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Mitochondrion 8 (2008) 35–46
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The process of RNA editing in plant mitochondria
Mizuki Takenaka *, Daniil Verbitskiy, Johannes A. van der Merwe,
Anja Zehrmann, Axel Brennicke
Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany
Received 15 August 2007; received in revised form 24 September 2007; accepted 26 September 2007
Available online 11 October 2007
Abstract
RNA editing changes more than 400 cytidines to uridines in the mRNAs of mitochondria in flowering plants. In other plants such as
ferns and mosses, RNA editing reactions changing C to U and U to C are observed at almost equal frequencies. Development of transfection systems with isolated mitochondria and of in vitro systems with extracts from mitochondria has considerably improved our
understanding of the recognition of specific editing sites in the last few years. These assays have also yielded information about the biochemical parameters, but the enzymes involved have not yet been identified. Here we summarize our present understanding of the process
of RNA editing in flowering plant mitochondria.
Ó 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Keywords: Plant mitochondria; RNA editing; Cytidine deaminase; RNA helicase
1. Introduction
In mitochondria and plastids of all flowering plants
examined, the sequences of many transcripts are altered
by numerous post-transcriptional conversions of C to U
(Fig. 1; Covello and Gray, 1989; Gualberto et al., 1989;
Hiesel et al., 1989). In plastids, including chloroplasts of
flowering plants, in total only about 35 Cs are found to
be deaminated to Us, details of which have recently been
summarized (Shinakai, 2006).
In Arabidopsis thaliana, Brassica napus and Oryza sativa,
the entire sets of editing sites in the protein-coding regions
of all identified mRNAs have been investigated. These
amount to 441, 427 and 491 editing sites per mitochondrial
transcriptome in these species, respectively (Giegé and
Brennicke, 1999; Notsu et al., 2002; Handa, 2003). The
total number of C to U changes will be somewhat higher,
since several events have been documented in non-protein-coding regions such as tRNAs and introns, as well as
*
Corresponding author. Tel.: +49 731 5022677; fax: +49 731 5022626.
E-mail address: [email protected] (M. Takenaka).
in leader and trailer regions, but not all of these regions
have been analyzed in any plant species. These are comparatively few editing sites, since by extrapolation, only about
20–50 are expected in a given transcriptome.
RNA editing is required for gene expression in plant
mitochondria. The genomic information encoding an open
reading frame or a tRNA is more often than not incomplete in these organelles and will not yield a functional
product. Therefore, gene expression in plant mitochondria
depends on RNA editing for its biological functionality. In
all flowering plant mitochondrial systems, RNA editing is
essential for the synthesis of correct proteins which, after
editing, generally exhibit closer sequence conservation with
their homologs in other systems. In addition, RNA editing
in plant mitochondria is required in several tRNAs to be
able to fold correctly and thus to become functional
(Marchfelder and Binder, 2004). RNA editing in plant
mitochondria is an essential step of RNA maturation without which no working respiratory chain and no functional
mitochondria can be assembled and maintained in the cell.
In this review, we will first summarize the observed features of RNA editing in plant mitochondria in the steadystate RNA population and their functional consequences.
1567-7249/$ - see front matter Ó 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
doi:10.1016/j.mito.2007.09.004
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M. Takenaka et al. / Mitochondrion 8 (2008) 35–46
Fig. 1. RNA editing in mitochondria of flowering plants is a posttranscriptional process which changes selected cytidines (C) in the primary
transcript to uridines (U) in the mature mRNA. The amino acid sequence
encoded by the fully edited, mature mRNA is different from the protein
sequence encoded by the genomic DNA and the primary transcript. In this
hypothetical sequence several examples of such amino acid changes are
included. About 400–500 such C to U RNA editing events are observed in
the mRNA and tRNA population in plant mitochondria of a given
angiosperm species. Since only some of the cytidines are altered by RNA
editing, these have to be specified and distinguished from cytidines which
are not changed.
In the second part, we will look at recent advances in the
analysis of the parameters of site recognition and of the
biochemistry involved.
2. How was RNA editing established in the plant kingdom?
In flowering plant mitochondria RNA editing involves
exclusively C to U changes, in which the C in the premRNA is presumably deaminated to a U moiety (Fig. 2).
Most RNA editing events are found in the coding regions
of mRNAs and less frequently in introns and other nontranslated regions. RNA editing in tRNA molecules can
restore essential base-pairings and only complete RNA
Fig. 2. The biochemical reaction of RNA editing in plant mitochondria is
in its effect a deamination step. The amino group from the base of the
cytidine may be removed by a modified cytidine deaminase like enzyme or
may be transferred by a modified transaminase to a receptor molecule.
This latter should be a larger moiety such as another RNA molecule, since
in the various functionally competent in vitro assays small molecules have
been removed and thus do not appear to be required.
editing will then allow correct folding and processing of
the tRNA precursors. In the ribosomal RNAs in plant
mitochondria as well as in chloroplasts no RNA editing
events have been observed so far and it is not clear why
these RNAs are exempt from editing.
In flowering plant species, U to C conversions do not
occur or are very rare events. In the plants A. thaliana,
B. napus and O. sativa, none have been identified (Giegé
and Brennicke, 1999; Notsu et al., 2002; Handa, 2003).
