Plant mitochondrial RNA editing: the Strasbourg chapter.

For 8 years, it was not understood why certain genes of plant mitochondria contain CGG (arginine) codons at positions where tryptophan codons (UGG) are present in the corresponding genes of nonplant species. Identification and sequencing of a tRNA(Trp) gene showed that it is not able to decode the CGG codon. Analysis of different discrepancies in the sequences of plant mitochondrial proteins prompted us to determine directly the corresponding RNA sequences. These experiments showed that plant mitochondrial transcripts are subject to RNA editing that changes C into U, resulting in a better phylogenetic conservation of protein sequences [Gualberto et al. (1989) Nature 341, 660-662].

GeneFarm, structural and functional annotation of Arabidopsis gene and protein families by a network of experts.

Genomic projects heavily depend on genome annotations and are limited by the current deficiencies in the published predictions of gene structure and function. It follows that, improved annotation will allow better data mining of genomes, and more secure planning and design of experiments. The purpose of the GeneFarm project is to obtain homogeneous, reliable, documented and traceable annotations for Arabidopsis nuclear genes and gene products, and to enter them into an added-value database. This re-annotation project is being performed exhaustively on every member of each gene family. Performing a family-wide annotation makes the task easier and more efficient than a gene-by-gene approach since many features obtained for one gene can be extrapolated to some or all the other genes of a family. A complete annotation procedure based on the most efficient prediction tools available is being used by 16 partner laboratories, each contributing annotated families from its field of expertise. A database, named GeneFarm, and an associated user-friendly interface to query the annotations have been developed. More than 3000 genes distributed over 300 families have been annotated and are available at http://genoplante-info.infobiogen.fr/Genefarm/. Furthermore, collaboration with the Swiss Institute of Bioinformatics is underway to integrate the GeneFarm data into the protein knowledgebase Swiss-Prot.

AtmtPNPase is required for multiple aspects of the 18S rRNA metabolism in Arabidopsis thaliana mitochondria.

Plant mitochondria contain three rRNA genes, rrn26, rrn18 and rrn5, the latter two being co-transcribed. We have recently identified a polynucleotide phosphorylase-like protein (AtmtPNPase) in Arabidopsis mitochondria. Plants downregulated for AtmtPNPase expression (PNP-plants) accumulate 18S rRNA species polyadenylated at internal sites, indicating that AtmtPNPase is involved in 18S rRNA degradation. In addition, AtmtPNPase is required to degrade the leader sequence of 18S rRNA, a maturation by-product excised by an endonucleolytic cut 5' to the 18S rRNA. PNP-plants also accumulate 18S rRNA precursors correctly processed at their 5' end but containing the intergenic sequence (ITS) between the 18S and 5S rRNA. Interestingly, these precursors may be polyadenylated. Taken together, these results suggest that AtmtPNPase initiates the degradation of the ITS from 18S precursors following polyadenylation. To test this, we overexpressed in planta a second mitochondrial exoribonuclease, AtmtRNaseII, that degrades efficiently unstructured RNA including poly(A) tails. This resulted also in the detection of 18S rRNA precursors showing that AtmtRNaseII is not able to degrade the ITS but can impede the action of AtmtPNPase in initiating the degradation of the ITS. These results show that AtmtPNPase is essential for several aspects of 18S rRNA metabolism in Arabidopsis mitochondria.

CcmF(C) involved in cytochrome c maturation is present in a large sized complex in wheat mitochondria.

In land plant mitochondria, c-type cytochromes are assembled via a mechanism similar to that found in Gram-negative bacteria and different from that used by mitochondria from other eukaryotes. The wheat mitochondrial genome encodes CCM (for cytochrome c maturation) proteins, among them CcmF(C), a protein similar to the C-terminal part of the bacterial CcmF. The gene is transcribed into a 1.7 kb transcript at steady state. However, the 3' termini of the transcript were found to occur upstream of the deduced gene termination codon. This discrepancy was solved by RNA editing that introduces a novel termination codon, thus shortening the reading frame by 27 codons. The processed transcript is translated into a protein integrated in the mitochondrial inner membrane. We also show that the protein is part of a large (700 kDa) protein complex, that possibly represents a cytochrome c assembly complex.

The three mitochondrial encoded CcmF proteins form a complex that interacts with CCMH and c-type apocytochromes in Arabidopsis.

