(Received for publication, March 20, 1995; and in revised form, May 25, 1995)
From the
Transcripts of higher plant mitochondria are modified
post-transcriptionally by RNA editing. To distinguish between the
mechanisms by which the cytidine to uridine transition could occur a
combined transcription/RNA editing assay and an in vitro RNA
editing system were investigated. Mitochondria isolated from etiolated
pea seedlings and potato tubers were supplied with
[
RNA editing in higher plant mitochondria is a process by which
genomically encoded cytidine residues are changed to uridines on the
RNA level(1, 2, 3) . Although most of the
conversions have been found in translated regions, structural RNAs,
intron sequences, and also leader and trailer sequences are
occasionally modified(4) . Polypeptides translated from edited
transcripts show a higher similarity to their homologues in animals or
fungi than those predicted from unedited
transcripts(5, 6) . Two biochemically distinct
processes have been identified as capable of changing the information
at the RNA level. One involves the insertion or deletion of
nucleotides(7) , and the other modifies specific nucleotides in
the mRNA sequence(8) . RNA editing in kinetoplastid protozoa
exemplifies the process of an insertional editing system with the
addition and deletion of uridine residues at specific sites. In the
current model this editing machinery is guided by small RNAs (guide
RNAs) that are complementary to the edited transcripts (9) and
also serve as a uridine donor for the editing activity(10) .
Modifications of nucleotides in tRNA or ribosomal RNA sequences,
although usually not called RNA editing, can affect the stability of
the RNA or modulate the RNA's conformation and identity. In the
alternative process RNA editing changes the information content via
modification, e.g. in creating a UAA stop-translation codon
from a CAA glutamine codon in apolipoprotein B mRNA or by modification
of a CAG codon specifying glutamine to an arginine codon (CGG) in
neural glutamate-gated calcium channel mRNA(11) . In the latter
example, a double-stranded RNA adenosine deaminase has been suggested
as the enzyme responsible for deaminating adenosine to inosine and thus
changing a CAG codon to CIG(12) . In apolipoprotein B mRNA
editing, a cytidine deaminase has been shown to be one component of the
enzymatic modification activity(13) . Neither biochemical
mechanism of the editing reaction in higher plant mitochondria nor the
components of the editing machinery are currently known. Three possible
mechanisms able to change a cytidine post-transcriptionally could
proceed by modification of the base, by base exchange, or by
replacement of the complete nucleotide (Fig. 1). The most simple
would be creation of a uridine by a site-specific hydrolytic
deamination of the cytosine at position 4. Modification of the base
pairing ability of the cytidine, e.g. by the attachment of the
amino acid lysine at position 2 of the cytidine, would create a
lysidine that is read by most enzymes as a uridine. Another possibility
is a transglycosylation reaction in which the cytosine is replaced by
uracil through breaking and reforming of the glycosyl bond.
Alternatively the cytidine nucleotide could be replaced by a uridine
nucleotide through cleavage and ligation of the phosphodiester backbone
of the RNA chain.
Figure 1:
Several biochemical mechanisms are
known that could change C to U nucleotide identities. Deamination of
the cytidine could create a uridine, while modification of the cytosine
base can result in a hypermodified base (e.g. lysidine), which
is recognized as uridine. Replacement of the cytosine base by uracil in
a transglycosylation reaction or complete replacement of the cytidine
nucleotide by uridine via deletion and insertion could also change the
nucleotide identity.
