INSERTIONAL EDITING IN ISOLATED PHYSARUM-MITOCHONDRIA IS LINKED TO RNA-SYNTHESIS

The mitochondrial RNAs of Physarum polycephalum are edited efficiently by nucleotide insertion both in vivo and in isolated mitochondria. Ou r recent studies have demonstrated that nucleotide addition can occur within 14-22 nt of the 3' end of a nascent RNA, suggesting that insert ional editing may be linked to transcription. To investigate the relat ionship between these processes, we have examined the effects of nucle otide concentration on templated and nontemplated nucleotide addition in isolated mitochondria. At very low CTP concentrations, transcriptio n and editing proceed with high fidelity, but the efficiency of cytidi ne insertional editing decreases. Insertion of single uridine and dinu cleotides is not diminished under conditions that yield unedited or pa rtially edited C insertion sites, indicating that editing events occur independently of one another. Moreover, analysis of partially edited RNA demonstrates that single nucleotides can be added at dinucleotide insertion sites. Importantly, pulse-chase experiments indicate that no ntemplated nucleotides are not inserted into previously synthesized RN A once editing conditions are restored, although RNA downstream of the unedited region is edited efficiently. This result indicates that ins ertional editing cannot occur posttranscriptionally under these condit ions, and suggests that there is only a small ''window of opportunity' ' in which nucleotide insertion can occur. Our data are consistent wit h an editing activity that functions in a strictly 5' to 3' direction and adds nucleotides at, or close to, the 3' end of nascent RNA in ass ociation with the transcription complex. Several possible models for t he mechanism of insertional editing in Physarum are discussed.