Three reading frames called ccmF(N1), ccmF(N2), and ccmF(c) are found in the mitochondrial genome of Arabidopsis. These sequences are similar to regions of the bacterial gene ccmF involved in cytochrome c maturation. ccmF genes are always absent from animal and fungi genomes but are found in mitochondrial genomes of land plant and several evolutionary distant eukaryotes. In Arabidopsis, ccmF(N2) despite the absence of a classical initiation codon is not a pseudo gene. The 3 ccmF genes of Arabidopsis are expressed at the protein level. Their products are integral proteins of the mitochondrial inner membrane with in total 11 to 13 predicted transmembrane helices. The conserved WWD domain of CcmF(N2) is localized in the inter membrane space. The 3 CcmF proteins are all detected in a high molecular mass complex of 500 kDa by Blue Native PAGE. Direct interaction between CcmF(N2) and both CcmF(N1) and CcmF(C) is shown with the yeast two-hybrid split ubiquitin system, but no interaction is observed between CcmF(N1) and CcmF(C). Similarly, interaction is detected between CcmF(N2) and apocytochrome c but also with apocytochrome c(1). Finally, CcmF(N1) and CcmF(N2) both interact with CCMH previously shown to interact as well with cytochrome c. This strengthens the hypothesis that CcmF and CCMH make a complex that performs the assembly of heme with c-type apocytochromes in plant mitochondria.

AtCCMA interacts with AtCcmB to form a novel mitochondrial ABC transporter involved in cytochrome c maturation in Arabidopsis.

ABC transporters make a large and diverse family of proteins found in all phylae. AtCCMA is the nucleotide binding domain of a novel Arabidopsis mitochondrial ABC transporter. It is encoded in the nucleus and imported into mitochondria. Sub-organellar and topology studies find AtCCMA bound to the mitochondrial inner membrane, facing the matrix. AtCCMA exhibits an ATPase activity, and ATP/Mg(2+) can facilitate its dissociation from membranes. Blue Native PAGE shows that it is part of a 480-kDa complex. Yeast two-hybrid assays reveal interactions between AtCCMA and domains of CcmB, the mitochondria-encoded transmembrane protein of a conserved ABC transporter. All these properties designate the protein as the ortholog in plant mitochondria of the bacterial CcmA required for cytochrome c maturation. The transporter that involves AtCCMA defines a new category of eukaryotic ABC proteins because its transmembrane and nucleotide binding domains are encoded by separate genomes.

Fate of a larch unedited tRNA precursor expressed in potato mitochondria.

In higher plant mitochondria, post-transcriptional C to U conversion known as editing mostly affects mRNAs. However, three tRNAs were also shown to be edited. Among them, three editing sites were identified in larch mitochondrial tRNA(His). We have previously shown that only the edited version can undergo maturation in vitro. In this paper, we introduced via direct DNA uptake the edited or unedited version of larch mitochondrial trnH into isolated potato mitochondria and expressed them under the control of potato mitochondrial 18 S rRNA promoter. As expected, the edited form of larch mitochondrial tRNA(His) precursor was processed in the isolated organelles. By contrast, no mature tRNA(His) was detected when using the unedited version of trnH. However, precursor molecules could be characterized by reverse transcription-PCR. These data demonstrate that the potato mitochondrial editing machinery is not able to recognize these "foreign" editing sites and confirm that these unedited tRNA precursor molecules are not correctly processed in organello. As a consequence, the fate of these RNA precursor molecules is likely to be degradation. Indeed, we detected by PCR two 3'-end truncated precursor RNAs. Interestingly, both RNA species exhibit poly(A) tails, a hallmark of degradation in plant mitochondria. Taken together, these data suggest that, in plant mitochondria, a defective unedited RNA precursor that cannot be processed to give a mature stable tRNA, is degraded through a polyadenylation-dependent pathway.

AtCCMH, an essential component of the c-type cytochrome maturation pathway in Arabidopsis mitochondria, interacts with apocytochrome c.