To investigate the biochemical nature underlying
the RNA editing process in mitochondria of higher plants, we traced the
fate of the
Spots containing hydrolyzed
Figure 2:
TLC separation of NMPs from potato
mitochondrial in organello transcripts. PanelA shows the one-dimensional TLC separation. Potato mitochondria were
incubated with [
Figure 3:
RNA editing in lysed pea mitochondria. PanelA shows the one-dimensional separation of NMPs
from pea mitochondrial run-on transcripts. Mitochondria of pea were
supplied with [
Figure 4:
In vitro transcripts are edited
in a pea mitochondrial lysate. PanelA, in vitro transcripts of the cox2 gene were incubated with pea
mitochondrial lysates. A region of the cox2(128-603)
from pea mitochondria, which is edited at 11 positions(17) ,
was amplified from pea mtDNA. The PCR product was used for in vitro transcription from the introduced T7 promoter to produce the
template for the editing assay. PanelB,
one-dimensional separation of NMPs from the cox2 transcripts
incubated with mitochondrial lysates of pea mitochondria. Cox2 in
vitro transcripts labeled with [
Figure 5:
Ring-labeled CMP is modified to labeled
UMP during incubation with mitochondrial lysates. T7 in vitro transcripts of the cox2 gene (Fig. 4A)
labeled with [5-
Figure 6:
RNA
editing modifies only unedited transcripts in the in vitro system. To analyze the specificity of the in vitro RNA
editing system transcripts of orf206 were incubated either in
the edited or in the unedited version together with pea mitochondrial
lysates. After incubation of 30 min the transcripts were reisolated and
digested with nuclease P1 to obtain the NMPs for TLC separation. The
unedited (orf206 genomic) template contains 36 and the edited
transcript (orf206 edited) 25 labeled cytidines, respectively. In the
TLC separation of the edited transcript (orf206 edited) two times more
radioactivity was loaded to show that no detectable amount of UMP was
produced during incubation. An arrow indicates the UMP
position, where only the unedited transcript shows a spot comigrating
with UMP after incubation.
Figure 7:
In vitro transcripts are edited
at specific editing sites. A, in vitro transcripts of orf206 were incubated with pea
mitochondrial lysate as shown in Fig. 6. After reisolation of
the template first strand cDNA was synthesized primed by T3-primer. PCR
amplification was done using the T7-orf206 and the T3 primers. B, PCR amplification products are shown from lysate without
template(1) , from incubated orf206 template with
mitochondrial lysate(2) , from unincubated orf206 template (3) , and from mtDNA of pea(4) . bp, base pairs. PanelC shows the orf206 sequence analyzed in the RT-PCR reaction. Stars indicate
the editing sites observed in fully edited orf206 transcripts.
PCR products obtained from the unincubated template showed only the
genomic sequence of orf206. Sequences from cDNA
clones(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) of
incubated templates are given under the listed nucleotide sequence.
Identical nucleotides are indicated by dots.
To analyze the biochemical nature of the RNA editing reaction
in plant mitochondria we followed the fate of the During the process of RNA editing the 5`-phosphate
of the modified cytidine is maintained, which is consistent with either
a deamination/transamination or a transglycosylation process. The
replacement of cytidine in the polynucleotide chain by uridine by a
deletion and insertion process can now be excluded, because no
enzymatic insertion mechanism is known that can introduce a nucleotide
into a polynucleotide chain without insertion of the 5`-phosphate of
the donor. In a transglycosylation reaction the phosphoribosyl chain is
maintained, but the base is exchanged. This mechanism is used to
post-transcriptionally modify preformed tRNAs, e.g. by the
introduction of hypoxanthine (21) and queosine(22) . A straightforward hydrolytic deamination appears to be most likely
the reaction in plant mitochondrial C to U RNA editing because
following a A site-specific
deaminase catalyzes the conversion from C to U in the apolipoprotein B
mRNA editing(13) . The catalytic subunit of this type of RNA
editing shares high homology to cytidine deaminases, while the target
site in the mRNA is determined by a second additional factor that is
proposed to bind downstream of the editing site at a conserved sequence
motif. A completely unrelated deamination activity, selective for
double-stranded RNAs, converts adenosyl residues to inosines in a
process termed double strand RNA unwinding(24) . This enzymatic
activity seems to be also involved in the editing process of the
mammalian neuronal glutamate-gated ion channel mRNA, where a CIG codon
is created from CAG(12) . In wheat mitochondria an in
