Download From gene to protein in higher plant mitochondria

C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 209–217
© 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés
S0764446900012932/REV
Genetics / Génétique
From gene to protein in higher plant mitochondria
Philippe Giegéa*, Axel Brennickeb
a
b
Department of Plant Sciences, Oxford University, South Parks Road, Oxford, OX1 3RB, UK
Universität Ulm, Allgemeine Botanik, Albert-Einstein-Allee 11, 89069 Ulm, Germany
Received 6 October 2000; accepted 27 November 2000
Communicated by Michel Thellier
Abstract – Higher plant mitochondria contain a genetic system with a genome, transcription and translation processes, which have to be logistically integrated with the two
other genomes in the nucleus and the plastid. In plant mitochondria, after transcripts
have been synthesised, at least in some cases by a phage-type RNA polymerase, they
have to go through a complex processing apparatus, which depends on protein factors
imported from the cytosol. Processing involves cis- and trans-splicing, internal RNA
editing and maturation at the transcript termini, these steps often occurring in parallel.
Transcript life is terminated by RNA degradation mechanisms, one of which involves
polyadenylation. RNA metabolism seems to be a key element of the regulation of gene
expression in higher plant mitochondria. © 2001 Académie des sciences/Éditions
scientifiques et médicales Elsevier SAS
plant mitochondria / gene expression / maturation / degradation
Résumé – Des gènes aux protéines dans les mitochondries de plantes supérieures. Les mitochondries de plantes supérieures contiennent un génome, une transcription et une traduction qui leur sont propres. Ce système génétique doit pouvoir s’inscrire
dans le cadre des deux autres génomes (nucléaire et chloroplastique) présent dans la
cellule végétale. Dans les mitochondries de plantes, après avoir été synthétisés, dans
certains cas par une ARN polymérase de type phagique, les transcrits sont pris en charge
par un complexe appareil de maturation dépendant de nombreuses protéines importés
du cytosol. Cette maturation implique des étapes d’épissage en cis et en trans, d’édition
des ARNs et de maturation des extrémités des transcrits. Ces étapes ont fréquemment
lieu en parallèle. La vie des transcrits s’achève par leur dégradation, un des mécanismes
de dégradation impliquant la polyadénylation. Le métabolisme de l’ARN semble être un
élément clé dans la régulation de l’expression des gènes des mitochondries de plante.
© 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS
mitochondrie de plante / expression génétique / maturation / dégradation
Version abrégée
La découverte de génomes dans les mitochondries a
fortement contribué à réactualiser la théorie proposant
une origine endosymbiotique aux cellules eucaryotes.
Les mêmes fonctions fondamentales sont codées dans
les génomes mitochondriaux. Néanmoins, ceux des
plantes se caractérisent par leur grande taille, souvent
20 fois supérieure à celles des animaux.
Considérant la trentaine de protéines seulement
codée dans les génomes mitochondriaux de plantes, la
plupart des métabolismes présent dans ces organelles,
incluant l’expression génétique, sont dépendant de
protéines importées du cytosol. De même, les mitochondries de plantes ne disposent pas d’un lot d’ARNts
* Correspondence and reprints.
E-mail address: [email protected] (P. Giegé).
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P. Giegé and A. Brennicke / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 209–217
suffisant pour traduire leurs gènes et doivent donc aussi
importer des ARNt du cytosol.
Contrairement aux animaux, la transcription des
génomes mitochondriaux de plantes est initiée à de
nombreux sites. Ces promoteurs se caractérisent par un
motif CRTA conservé. Ce motif seul est néanmoins
insuffisant pour initier la transcription. Cette transcription est catalysée, aux minimum dans certain cas, par
des ARN polymérases de type bactériophagique. L’hétérogénéité des promoteurs suggère néanmoins que
d’autres enzymes ou facteurs de transcription doivent
être présent dans les mitochondries de plante.
Les transcrits mitochondriaux de plante sont très
fréquemment interrompus par des introns, tous à une
exeption près de groupe II. Ces introns, dont certains
comportent un gène codant une maturase dans leur
séquence, sont épissés le plus souvent en cis, mais
aussi en trans (quand les exons ne sont pas linéairement séparés par l’intron et qu’ils sont distants l’un de
l’autre dans le génome). La structure des introns de
groupe II est essentielle pour l’épissage, mais des
facteurs protéique sont aussi requis.
Au cours de leur maturation, les transcrits mitochondriaux de plante sont aussi très souvent édités. Dans ce
cas l’édition de l’ARN consiste en des modifications de
C en U, et plus rarement de U en C. L’édition, qui
affecte majoritairement les ARNm, corrige des codons
conservés dans d’autres espèces où l’édition n’a pas
lieu. Des expériences in vitro ont montrées que l’édition opérait par désamination ou transamination des
C’s. Une cytidine désaminase ou transaminase est donc,
semble-t-il, requise. La comparaison des sites d’édition
de l’ARN à l’échelle d’un génome a révélée qu’il
n’existe pas de séquence ou de repliement de l’ARN
conservé. Ceci suggère que les sites d’édition sont
reconnus individuellement ou par petit groupes par
des facteurs agissant probablement en trans. L’édition
n’est pas un phénomène séquentiel et s’effectue indépendamment de l’épissage e.g.
1. Structure of plant mitochondrial DNA
density is comparatively low in plant mitochondria. The
16.5-kb human mitochondrial genome [8], though 22
times smaller than Arabidopsis mt genome, encodes a
quarter of its genes. The actual coding regions represent
only about 10 % of plant mt genomes, with large parts of
these genomes (7 % in Arabidopsis) being taken up by
repeated sequences, which are responsible for the physical organisation of the mitochondrial genome. In Arabidopsis, two large repeats of 6.5 and 4.2 kb have been
shown to be active in recombination, resulting in the
production of sub-genomic circular molecules [9]. Further
large portions of the plant mtDNAs are covered by probably functionally insignificant open reading frames, by
introns and by sequences imported from the plastid and
nuclear genomes. All in all, just over half of plant mitochondrial DNA is of as yet unaccounted function [6, 7].