The maturation of c-type cytochromes requires the covalent ligation of the heme cofactor to reduced cysteines of the CXXCH motif of apocytochromes. In contrast to mitochondria of other eukaryotes, plant mitochondria follow a pathway close to that found in alpha- and gamma-proteobacteria. We identified a nuclear-encoded protein, AtCCMH, the Arabidopsis thaliana ortholog of bacterial CcmH/CycL proteins. In bacteria, CcmH and the thioredoxin CcmG are components of a periplasmic thio-reduction pathway proposed to maintain the apocytochrome c cysteines in a reduced state. AtCCMH is located exclusively in mitochondria. AtCCMH is an integral protein of the inner membrane with the conserved RCXXC motif facing the intermembrane space. Reduction assays show that the cysteine thiols in the RCXXC motif of AtCCMH can form a disulfide bond that can be reduced by enzymatic thiol reductants. A reduced form of AtCCMH can reduce the intra-disulfide bridge of a model peptide of apocytochrome c. When expressed in Escherichia coli, AtCCMH coimmunoprecipitates with the bacterial CcmF, a proposed component of the heme lyase. Blue-native PAGE of mitochondrial membrane complexes reveals the colocalization of AtCCMH and AtCcmF(N2) in a 500-kDa complex. Yeast two-hybrid assays show an interaction between the AtCCMH intermembrane space domain and A. thaliana apocytochrome c. A. thaliana ccmh/ccmh knockout plants show lethality at the torpedo stage of embryogenesis. Our results show that AtCCMH is an essential mitochondrial protein with characteristics consistent with its proposed apocytochrome c-reducing and heme lyase function.

Plant mitochondrial rps2 genes code for proteins with a C-terminal extension that is processed.

A gene (rps2) coding for ribosomal protein S2 (RPS2) is present in the mitochondrial (mt) genome of several monocot plants, but absent from the mtDNA of dicots. Confirming that in dicot plants the corresponding gene has been transferred to the nucleus, a corresponding Arabidopsis thaliana nuclear gene was identified that codes for mitochondrial RPS2. As several yeast and mammalian genes coding for mt ribosomal proteins, the Arabidopsis RPS2 apparently has no N-terminal targeting sequence. In the maize mt genome, two rps2 genes were identified and both are transcribed, although at different levels. As in wheat and rice, the maize genes code for proteins with long C-terminal extensions, as compared to their bacterial counterparts. These extensions are not conserved in sequence. Using specific antibodies against one of the maize proteins we found that a large protein precursor is indeed synthesized, but it is apparently processed to give the mature RPS2 protein which is associated with the mitochondrial ribosome.

A family of RRM-type RNA-binding proteins specific to plant mitochondria.

Expression of higher plant mitochondrial (mt) genes is regulated at the transcriptional, posttranscriptional, and translational levels, but the vast majority of the mtDNA and RNA-binding proteins involved remain to be identified. Plant mt single-stranded nucleic acid-binding proteins were purified by affinity chromatography, and corresponding genes have been identified. A majority of these proteins belong to a family of RNA-binding proteins characterized by the presence of an N-terminal RNA-recognition motif (RRM) sequence. They diverge in their C-terminal sequences, suggesting that they can be involved in different plant mt regulation processes. Mitochondrial localization of the proteins was confirmed both in vitro and in vivo and by immunolocalization. Binding experiments showed that several proteins have a preference for poly(U)-rich sequences. This mt protein family contains the ubiquitous RRM motif and has no known mt counterpart in non-plant species. Phylogenetic and functional analysis suggest a common ancestor with RNA-binding glycine-rich proteins (GRP), a family of developmentally regulated proteins of unknown function. As with several plant, cyanobacteria, and animal proteins that have similar structures, the expression of one of the Arabidopsis thaliana mt RNA-binding protein genes is induced by low temperatures.

Two exoribonucleases act sequentially to process mature 3'-ends of atp9 mRNAs in Arabidopsis mitochondria.

In plant mitochondria, transcription proceeds well beyond the region that will become mature 3' extremities of mRNAs, and the mechanisms of 3' maturation are largely unknown. Here, we show the involvement of two exoribonucleases, AtmtPNPase and AtmtRNaseII, in the 3' processing of atp9 mRNAs in Arabidopsis thaliana mitochondria. Down-regulation of AtmtPNPase results in the accumulation of pretranscripts of several times the size of mature atp9 mRNAs, indicating that 3' processing of these transcripts is performed mainly exonucleolytically by AtmtPNPase. This enzyme is however not sufficient to completely process atp9 mRNAs, because with down-regulation of another mitochondrial exoribonuclease, AtmtRNaseII, about half of atp9 transcripts exhibit short 3' nucleotide extensions compared with mature mRNAs. These short extensions can be efficiently removed by AtmtRNaseII in vitro. Taken together, these results show that 3' processing of atp9 mRNAs in Arabidopsis mitochondria is, at least, a two-step phenomenon. First, AtmtPNPase is involved in removing 3' extensions that may reach several kilobases. Second, AtmtRNaseII degrades short nucleotidic extensions to generate the mature 3'-ends.