Critical Review
Plant Mitochondrial RNA Editing: The Strasbourg Chapter
Jean Michel GrienenbergerInstitut de Biologie Moleculaire des Plantes du CNRS, Universite de Strasbourg, 12 Rue du General Zimmer,67084 STRASBOURG, France
Summary
For 8 years, it was not understood why certain genes ofplant mitochondria contain CGG (arginine) codons at positionswhere tryptophan codons (UGG) are present in the correspond-ing genes of nonplant species. Identification and sequencing of atRNATrp gene showed that it is not able to decode the CGGcodon. Analysis of different discrepancies in the sequences ofplant mitochondrial proteins prompted us to determine directlythe corresponding RNA sequences. These experiments showedthat plant mitochondrial transcripts are subject to RNA editingthat changes C into U, resulting in a better phylogenetic conser-vation of protein sequences [Gualberto et al. (1989) Nature 341,660–662]. � 2009 IUBMB
IUBMB Life, 61: 1110–1113, 2009
Keywords RNA editing; plant mitochondria; cDNA; genetic code;
tRNA genome evolution; RNA editing in plants; transfer
RNAs; aminoacyl-tRNA synthetases.
PROBLEM OF THE PLANT MITOCHONDRIALGENETIC CODE
Following a doctoral thesis in the Laboratoire de Biologie
Moleculaire vegetale in Orsay (France), I spent a year (1978–
1979) in the lab of Chris Leaver (University of Edinburgh)
working on the molecular biology of glyoxysomes. It is fun to
recall this period, when I shared a lab with Brian Forde (now at
the University of Lancaster, UK), who had begun investigating
protein synthesis in plant mitochondria the year before I started
studying plant mitochondria tRNA genes. I then moved to
Strasbourg University (Institut de Biologie Moleculaire et Cellu-
laire du CNRS) where I was asked to investigate genes encod-
ing plant mitochondrial tRNAs.
Very soon after my arrival in Strasbourg, the 1981 Cell
article by Fox and Leaver (1) appeared, which indicated that
the plant mitochondrial genetic code could not be the standard
one, because predicted arginine (Arg) residues were located at
the positions of three conserved tryptophan (Trp) residues in the
sequence of the maize cytochrome oxidase subunit 2 protein
(Cox2p). At the time, because modifications of the genetic code
had been documented previously in animal and yeast mitochon-
dria (2, 3), it appeared reasonable that this might also be the
case in plant mitochondria. However, it was clear that we had
to take that inference into account when working with tRNAs,
which are the adaptor molecules in protein synthesis.
I engaged myself rapidly in the identification and sequencing
of tRNA genes from plant mitochondria, using total plant mito-
chondrial tRNAs as probes for hybridization to clones of
mtDNA from wheat (provided by F. Quetier and B. Lejeune of
Orsay University) (4) and maize (provided by D. Lonsdale, PBI
Cambridge, UK and later by C. Fauron, University of Utah) (5,
6). The overall aim was to map and obtain the sequences of the
full set of mtDNA-encoded tRNA genes (7). A complementary
strategy was implemented by Pierre Guillemaut and Laurence
Marechal of the same lab, who wanted to determine the RNA
sequences of the tRNAs directly, after isolating individual spe-
cies and determining their identity by aminoacylation experi-
ments. At the time these were experiments that required large
amounts of very pure mitochondria, and a two-meter long frac-
tionation column to isolate the individual tRNA species. The
tRNA isolation was easier with bean mitochondria.
