ACCURATE AND EFFICIENT INSERTIONAL RNA EDITING IN ISOLATED PHYSARUM MITOCHONDRIA

RNA editing is a process whereby nucleotide insertion, deletion, or ba se substitution results in the production of an RNA whose sequence dif fers from that of its template. The mitochondrial RNAs of Physarum pol ycephalum are processed specifically at multiple sites by both mono- a nd dinucleotide insertions, as well as apparent cytidine (C) to uridin e (U) changes. The precise mechanism and timing of these processing ev ents are currently unknown. We describe here the development of an iso lated mitochondrial system in which exogenously supplied nucleotides c an be incorporated into RNAs under defined conditions. The results of S1 nuclease protection, nearest neighbor and RNase Tt fingerprint anal yses indicate that the vast majority of these newly synthesized mitoch ondrial RNAs have been accurately and efficiently processed by both mo no- and dinucleotide insertions. This work provides a direct demonstra tion of faithful nucleotide insertion in a mitochondrial editing syste m. In contrast, the newly synthesized RNAs are not processed by C to U changes in the isolated mitochondria, suggesting that the base change s observed in Physarum are unlikely to occur via a deletion/insertion insertion mechanism.