In non-flowering plants, the amination of U to C is
much more common. In hornworts, for example, this reaction almost reaches the frequency of the C to U editing
events (Malek et al., 1996; Knoop, 2004). The only land
plants in which no RNA editing have been observed is
the liverwort Marchantia polymorpha (Oda et al., 1992)
and species of the Marchantiales closely related to M. polymorpha (Malek et al., 1996). Liverworts of other orders, for
example, the Metzgeriales, do show frequent RNA editing
at least in their mitochondrial mRNAs (Malek et al., 1996).
Interestingly none of the closest relatives to the land plants
in the group of the green algae, such as the Charales, show
any RNA editing (Turmel et al., 2003). From these observations it has been postulated that the establishment of
RNA editing in plant mitochondria may have occurred at
some point of the early evolution of land plants, the Marchantiales presently being considered as branching very
near or at the very root of the land plant tree (Malek
et al., 1996; Knoop, 2004).
The presence of frequent U to C aminations among the
mosses and ferns, for example in hornwort species and in
the Lycopodiopsida (Knoop, 2004; Groth-Malonek and
Knoop, 2005) has consequences for the biochemical reactions and therefore the enzymes possibly involved. The
characteristics of the previously characterized cytidine
deaminases and cytidine synthases make it unlikely that a
single enzyme of either of the two classes can perform both
reactions. Details of these groups of enzymes are discussed
further below.
The asymmetric distribution of editing sites between
coding and non-coding regions in RNAs suggests that
there is some selective pressure at work. Without a functional necessity to maintain an editing site, this would soon
be lost. The requirement to produce proteins with certain
amino acid sequences may be this selective pressure in
the open reading frames of mRNAs, where only a U at a
given editing position will yield a specific codon identity.
In introns, RNA editing is sometimes a prerequisite of correct folding of essential secondary structures and, thus, efficient intron excision (Carillo and Bonen, 1997), which
latter is of course required to produce a functional protein
product.
This requirement for RNA editing in plant mitochondria has most likely evolved after the biochemical potential
to post-transcriptionally modify certain nucleotide positions was established. This order of events was first formulated by Covello and Gray (1993), where the authors
suggest that first a deaminating/aminating enzyme had
M. Takenaka et al. / Mitochondrion 8 (2008) 35–46
mutated from acting only on mononucleotides or whatever
other substrates to also accept polynucleotide templates. In
the second step, the mitochondrial DNA was then allowed
to mutate by changing a T to a C (or a C to a T) in those
positions that could be addressed and corrected post-transcriptionally by the novel RNA editing enzyme. Once the
first such mutation requiring this compensating correction
in the RNA had occurred, RNA editing was established
and had to be maintained. This order of events was aptly
named the bureaucracy model (Mulligan, 2004), because
of its similarity to any administration which will introduce
often unnecessary complexities with the sole aim of making
itself indispensable.
3. What can RNA editing in plant mitochondria be good for?
In this bureaucracy model RNA editing is just a waste of
time and energy, because, without exception, all of the
RNA editing events described to date would be rendered
unnecessary if the sequence of the mature mRNA were
encoded in the genome, as is the case for example in bacteria. If there is no obvious advantage in the existence of
RNA editing, there may be a hidden biological importance
of RNA editing. One possibility of such a camouflaged
agenda might be another level of regulatory control of gene
expression among the various post-transcriptional
processes.
For example, the introduction of an AUG translational
start codon from an ACG codon could make an mRNA
rapidly accessible for translation. This would be much faster than the complete de novo synthesis of the affected transcript (Fig. 1).
RNA editing could confer an advantage with its potential of generating mRNA variants, which will allow to synthesize two or more different polypeptide products from a
single gene. This potential is realized by some viral editing
systems and most notably in the tissue-specific apolipoprotein B editing in mammals. The site specific C to U deamination changing a CAA codon in the apolipoprotein B
mRNA to a UAA stop codon is controlled tissue specifically. In different cells two distinct lipid carrier proteins
of 100 kDa and 49 kDa, respectively, are synthesized from
one and the same genomic coding region (Greeve et al.,
1991; Chang et al., 1998). On a larger scale, the A to I
RNA editing in the GluR receptor channels results in a
large array of variant proteins available for the combinatory assembly of these channels which are involved in
fine-tuning the properties of the chemo-electric potential
transmission (O’Connell, 1997; Sommer et al., 1991).
This latter option of generating polypeptide variants
should be reflected in the access of the translation system
to the partially edited mRNAs observed in plant mitochondria for some mRNA species. Indeed, experimental examinations showed that partially edited mRNAs are present
in polysomal fractions of plant mitochondria and can
apparently be translated into a family of variant proteins
(Mulligan, 2004). However, these variant proteins are most
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likely non-functional and get rapidly degraded, since only
one type of protein sequence appears to be incorporated
into the polypeptide complexes of the respiratory chain
(Grohmann et al., 1994). These integrated polypeptide
sequences correspond to the protein specified by the fully
edited mRNA with the omission of the rare sporadic editing events which are most likely errors of the editing
machinery (Grohmann et al., 1994; Phreaner et al., 1996;
Mulligan, 2004).
In fact, it is unlikely that polypeptides synthesized from
unedited RNAs would function properly. These variant
proteins would rather impair the efficiency of mitochondrial respiration, since the polypeptide sequences derived
from the fully edited mRNAs are generally better conserved with the homologs in other organisms than the proteins encoded by the genomic sequences without editing or
by any of the editing intermediates. Therefore, the proteins
encoded by the fully edited mRNAs have been extensively
selected for their physiological and biochemical
functionality.
The possible functionality of proteins made from unedited mRNAs has been experimentally investigated for the
atp9 gene. The (unedited) mitochondrial genomic sequence
was integrated into the nuclear genome of tobacco with the
proper expression signals as well as with correct mitochondrial import addresses fused to the open reading frame.