The finding of unique genomes in mitochondria strongly
helped to reactualise the endosymbiont theory for the
evolution of the eukaryotic cell [1–3]. Sequence analysis
has revealed that higher plant mitochondrial genomes,
similar to their animal, fungal or protist counterparts, show
a basal stability of gene content, but often differ in the
presence or absence of individual genes. The same fundamental functions are found encoded in the mtDNAs of all
eukaryotic cells. These are proteins involved in mitochondrial metabolism, i.e. genes encoding components of the
complexes forming the respiratory chain, and in plants
and protists also genes encoding proteins involved in the
c-type cytochrome biogenesis, and components of the
mitochondrial translational apparatus, i.e. a set of about
20 tRNAs, ribosomal RNAs and, mostly in plants and
protists, genes for ribosomal proteins [4].
A major common characteristic of plant mitochondrial
genomes is their large size, with an estimated 180–2 400
kb for the land plant genomes [5], with precise data
available for the three completely sequenced genomes
covering 187 kb in the liverwort Marchantia, 367 kb in
Arabidopsis [6] and 369 kb in the sugar beet [7]. Gene
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Dans la région 3' non traduite des gènes mitochondriaux de plante, des séquences en double répétition
inverse sont souvent trouvées. Dans certain cas, il a été
montré qu’il s’agit la de signaux de maturation en 3',
plutôt que de terminateurs de la transcription.
Contrairement aux ARNs cytoplasmiques, où la polyadénylation joue un rôle dans la stabilité des transcrits,
il a récemment été montré que dans les mitochondries
de plante les ARNs polyadénylés étaient préférentiellement dégradés. La polyadénylation dans les mitochondries de plante est donc, tout comme dans les chloroplastes et les bactéries, associée à la dégradation des
ARNs.
Après toute les étapes de maturation les transcrits
mitochondriaux de plante sont traduits. La traduction
parfois initiée à des codons autres que AUG, s’effectue
indépendamment de l’édition des ARNs. Néanmoins,
seules les protéines issues de transcrits complètement
édités sont fonctionnelles, les autres sont rapidement
dégradées.
2. Proteins and tRNAs have to be
imported into the mitochondria from
the cytosol
Given the small number of proteins (approximately 30)
encoded in plant mitochondrial genomes, most metabo-
P. Giegé and A. Brennicke / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 209–217
lisms taking place in this organelle, including genome
expression, depend on imported proteins. About 4 000
proteins [10] encoded in the nucleus are estimated to be
imported from the cytosol to the mitochondrial compartment. Nuclear encoded mt proteins contain a signal peptide at their N-terminus, which is recognised by translocases of the outer (TOM) and inner mitochondrial
membranes (TIM) at contact points of the two bilayers.
Once translocated, the pre-proteins are released into the
matrix by processing peptidases (MPP) that cleave off the
signal peptides. This whole process is associated with
chaperones involved in pre-protein unfolding, translocation (HSP70, MGE), refolding and assembly (MDJ, HSP60
and HSP10) (for review, see e.g. [11]).
It is very likely that an additional protein import apparatus exists in plant mitochondria, since the long time
elusive orfx gene conserved in higher plant mitochondria
[12] was identified to encode Mtt2, a component of a
non-secretory protein transport pathway, analogous to
one described in Escherichia coli [13].
Similarly to proteins, the set of tRNAs encoded in plant
mitochondria is not sufficient to perform mitochondrial
translation and tRNAs have to be imported from the cytosol [14, 15]. In Arabidopsis, 13 different tRNAs are missing
to decode all of the codons used [4]. Several studies
suggest that tRNAs may be imported in association with
precursor aminoacyl-tRNA synthetases, e.g. [16, 17], this
interaction, though necessary, alone, however, is sufficient
to trigger tRNA import in mitochondria [18].
3. Mitochondrial RNA synthesis,
transcription
3.1. Promoter sequences and cis-acting factors
Contrary to vertebrate mitochondria, where transcription initiation occurs at only one promoter per DNA
strand, plant mitochondrial genome transcription is initiated at numerous sites. In the higher plant Oenothera, in
vitro capping experiments showed that at least 15 promoters are active in the genome [19]. The individual promoters in plant mitochondria are of different strength as documented by the very heterogeneous run-on transcription
levels observed for different genes in Arabidopsis [20].
Several promoters can be active for a single gene as
described for atp9 and cox2 in maize mitochondria [21,
22]. Comparison between primer extension, nuclease S1
analysis and in vitro capping experiments revealed that
higher plant mitochondrial transcripts do not always
undergo processing in their 5'-regions, steady-state mRNA
termini thus being in several instances true transcription
initiation start points.
In vitro transcription assays and sequence analysis
around 5'-transcript termini revealed that higher plant
mitochondrial promoters are characterised by a conserved
CRTA sequence (R being purines). For monocots, a little
conserved 15 nucleotides long sequence containing the
CRTA motif was shown to be sufficient for in vitro transcription initiation. In dicot mitochondria, a nonanucleotide motif was found conserved at transcription
initiation sites [23]. This sequence is, however, not sufficient to give full in vitro transcription initiation. Comparison of various promoter structures from pea, Oenothera,
soybean and potato revealed that an 18-nucleotide-long
sequence is necessary and sufficient to initiate transcription at least for the genes investigated [24]. This sequence
between positions –14 and +4 around the initiation point,
the first transcribed nucleotide, contains an AT box in the
5'- and a purine-rich stretch in the 3'-region. This type of
promoter sequence may not be responsible for all of the
transcription initiation events, since these conserved motifs
are not present in all promoters of plant mitochondria.