vitro RNA editing system has been reported (14) that edits atp9 transcripts and requires no exogenous nucleotides. This
observation supports a deamination reaction for RNA editing in plant
mitochondria, although it has to be taken into account that in this
lysate endogenous nucleotides have not been depleted. Another recent
report, also based on a similar experimental approach, likewise
suggests the conversion of cytidine to uridine in intact mitochondria
of maize and Petunia by either a deamination or
transglycosylation mechanism(20) . To distinguish between
deamination and transglycosylation we followed the fate of a To determine the specificity of the in vitro reaction
edited and unedited templates were tested in the mitochondrial lysate
and analyzed by RT-PCR after incubation. The data show that the in
vitro RNA editing system of pea mitochondria described here is
specific for cytidines at editing positions while other cytidines in
the polynucleotide chain are no targets for C to U RNA editing. How
site selection is determined is still enigmatic, while the RNA editing
patterns observed in in vitro incubated templates clearly
indicate the involvement of site-specific factor(s) for individual
editing sites.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-
P]CTP to radiolabel the mitochondrial
run-on transcripts. High molecular weight run-on transcripts were
isolated and hydrolyzed, and nucleotide identities were analyzed by
one- and two-dimensional thin layer chromatography. The amount of label
comigrating with UMP nucleotides increases with extended incubation
times. Analogous products were obtained by incubation of
[
-
P]CTP or [5-
H]CTP
radiolabeled in vitro transcripts with a mitochondrial lysate
from pea mitochondria. 5-
H label of the cytosine base was
detected in the UMP spot after incubation of in vitro transcripts with mitochondrial lysate. These results are
consistent with a deamination reaction involved in this
post-transcriptional C to U modification process. To prove that
cytidines are deaminated specifically in vitro transcripts
were reisolated after incubation and analyzed by reverse
transcription-polymerase chain reaction. Sequence analysis clearly
shows that only cytidines at editing sites are edited while residual
cytidines are not modified and suggests that site-specific factors are
involved in RNA editing of plant mitochondria.
-phosphate of the cytidine and a 5-
H label
in the cytosine base after incorporation into high molecular weight
RNA. A system for coupled transcription/RNA editing was established for
this purpose. Results obtained by this system are confirmed by an in vitro RNA editing analysis, in which
P or
H radiolabeled in vitro transcripts were incubated
with lysates from pea mitochondria. To show that the editing reaction
is site-specific we compared edited and unedited templates after
incubation with mitochondrial lysate.
Isolation of Plant Mitochondria
Mitochondria
were isolated from potato tubers (Solanum tuberosum var. Bintje) or from dark grown shoots of 4-day-old pea seedlings (Pisum sativum var. Progress) by four 5-s pulses on a
high speed blender in extraction buffer containing 400 mM mannitol, 1 mM EGTA, 25 mM Tricine ()(pH 7.2), 10 mM
-mercaptoethanol, 50
µM phenylmethylsulfonyl fluoride, 0.1% bovine serum
albumin, and subsequent filtering through two layers of Miracloth. The
filtrate was spun at 2,000
g for 15 min at 4 °C to
remove cellular debris and then at 15,000
g for 20 min
at 4 °C to pellet mitochondria. After several washes in a buffer
containing 400 mM mannitol, 10 mM Tricine (pH 7.2), 1
mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride,
mitochondria were layered onto a 14 to 28 to 45% Percoll step gradient
and spun at 70,000
g for 45 min in a Beckman SW-28
rotor at 4 °C. The interface band between 28 and 45% was collected
and washed several times. Mitochondria were used on the day of
isolation for the in organello run-on transcription/RNA
editing analysis.
Preparation of Mitochondrial RNA Editing
Extracts
The RNA editing extract of pea mitochondria was
prepared according to a modification of the method used to isolate
wheat mitochondrial editing activity(14) . Briefly,
mitochondria were lysed in the presence of 1 M ammonium
sulfate and 0.2% Triton X-100. Centrifugation of the lysate at 100,000
g yielded a membrane-free soluble protein fraction
(S100). The S100 fraction was subjected to anion-exchange
chromatography using DEAE-cellulose, and proteins were eluted with 50
mM KCl. The fractions were dialyzed against 10 mM Tris-HCl (pH 8.0), 10 mM KCl, 1 mM
dithiothreitol, 0.1 mM EDTA, and 10% (w/v) glycerol,
concentrated using a Centricon-3 microconcentrator (Amicon), and stored
at -80 °C.