The synthesized and imported ATP9 protein variant is
not functional since its presence disturbs mitochondrial
function, presumably by competing with the endogenous
native, mitochondrially encoded protein. The resulting
phenotype manifests itself in a dysfunction of the pollen
development and results in male sterility (Hernould et al.,
1993; Zabaleta et al., 1996).
The large size of the plant mitochondrial genomes with
their ample space to encode additional genes or their variants make it unlikely that RNA editing would be advantageous to increase the coding capacity of a given
mitochondrial gene just to save space. Furthermore, such
a presumed advantage would be illusionary because the
RNA editing process itself requires a number of additional
polypeptides.
Thus we are presently left with the bureaucracy model of
RNA editing being a waste of time and energy, with RNA
editing being a biologically selfish process, which once it
has become established now has to be perpetuated.
However, there is still a third possible raison d’etre, a
long term evolutionary advantage. This may be to prevent
or at least to complicate transfer of mitochondrial genes to
the nuclear genome. The mitochondrion may profit from
the establishment of RNA editing as a means to protect
its genome. Since there is no evidence of editing in the
nucleus/cytoplasm of plants, a gene sequence requiring
editing in the mitochondrion could not immediately
become functional upon integration in the nuclear genome.
A successful transfer would be most easily achieved by
reverse transcribing the respective coding region from a
fully edited mitochondrial mRNA molecule. The resulting
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M. Takenaka et al. / Mitochondrion 8 (2008) 35–46
cDNA sequence would encode the correct protein sequence
upon appropriate activation in the nuclear genome. However, the reverse transcription step is not straightforward
and so far mostly unedited genomic mitochondrial
sequences have been found to be inserted into the nuclear
genomes of various plants (Bergthorsson et al., 2003; Blanchard and Schmidt, 1995; Brennicke et al., 1993; Stupar
et al., 2001).
The protective effect of the editing requirement may be
real since mitochondria in many other species protect their
genes from transfer by using variants of the codon translation system. In these organisms (such as mammals) genes
from the mitochondrion cannot be directly translated into
functional proteins in the nucleus/cytoplasm since several
essential amino acids will be decoded wrongly. The coincidence of identical decoding systems with the same translational cipher in plant mitochondria (and plastids) and in
the cytoplasmic system is probably unrelated to the advent
of RNA editing, but may be a fortuitous consequence of
the established import of various tRNA species from the
cytoplasm into mitochondria. Since the mitochondrion
and the cytoplasm share several of these tRNAs, they will
impose a stringent selection pressure to maintain the standard code in both compartments.
4. How is an RNA editing site identified in plant
mitochondria?
As the name implies, RNA editing in plant mitochondria and in chloroplasts is a post-transcriptional process.
The identification of partially edited mRNA molecules
was the first hint that RNA editing is a process separated
from transcription and that the nucleotide changes are
not introduced by canonic signals in the genomic DNA.
In such partially edited transcripts, some of the Cs at
potential editing sites have been altered into Us while some
the Cs encoded by the genome, but found edited to Us in
most mRNA molecules, are (still) unedited. Subsequently
the successful in vitro assays in which respective changes
were observed in added RNA molecules clearly confirmed
this conclusion.
These partially edited RNA molecules furthermore suggest that each editing site is identified individually with
some being more rapidly edited than others. To differentiate a C to be edited from a C-moiety not to be altered,
these need to be discriminated. The respective information
to identify a given editing site can only be encoded in its
specific nucleotide sequence context. Theoretical calculations taking into account the genome size and the number
of RNA editing sites suggest that ten nucleotides would be
sufficient to identify a nucleotide sequence motif unambiguously in a plant mitochondrial genome.
In the past few years detailed studies of the parameters
selecting RNA editing sites within an RNA molecule by
in organello and in vitro assays have yielded considerable
advances in our understanding of this process. In the following we summarize these insights gathered in various
plant species. The very similar observations made in the
different flowering plants suggest that one and the same
recognition process has been maintained through the evolution of the higher plants.
5. In vivo information on the specificity determinants
Genome internal recombinations in the monocot plant
rice and in the dicot plant Oenothera created duplicated
fragment of genes. From these fragments transcripts are
generated in vivo in which some of the editing sites of the
respective original open reading frame are present but have
retained only few of the nucleotides surrounding this editing site in the original context (Lippok et al., 1994; Kubo
and Kadowaki, 1997). In those instances where upstream
sequences were retained, these duplicated sites were seen
edited. Conversely, when only the downstream context
was duplicated, no editing was observed. These natural chimeric sequences had thus already indicated that most of the
specificity determinants must be located upstream and that
downstream as little as three conserved nucleotides could
be sufficient to identify a given editing site (Lippok et al.,
1994).
In silico comparisons of the sequences surrounding editing sites, e.g. all the 441 sites in the protein-coding regions
of the A. thaliana mitochondrial RNA, did not reveal common sequence motif(s) in all, many or even only several
editing sites (Giegé and Brennicke, 1999). The only common feature observed in the vicinities of editing sites is
preferences for the nucleotide identity immediately adjacent to the edited C. Most notably are guanosines rarely
observed immediately upstream of the edited C. The editing enzyme or complex thus seems to sterically prefer
pyrimidines at this position while a guanosine may make
access more difficult.
