of these
plants.

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Fig. 3.
Sequencing ladders showing editing of
accD transcripts in seedlings of G. max, B. napus, A. thaliana, and P. thunbergii.
Arrows indicate editing sites.
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DISCUSSION |
The experiments reported here are the first demonstrations that
chloroplast RNA editing is required for a functional ACCase, using two
different approaches. One is the biochemical demonstration that the
unedited recombinant enzyme is inactive in contrast to the edited
enzyme. This means that editing is essential for in vitro
enzyme activity. The second is the demonstration that chloroplast RNA
editing takes place to create the leucine codon in all the examined
accD gene not having leucine or its similar amino acid codon
necessary for a functional enzyme. This suggests the necessity of RNA
editing for a functional enzyme in vivo. We propose here that in plants RNA editing to create the leucine codon is essential in vivo.
The most frequently edited chloroplast RNAs are probably the
ndh transcripts encoding a putative chloroplast NADH
dehydrogenase. For example, tobacco ndhA, ndhB, and
ndhD transcripts have been reported to have two, nine, and
two editing sites, respectively (11, 13). The extent of editing in the
ndhD transcript depends on developmental and environmental
conditions (24). These genes are shown to be dispensable for plant
growth under mild environmental conditions (25, 26), and the biological
significance of RNA editing in ndh transcripts is not yet understood.
A possible role of chloroplast RNA editing has been reported for the
tobacco rpoA transcript encoding the
subunit of
chloroplast-encoded RNA polymerase (13). This editing is believed to be
involved in the regulation of RNA polymerase activity, because the
extent of editing depends on developmental conditions. However, it is not known whether both the unedited and edited enzymes are functional. Further experiments are needed to characterize the biological significance.
In contrast to ndh transcripts, the biological significance
has been demonstrated for psbF (27) encoding a core
component of the photosystem II and for petB (28) encoding a
subunit of the cytochrome b6f
complex. psbF is required for the functional photosystem II
(29). Spinach and tobacco psbF proteins are 100% identical,
but only a nucleotide of the spinach transcript is edited. The
introduction of spinach psbF into the tobacco plastid genome
that lacks the capacity to edit the introduced site resulted in a
mutant phenotype of slower growth, lowered chlorophyll content, and
high chlorophyll fluorescence. This lack of editing resulted in reduced
protein activity, but not a complete loss of function, indicating that
this RNA editing is not essential for in vivo function.
Chlamydomonas and maize petB proteins are 88%
identical (94% similar). A codon at position 204 of the
petB gene is leucine in Chlamydomonas, but in
maize it is proline, which is changed to a leucine by RNA editing. To
examine a possible role of proline at the position 204, a proline codon
was introduced in place of a leucine codon at position 204 of the
petB gene of Chlamydomonas. Chloroplast
transcripts are not edited in Chlamydomonas. The
Chlamydomonas transformants obtained were nonphototrophic
mutant. They lacked photosynthetic electron transfer and cytochrome
b6f activity, indicating that the
proline is not fit for Chlamydomonas cytochrome b6f. This result strongly suggests
that this RNA editing is essential for photosynthesis in maize.
Plants have two forms of ACCase, the heteromeric, prokaryotic form
composed of four subunits in chloroplasts, and the homomeric, eukaryotic form composed of a single polypeptide in cytosol, except for
Gramineae, which lacks the accD gene (30, 31). Although rice
has an accD gene remnant, wheat and maize do not have a
counterpart to this. Gramineae does not have the prokaryotic form of
ACCase in chloroplasts but has the nuclear-encoded eukaryotic form of ACCase in both chloroplasts and cytosol. Each ACCase supplies malonyl-CoA for the synthesis of fatty acids in chloroplasts or for the
synthesis of flavonoid and the chain elongation of fatty acids in
cytosol. To our knowledge no evidence exists that malonyl-CoA synthesized in cytosol enters into plastids. ACCase is necessary in
chloroplasts for de novo fatty acid synthesis. Probably the prokaryotic form of ACCase is essential for plants. E. coli
accD is an essential gene (32). Both accA and
accD are present as a single copy in Arabidopsis
nuclear and chloroplast genomes, respectively, and are essential genes.
Thus, chloroplast RNA editing is necessary not only for ACCase but also
for the survival of these plants.