Evidence for a Site-specific Cytidine Deamination Reaction Involved in C to U RNA Editing of Plant Mitochondria

Transcripts of higher plant mitochondria are modified post-transcriptionally by RNA editing. To distinguish between the mechanisms by which the cytidine to uridine transition could occur a combined transcription/RNA editing assay and an in vitro RNA editing system were investigated. Mitochondria isolated from etiolated pea seedlings and potato tubers were supplied with [alpha-32P]CTP to radiolabel the mitochondrial run-on transcripts. High molecular weight run-on transcripts were isolated and hydrolyzed, and nucleotide identities were analyzed by one- and two-dimensional thin layer chromatography. The amount of label comigrating with UMP nucleotides increases with extended incubation times. Analogous products were obtained by incubation of [alpha-32P]CTP or [5-3H]CTP radiolabeled in vitro transcripts with a mitochondrial lysate from pea mitochondria. 5-3H label of the cytosine base was detected in the UMP spot after incubation of in vitro transcripts with mitochondrial lysate. These results are consistent with a deamination reaction involved in this post-transcriptional C to U modification process. To prove that cytidines are deaminated specifically in vitro transcripts were reisolated after incubation and analyzed by reverse transcription-polymerase chain reaction. Sequence analysis clearly shows that only cytidines at editing sites are edited while residual cytidines are not modified and suggests that site-specific factors are involved in RNA editing of plant mitochondria.