In chloroplasts, in vivo analysis by transformation has
been established for tobacco and has yielded important
insights into the structure and extension of cis-elements
in this organelle (Chaudhuri et al., 1995; Chaudhuri and
Maliga, 1996; Bock et al., 1996, 1997; Reed et al., 2001;
Chateigner-Boutin and Hanson, 2002). This approach is
so far not possible for plant mitochondria since no successful transformation has as yet been reported mostly due to
the lack of a selectable marker gene specific for
mitochondria.
6. Experimental investigations of the specificity determinants
The requirements of size and location of the specificityconferring nucleotide context relative to the edited nucleotide in plant mitochondria have recently been experimentally investigated in detail by in organello and in vitro
approaches.
The successful establishment of in organello editing systems for wheat and maize (Farré and Araya, 2001) allowed
the characterization of the cis-requirements at individual
sites in these two monocot plant species. In vitro RNA edit-
M. Takenaka et al. / Mitochondrion 8 (2008) 35–46
ing systems were first developed for chloroplasts (Hirose
and Sugiura, 2001; Miyamoto et al., 2002, 2003). Analogous to these, reliable in vitro activities could be established
for pea and cauliflower mitochondria (Takenaka and Brennicke, 2003).
6.1. In organello RNA editing in wheat and maize
The first in organello assay was successfully established
by the group of Alejandro Araya for wheat mitochondria
(Farré and Araya, 2001; Choury and Araya, 2006). The
procedure was successfully adapted to maize mitochondria
in the laboratory of Frank Kempken (Staudinger and
Kempken, 2003). Briefly, DNA molecules coding appropriate promoter and RNA stability structures and non-mitochondrial sequences for discrimination from the
endogenous mRNAs are introduced into intact isolated
mitochondria by electroporation. The DNA is transcribed
in the organelles, and the RNA is processed by correct
intron excision and RNA editing at a number of the bona
fide in vivo RNA editing sites.
Several of these actively edited sites have been investigated in detail by various mutations of the nucleotide
sequences surrounding the analyzed editing site. In the
two monocot plants wheat and maize about 16 nucleotides
in the upstream region and at most six nucleotides downstream are sufficient to determine at least some of the
nucleotide positions to be edited (Farré et al., 2001; Choury
et al., 2004). For example, the extensively analyzed editing
sites 77 and 259 in the cox2 mRNA are correctly recognized in wheat mitochondria when this window of surrounding nucleotides is present. More distant nucleotides
have little influence, since relocation of these 23 respective
nucleotides into a different further sequence context in the
mRNA still confers correct RNA editing at this C (Choury
et al., 2004).
The sequence window requirement of 23 nucleotides is
similar for these two editing sites in the cox2 mRNA.
The observation that the importance of individual nucleotide identities (such as the nucleotide immediately 3 0 of the
edited C) differ between the two sites, suggests that editing
sites in plant mitochondria are identified by individual and
specific trans-recognition factors in the assembly of the
hypothetical editosome.
In maize mitochondria the in organello editing system
shows that, in some instances, more than the immediate
vicinity of an editing site may be required for efficient editing (Staudinger and Kempken, 2003; Staudinger et al.,
2005). While transcripts synthesized in the purified mitochondria from an introduced cox2 gene from the rather distant dicot A. thaliana were edited at several sites, an atp6
mRNA from the more closely related plant Sorghum bicolor was not edited at all, although in these transcripts all
editing sites are identical with maize and their surrounding
sequences are also largely conserved (Bolle and Kempken,
2006). These results suggest that variations in distant
39
regions, possibly even the structures of 5 0 - and/or 3 0 untranslated sequences, might influence RNA editing.
6.2. In vitro RNA editing in pea and cauliflower
The in vitro RNA editing systems from pea and cauliflower mitochondria overall yielded similar results on the
recognition of RNA editing sites in mRNAs of the two
dicots as in the in organello systems of the two monocots.
This suggests that the basic parameters of the RNA editing
machinery have been conserved in flowering plants. In both
dicot plants about 20 nucleotides are essential upstream, 40
are optimal, and none are necessary downstream of the first
RNA editing site in the atp9 mRNA. Analysis of the cisrequirements by targeted mutations of the template and
competition experiments narrowed the sequence requirements for the site specificity to the region 5–20 nucleotides
upstream of this atp9 mRNA site (Takenaka et al., 2004).
The region up to 40 nucleotides upstream influences the
efficiency of in vitro editing (Neuwirt et al., 2005). If this
sequence region is deleted, in vitro editing is still observed,
but with less than 50% of the activity recorded with the
full-length template. This region surprisingly also influences editing at the next downstream site, which is 30
nucleotides 3 0 of the first site, and thus acts across a total
distance of about 70 nucleotides (van der Merwe et al.,
2006). Here species-specific adaptations are observed, since
the native cauliflower 20 to 40 sequence region
enhances the reaction, while the heterologous pea sequence
inhibits editing in the cauliflower lysate.
This long-distance effect is analogous to the observations made with a heterologous sorghum atp6 sequence in
electroporated maize mitochondria (Staudinger et al.,
2005). A homologous maize sequence is required far
upstream of the conserved atp9 reading frame of sorghum
origin to confer correct and efficient RNA editing in organello. These observations suggest that there is a species-specific RNA editing entry motif in this upstream region. The
results furthermore indirectly suggest that the RNA editing
factor(s) move in some mode of linear progression along
the mRNA molecule. However, only few and apparently
randomly distributed conserved nucleotide sequence motifs
have been found upstream of open reading frames in plant
mitochondria (Schuster and Brennicke, 1989; Pring et al.,
1992), and it is questionable whether these might play a
role in such a function.
