RNA processing in plant mitochondria is independent of transcription.

We analyzed the ability of plant mitochondria to process introduced RNA. Arabidopsis thaliana cox2 transcripts were synthesized in vitro. The in vitro transcribed mRNA was electroporated into maize and cauliflower mitochondria and incubated in organello. RNA was isolated and RT-PCR was carried out to analyze RNA processing. Our data indicate that cox2 transcripts introduced into isolated plant mitochondria are processed completely. This is the first report of in organello editing of introduced transcripts. We also found that none of the transcription, translation, or respiration inhibitors we used influenced RNA splicing or RNA editing of the cox2 transcript. Thus, our data also demonstrate that plant mitochondrial RNA processing may be independent of both transcription and respiratory regulation.

Plastid mRNAs are neither spliced nor edited in maize and cauliflower mitochondrial in organello systems.

The process of RNA editing in chloroplasts and higher plant mitochondria displays some similarities, raising the question of common or similar components in editing apparatus of these two organelles. To investigate the ability of plant mitochondria to edit plastid transcripts, we employed a previously established mitochondrial maize and cauliflower in organello system. Two plastid genes, Zea mays ndhB and ycf3 containing group II introns and several editing sites, were introduced into mitochondria. The genes were transcribed in organello. However, these transcripts of the plastid genes are neither spliced nor edited in plant mitochondria. A comparison of maize ndhB editing sites and maize mitochondrial editing sites reveals considerable sequence similarities between three ndhB editing sites and several mitochondrial sites. Nevertheless, these ndhB editing sites were not recognized in the mitochondria. Thus, we present for the first time direct evidence that the factors present in the plant mitochondria are not sufficient to allow editing and splicing of plastid transcripts.

Transcript end mapping and analysis of RNA editing in plant mitochondria.

Mitochondria are genetic compartments with their own enzymatic equipment for maintenance and expression of their genetic information. As in all genetic systems, gene expression has to be regulated, and in mitochondria this also has to be coordinated with the expression of nuclear-encoded mitochondrial proteins. Presently, there is virtually no information available about the mechanistic details and the enzymes involved in these processes. There is still much to be learned about how plant mitochondrial gene expression is managed and to what extent the contribution of transcription initiation and posttranscriptional processes, respectively, contribute to this control. As one prerequisite for better understanding of the mechanisms and regulatory controls, more fundamental data on mitochondrial transcription initiation and posttranscriptional RNA processing are necessary. As part of the essential methodology, we present methods for the analysis of the 5' and 3' extremities of mitochondrial transcripts and the identification of transcription initiation sites. An in organello system is described for the functional investigation of ribonucleic acid editing in plant mitochondria.

Mono- and dicotyledonous plant-specific RNA editing sites are correctly edited in both in organello systems.

We set out to analyse the phylogenetic distribution of cox2 RNA editing sites. Database searches have revealed the presence of mono- and dicotyledonous-specific RNA editing sites. Therefore, to better understand tRNA editing system in plants, we developed a new dicotyledonous in organello RNA editing system using cauliflower mitochondria and analysed the transcription of the cox2 gene for both maize and Arabidopsis. These results were compared with those obtained from a maize mitochondrial in organello system. Surprisingly, both the mono- and dicotyledonous cox2 transcripts were efficiently edited in the mitochondrial cauliflower and maize in organello systems, respectively, even for RNA editing sites not present in the endogenous cox2 sequences. Taken together, our observations support a self-guiding-transcript model for RNA editing in higher plants.

Mitochondrial electroporation and in organello RNA editing of chimeric atp6 transcripts.

The Sorghum bicolor atp6-1 gene and chimeric atp6 genes with additional maize sequences were introduced into isolated maize mitochondria via electroporation. Transcripts isolated after in vitro incubation of the transformed organelles were then analysed for RNA editing. Transcripts of the S. bicolor atp6-1 gene, and the RNAs obtained from most of chimeric sorghum-maize atp6 gene constructs tested, were not edited. However, the transcript of one engineered chimeric gene comprising the 5'untranslated sequence and a segment of the N-terminal ORF of the maize atp6 combined with the sorghum atp6 core ORF and 3'untranslated sequence was found to be partially edited. We were able to exclude low RNA stability or insufficient editing capacity as the reason for failure to edit in the other instances. Instead, the data indicate that the maize sequence in the edited fusion transcript provides a structural motif or binding site for a transcript-specific editing factor.