RNA editing in higher plant mitochondria: analysis of biochemistry and specificity.

RNA editing alters genomically encoded cytidines to uridines posttranscriptionally in higher plant mitochondria. Most of these editing events occur in translated regions and consequently alter the amino acid sequence. In Oenothera berteriana more than 500 editing sites have been detected and the total number of editing sites exceeds 1000 sites in this mitochondrial genome. To identify the components involved in this process we investigated the factors determining the specificity of RNA editing and the apparent conversion of cytidine to uridine residues. The possible biochemical reactions responsible for RNA editing in plant mitochondria are de- or transamination, base substitution and nucleotide replacement. In order to discriminate between these different biochemical mechanisms we followed the fate of the sugar-phosphate backbone by analysing radiolabeled nucleotides after incorporation into high molecular mass RNA, Plant mitochondria were supplied with [alpha-P-32]CTP to radiolabel CMP residues in newly synthesized transcripts. Radiolabeled mtRNA was extracted and digested with nuclease P1 to hydrolyse the RNA to monophosphates. The resulting monophosphates were analysed on one- and two-dimensional TLC systems to separate pC from pU. Radiolabeled pU was detected in increasing quantities during the course of incubation. These results suggest that RNA editing in plant mitochondria involves either a deamination or a transglycosylation reaction. The editing product was identified as uridine and not as a hypermodified nucleotide which is recognized as uridine. Similar results have been obtained by incubating in vitro transcribed mRNAs with mitochondrial lysates indicating that RNA editing and transcription is not directly linked in plant mitochondria. The mechanism of editing site selection is still unclear. The nucleotide distribution around editing sites in Oenothera mitochondrial transcripts gives no indication of conserved primary or secondary structure motifs involved in editing site determination. However, experiments with labeled mtRNA show that edited transcript regions hybridize selectively with mitochondrial RNA fractions suggesting that RNA molecules may act as site determination factors.