6.3. Long distance sequence effects
How can the observed long distance sequence effects be
explained mechanistically? A straightforward explanation
would involve the editosome directly and would postulate
some type of scanning progression of the editing complex.
This model would, however contradict the observed individual and independent addressing of each editing site
through random recognition of the surrounding specific
sequence motifs.
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M. Takenaka et al. / Mitochondrion 8 (2008) 35–46
An alternative, more indirect explanation might be the
engagement of RNA helicases and potential cofactors,
which are required to clear the template RNA for access
of the editosome (Fairman et al., 2004). A sequence motif
active in one species may not be sufficient, or may be a hindrance, for a helicase (complex) from another plant species.
The resulting mode of action of the RNA editing process
would appear as an intermediate between a scanning and
a hit-and-run mechanism, and would show characteristics
of either. Depending on the experimental assay, the scanning of the RNA helicase would appear as dominant characteristic or the apparently random editing of individual
sites such as manifested in the appearance of partially edited sites in the in vivo mRNA population would be the
prominent feature observed.
6.4. Site recognition
The hit-and-run model, according to which specific
regions of the RNA molecule are addressed individually
and randomly by the RNA editing machinery, was tested
by detailed in vitro experiments (Verbitskiy et al., 2006).
This investigation showed that partially edited transcripts
are true intermediates, which can serve as substrates for
further rounds of RNA editing. It is particularly striking
that even substrate RNA molecules, in which one of two
closely spaced editing sites was already modified, are
accepted as substrates for further rounds of RNA editing.
In fact, editing efficiencies in such partially edited RNAs
are comparable to those in completely unedited RNA molecules. This result shows that recognition of a specific editing site by the respective trans-factors is independent of the
nucleotide identity at the actual editing site.
Site recognition and site specificity generally appear to
occur at random contact points along the mRNA molecule. The vicinity of each site carries its own recognition
context, resulting in an apparent hit-and-run process of
RNA editing. Most notably in this respect are the observed
efficiencies of competition experiments of in vitro assays
and the results of the transfection assays with isolated
wheat mitochondria (Choury et al., 2004; Verbitskiy
et al., 2006). These in vitro and in organello competition
assays show that a homologous sequence can compete
RNA editing at a given site, while no competition with
the sequence contexts of different editing sites is observed.
These competition assays further underline the importance of the specific upstream elements. A wild type
upstream sequence can compete as long as it is competent
as a template. The competition effect is lost for mutated
sequences which are not edited in vitro since they no longer
contain the individual site-specific nucleotide positions
essential for recognition. Position and extent of these essential nucleotides upstream (and at very few nucleotides
downstream) have been found to vary between different
editing sites. This observation suggests that not only the
variant primary sequences but also the extent of the contact
points of the specific trans-factors can vary between indi-
vidual recognition elements and is crucial for efficient
editing.
The experimental results from in vitro assays with dicots
and in organello investigations with monocots furthermore
suggest that while the recognition parameters can vary
between individual sites, the characteristics of the recognition of a given RNA editing site may have maintained similar features of primary sequence and extension between
different plant species. Since most of the 400 RNA editing
sites in flowering plant mitochondria are conserved
between different species and since most of these sites are
in conserved mRNAs or tRNAs, the sequence vicinities
of homologous sites are also very similar. Consequently,
it can be postulated that in different plants these sites are
recognized by likewise conserved trans-factors.
Therefore, to identify an individual conserved RNA
editing site, distinct trans-factors have to be involved which
may have been conserved to some extent during the evolution of the flowering plants. Consequently, several hundred
distinct, but at least partially conserved trans-factors have
to be postulated to address each of the 400–500 editing
events in plants mitochondria.
6.5. Recognition of RNA editing sites by protein-factors?
To correctly and unambiguously identify this large number of editing sites in mitochondrial transcripts, a correspondingly large number of trans-factors will be required.
Since the search for guide-RNA like molecules has been
unsuccessful so far, sequence-specific recognition may be
mediated by proteins alone as trans-factors. The number
of different site-specific proteins required to specify all of
the sites will require several hundreds of corresponding
genes in the nuclear genome of plants.
Recently, the protein (and corresponding gene) family of
the so-called PPR proteins has been proposed as a candidate group for these specificity factors. Its name derives
from the common presence of numerous pentatricopeptide
repeats, which vary in number and in their combinations
with other domains in various subgroups. In plants, this
family of proteins is highly amplified and contains several
hundred members (Small and Peeters, 2000; Lurin et al.,
2004). The profiles of prediction programs suggest that a
large proportion of the encoded proteins is indeed targeted
to either plastids or mitochondria, or both (Lurin et al.,
2004). Experimental evidence for several members with
full-length reading frames or with their N-terminal regions
in transgenic plants or in transient intracellular assays confirms that these are competent to target the respective proteins to these organelles.
The first theoretical considerations that these proteins
could be nucleic acid binding are confirmed by genetic
and biochemical evidence, which has shown that several
of these PPR proteins are involved in various processing
steps of RNAs in plastids. For these functions in intron
excision and endonucleolytic processing binding of the
RNA substrate is essential.
M. Takenaka et al. / Mitochondrion 8 (2008) 35–46
The first clear connection between the PPR proteins and
RNA editing was also made in the chloroplast, where inactivation of a specific PPR protein encoding gene in A. thaliana leads to the loss of one specific RNA editing site
(Kotera et al., 2005). A second example of such a connection has recently been found between a second such PPR
protein and another editing site in plastids (Okuda et al.,
2007). The family of PPR proteins is thus presently the
favored candidate to contribute the site-specific recognition
factors for RNA editing in plastids as well as in
mitochondria.
