Accurate and efficient insertional RNA editing in isolated Physarum mitochondria.
RNA editing is a process whereby nucleotide insertion, deletion, or base substitution results in the production of an RNA whose sequence differs from that of its template. The mitochondrial RNAs of Physarum polycephalum are processed specifically at multiple sites by both mono- and dinucleotide insertions, as well as apparent cytidine (C) to uridine (U) changes. The precise mechanism and timing of these processing events are currently unknown. We describe here the development of an isolated mitochondrial system in which exogenously supplied nucleotides can be incorporated into RNAs under defined conditions. The results of S1 nuclease protection, nearest neighbor and RNase T1 fingerprint analyses indicate that the vast majority of these newly synthesized mitochondrial RNAs have been accurately and efficiently processed by both mono- and dinucleotide insertions. This work provides a direct demonstration of faithful nucleotide insertion in a mitochondrial editing system. In contrast, the newly synthesized RNAs are not processed by C to U changes in the isolated mitochondria, suggesting that the base changes observed in Physarum are unlikely to occur via a deletion/insertion mechanism.
Visomirski-Robic
L M
LM
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Gott
J M
JM
D000818:Animals; D001483:Base Sequence; D008928:Mitochondria / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000378:metabolism; D016839:Physarum polycephalum / Q000235:genetics*; D017393:RNA Editing; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics*; D016053:RNA, Protozoan / Q000096:biosynthesis / Q000235:genetics*; D006163:Ribonuclease T1 / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic
D009711:Nucleotides; D012331:RNA, Fungal; D016053:RNA, Protozoan; D006163:Ribonuclease T1