This was observed first for the atp1 promoter in maize
mitochondria and subsequently also seen for cox3 and
atp6 promoters in the same plant [25, 26]. Similarly in
dicots, e.g. in the Arabidopsis mitochondrial genome,
numerous genes lack these or any other detectable consensus structures at their promoter sites. One or more
other types of promoters must thus be active in plant
mitochondrial transcription initiation.
3.2. RNA polymerase and other predicted trans-acting
factors
In chloroplasts transcription is performed by at least
three different RNA polymerases, a bacterial-type RNA
polymerase (PEP) encoded in the plastid genome (i.e. a
chloroplastic enzyme having a sequence homologous to
RNA polymerases found in bacteria) and two nuclearencoded bacteriophage-type RNA polymerases (NEP) (i.e.
enzymes having their sequence homologous to bacteriophage RNA polymerases). In contrast, transcription in
higher plant mitochondria seems to be catalysed by at
least two nuclear-encoded phage-type enzymes; for a
review see [27]. Detailed investigations in Arabidopsis
have yielded in vitro as well as in vivo data demonstrating
that three such single-subunit enzymes are coded by the
nuclear genome, one of each being targeted to plastids
and mitochondria and the third being imported into both
organelles [28–30]. In the monocots maize and wheat
similar mitochondrial RNA polymerases have been characterised, but their total number is not yet clear [31, 32].
As discussed above, dicot and particularly monocot
promoters do not all conform to a single strict consensus
sequence. Therefore, promoter recognition will require
the different RNA polymerases to be specific for one type
of promoter, and/or very likely also the additional presence of various transcription factors to modulate promoter
specificity. One of these could be a DNA-binding protein
recently characterised to attach to transcription initiation
sites in wheat mitochondria [33]. In the dicot pea a different, apparently unrelated promoter-binding protein was
identified in mitochondria, but its functional involvement
in promoter recognition still needs to be determined [34].
Proteins similar to CDF2, which interacts with the phagetype RNA polymerase in the chloroplast, are possibly also
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P. Giegé and A. Brennicke / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 209–217
involved in promoter recognition and activation in plant
mitochondria [35].
4. Maturation of mitochondrial RNA
not account for splicing by itself, but including the maturases sometimes internally encoded, introns require protein factors to catalyse splicing. Recent studies have shown
in tobacco that mitochondrial splicing is dependent on the
expression of at least one nuclear-encoded factor, a probable member of the predicted splicing machinery [46].
4.1. Splicing
4.1.1. Cis- and trans-splicing introns
Two classes of introns, group I and group II, are characteristic of organelles, first distinguished by their characteristic RNA secondary structures in fungal mitochondria by
Michel et al. [36]. In higher plant mitochondria group II
introns are generally found in several genes, while only
one instance of a recently acquired, unique group I intron
has been found in the cox1 genes of Peperomia and some
other higher plants [37, 38].
Group II (and group I) introns sometimes encode proteins in their sequences, which function in the dispersion
of the intron into new sites in the DNA or in its excision
from the mRNA (maturases). Of the approximately 100
group II introns known up to now, approximately onefourth potentially encode proteins, most of them maturases, which are usually necessary for splicing of at least
their respective intron.
The genes encoded in the Arabidopsis mitochondrial
genome are interrupted by altogether 23 group II introns
with sizes varying from 485 to approximately 4 000 nucleotides [6]. Some genes are interrupted by more than one
intron, e.g. nad7 has four introns, but only the intron
between exons d and e of nad1 encodes a maturase-like
open reading frame in most of the plants.
Trans-splicing is found in plant mitochondria in several
instances, where the physically separated exons are
flanked by partial group II intron sequences [39–41].
Trans-splicing designates splicing events where consecutive exons are not linearly separated by a continuous
intron, but are present at distant positions on the genome,
sometimes on the other strand. These trans-splicing events
occur via the group II intron fragments, which connect the
separated exons in two- or three-molecule interactions. In
Arabidopsis mitochondria, five trans-splicing events are
detected in nad1 [40], nad2 [42] and in nad5 [43], in the
latter an only 22-nucleotide-long exon being integrated by
two trans-splicing events.
4.1.2. Splicing mechanisms and factors involved
Similarly to nuclear introns, group I and II intron splicing occurs by transesterification reactions. In group I
introns, free GTP is needed as cofactor with its 3'-OH
group attacking the 5'-end of the intron. In group II introns,
the 5'-end of the intron is attacked by an OH group of a
conserved internal A residue. The highly conserved structure of the group II introns has been shown to be essential
for splicing activity [44, 45]. Several introns of both
organellar groups are characterised by their ability to
perform autocatalytic splicing in vitro under essentially
non-physiological conditions. In vivo, RNA structure can
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4.2. RNA editing
4.2.1. Global effect
The discovery of RNA editing in plant mitochondria,
manifested as mainly C to U conversions in mRNAs
[47–49], provided an answer to the long-term puzzle
concerning codon conservation in plant mitochondrial
genes, including the use of a potentially specific genetic
code. Besides C to U alterations in RNAs, U to C ‘reverse
editing’ events have also been observed in both plant
organelles, although rarely in flowering plants, e.g.
[50–52]. RNA editing affects preferentially protein coding
transcripts, but is occasionally also found in structural
RNAs. In mRNAs, editing modifications result in increasing the conserved similarity of the deduced protein
sequences with homologous proteins in other species.