Recently genetic approaches have been initiated to identify the trans-acting specificity factors in A. thaliana (Bentolila et al., 2005; Anja Zehrmann, Johannes A. van der
Merwe, Mizuki Takenaka, unpublished results). These
assays take advantage of ecotype-specific variations in the
RNA editing efficiency at some of the sites. Several essential sites have been identified beyond the one previously
reported (Bentolila et al., 2005), which are in one ecotype
edited completely or nearly completely in the in vivo
mRNA population, but are edited to only about 40–60%
in another ecotype (Anja Zehrmann, Johannes A. van der
Merwe, Mizuki Takenaka, unpublished results). These
QTL-traits are presently being followed through their pattern of inheritance and true nuclear inheritance may eventually lead to the identification of the loci responsible and
may identify one or more of the genes encoding these specificity-mediating proteins in flowering plants.
7. Which enzymes may do the RNA editing in plant
mitochondria?
In plant mitochondria (and plastids) no nucleotide insertions or other changes of nucleotide identities, such as
those identified in kinetoplasts (Benne et al., 1986; Chang
et al., 1998; Hajduk and Sabatini, 1998) or in mitochondria
of the slime mould Physarum polycephalum (Mahendran
et al., 1991; Gott and Visomirski-Robic, 1998) have been
observed. Therefore, the most parsimonious explanation
of the enzymatic mechanism of RNA editing in plants
would be to postulate the involvement of an RNA-specific
C-deaminase analogous to the APOBEC-1 enzyme
described in the apolipoprotein B mRNA editing of mammals (Greeve et al., 1991; Navaratnam et al., 1998). Several
lines of experimental evidence indeed suggest that a deamination reaction may be performing the C to U conversions
in plant mitochondria (Araya et al., 1992; Rajasekhar and
Mulligan, 1993; Yu and Schuster, 1995; Mulligan, 2004).
Assays with in organello and in vitro systems have investigated the fate of the sugar-phosphate backbone as well as
the nucleotide base (Rajasekhar and Mulligan, 1993; Yu
and Schuster, 1995). These experiments have shown that
the sugar-phosphate backbone and the nucleotide base
are retained intact during and after the editing process,
which excludes nucleotide excision and base exchange as
possible reactions. These results leave deamination or
41
transamination to mediate the resulting C to U change in
flowering plant mitochondria (Fig. 2).
7.1. Are transamination reactions involved?
Transamination reactions transferring an amino group
from one molecule to another occur in many of the various
pathways of amino acid biosynthesis. Usually these reactions result in the amination or deamination of an intermediate of the compound to be synthesized and often require
another compound as acceptor of the amino group. In the
in vitro RNA editing system we have tested several of these
acceptor molecules such as oxaloacetate and a-ketoglutarate for their effect on the editing reaction (Mizuki Takenaka, unpublished data). None of these low molecular
weight molecules showed any influence on the efficiency
of the in vitro editing reaction. In addition, these small molecules such as oxaloacetate and a-ketoglutarate with
molecular weights below 10 kDa should be quantitatively
removed from the in vitro plant mitochondrial lysates during the extensive dialysis step of the extract. Since the RNA
editing reaction proceeds in vitro without their addition to
the assay, these or similar compounds should not be
required as amino group receptors. However, if these
enzymes had evolved to accept as substrates polynuclotides
such as a long RNA molecule they may also have evolved
to accept different amino group receptors, for example
longer molecules such as another RNA. Therefore such a
transamination reaction cannot be excluded per se to be
involved in the C to U deamination observed in flowering
plant mitochondria.
One such candidate enzyme is the enzyme glutamate
dehydrogenase (GDH). GDH can catalyze amination as
well as deamination reactions depending on the presence
and concentration of various allosteric and isosteric regulators (Fig. 3). There is thus a (albeit remote) possibility that
one or more of these enzymes have been recruited into the
RNA editing reaction to catalyze the deamination step
from C to U. Surprisingly representatives of the glutamate
dehydrogenase group of proteins, i.e. GDH1 and GDH2,
were found among a number of general plant mitochon-
Fig. 3. The household enzyme glutamate dehydrogenase catalyzes reactions which include deamination and amination steps. The compounds
NAD(P) or NAD(P)H are cofactors for the GDH enzymes and are
required for the respective directions of the activity. The equilibrium of the
reaction is allosterically influenced by the ambient concentrations of ATP.
One of the GDH proteins or a similar enzyme could be a candidate for the
evolutionary origin of the plant mitochondrial and plastid RNA editing
enzyme. Mutations could have altered such an enzyme to be able to
contact RNA molecules and to deaminate specific cytidines and in
addition to aminate certain uridines.
42
M. Takenaka et al. / Mitochondrion 8 (2008) 35–46
drial proteins identified by binding to RNA editing templates (Takenaka et al., 2007).
The potential participation of the GDH in the C to U
deamination reaction was investigated by testing the influence of various cofactors of this enzyme on the in vitro editing assay. The most prominent and essential cofactors of
the normal GDH catalyzed reactions are NADH and
NADP for the deamination and amination reactions,
respectively. However, NADH as well as NADPH stimulated the reaction while the oxidized dinucleotides NAD
and NADP did not.
