Discovery of new genes and deletion editing in Physarum mitochondria enabled by a novel algorithm for finding edited mRNAs

Gene finding is complicated in organisms that exhibit insertional RNA editing. Here, we demonstrate how our new algorithm Predictor of Insertional Editing (PIE) can be used to locate genes whose mRNAs are subjected to multiple frameshifting events, and extend the algorithm to include probabilistic predictions for sites of nucleotide insertion; this feature is particularly useful when designing primers for sequencing edited RNAs. Applying this algorithm, we successfully identified the nad2, nad4L, nad6 and atp8 genes within the mitochondrial genome of Physarum polycephalum, which had gone undetected by existing programs. Characterization of their mRNA products led to the unanticipated discovery of nucleotide deletion editing in Physarum. The deletion event, which results in the removal of three adjacent A residues, was confirmed by primer extension sequencing of total RNA. This finding is remarkable in that it comprises the first known instance of nucleotide deletion in this organelle, to be contrasted with nearly 500 sites of single and dinucleotide addition in characterized mitochondrial RNAs. Statistical analysis of this larger pool of editing sites indicates that there are significant biases in the 2 nt immediately upstream of editing sites, including a reduced incidence of nucleotide repeats, in addition to the previously identified purine-U bias.