Editing thus ‘corrects’ affected codons to those conserved
in ‘non-editing’ genetic systems. It has also been observed
that the 441 editing modifications found in Arabidopsis
mRNAs lead to an overall increase of the hydrophobicity
of the mitochondrially encoded proteins [53]. In structural
RNAs, usually tRNAs, RNA editing is believed to improve
the spatial folding of these molecules, and thus to improve
their functionality.
4.2.2. The editase
In order to identify the enzyme(s) catalysing the RNA
editing activity in plant organelles, the first question
addressed was the biochemical nature of the C to U and U
to C conversions. Different in vitro and in organello experimental assays such as internal labelling with radioactive
CTP of RNAs in purified mitochondria [54] and incubation
of synthetic RNAs containing radiolabelled Cs with in
vitro active extracts [55] have shown that the RNA backbone is not disrupted by the editing activity, the phosphate
and ribose groups remaining unchanged. Other experiments showed that the amino group of the cytidine is
removed by editing while the base labelled on its
α-phosphate remains in the RNA [54, 56]. Zinc ions,
typical co-factors of deaminases, are found to be necessary for the in vitro editing activity [55]. The present
working hypothesis is that RNA editing occurs via a deamination or a transamination reaction. If deaminases are the
editing enzymes, the rare ’reverse’ editing should have to
be catalysed by a separate CTP synthase activity. On the
other hand, a transaminase could catalyse both C to U and
U to C editing activities possibly by using different cofactors. The cytidine deaminases and transaminases found in
plant genomes have characteristic conserved sequences.
It is believed that the editing enzyme(s) must be encoded
in the plant cell nucleus since no such characteristic
P. Giegé and A. Brennicke / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 209–217
sequences has been found up to now in the complete
mitochondrial genome sequences available [6, 7].
4.2.3. Editing site recognition
When comparing the editing site sequences at the
genome level [53] no conserved sequence or RNA folding
pattern can be observed. A bias towards an elevated
frequency of pyrimidines directly in 5' of the sites is
observed, but insufficient to label the respective Cs as
‘editable’. Individual RNA editing sites are thus most likely
targeted one by one (or as small families) by separate
specificity factors, probably acting in trans. Chaudhuri et
al. [57] showed that for chloroplasts the introduction of a
transgene inducing additional RNA copies had the consequence of decreasing the editing frequency. This suggests
that a trans-acting factor may be present in limited
amounts. This factor could a priori be a protein or a
nucleic acid, but at least in plant mitochondrial transcripts
recognition solely by site-specific proteins seems disfavoured by the large number of different editing sites.
Indeed, an in vitro extract described by Araya et al. [58]
appeared to be vulnerable to both protease and micrococcal nuclease activities. This suggests that nucleic acids are
involved in the RNA editing mechanism, which may be
small RNA molecules in antisense to editing site sequences.
The search for such molecules guiding the editing activity
(gRNAs) has identified in Oenothera fragments of the 26S
ribosomal RNA with limited antisense pairing to editing
sites [59]. These similarities could, however, be due to
statistically random nucleotide combinations and thus
will have to be confirmed with other editing sites and in
other plants.
4.2.4. RNA editing and the control of gene expression
The analysis of partially edited transcripts revealed that
in plant mitochondria editing is not a sequential process
scanning along the RNA thread, but that the editing
machinery probably randomly homes in on the site to be
edited. Analyses of large numbers of individual cDNA
clones for a given gene reveal site-specific variations in
editing frequencies, which result in a heterogeneously
edited steady-state RNA population. The frequency of
partial editing differs in mature RNAs, precursor RNAs and
polysome-enriched fractions. Mature RNAs are found to
be more frequently edited and polysome-enriched fractions are nearly all completely edited [60], suggesting that
editing is a post-transcriptional process. The detection of
editing in non-spliced pre mRNAs indicates that editing
probably happens independently of splicing. In Arabidopsis mitochondria no correlation could be observed
between the population of edited codons and the codon
usage, suggesting that the RNA editing machinery acts
independently of the translation apparatus [53]. RNA editing thus appears to take place anywhere between transcription and translation and could there play a role in
regulation.
4.3. Transcript 3'-processing
In plant mitochondria, the 3'-untranslated regions often
contain inverted repeat sequences [61]. In animal mitochondria, such structures are found to be regulated terminators of transcription [62] similar to bacteria, where
inverted repeats have been found to act as rho-independent
terminators [63]. In chloroplasts, stem-loop structures in
the 3'-region of mRNA genes act as both processing signals and stability elements [64]. Proteins binding to such
structures have been identified in these organelles to
include both exo- and endonucleases [65]. Differential
binding of these factors plays a role in the stability and in
the turnover of the respective transcripts. In mitochondria
of pea, the function of such an inverted repeat structure
located in 3' of atp9 has been investigated [66]. In vitro
transcription is not inhibited at this structure, but an in
vitro processing system recognises the inverted repeat,
which in this assay checks and stops the progressive
exonucleolytic removal of nucleotides from the
3'-terminus. Thus, in plant mitochondria, inverted repeats
are believed to be processing signals rather than transcription terminators. The DexH box RNA helicase recently
characterised in Arabidopsis mitochondria [67] may help
to solve such RNA structures at transcript 3'-ends, and
may be part of the predicted machinery responsible for
mRNA 3'-maturation. It is thus possible that this protein is
involved in the control of transcript degradation in plant
mitochondria.