To resolve this contradiction and to determine whether
the GDH is indeed required for the RNA editing reaction,
the effect of a direct blocker of the GDH proteins, the compound phosphinothricin, was analyzed. Surprisingly addition of phosphinothricin to the in vitro RNA editing
reaction did not inhibit, but rather stimulated the in vitro
editing activity (Fig. 4; Takenaka et al., 2007). This result
clearly shows that the GDH polypeptides are not the active
enzymes involved in RNA editing in plant mitochondria.
7.2. Is a cytidine deaminase involved?
These recent results and arguments make the participation of a modified cytidine deaminase as the responsible
enzyme more likely. A family of nine cytidine deaminase
genes has been described in the nuclear genome of A. thaliana (Faivre-Nitschke et al., 1999). One or more of these
enzyme homologs may be targeted to mitochondria (and/
or plastids) and could thus be involved in the RNA editing
reactions in mitochondria (and plastids), leaving others
available for the classic role of cytidine deaminase in the
Fig. 4. The enzyme glutamate dehydrogenase (GDH) is not involved in
RNA editing in plant mitochondria. This is evidenced by experimental
results, which show that the GDH-specific inhibitor phosphinothricin does
not inhibit in vitro editing. On the contrary, GDH supresses in vitro
editing, since phosphinothricin substitutes the requirement for NTP in the
in vitro RNA editing reaction. In this experiment, RNA editing of the first
editing site in an atp4 template RNA is observed in the presence of 15 mM
CTP (left lane; the fluorescent cDNA molecules are derived from TDGrecognized mismatches due to the C to U editing reaction in the template
RNA; Takenaka and Brennicke, 2003). In the control in vitro incubation
without any added nucleotide no editing signal is detected (lane marked 0).
In the presence of 10 or 15 mM phosphinothricin (lanes labeled ‘‘phosphinothricin’’) the products of in vitro editing reactions are detected
although no NTP or dNTP had been added. Only the relevant portion of
the gel image is shown.
nucleotide biosynthesis pathway from cytidine towards
uridine.
Presently there are two circumstantial arguments against
the participation of such a classic type of cytidine deaminase. The first difficulty derives from the experimental failure to inhibit the in vitro RNA editing activity by
extraction of the usually required and usually accessible
zinc ions from the reaction. All classic cytidine deaminases
contain reaction-centre bound zinc and can be readily incapacitated by specific zinc chelators such as 1,10 o-phenanthroline. The bacterial cytidine deaminases as well as the
apolipoprotein B cytidine deaminase are both completely
blocked by addition of this chelator (Carter, 1998). Similarly, in chloroplast in vitro assays, the zinc chelator has
some detrimental effect on the editing reaction (Hegemann
et al., 2005). The in vitro plant mitochondrial RNA editing
reaction is however not inhibited by the addition of this
compound (Takenaka and Brennicke, 2003). The plant
mitochondrial enzyme thus would have to have evolved
differently to protect the central zinc ion. Indeed, other
enzymes have been identified, in which the essential zinc
atoms are bound so tightly inside the folded protein that
none of the chelators tested can completely remove these
metal atoms (Späth et al., 2007). Alternatively, a different
C-deaminating enzyme may not require a bound zinc atom.
The second, less direct argument against the involvement of a classic cytidine deaminases derives from the
occurrence of the ‘reverse’ editing events of U to C so frequently observed in ferns and mosses. For energetic considerations, cytidine deaminases are unlikely to be able to
catalyze this reaction and add an amino group to a U residue to generate a C. Thus, a second unrelated enzyme
would have to be invoked for these reverse reactions. However, the more parsimonious solution would be a single
enzyme able to perform both reactions. Slightly diverging
evolutionary paths may have lead to an enzyme in hornworts and related plant species which is able to catalyze
the U to C reaction, while the variant protein in the flowering plants would be less suited to facilitate this reaction.
On the other hand parallel evolution of various enzymes
involved in RNA editing has been observed in mitochondria of P. polycephalum and also in kinetoplasts of Trypanosomes and could also have established two distinct
activities in non-flowering plants. One of these, the U to
C activity, may subsequently have been lost in the lineage
of the flowering plants.
7.3. Is a CTP synthase involved?
An analogous problem arises when the family of CTP
synthases is considered as alternative evolutionary source
for the RNA editing enzyme in plant mitochondria. CTP
synthases might have been adapted to perform the amination reaction changing U to C in polynucleotides, but are
unlikely to be able to catalyze the cytidine deamination.
So far none of the CTP synthase enzymes described for
the mononucleotide amination from UTP to CTP has been
M. Takenaka et al. / Mitochondrion 8 (2008) 35–46
reported to also catalyze the deamination step. A second
problem with the CTP synthases is their requirement for
low molecular weight cofactors. Generally ammonia or
glutamine serve as amino group donors, which, as detailed
above for the transaminases, should have been quantitatively eliminated by the extensive dialysis step from the
editing-competent in vitro lysates. For the in vitro assayed
C to U changes, an amino group acceptor would be
required, which, considering a strictly reverse reaction scenario, would also be such a smaller molecule.
Alternatively any of the various tRNA modifying
enzymes such as one of the nucleotide-specific deaminases
or transaminases may have evolved into the RNA editing
enzyme. Less likely, but still possible are DNA repair
enzymes as an evolutionary source of the editing activities
in plastids and mitochondria of plants. Identification of the
enzyme involved in plant mitochondrial RNA editing
remains the foremost problem in this field for the near
future, and several approaches by genetic analysis and
experimental protein identification are presently being pursued by several laboratories around the world.