During these studies, we were able to sequence the bean
mt-tRNATrp (8) and the wheat and maize mt-tRNATrp genes
(9, 10). It appeared that all of these sequences shared a high
degree of similarity with their chloroplast counterparts. This
conclusion was confirmed at the gene level for the mt-tRNATrp
(11) from Oenothera berteriana, in collaboration with the team
of Axel Brennicke in Tubingen (Germany). For all of these
plants, a single mitochondrial gene encodes a tRNA that is spe-
cific for Trp. In all cases this tRNA is of chloroplast origin,
originating from insertion of a portion of the chloroplast DNA
Address correspondence to: Jean Michel Grienenberger, 17 rue
Valerien, 67200 Strasbourg, France. E-mail: jeanm.grienenberger@
free.fr
Received 14 September 2009; accepted 30 September 2009
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.277
IUBMB Life, 61(12): 1110–1113, December 2009
into the mitochondrial genome. This insertion also contains a
tRNAPro gene or pseudo-gene from the same part of the chloro-
plast genome. Moreover, in maize mitochondria both genes
were found on a plasmid and not in the main mitochondrial ge-
nome, with the observed mt-tRNATrp the only one functioning
in maize mitochondria. The bean mt-tRNATrp was sequenced,
taking into account post-transcriptional modifications, and it
was possible to show that the anticodon was CmCA, which is
unable to recognize the CGG codon.
Soon after that, we were very puzzled by another result
involving the CGG codon. Jose Gualberto, at that time a PhD
student in the lab, was sequencing the gene coding for Cox3p
(subunit 3 of cytochrome oxidase) of wheat (12). In this gene
we found the presence of two CGG codons that mapped to con-
served positions in the protein sequence. Upon comparison with
homologous proteins (either plant or nonplant), we obtained the
perplexing result that in one case the CGG codon aligned with
codons specifying a conserved Arg, as in the standard genetic
code, whereas in the second case CGG aligned with codons
specifying a conserved Trp, as previously described for the
cox2 gene of maize mitochondria. In fact, we had already
observed an analogous situation for the co-transcribed nad3 and
rps12 (13, 14) genes, but in this case the ‘Arg-encoding’ and
‘Trp-encoding’ CGG codons were in different genes. The cox3
case was the first time we saw this anomaly in the same gene.
We were not able to interpret this situation, concluding that
we needed more data to understand it. We planned several
experiments that we felt should answer the fundamental ques-
tion of the identity of the CGG codon in plant mitochondria.
One of them involved preparing antibodies specific for Rps12p,
to identify the size of specific proteolytic fragments. These anti-
bodies were prepared, but in the end were never used for their
intended purpose.
THE FIRST EXPERIMENTAL FACT AND THE LONGWAY TO RNA EDITING
The experiment that marked the beginning of the Strasbourg
editing story was carried out at the end of 1988. We had been
able to obtain a probe containing part of the gene coding for
Nad4p (subunit 4 of the mitochondrial NADH dehydrogenase of
respiratory Complex I) of soybean (15). This probe was pro-
vided by Henri Wintz, a former PhD student in the lab, at that
time a postdoctoral fellow at Cornell University. The probe
comprised a middle portion of nad4, namely exon 3 of a gene
whose transcripts appeared to be spliced. We therefore set out
to determine the nad4 gene structure, sequence, and transcrip-
tion pattern using the cloned library of wheat mtDNA. This
work was undertaken by a postdoctoral fellow, Lorenzo Lamat-
tina from Mar del Plata in Argentina.
Hybridization with the labelled probe indicated that this gene
is unique in the wheat mitochondrial genome, located on a SalI
fragment named C3. The genomic region corresponding to exon
3 was sequenced, and to map the upstream exons we set up an
experiment to obtain the sequence of the mature mRNA. Total
mt-RNA was hybridized with a labelled antisense oligonucleo-
tide 49 nucleotides downstream of a potential intron/exon
boundary. The mRNA was sequenced using AMV reverse tran-
scriptase as a polymerase and dideoxynucleotides. This experi-
ment allowed us to identify exon 2, which appeared to be
located 3405 nucleotides upstream in the mtDNA. Following
this experiment, Lorenzo was able to reconstruct the structure
of nad4. Upon analysis, he found that there was one difference
between the DNA sequence and the RNA sequence (16, 17). At
the position corresponding to the first nucleotide of exon 3 we
found a U in the RNA that could not be accounted for in the
genomic DNA sequence. This residue could either result from
the insertion of an additional U at the exon 2-exon 3 border or
correspond to the very first nucleotide of the genomic sequence
of exon 3, which is a C. The determination of this sequence
was repeated several times without any change in this discrep-
ancy. These experiments were performed at the beginning of
1989, and we spent some months trying to understand how the
splicing mechanism for a Group II intron could generate such a
modification. The exon borders of additional wheat mitochon-
drial transcript genes were also sequenced, but no additional U
was found in those cases.