Run-on Transcription/in Organello
Incubation
Highly purified mitochondria (200 µg) were
resuspended in 1 ml of run-on buffer containing 10 mM Tris-HCl
(pH 8.0), 5 mM MgCl, 1 mM dithiothreitol,
50 mM KCl, 1 mM EDTA, 200 units of RNasin, 0.5 mM each of UTP, ATP, and GTP, and 250 µCi of
[
-
P]CTP(15) . The reaction mixture
was incubated at 37 °C, and aliquots were taken after different
times up to 1 h. In some experiments, radiolabeled UTP was used instead
of CTP to follow the run-on transcription. To investigate the
possibility of cytidine to uridine conversion at the nucleotide level
high concentrations of cold UTP (5 mM) were used in some
run-on transcription assays. After 10, 30, and 60 min of incubation,
aliquots were taken and nucleic acids were isolated from mitochondria
by two phenol/chloroform extractions. The high molecular weight nucleic
acid fraction was purified by a NAP 5 column (Pharmacia Biotech Inc.)
and precipitated with ethanol. Nucleic acids were digested to
monophosphate nucleotides with 10 units of nuclease P1 (Life
Technologies, Inc.) in 10 µl of P1 buffer (50 mM sodium
acetate (pH 5.3)) at 37 °C for 2-6 h. To verify that
transcripts obtained by in organello transcription are real
mitochondrial transcripts, and not bacterial contaminations, blots of
digested cosmids with mtDNA of Arabidopsis thaliana were
hybridized with the transcriptional products of the in organello synthesis (data not shown).
Thin Layer Chromatography
Aliquots (5-10
µl) of the nuclease P1 hydrolyzed products were spotted onto
cellulose TLC plates (CEL 300-10, Macherey-Nagel, Germany), and
one- and two-dimensional chromatography was carried out at 20 °C.
The solvent systems used for two-dimensional TLC were isobutyric
acid/water/25% ammonia (66:33:1 by volume) for the first dimension and
2-propanol/37% HCl/water (70:15:15 by volume) for the second
dimension(16) . One-dimensional TLC was developed in t-butanol/37% HCl/water (70:15:15 by volume). To identify the
positions of the 5`-monophosphates by UV shadowing 2-5 µl
each of 100 mM solutions of cytidine, adenosine, uridine, and
guanosine 5`-monophosphates (NMPs) were added. The relative positions
of the nucleotides varied slightly depending on the time of development
and the condition of the solvents, e.g. after repeated use. H-labeled CMP and UMP were
identified after TLC separation by UV shadowing and removed from the
TLC plate. After elution of the NMPs from the cellulose material 2 ml
of liquid scintillation mixture (Ready Safe, Beckman) were added, and
the radioactivity was determined by liquid scintillation counting.
PCR, in Vitro Transcription, and Incubation
The
templates for in vitro transcription were synthesized by
polymerase chain reaction (PCR) using the following primers:
T7-5`-primer (cox2): 5`-AGGATC
CTAATACGACTCACTATAGGGAGATCTTCCTCATTCTTATTTTGG-3`; 3`-primer (cox2): 5`-AGGATCCAGGTACAGCATCACATTTGAC-3`; T7-5`-primer (orf206): 5`-TAATACGACTCACTATAGGGAGACCACTTCACCTACTTCA-3`;
3`-primer (orf206):
5`-AAATTAACCCTCACTAAAGGGAAAAAGAGATACGAAC-3`. The PCR products obtained
represent either an internal part of the cox2 gene (542
nucleotides) from pea(17) , which is linked upstream with a
promoter sequence of the T7 RNA polymerase or a highly edited region
(114 nucleotides) of the orf206 gene(6) . For the PCR
reaction mtDNA or random primed cDNA from pea mitochondria was used.
RT-PCR of orf206 in vitro transcripts was initiated by first
strand cDNA synthesis primed by a T3-primer
(5`-AATTAACCCTCACTAAAGGG-3`). The PCR reaction was done using the
T7-orf206- and the T3-primers, and the PCR amplification
products were cloned into the pCRII vector using a TA
cloning kit (Invitrogen). The reaction mixture (100 µl) contained
50 mM KCl, 1.5 mM MgCl
, 10 mM Tris-HCl (pH 8.3), 0.5 µg of each primer, 50 fmol of each
dNTP, 10 ng of cDNA, and 2.5 units of Taq polymerase
(Boehringer Mannheim). PCR was performed on a Biomed cycler under the
following conditions: cycle 1, 1 min at 94 °C; cycle 2, 1 min at 45
°C; and cycle 3, 3 min at 72 °C. All cycles were repeated 30
times, and an extension of 10 min at 72 °C was added at the end. In
the in vitro transcription reactions 2 µg of PCR product
were used. Labeled transcripts were prepared under standard conditions
using [
-
P]CTP,
[
-
P]UTP, or [5`-
H]CTP
and T7 RNA polymerase (Boehringer Mannheim). To obtain RNA of maximal
specific activity, 500 µCi of [5`-
H]CTP at 22
Ci/mmol was dried and used per transcription. After transcription,
template DNA was removed by DNase I, and the synthesized RNA was
precipitated. Aliquots of each reaction step were analyzed on agarose
gels to monitor the reactions and to verify the transcription products.