7.4. Does an RNA helicase participate?
The participation of an RNA helicase in plant mitochondrial RNA editing has been proposed from the observation of a strict requirement of ATP in the in vitro
reactions. Particularly the finding that in plant mitochondrial in vitro assays, this ATP can be fully substituted by
any of the dNTPs (and any of the NTPs) has prompted
speculations about an RNA helicase (Takenaka and Brennicke, 2003).
Several members of this class of enzymes have been
shown to tolerate such a broad spectrum of nucleotides,
which is rarely seen in other enzymes. RNA helicases participate in numerous processes of RNA maturation and
might very well also be involved in RNA editing in plant
mitochondria.
One possible function of the RNA helicase would be to
resolve inhibitory secondary structures in the template
43
mRNAs and present the RNA molecule in a form accessible to the specific trans-acting RNA editing factors.
Another function of the RNA helicase could be to clear
the RNA template from RNA binding proteins which
adhere unspecifically to any RNA sequence and thus block
access to the RNA editing proteins (Fairman et al., 2004).
The main function of such general RNA binding proteins is
of course the protection of the RNA against nucleases. The
RNA helicase would dissociate the bound unspecific proteins along the RNA molecule, the energy which it requires
for this task provided by the nucleotidetriphosphates which
have to be added in vitro. This cleaning of the RNA would
free the recognition site and the nucleotide to be edited
and, thus, allow access of the editing activity (Fig. 5).
This involvement of an RNA helicase seems to be the
most likely scenario to explain the various observations
made for NTP requirements in plant mitochondrial and
plastid in vitro assays. In the latter the optimal concentration of the NTP/dNTP which has to be added in vitro
appears to vary depending on the plant species and sometimes on the lysate preparation. The substitution of the
NTP/dNTP requirement in the mitochondrial in vitro reaction by the GDH blocker phosphinothricin suggests that
the observed strict nucleotide dependence of the reaction
is indeed due to this auxiliary reaction of an RNA helicase,
rather than a direct requirement of NTP as an energy
source for the biochemical deamination reaction of C to
U (Fig. 5).
Participation of an RNA helicase as a separate but necessary auxiliary function in the RNA editing complex in
plant mitochondria thus appears likely, but will have to
be confirmed by direct identification of the RNA helicase
enzyme involved.
8. Outlook on RNA editing in plant mitochondria
The still open question, which has not been clarified in
the 15 years since RNA editing in plant mitochondria has
been first recognized, is which enzyme(s) is/are involved
in the C to U alterations in the flowering plants and what
Fig. 5. Cartoon of our current model of the process of RNA editing in plant mitochondria. The C to-be-edited is indicated in the RNA template molecule.
The RNA is represented by a horizontal line. The trans-factor recognizes the sequence region located between 5 and 10 to about 20 and 25 nucleotides
upstream of the edited C. This specificity factor cannot access the RNA which is covered by unspecific RNA binding proteins, the most prominent of
which is in vitro the enzyme glutamate dehydrogenase (GDH). The GDH can be directly inhibited from binding to the RNA by the specific blocker
phosphinothricin or by NADH. The analogous effect of added NTP or dNTP such as ATP or dGTP is either generated also directly through binding to
the GDH or is indirectly mediated by activation of an RNA helicase. The activated helicase removes the GDH and other RNA binding proteins attached
to the template RNA and makes this molecule accessible for the RNA editing activity.
44
M. Takenaka et al. / Mitochondrion 8 (2008) 35–46
is the underlying biochemistry? Is the C to U transition in
flowering plants a straightforward deamination reaction?
Or is a transamination process involved which moves the
amino group to large acceptor molecules? Could such
acceptor molecules be unspecific or dedicated RNA molecules? Are modified tRNA modifying enzymes involved
such as specific deaminases which have lost their tRNA
specificity? Or has one or more of the DNA repair enzymes
altered its structure to also accept RNA as substrate? Connected with this unresolved problem is the question how
the U to C alteration is performed in the mitochondrial
(and plastid) RNAs of non-flowering plants?
Recognition of the PPR protein family seems to have
brought the question of the mediators of specificity in
RNA editing to a realistic solution. Although these proteins are presently the most likely candidates for the specificity-mediating trans-factors, their actual involvement in
RNA editing in plant mitochondria and their individual
assignment to specific RNA editing sites now needs to be
proven experimentally.
The identity and structure of the components of the
potential ‘editosome’ have to be resolved. For example, it
will have to be clarified, if and how the PPR proteins interact with the enzyme? Are other proteins involved and if so,
which are these?
Is an RNA helicase directly involved in the RNA editing
machinery? Do unspecific RNA binding proteins such as the
RBP proteins, which are abundant in plant mitochondria
(Vermel et al., 2002) and presumably can bind to RNA
unspecifically, play a role in editing? Answers to these questions are steps towards the assembly of a model of RNA maturation in plant mitochondria. This will have to include
information of the location of the mRNA molecules and
the order of the various processes in the mitochondrial organelle. The model will have to assign the in vivo coupling and
channeling of transcription and RNA maturation (and possibly translation) within the organelle to understand how the
protein products required for plant mitochondrial functions
are provided by the organelle.
Acknowledgements
We are grateful to the editors and the anonymous reviewer for very helpful comments on our presentation.
Mizuki Takenaka was a fellow of the JSPS and Johannes
Andreas van der Merwe was a fellow of the DAAD. Grants
from the Deutsche Forschungsgemeinschaft supported the
work in the laboratory of the authors.
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