The shift toward recognition of RNA editing occurred in two
steps during the first semester of 1989. First, a visit was organ-
ized at the Institute for Gottfried Schatz (University of Basel)
during the spring of 1989. I had a very interesting discussion
with him about the question of the CGG codon and the pro-
posed coding for Trp as described by Fox and Leaver. During
this discussion Schatz suggested an analysis of distribution of
this amino acid in plant mitochondrial proteins taking advantage
of the inherent fluorescence of Trp. We did not follow up this
suggestion, but the discussion opened the possibility that the
Central Dogma of Molecular Biology might support exceptions
such as the insertion of Trp directed by a CGG codon.
At the same time, we had a long discussion with Jose Gual-
berto (presently ‘‘Directeur de Recherche’’ at IBMP-CNRS)
about the possibility that RNA editing might be occurring in
plant mitochondria. Jose had recently presented a journal club
talk about editing in trypanosome mitochondria, and during this
discussion he pointed me to a review article on the subject,
authored by Rob Benne (18, 19).
TOWARD THE SOLUTION OF THE CGGCODON PROBLEM
Taking into account these discussions, we decided to test the
idea that the CGG codon problem might be explained by a modi-
fication of the mt-mRNA that changes the CGG codon into UGG,
the normal Trp codon. To achieve this goal, I designed 10 oligo-
nucleotides that were complementary to the sequence of the mt-
mRNA of several genes that we were studying in the lab (cox2,
cox3, cob, nad3, rps12, and nad4). These oligonucleotides were
all targeted to identify the corresponding RNA sequence in the
1111PLANT MITOCHONDRIAL RNA EDITING
vicinity of a CGG codon predicted to be translated as either Trp
or Arg. Total mt-mRNA was used as a template to determine the
cDNA sequence using AMV reverse transcriptase as polymerase.
The result was impressive. At each position of a predicted ‘Trp-
encoding’ CGG codon in the gene sequence, a UGG codon was
present in the RNA; conversely, wherever a CGG codon was pre-
dicted to specify Arg, it remained CGG in the RNA. These data
demonstrated the RNA editing phenomena at select CGG codons
by the conversion of C to U, validating our hypothesis. These
results also indicated that plant mitochondria use the standard
genetic code.
Moreover, when analyzing the cDNA sequences we found
that other C to U conversions were present, modifying the
genetic message at the first or second nucleotides of codons
containing C at these positions. In fact, it began to be clear that
the CGG codon emerged as an issue in the work of Fox and
Leaver because UGG is the only codon for Trp, thus, any modi-
fication of this codon is readily evident, in contrast to editing of
other codons.
Further analysis of the results showed that at some positions
it was impossible to determine the sequence of the cDNA, the
reading giving a mix of CGG and UGG sequences. This obser-
vation raised the possibility that the total mt-RNA population
contains template for both the edited and nonedited versions of
the same message, at different ratios.
To evaluate the effect of RNA editing on the sequences of
plant mitochondrial proteins, we compared these sequences with
the corresponding ones from nonplants. We obtained a consen-
sus from a mix of animal and yeast protein mitochondrial
sequences that showed the presence of invariant amino acids at
specific positions. When we compared this consensus with plant
mitochondrial protein sequences deduced from edited and non-
edited RNA sequences, we deduced that RNA editing enhances
the phylogenetic conservation of the protein sequences. The
editing process is still not completely understood, but the recent
discovery in land plants of a large gene family coding for PPR
(pentatricopeptide repeat) proteins likely has important implica-
tions for elucidating the mechanism of C-to-U RNA editing in
plant mitochondria (20).
