The kinetoplastids are unicellular flagellates that derive their name from the 'kinetoplast', a region within their single mitochondrion harboring its organellar genome of high DNA content, called kinetoplast (k) DNA. Some protein products of this mitochondrial genome are encoded as cryptogenes; their transcripts require editing to generate an open reading frame. This happens through RNA editing, whereby small regulatory guide (g)RNAs direct the proper insertion and deletion of one or more uridines at each editing site within specific transcript regions. An accurate perspective of the kDNA expansion and evolution of their unique uridine insertion/deletion editing across kinetoplastids has been difficult to achieve. Here, we resolved the kDNA structure and editing patterns in the early-branching kinetoplastid Trypanoplasma borreli and compare them with those of the well-studied trypanosomatids. We find that its kDNA consists of circular molecules of about 42 kb that harbor the rRNA and protein-coding genes, and 17 different contigs of approximately 70 kb carrying an average of 23 putative gRNA loci per contig. These contigs may be linear molecules, as they contain repetitive termini. Our analysis uncovered a putative gRNA population with unique length and sequence parameters that is massive relative to the editing needs of this parasite. We validated or determined the sequence identity of four edited mRNAs, including one coding for ATP synthase 6 that was previously thought to be missing. We utilized computational methods to show that the T. borreli transcriptome includes a substantial number of transcripts with inconsistent editing patterns, apparently products of non-canonical editing. This species utilizes the most extensive uridine deletion compared to other studied kinetoplastids to enforce amino acid conservation of cryptogene products, although insertions still remain more frequent. Finally, in three tested mitochondrial transcriptomes of kinetoplastids, uridine deletions are more common in the raw mitochondrial reads than aligned to the fully edited, translationally competent mRNAs. We conclude that the organization of kDNA across known kinetoplastids represents variations on partitioned coding and repetitive regions of circular molecules encoding mRNAs and rRNAs, while gRNA loci are positioned on a highly unstable population of molecules that differ in relative abundance across strains. Likewise, while all kinetoplastids possess conserved machinery performing RNA editing of the uridine insertion/deletion type, its output parameters are species-specific.

• Keywords
• ATPase 6, Euglenozoa, Maxicircle, Metakinetoplastina, Mitochondrion, RNA editing, U-indel editing, Uridine insertion/deletion editing, guide RNA,
• Publication type
• Journal Article MeSH

Uridine insertion/deletion (U-indel) editing of mitochondrial mRNA, unique to the protistan class Kinetoplastea, generates canonical as well as potentially non-productive editing events. While the molecular machinery and the role of the guide (g) RNAs that provide required information for U-indel editing are well understood, little is known about the forces underlying its apparently error-prone nature. Analysis of a gRNA:mRNA pair allows the dissection of editing events in a given position of a given mitochondrial transcript. A complete gRNA dataset, paired with a fully characterized mRNA population that includes non-canonically edited transcripts, would allow such an analysis to be performed globally across the mitochondrial transcriptome. To achieve this, we have assembled 67 minicircles of the insect parasite Leptomonas pyrrhocoris, with each minicircle typically encoding one gRNA located in one of two similar-sized units of different origin. From this relatively narrow set of annotated gRNAs, we have dissected all identified mitochondrial editing events in L. pyrrhocoris, the strains of which dramatically differ in the abundance of individual minicircle classes. Our results support a model in which a multitude of editing events are driven by a limited set of gRNAs, with individual gRNAs possessing an inherent ability to guide canonical and non-canonical editing.

• MeSH
• RNA Editing * MeSH
• Phylogeny MeSH
• Genome, Protozoan * MeSH
• RNA, Messenger metabolism   MeSH
• RNA, Mitochondrial metabolism   MeSH
• Transcriptome MeSH
• Trypanosomatina genetics   metabolism   MeSH
• RNA, Guide, CRISPR-Cas Systems MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Messenger MeSH
• RNA, Mitochondrial MeSH

Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented.

• MeSH
• Adenosine Triphosphatases genetics   MeSH
• RNA Editing MeSH
• Phylogeny MeSH
• Genome, Mitochondrial MeSH
• RNA, Guide, Kinetoplastida genetics   MeSH
• Mitochondria genetics   MeSH
• Protein Subunits genetics   MeSH
• DNA, Protozoan genetics   MeSH
• RNA, Protozoan genetics   MeSH
• Trypanosoma lewisi genetics   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• RNA, Guide, Kinetoplastida MeSH
• Protein Subunits MeSH
• DNA, Protozoan MeSH
• RNA, Protozoan MeSH