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-P-32]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-P-32]CTP or, [5-H-3]CTP radiolabeled in vitro transcripts with a mitochondrial lysate from pea mitochondria. 5-H-3 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.