The labeled in vitro RNAs were either incubated with
mitochondrial lysates or used directly as a control after nuclease P1
digestion in the TLC to analyze the separation conditions. A typical in vitro RNA editing assay contained 50 mM Tris
acetate (pH 7.8), 10 mM magnesium acetate, and 25 units of
RNasin in a final volume of 50 µl. 40 µg of mitochondrial
protein was used per assay.
Run-on Transcription in Mitochondria
To study
the biochemistry of the RNA editing process in higher plants, isolated
mitochondria were initially chosen as the most intact system. Attempts
to transfer in vitro transcripts as editing templates into
isolated mitochondria by electroporation had failed to give clear
results. ()A run-on transcription system (15, 19) was therefore chosen to approach in vitro the RNA editing process in plant mitochondria. This system allows
direct in organello labeling of the editing substrate
(mitochondrial RNA) coupled with the possibility to follow the RNA
editing process in a time-dependent manner.
Run-on Transcription/RNA Editing in Intact Potato
Mitochondria
To analyze RNA editing in intact mitochondria,
purified potato mitochondria were supplied with
[-
P]CTP to label the newly transcribed
mitochondrial transcripts. Aliquots were taken after 0, 15, 30, 45, and
60 min of incubation, and nucleic acids were extracted. Nucleic acids
were treated with DNase I, and the bulk of the unincorporated label was
removed by gel filtration chromatography on NAP 5 columns (Pharmacia).
After digestion of the labeled high molecular weight RNA with nuclease
P1 the resulting 5`-monophosphate nucleotides were separated by one-
and two-dimensional TLC (Fig. 2). After 15 min of incubation new
discrete spots begin to appear and increase in intensity with the time
of incubation. One of the spots comigrates with UMP in both
one-dimensional and two-dimensional TLC. Two additional spots (x and y) that are not specific to RNA editing generally
appear in relatively constant proportion and are most likely
incompletely digested dinucleotides, since they also appear after
nuclease P1 digestion of in vitro synthesized transcripts not
incubated with mitochondrial lysates. Similar results have been
previously reported by other investigators for the human apolipoprotein
B and maize mitochondrial editing systems(18, 20) . To
investigate whether these results are restricted to potato mitochondria
we tested mitochondria of several other plant species. Pea mitochondria
showed the highest in organello transcriptional activity of
the species investigated (data not shown) and were therefore used for
further studies.
-
P]CTP, and aliquots were
taken after 15, 30, 45, and 60 min. Nucleic acids were hydrolyzed with
nuclease P1, and the resulting NMPs were separated by one-dimensional
TLC. Inorganic phosphate (Pi) is the product with the highest
mobility, followed by UMP (pU), a product of unknown identity (x), CMP (pC), and another unknown product (y). The mobility of products x and y is consistent
with the dinucleotides pCpA and pApC derived by incomplete hydrolysis. PanelB shows the two-dimensional separation of NMPs
from potato mitochondria. Hydrolysis products from the 30-min
incubation separated on one-dimensional TLC were reisolated from the
plate and separated by two-dimensional TLC to verify the mobility of
the hydrolysis products. The mobility of unlabeled NMPs identified by
UV shadowing is shown schematically above the two-dimensional TLC
separation. The spots of CMP (pC) and UMP (pU) are
indicated by arrows.
RNA Editing in a Lysed System of Pea
Mitochondria
A lysed in organello system was used to
study run-on transcription (19) and RNA editing in disrupted
mitochondria. Purified pea mitochondria were supplied with
[-
P]CTP, analogous to intact mitochondria,
without added osmoticum. After 30 and 60 min aliquots were taken,
nucleic acids were extracted, and the purified high molecular weight
RNAs were analyzed by TLC after nuclease P1 digestion (Fig. 3).