The definitive evidence of RNA editing in plant mitochon-
dria came essentially on 13 July 1989, the day before Bastille
Day in France. This was a fantastic day in the lab because we
were very pleased to have made this discovery, and we under-
stood that it would open up a new field of scientific research in
the domain of the molecular biology of plant mitochondria.
After repeating the experiments it was time to prepare the
publications to disseminate these results. The first manuscript
that was written described the nad4 story and was sent as a let-
ter to Nature. At the end of July, the reviewed manuscript was
back, with a negative decision (16). In fact, the reviewers con-
sidered that the demonstration was not compelling because this
single C-to-U change was unique to nad4 and also because we
were not able to eliminate the possibility that the nad4 gene
was redundant and that there might be some sub-stoichiometric
genomic versions containing the ‘correct’ nad4 sequence (21).
This objection was in fact resolved by the later publication of
the full genomic sequence of wheat mitochondria (22).
We therefore decided to try to publish the entire story with
the documentation of multiple editing sites together with Dr. G.
Bonnard (presently Directeur de Recherche at IBMP-CNRS),
who contributed to the experiments. This new manuscript was
sent to Nature around the 10th of August, largely in the form in
which it was eventually published. This time we were pleased
that it was accepted with few criticisms. The reviewers’ com-
ments indicated that the discovery of RNA editing had substan-
tial biological significance. One of the reviewers was, in fact,
also a reviewer of the first (rejected) manuscript, but was con-
vinced by this second version.
Soon after mailing the manuscript to Nature, I attended the
19th Linderstrøm-Lang Conference on Plant Mitochondria and
Respiration near Stockholm, 11–13 August 1989, to which I
was invited by Elzbieta Glaser. This meeting gathered together
a number of plant mitochondrial specialists, with a Molecular
Biology session chaired by Axel Brennicke. For that session I
had announced that my talk would be devoted to the structure
of co-transcribed mitochondrial genes nad3 and rps12. During
his introductory remarks as chairman, Axel took the opportunity
to announce that his lab had discovered that RNA editing oper-
ates in plant mitochondria. As it is easy to imagine, this
announcement came as a real shock and prompted me to show
the RNA editing data we had obtained in Strasbourg, establish
our independent discovery of the same phenomenon. Luckily, I
had brought transparencies summarizing all these data, and
spent double the time originally allocated to my talk describing
them. Afterward I informed Axel that our paper had been sub-
mitted to Nature, and he then told me that he would be sending
a similar paper to Science (23).
Another surprise came when we received the proofs of our
Nature paper at the beginning of September 1989. Very soon
after we received a parcel from Mike Gray (Dalhousie Univer-
sity, Halifax, Canada) containing the proofs of a manuscript
from his team with a description of the same phenomenon of
RNA editing in plant mitochondria. Mike told us that his article
had been accepted by Nature and that it would appear soon. It
was clear that Mike was not aware of our results and therefore,
we sent back a copy of our proofs to him. We realized at the
same time that the two articles were likely to be published to-
gether, which they were (back-to-back) in the same issue of Na-
ture (24, 25).
Finally, I would like to say that the main difficulty I had
during the writing of the Nature article was a question of
authorship. As aforementioned, the discovery of RNA editing in
plant mitochondria was possible, thanks to the contributions of
two researchers. Lorenzo Lamattina performed the first experi-
ments on the nonencoded U at the very 50 end of exon 3 of
nad4, but without understanding its significance. Jose Gualberto
was the one who proposed that this modification might be
achieved through an RNA editing mechanism. In making the
1112 GRIENENBERGER
decision about order of authorship, I placed more emphasis on
the conceptual side of the research. I only regret that at the
time it was not customary to designate two first authors, as it is
now.
ACKNOWLEDGEMENTS
This research was fully supported by the CNRS. Collaboration
with the group of Axel Brennicke and Rudolf Hiesel, and their
colleagues was supported by a grant from the Human Frontier
Science Program (HSFP).
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