Diverse RNA Editing Systems Across Heterolobosea (Discoba)

Mitochondrial RNA editing events were previously reported in only three unrelated species within the phylum Heterolobosea (Discoba): Naegleria gruberi and Acrasis kona (which performs C-to-U substitutions) and Pharyngomonadida sp. BB2 (which inserts single nucleotides by a yet unknown mechanism). We present novel high-quality genome assemblies obtained via a combination of whole-genome and targeted sequencing approaches and predicted proteomes for two species, Paravahlkampfia ustiana and Heteramoeba clara, representing an understudied clade of the phylum. Surprisingly, we discovered RNA editing events of C-to-U substitutions and mononucleotide insertions co-existing in the mitochondria of P. ustiana, while the two systems were both absent in H. clara. In addition, P. ustiana also exhibits A-to-I editing sites in rRNA transcripts encoded in multicopy circular rDNA plasmids. RNA editing events in P. ustiana were verified by proteomics, Illumina short RNA reads, ONT long cDNA and direct RNA reads. We elaborate on the potential mechanisms of mitochondrial insertional editing by investigating several aspects of the RNA transcription machinery and base modifications in template DNA and mRNA products. We report additional cases of C-to-U RNA editing in other species of Heterolobosea and explore the distribution of various putative components of the C-to-U editosome, including the DYW domain-containing pentatricopeptide repeat (PPR) proteins, across the phylum expanding the understanding of the evolutionary dynamics underlying this phenomenon, which remains understudied outside land plants. The comparative analysis of available sequence data from Heterolobosea revealed that C-to-U RNA editing was acquired relatively deep in the evolution of Heterolobosea and subsequently underwent repeated losses.