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).

REFERENCES1. Fox, T. D. and Leaver, C. J. (1981) The Zea mays mitochondrial gene

coding cytochrome oxidase subunit II has an intervening sequence and

does not contain TGA codons. Cell 26, 315–323.

2. Barrell, B. G., Bankier, A. T. and Drouin, J. (1979) A different genetic

code in human mitochondria. Nature 282, 189–201.

3. Macino, G., Coruzzi, G., Nobrega, F. G., Li, M. and Tzagoloff, A.

(1979) Use of the UGA terminator as a tryptophan codon in yeast mito-

chondria. Proc. Natl. Acad. Sci. USA 76, 3784–3785.

4. Quetier, F. and Vedel, F. (1977) Heterogenous population of mitochon-

drial DNA molecules in higher plants. Nature 268, 365–368.

5. Lonsdale, D. M. (1984) A review of the structure and organization of the

mitochondrial genome of higher plants. Plant Mol. Biol. 3, 201–2006.

6. Fauron, C. M. R. and Havlik, M. (1988) The BamHI, XhoI, SmaI

restriction enzyme maps of the normal maize mitochondrial genome

genotype B37. Nucleic Acids Res. 16, 10395–10396.

7. Sangare, A., Weil, J. H., Grienenberger, J. M., Fauron, C., and Lons-

dale, D. (1990) Localization and organization of tRNA genes on the

mitochondrial genomes of fertile and male-sterile lines of maize. Mol.

Gen. Genet. 223, 224–232.

8. Marechal, L., Guillemaut, P., Grienenberger, J. M., Jeannin, G., and

Weil, J.-H. (1985) Sequence and codon recognition of bean mitochon-

dria and chloroplast tRNAsTrp: evidence for a high degree of homology.

Nucleic Acids Res. 13, 4411–4416.

9. Marechal, L., Runeberg-Roos, P., Grienenberger, J. M., Colin, J., Weil, J. H.,

Lejeune, B., Quetier, F., and Lonsdale, D. M. (1987) Homology in the region

containing a tRNATrp gene and a (complete or partial) tRNAPro gene in wheat

mitochondrial and chloroplast genomes. Curr. Genet. 12, 91–98.

10. Leon, P., Walbot, V. and Bedinger, P. (1989) Molecular analysis of the

linear 2.3 kb plasmid of maize mitochondria: apparent capture of tRNA

genes. Nucleic Acids Res. 17, 4089–4099.

11. Schuster, W., Grienenberger, J. M., Weil, J. H. and Brennicke, A.

(1988) Comparison of tRNATrp and tRNAPro gene sequences in the

chloroplast and mitochondrial genomes of Oenothera. Nucleic Acids

Res. 16, 7737.

12. Gualberto, J. M., Weil, J. H., and Grienenberger, J. M. (1990) 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, 3771–3776.13. Gualberto, J. M., Wintz, H., Weil, J. H., and Grienenberger, J. M.

(1988) The genes coding for subunit 3 of NADH dehydrogenase and for

ribosomal protein S12 are present in the wheat and maize mitochondrial

genomes and are co-transcribed. Mol. Gen. Genet. 215, 118–127.14. Gualberto, J. M., Bonnard, G., Lamattina, L., and Grienenberger, J. M.

(1991) Expression of the wheat mitochondrial nad3-rps12 transcription

unit: correlation between editing and mRNA maturation. Plant Cell 3,

1109–1120.

15. Wintz, H., Chen, H. C. and Pillay, D. T. N. (1989) Partial characteriza-

tion of the gene coding for subunit IV of soybean mitochondrial NADH

dehydrogenase. Curr. Genet. 15, 155–160.16. Lamattina, L., Weil, J. H., and Grienenberger, J. M. (1989) RNA

editing at a splicing site of NADH dehydrogenase subunit IV gene

transcript in wheat mitochondria. FEBS Lett. 258, 79–83.

17. Lamattina, L. and Grienenberger, J. M. (1991) RNA editing of the tran-

script coding for subunit-4 of NADH dehydrogenase in wheat mitochon-

dria: uneven distribution of the editing sites among the 4 exons. Nucleic

Acids Res. 19, 3275–3282.

18. Benne, R. (1989) RNA-editing in trypanosome mitochondria. Biochim.Biophys. Acta 1007, 131–139.

19. Benne, R., Van den Burg, J., Brakenhoff, J., Sloof, P., Van Boom, J.

H., and Tromp, M. C. (1986) Major transcript of the frameshift coxIIfrom trypanosome mitochondria contains four nucleotides that are not

encoded in the DNA. Cell 46, 819–826.

20. Lurin, C., Andres, C., Aubourg, S., Bellaoui, M., Bitton, F., Bruyere,

C., Caboche, M., Debast, C., Gualberto, J., Hoffmann, B., Lecharny, A.,

Le Ret, M., Martin-Magniette, M. L., Mireau, H., Peeters, N., Renou, J.

P., Szurek, B., Taconnat, L., and Small, I. (2004) Genome-wide analysis

of Arabidopsis pentatricopeptide repeat proteins reveals their essential

role in organelle biogenesis. Plant Cell 16, 2089–2103.21. Small, I. D., Suffolk, R., and Leaver, C. J. (1989) Evolution of plant

mitochondrial genomes via substoichiometric intermediates. Cell 58,

69–76.

22. Ogihara, Y., Yamazaki, Y., Murai, K., Kanno, A., Terachi, T., Shiina, T.,

Miyashita, N., Nasuda, S., Nakamura, C., Mori, N., Takumi, S., Murata,

M., Futo, S., and Tsunewaki, K. (2005) Structural dynamics of cereal mi-

tochondrial genomes as revealed by complete nucleotide sequencing of

the wheat mitochondrial genome. Nucleic Acids Res. 33, 6235–6250.

23. Hiesel, R., Wissinger, B., Schuster, W. and Brennicke, A. (1989) RNA

editing in plant mitochondria. Science 246, 1632–1634.

24. Gualberto, J. M., Lamattina, L., Bonnard, G., Weil, J. H., and Grienen-

berger, J. M. (1989) RNA editing in wheat mitochondria results in the

conservation of protein sequences. Nature 341, 660–662.

25. Covello, P. S. and Gray, M. W. (1989) RNA editing in plant mitochon-

dria. Nature 341, 662–666.

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