5. Mitochondrial RNA degradation
In the nucleus and the cytoplasm polyadenylation
enhances the stability of steady-state transcripts, whereas
in plant mitochondria and in plastids polyadenylation
rather destabilises affected RNAs. The large 3'-untranslated
regions present in some of the mitochondrial mRNAs
probably play individually distinct roles in modulating
stability and maturation of transcripts. Transcript polyadenylation has recently been detected in plant mitochondrial
mRNAs [68, 69]. For example, in a CMS (cytoplasmic
male sterile) sunflower line, an atpA-orf522 transcript is
polyadenylated in vivo. An increase in this polyadenylation activity can be correlated with an increase in transcript instability rather than with an prolonged lifespan.
Nicely complementing these results is the ribonuclease
activity detected in sunflower mitochondria which preferentially degrades polyadenylated transcripts [68]. Polyadenylation of transcripts has also been described in maize
cox2 mRNAs [69]. These observations suggest that polyadenylation, as previously observed for chloroplasts [70]
and bacteria [71], is also involved in the degradation of
transcripts in plant mitochondria. The trans-acting signals
predicted to trigger selective and regulatory polyadenylation remain as yet elusive. Such polyadenylation signals
do not appear to be affected by RNA editing, since edited
and unedited transcripts are equally found polyadenylated
[69]. It is unclear whether any such specificity for poly-
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P. Giegé and A. Brennicke / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 209–217
Figure 1. This cartoon summarises gene expression mechanisms presently characterised or envisaged to play a role in the genetic system of plant
mitochondria.
Predicted but not yet identified factors are indicated by question marks. Black arrows show actions of proteins, black dashed arrows suggest
potential protein actions and grey dashed arrows show the movement and localisation of proteins and tRNAs. The bold grey arrows separate the
major stages of gene expression, i.e. transcription, transcript maturation, translation and transcript degradation processes.
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P. Giegé and A. Brennicke / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 209–217
adenylation is required at all, since only a few transcripts
have been investigated for poly-A addition and it is still
feasible to propose a non-selective addition of tails to all
RNAs in plant mitochondria.
6. Translation and regulation at
the protein level
with the steady-state transcription levels than with the
respective run-on transcription levels [20]. This suggests
that regulation of gene expression occurs at the posttranscriptional and post-translational levels.
7. Origin and evolution of unique
features
6.1. Translation initiation sites
Sequence analysis has shown that translation is usually,
but not always, initiated with an AUG codon in higher
plant mitochondria. In Arabidopsis GGG, AAU and GUG
are possible additional translation initiator triplets [6].
Similarly, in Oenothera GUG and in radish ACG were
found to be potential translation initiation sites [72, 73].
Unlike in the chloroplast, where Shine-Dalgarno-like
sequences have been found for some genes, the ribosomebinding sites presently remain elusive in plant mitochondria, e.g. [74].
6.2. Regulation of gene expression at the protein level
Protein sequence comparisons have shown that only
proteins encoded by fully edited transcripts should yield
evolutionary conserved and functional proteins. Translation of fully, partially and non-edited transcripts would
produce a family of protein isoforms deviating at nonsilent editing sites. Protein analysis in maize mitochondria
using specific antibodies produced against proteins coded
by non-edited, partially edited and fully edited rps12
fragments, revealed that ‘partially edited proteins’ are
indeed synthesised [75]. On the other hand, direct
sequencing of the proteins ATP9 from wheat [76], NAD9
from potato [77] and ATP6 from petunia [78] revealed that
only ‘fully edited proteins’ are present in the respective
multi-subunit polyprotein complexes. These observations
suggest that while all the mRNAs are translated indiscriminately of their editing status, selection occurs posttranslationally, and non-functional proteins are not incorporated into the complexes and are presumably rapidly
degraded.
Other recent studies in Arabidopsis have shown that the
protein complex subunit stoichiometries correlate better
References
[1] Margulis L., Recombination of non-chromosomal genes in Chlamydomonas: assortment of mitochondria and chloroplasts? J. Theor. Biol. 26
(1970) 337–342.
[2] Gray M.W., Doolittle W.F., Has the endosymbiont hypothesis been
proven? Microbiology 46 (1982) 1–42.
[3] Gray M.W., Burger G., Lang B.F., Mitochondrial evolution, Science
283 (1999) 1476–1481.
[4] Marienfeld J., Unseld M., Brandt P., Brennicke A., The mitochondrial genome of the flowering plant Arabidopsis thaliana is composed of
both native and immigrant information, Trends Plant Sci. 4 (1999) 495–
502.
The evolutionary histories of some of the specific features in the genetic system of plant mitochondria reflect
the fluidity and flexibility of genetic systems, particularly
of such comparatively small genomes (figure 1). The two
presently identified RNA polymerases appear to be derived
from phage-type ancestral enzymes, which in the emerging eukaryotic cells presumably replaced the original
bacteria-like polymerase. Evidence for this scenario has
been found in the protozoon Reclinomonas americana,
which harbours the only known mitochondrial genome
still coding for the bacterial-type enzyme [79].
The trans-splicing introns have evolved from originally
cis-arranged continuous exon–intron structures, which
were disrupted by genome recombination and reshuffling
events during the evolution of the higher plants. In depth
investigations of intron structures in ferns, fern allies and a
hornwort revealed cis-homologues for all of the higher
plant trans-splicing introns and showed that some of these
introns invaded the plant line very early during evolution
[80].
RNA editing is also a feature that evolved during the
evolution of plants, most likely in the early land plants.
The presence of this type of editing in all land plants but
the Marchantiidae may reflect the evolutionary history of
this process, its origin thus being placed within the line of
the liverworts [81, 52].
Acknowledgements. We apologise to all colleagues
whose work could not be cited because of space limitations. We thank Professor C.J. Leaver for his continuous encouragement. P.G. was funded by an EMBOfellowship. Support was provided by the Fonds der
chemischen Industrie.
