Non-templated addition of nucleotides to the 3' end of nascent RNA during RNA editing in Physarum.

RNAs in Physarum: mitochondria contain extra nucleotides that are not encoded by the mitochondrial genome, at least in the traditional sense. While it is known that insertion of non-encoded nucleotides is linked to RNA synthesis, the exact nature of this relationship remains unclear. Here we demonstrate that the efficiency of editing is sensitive not only to the concentration of the nucleotide that is inserted, but also to the concentration of the nucleotide templated just downstream of an editing site. These data strongly support a co-transcriptional mechanism of Physarum: RNA editing in which non-encoded nucleotides are added to the 3' end of nascent RNAs. These results also suggest that transcription elongation and nucleotide insertion are competing processes and that recognition of editing sites most likely involves transient pausing by the Physarum: mitochondrial RNA polymerase. In addition, the pattern of nucleotide concentration effects, the context of editing sites and the accuracy of the mitochondrial RNA polymerase argue that the mechanism of Physarum: editing is distinct from that of other co-transcriptional editing systems.

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. Our recent studies have demonstrated that nucleotide addition can occur within 14-22 nt of the 3' end of a nascent RNA, suggesting that insertional editing may be linked to transcription. To investigate the relationship between these processes, we have examined the effects of nucleotide concentration on templated and nontemplated nucleotide addition in isolated mitochondria. At very low CTP concentrations, transcription and editing proceed with high fidelity, but the efficiency of cytidine insertional editing decreases. Insertion of single uridine and dinucleotides is not diminished under conditions that yield unedited or partially 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 nontemplated nucleotides are not inserted into previously synthesized RNA once editing conditions are restored, although RNA downstream of the unedited region is edited efficiently. This result indicates that insertional editing cannot occur posttranscriptionally under these conditions, and suggests that there is only a small "window of opportunity" in which nucleotide insertion can occur. Our data are consistent with 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 association with the transcription complex. Several possible models for the mechanism of insertional editing in Physarum are discussed.

Insertional editing of nascent mitochondrial RNAs in Physarum.

Maturation of Physarum mitochondrial RNA involves the highly specific insertion of nonencoded nucleotides at multiple locations. To investigate the mechanism(s) by which this occurs, we previously developed an isolated mitochondrial system in which run-on transcripts are accurately and efficiently edited by nucleotide insertion. Here we show that under limiting concentrations of exogenous nucleotides the mitochondrial RNA polymerases stall, generating a population of nascent RNAs that can be extended upon addition of limiting nucleotide. Several of these RNA species have been characterized and were found to be fully edited, indicating that nascent RNA is a substrate for nucleotide insertion in isolated Physarum mitochondria. Remarkably, these RNAs are edited at positions located within 14-22 nucleotides of the polymerase active site, suggesting that insertional editing may be physically or functionally associated with transcription. The absence of unedited RNA in these experiments indicates that large tracts of RNA downstream of editing sites are not required for nucleotide addition, and argues that insertional editing in Physarum occurs with a 5' to 3' polarity. These data also provide strong evidence that insertional editing in Physarum is mechanistically distinct from editing in kinetoplastid systems.

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.