After incubation for 30 min an additional spot appeared comigrating
with UMP in the TLC mononucleotide analysis. To exclude that the
radiolabeled UMP detected by the TLC analysis is due to a CTP deaminase
activity produced at the mononucleotide level rather than modification
at the polynucleotide level, we added a 10-fold excess of unlabeled UTP
to compete any such reaction. The radiolabeled nucleotide comigrating
with UMP retained about the same intensity in both high or low UTP (Fig. 3). These results show that labeled UMP was not integrated
into the nascent RNA chain as [
-
P]UTP.
Furthermore, this observation indicates that there is no detectable
level of CTP deaminase activity in plant mitochondria. RNA editing
activity was thus detected in both intact and lysed mitochondria by a
coupled transcription/RNA editing system.
-
P]CTP in an osmotically
lysed system. Different concentrations of UTP (low UTP, 0.5
mM; high UTP, 5 mM) were used for competition to
exclude potential C to U alterations on the NTP level. The mobility of
the labeled UMP was determined by separation of a hydrolyzed
[
-
P]UTP-labeled in vitro transcript (laneUMP). As control, labeled UMP
was added to the nuclease P1-digested products of the 60-min reaction
and confirms the mobility of the UMP (pU). PanelB shows the two-dimensional separation of the NMPs from
pea mitochondria. The mobility of the products of the one-dimensional
TLC separation from the run-on transcription/RNA editing assay was
confirmed by a two-dimensional separation. The UMP spots are indicated
by arrows. In a control separation the UMP spot was verified
by addition of labeled UMP (Control (30 min + UMP*)).
Incubation of in Vitro Transcripts with Pea Mitochondrial
Lysates
To investigate the editing activity in mitochondrial
lysates we incubated [-
P]CTP-labeled in
vitro transcripts of the cox2 and orf206 genes
with lysates from pea mitochondria. The cox2 template (Fig. 4) selected for in vitro transcription contains
11 in vivo editing sites (17) and was amplified by PCR
from mtDNA of pea. The labeled in vitro synthesized cox2 transcripts were incubated with S100 supernatant and extracts
obtained after DEAE anion-exchange chromatography. After incubation the in vitro transcripts were reisolated, and the monophosphate
nucleotide composition was analyzed by TLC (Fig. 4). A small
amount of radiolabeled UMP (pU) was visible in the S100 incubation
while no pU could be detected in a heat-treated S100 fraction. Heat
inactivation thus appears to completely abolish the editing activity. A
strong pU signal was observed after incubation of the cox2 transcript with the DEAE fraction.
-
P]CTP
were incubated for 30 min with heat-inactivated S100 (template
(CMP*)), S100, and a lysate after DEAE chromatography from pea
mitochondria. In the control lane labeled UMP (pU) was added to the
DEAE hydrolysis products to confirm the mobility of the UMP spot. The
mobility of the pU spot is consistent with the spot of a nuclease
P1-digested in vitro transcript that was labeled with
[
-
P]UTP. C, two-dimensional
separation of the NMPs from the cox2 transcript incubated with
mitochondrial lysates. After nuclease P1 digestion of the cox2 transcripts incubated with mitochondrial lysates the NMPs were
separated on two-dimensional TLC. The spots comigrating with UMP are
indicated by arrows. The mobility of the NMPs (pA, pC, pU, pG)
was visualized by UV shadowing, and the relative mobility of the NMP
spots is given schematically on the right.
Evidence for Cytidine Deamination
To follow the
fate of the cytosine base in order to distinguish between
transglycosylation and deamination we used
[5`-H]CTP to label in vitro transcripts.
After incubation with pea mitochondrial lysates we determined the
amount of radioactivity appearing in the UMP spot after TLC separation (Fig. 5). No label is expected in the UMP spot if the CMP to UMP
conversion occurs by a transglycosylation reaction, while
[5`-
H]UMP is the expected product of a
deamination process. Although [5`-
H]CTP is
available at a much lower specific activity than
[
-
P]CTP and detection is more difficult, we
did observe a significant increase of radioactivity in the UMP spot
after incubation of
H-labeled transcripts with
mitochondrial lysates for 60 min at 30 °C (Fig. 5). The
radioactivity of the CMP spot was determined in independent experiments
with 2,360 and 3,800 cpm, respectively. The UMP spot of the incubated
transcript was labeled with 140 and 170 cpm. Only background
radioactivity (20 ± 5 cpm) could be measured in UMP spots not
incubated with mitochondrial lysate. About 10% of the cytidines
represent editing sites in the cox2 in vitro transcript, and
accordingly only between 236 and 380 cpm are expected for a fully
edited transcript. From these data we conclude that only about
30-40% of the editing sites have been changed from C to U in this
system.