[5] Ward B.L., Anderson R.S., Bendich A.J., The mitochondrial genome
is large and variable in a family of plants, Cell 25 (1981) 793–803.
[6] Unseld M., Marienfeld J.R., Brandt P., Brennicke A., The mitochondrial genome of Arabidopsis thaliana contains 57 genes in 366 924
nucleotides, Nat. Genet. 15 (1997) 57–61.
[7] Kubo T., Nishizawa S., Sugawara A., Itochoda N., Estiat A.,
Mikami T., The complete nucleotide sequence of the mitochondrial
genome of sugar beet (Beta vulgaris L.) reveals a novel gene for tRNA(Cys)
(GCA), Nucleic Acids Res. 28 (2000) 2571–2576.
[8] Anderson S., Bankier A.T., Barrell B.G., de Brujn M.H.L., Coulson A.R., Drouin J., Eperon I.C., Nierlich D.P., Roe B.A., Sanger F.,
Schreier P.H., Smith A.J.H., Staden R., Young I.G., Sequence and organization of the human mitochondrial genome, Nature 290 (1981) 457–465.
215
P. Giegé and A. Brennicke / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 209–217
[9] Klein M., Eckert-Ossenkopp U., Schmiedeberg I., Brandt P.,
Unseld M., Brennicke A., Schuster W., Physical mapping of the mitochondrial genome of Arabidopsis thaliana by cosmid and YAC clones, Plant J.
6 (1994) 447–455.
[10] Emanuelsson O., Nielsen H., Brunak S., von Heijne G., Predicting
subcellular localization of proteins based on their N-terminal amino acid
sequence, J. Mol. Biol. 300 (2000) 1005–1016.
[11] Braun H.P., Schmitz U.K., The protein-import apparatus of plant
mitochondria, Planta 209 (1999) 267–274.
[12] Sünkel S., Brennicke A., Knoop V., RNA editing of a conserved
reading frame in plant mitochondria increases its similarity to two overlapping reading frames in Escherichia coli, Mol. Gen. Genet. 242 (1994)
65–72.
[13] Weiner J.H., Bilous P.T., Shaw G.M., Lubitz S.P., Frost L., Thomas G.H., Cole J.A., Turner R.J., A novel ubiquitous system for membrane
targeting and secretion of cofactor-containing proteins, Cell 93 (1998)
93–101.
[14] Kumar R., Maréchal-Drouard L., Akama K., Small I., Striking
differences in mitochondrial tRNA import between different plant species,
Mol. Gen. Genet. 252 (1996) 404–411.
[15] Maréchal-Drouard L., Small I., Weil J.H., Dietrich A., Transfer R.N., A import into plant mitochondria, Methods Enzymol. 260 (1995)
310–327.
[16] Dietrich A., Maréchal-Drouard L., Carneiro V., Cosset A., Small I.,
A single base change prevents import of cytosolic tRNA (Ala) into mitochondria in transgenic plants, Plant J. 10 (1996) 913–918.
[17] Ramamonjisoa D., Kauffmann S., Choisne N., MaréchalDrouard L., Green G., Wintz H., Small I., Dietrich A., Structure and
expression of several bean (Phaseolus vulgaris) nuclear transfer RNA
genes: relevance to the process of tRNA import into plant mitochondria,
Plant Mol. Biol. 36 (1998) 613–625.
[18] Mireau H., Cosset A., Maréchal-Drouard L., Fox T.D., Small I.D.,
Dietrich A., Expression of Arabidopsis thaliana mitochondrial alanyltRNA synthase is not sufficient to trigger mitochondrial import of tRNAAla
in yeast, J. Biol. Chem. 275 (2000) 13291–13296.
[19] Binder S., Brennicke A., Transcription initiation sites in mitochondria of Oenothera berteriana, J. Biol. Chem. 268 (1993) 7849–7855.
[20] Giegé P., Hoffmann M., Binder S., Brennicke A., RNA degradation
buffers asymmetries of transcription in Arabidopsis mitochondria, EMBO
Rep. 1 (2000) 164–170.
[21] Mulligan R.M., Maloney A.P., Walbot V., RNA processing and
multiple transcription initiation sites result in transcript size heterogeneity
in maize mitochondria, Mol. Gen. Genet. 211 (1988) 373–380.
[22] Newton K.J., Winberg B., Yamato K., Lupold S., Stern D.B.,
Evidence for a novel mitochondrial promoter preceding the cox2 gene of
perennial Teosintes, EMBO J. 14 (1995) 585–593.
[23] Giese A., Thalheim C., Brennicke A., Binder S., Correlation of
nonanucleotide motifs with transcript initiation of 18S rRNA genes in
mitochondria of pea, potato and Arabidopsis, Mol. Gen. Genet. 252
(1996) 429–436.
[24] Dombrowski S., Hoffmann M., Guha C., Binder S., Continuous
primary sequence requirements in the 18-nucleotide promoter of dicot
plant mitochondria, J. Biol. Chem. 274 (1999) 10094–10099.
[25] Rapp W.D., Lupold D.S., Mack S., Stern D.B., Architecture of the
maize mitochondrial atp1 promoter as determined by linker scanning and
point mutagenesis, Mol. Cell Biol. 13 (1993) 7232–7238.
[26] Trac R.L., Stern D.B., Mitochondrial transcription initiation: promoter structures and RNA polymerases, Curr. Genet. 28 (1995) 205–216.
[27] Gray M.W., Lang B.F., Transcription in chloroplasts and mitochondria: a tale of two polymerases, Trends Microbiol. 6 (1998) 1–3.
[28] Hedtke B., Börner T., Weihe A., Mitochondrial and chloroplast
phage-type RNA polymerases in Arabidopsis, Science 277 (1997) 809–
811.
