RNA EDITING IN HIGHER-PLANT MITOCHONDRIA - ANALYSIS OF BIOCHEMISTRY AND SPECIFICITY

RNA editing alters genomically encoded cytidines to uridines posttrans criptionally in higher plant mitochondria. Most of these editing event s occur in translated regions and consequently alter the amino acid se quence. In Oenothera berteriana more than 500 editing sites have been detected and the total number of editing sites exceeds 1000 sites in t his 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 plan t mitochondria are de- or transamination, base substitution and nucleo tide replacement. In order to discriminate between these different bio chemical mechanisms we followed the fate of the sugar-phosphate backbo ne 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. Radi olabeled mtRNA was extracted and digested with nuclease P1 to hydrolys e the RNA to monophosphates. The resulting monophosphates were analyse d on one- and two-dimensional TLC systems to separate pC from pU. Radi olabeled pU was detected in increasing quantities during the course of incubation. These results suggest that RNA editing in plant mitochond ria involves either a deamination or a transglycosylation reaction. Th e 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 l ysates indicating that RNA editing and transcription is not directly l inked in plant mitochondria. The mechanism of editing site selection i s still unclear. The nucleotide distribution around editing sites in O enothera mitochondrial transcripts gives no indication of conserved pr imary or secondary structure motifs involved in editing site determina tion. However, experiments with labeled mtRNA show that edited transcr ipt regions hybridize selectively with mitochondrial RNA fractions sug gesting that RNA molecules may act as site determination factors.