H]CTP were incubated with pea
mitochondrial extracts for 60 min. After incubation with mitochondrial
lysate transcripts were reisolated and hydrolyzed, and the resulting
NMPs were separated by TLC. After separation the UMP and CMP spots were
identified by UV shadowing and removed from the plate, and the activity
of the spots was determined. Only background counts were detectable in
the UMP spot that was not incubated with mitochondrial lysate
(-lysate), while incubation with the lysate
(+lysate) revealed significant radiolabeling of the UMP
spot.
Edited Transcripts Are Not Modified
To investigate
whether the in vitro RNA editing system works at specific
cytidines we compared edited with unedited templates of orf206 after incubation with pea mitochondrial lysates. The orf206 gene was selected because it is one of the most highly edited
genes detected to date in plant mitochondria(6) . The unedited in vitro transcript covers 11 editing sites that are replaced
by uridines in the edited RNA (Fig. 6). After incubation with
mitochondrial pea lysates for 30 min the incubated in vitro transcripts were hydrolyzed and the resulting NMPs were separated
by TLC. Radiolabeled UMP was detected only from unedited templates
indicating that only specific editing sites have been modified.
Incubation of the edited template revealed no label comigrating with
UMP although 25 P-labeled cytidines are still present in
the polynucleotide chain.
In Vitro Transcripts of orf206 Are Edited Correctly by
the Mitochondrial Lysate
After incubation of T7-transcribed in vitro transcripts of unedited orf206 first strand
cDNA was synthesized from a T3 primer. The PCR product obtained with
the T7-orf206- and T3-primers was cloned, and 20 cDNA clones
were sequenced (Fig. 7). No PCR product was obtained with either
the lysate alone or with pea mtDNA. None of the cDNA clones was fully
edited; however, 6 out of 20 clones analyzed showed C to U transitions
indicating RNA editing at some of the expected cytidine positions. One
clone showed 7 out of the 8 expected C to U changes of the fully edited orf206 transcript. Two clones showed 3 changes, one clone was
edited at two sites, and two clones were modified at only a single
site. In the 20 cDNA clones sequenced no other modification was
observed. The editing pattern of the in vitro edited
transcripts indicates that RNA editing occurs at individual sites
independently. From this observation it is tempting to speculate that
site-specific factors may be involved for individual sites.
-phosphate of
cytidine nucleotides and the labeled cytosine base to distinguish
between the possible reaction mechanisms. The experiments show that
intact and lysed mitochondrial systems are useful tools to study
organellar run-on transcription (15, 20) and RNA
editing in plants (19) . The results obtained by in
organello and in vitro systems show that in the RNA
editing process the production of uridine from cytidine occurs at the
polynucleotide level. In coupled run-on transcription/RNA editing
systems of higher plants the product of the RNA editing process in
mitochondria was determined as a genuine uridine. Furthermore,
incubation of in vitro transcripts with mitochondrial lysates
showed that the editing process is not directly linked to transcription
in mitochondria.
H-labeled cytosine base led to labeled UMP. The
occasional reverse editings from uridine to cytidine(23) ,
however, cannot as easily be explained by a straightforward reverse
deamination process. Either the reverse U to C editing is catalyzed by
a different mechanism, e.g. by a CTP synthase, or the
deamination process involves the transfer of the released amine to
another molecule in a transamination reaction.
H-labeled cytosine base, which led to
H-labeled
UMP. These results are consistent with a deamination reaction because
in a transglycosylation reaction unlabeled UMP is expected as product.
We are grateful to Dr. Axel Brennicke for advice
throughout this study and for helpful discussions. We thank Dr. Charles
Andr and Dr. Hugo Sanchez for critical reading
of the manuscript. We also thank Waltraut Jekabsons and Iris Gruska for
excellent technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.