[29] Hedtke B., Meixner M., Gillandt S., Richter E., Börner T.,
Weihe A., Green fluorescent protein as a marker to investigate targeting of
organellar RNA polymerase of higher plants in vivo, Plant J. 17 (1999)
557–561.
[30] Hedtke B., Börner T., Weihe A., One RNA polymerase serving two
genomes, EMBO Rep. (2000) (in press).
[31] Young D.A., Allen R.L., Harvey A.J., Lonsdale D.M., Characterisation of a gene encoding a single-subunit bacteriophage-type RNA
216
polymerase from maize which is alternatively spliced, Mol. Gen. Genet.
260 (1998) 30–37.
[32] Ikeda T.M., Gray M.W., Characterization of a DNA-binding protein implicated in transcription in wheat mitochondria, Mol. Cell Biol. 19
(1999) 8113–8122.
[33] Ikeda T.M., Gray M.W., Identification and characterization of
T3/T7 bacteriophage-like RNA polymerase sequences in wheat, Plant
Mol. Biol. 40 (1999) 567–578.
[34] Hatzak F., Dombrowski S., Brennicke A., Binder S., Characterization of DNA-binding proteins from pea mitochondria, Plant Physiol. 116
(1998) 519–527.
[35] Bligny M., Courtois F., Thaminy S., Chang C., Lagrange T., BaruahWolff J., Stern D., Lerbs-Mache S., Regulation of plastid rDNA transcription by interaction of CDF2 with two different RNA polymerases, EMBO J.
19 (2000) 1851–1860.
[36] Michel F., Jacquier A., Dujon B., Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure,
Biochimie 64 (1982) 867–881.
[37] Vaughn J.C., Mason M.T., Sper-Whitis G.L., Kuhlman P.,
Palmer J.D., Fungal origin by horizontal transfer of a plant mitochondrial
group I intron in the chimeric coxI gene of Peperomia, J. Mol. Evol. 41
(1995) 563–572.
[38] Cho Y., Qiu Y.L., Kuhlman P., Palmer J.D., Explosive invasion of
plant mitochondria by a group I intron, Proc. Natl. Acad. Sci. USA 95
(1998) 14244–14249.
[39] Chapdelaine Y., Bonen L., The wheat mitochondrial gene for
subunit I of the NADH dehydrogenase complex: a trans-splicing model for
this gene-in-pieces, Cell 65 (1991) 465–472.
[40] Wissinger B., Schuster W., Brennicke A., Trans-splicing in
Oenothera mitochondria: nad1 mRNAs are edited in exon and transsplicing group II intron sequences, Cell 65 (1991) 473–482.
[41] Malek O., Brennicke A., Knoop V., Evolution of trans-splicing
plant mitochondrial introns in pre-Permian times, Proc. Natl. Acad. Sci.
USA 94 (1997) 553–558.
[42] Lippok B., Brennicke A., Unseld M., The rps4-gene is encoded
upstream of the nad2-gene in Arabidopsis mitochondria, Biol. Chem. 377
(1996) 251–257.
[43] Knoop V., Schuster W., Wissinger B., Brennicke A., Trans-splicing
integrates an exon of 22 nucleotides into the nad5 mRNA in higher plant
mitochondria, EMBO J. 10 (1991) 3483–3493.
[44] Knoop V., Kloska S., Brennicke A., On the identification of group
II introns in nucleotide sequence data, J. Mol. Biol. 242 (1994) 389–396.
[45] Michel F., Ferrat J.L., Structure and activities of group II introns,
Annu. Rev. Biochem. 64 (1995) 435–461.
[46] Brangeon J., Sabar M., Gutierres S., Combettes B., Bove J.,
Gendy C., Chetrit P., Des Francs-Small C.C., Pla M., Vedel F., De Paepe R.,
Defective splicing of the first nad4 intron is associated with lack of several
complex I subunits in the Nicotiana sylvestris NMS1 nuclear mutant, Plant
J. 21 (2000) 269–280.
[47] Covello P.S., Gray M.W., RNA editing in plant mitochondria,
Nature 341 (1989) 662–666.
[48] Gualberto J.M., Lamattina L., Bonnard G., Weil J.H., Grienenberger J.M., RNA editing in wheat mitochondria results in the conservation of protein sequences, Nature 341 (1989) 660–662.
[49] Hiesel R., Wissinger B., Schuster W., Brennicke A., RNA editing in
plant mitochondria, Science 246 (1989) 1632–1643.
[50] Gualberto J.M., Weil J.H., Grienenberger J.M., Editing of the
wheat coxIII transcript: evidence for twelve C to U and one U to C
conversions and for sequence similarities around editing sites, Nucleic
Acids Res. 18 (1990) 3771–3776.
[51] Schuster W., Hiesel R., Wissinger B., Brennicke A., RNA editing in
the cytochrome b locus of the higher plant Oenothera berteriana includes
a U to C transition, Mol. Cell Biol. 10 (1990) 2248–2431.
[52] Steinhauser S., Beckert S., Capesius I., Malek O., Knoop V., Plant
mitochondrial RNA editing, J. Mol. Evol. 48 (1999) 303–312.
[53] Giegé P., Brennicke A., RNA editing in Arabidopsis mitochondria
effects 441 C to U changes in open reading frames, Proc. Natl. Acad. Sci.
USA 96 (1999) 15324–15329.
[54] Blanc V., Jordana X., Litvak S., Araya A., Control of gene expression by base deamination: the case of RNA editing in wheat mitochondria,
Biochimie 78 (1996) 511–517.
P. Giegé and A. Brennicke / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 209–217
[55] Yu W., Schuster W., Evidence for a site specific cytidine deamination reaction involved in C to U RNA editing of plant mitochondria, J. Biol.
Chem. 270 (1995) 18227–182233.
[56] Blanc V., Litvak S., Araya A., RNA editing in wheat mitochondria
proceeds by a deamination mechanism, FEBS Lett. 373 (1995) 56–60.
[57] Chaudhuri S., Carrer H., Maliga P., Site specific factor involved in
the editing of the psbL mRNA in tobacco plastids, EMBO J. 14 (1995)
2951–2957.
[58] Araya A., Domec C., Bégu D., Litvak S., An in vitro system for the
editing of ATP synthase subunit 9 mRNA using wheat mitochondrial
extracts, Proc. Natl. Acad. Sci. USA 89 (1992) 1040–1044.
[59] Yu W., Sanchez H., Schuster W., Assays for investigating RNA
editing in plant mitochondria, Methods 15 (1998) 63–74.
[60] Lu B., Hanson M., Fully edited and partially edited nad9 transcripts differ in size and both are associated with polysomes in potato
mitochondria, Nucleic Acids Res. 24 (1996) 1369–1374.
[61] Schuster W., Hiesel R., Isaac P.G., Leaver C.J., Brennicke A.,
Transcript termini of messenger RNAs in higher plant mitochondria,
Nucleic Acids Res. 14 (1986) 5943–5954.
[62] Clayton D.A., Transcription of the mammalian mitochondrial
genome, Annu. Rev. Biochem. 53 (1984) 573–594.
[63] Cohen S.N., Surprises at the 3'-end of prokaryotic RNA, Cell 80
(1995) 829–832.
[64] Stern D.B., Gruissem W., Control of plastid gene expression:
3'-inverted repeats act as mRNA processing and stabilizing elements, but
do not terminate transcription, Cell 51 (1987) 1145–1157.
[65] Chen H.C., Stern D.B., Specific ribonuclease activities in spinach
chloroplasts promote mRNA maturation and degradation, J. Mol. Biol.
266 (1991) 24205–24211.
[66] Dombrowski S., Brennicke A., Binder S., 3'-Inverted repeats in
plant mitochondrial mRNAs are processing signals rather than transcription terminators, EMBO J. 16 (1997) 5069–5076.
[67] Gagliardi D., Kuhn J., Spadinger U., Brennicke A., Leaver C.J.,
Binder S., An RNA helicase (AtSUV3) is present in Arabidopsis thaliana
mitochondria, FEBS Lett. 458 (1999) 337–342.
[68] Gagliardi D., Leaver C., Polyadenylation accelerates the degradation of the mitochondrial mRNA associated with cytoplasmic male sterility in sunflower, EMBO J. 18 (1999) 3757–3766.
[69] Lupold D.S., Caoile A.G., Stern D.B., Polyadenylation occurs at
multiple sites in maize mitochondrial cox2 mRNA and is independent of
editing status, Plant Cell 11 (1999) 1565–1578.
[70] Kudla J., Hayes R., Gruissem W., Polyadenylation accelerates
degradation of chloroplast mRNA, EMBO J. 15 (1996) 7137–7146.
[71] Sarkar N., Polyadenylation of mRNA in prokaryotes, Annu. Rev.
Biochem. 66 (1997) 173–197.
[72] Bock H., Brennicke A., Schuster W., Rps3 and rpl16 genes do not
overlap in Oenothera mitochondria: GTG as a potential translation initiation codon in plant mitochondria? Plant Mol. Biol. 24 (1994) 811–818.
[73] Dong F.G., Wilson K.G., Makaroff C.A., The radish (Raphanus
sativus L.) mitochondrial cox2 gene contains an ACG at the predicted
translation initiation site, Curr. Genet. 34 (1998) 79–87.
[74] Hirose T., Kusumegi T., Sugiura M., Translation of tobacco chloroplast rps14 mRNA depends on a Shine-Dalgarno-like sequence in the
5'-untranslated region but not on internal RNA editing in the coding
region, FEBS Lett. 430 (1998) 257–260.
[75] Phreaner C.G., Williams M.A., Mulligan M., Incomplete editing of
rps12 transcripts results in the synthesis of polymorphic polypeptides in
plant mitochondria, Plant Cell 8 (1996) 107–117.
[76] Bégu D., Graves P.V., Domec C., Arselin G., Litvak S., Araya A.,
RNA editing of wheat mitochondrial ATP synthase subunit 9: direct
protein and cDNA sequencing, Plant Cell 2 (1990) 1283–1290.
[77] Grohmann L., Thieck O., Herz U., Schröder W., Brennicke A.,
Translation of nad9 mRNA in mitochondria from Solanum tuberosum is
restricted to completely edited transcripts, Nucleic Acids Res. 22 (1994)
3304–3311.
[78] Lu B., Hanson M., A single homogeneous form of ATP6 protein
accumulates in petunia mitochondria despite the presence of differentially edited atp6 transcripts, Plant Cell 6 (1994) 1955–1968.
[79] Lang B.F., Burger G., O’Kelly C.J., Cedergren R., Golding G.B.,
Lemieux C., Sankoff D., Turmel M., Gray M.W., An ancestral mitochondrial DNA resembling a eubacterial genome in miniature, Nature 387
(1997) 493–497.
[80] Malek O., Knoop V., Trans-splicing group II introns in plant
mitochondria: The complete set of cis-arranged homologs in ferns, fernallies, and a hornwort, RNA 4 (1998) 1599–1609.
[81] Malek O., Lättig K., Brennicke A., Knoop V., RNA editing in
bryophytes and a molecular phylogeny of land plants, EMBO J. 15 (1996)
1403–1411.
217