rKOMICS:An R package for processing mitochondrial minicircle assemblies in population-scale genome projects

Abstract Background The advent of population-scale genome projects has revolutionized our biological understanding of parasitic protozoa. However, while hundreds to thousands of nuclear genomes of parasitic protozoa have been generated and analyzed, information about the diversity, structure and evolution of their mitochondrial genomes remains fragmentary, mainly because of their extraordinary complexity. Indeed, unicellular flagellates of the order Kinetoplastida contain structurally the most complex mitochondrial genome of all eukaryotes, organized as a giant network of homogeneous maxicircles and heterogeneous minicircles. We recently developed KOMICS, an analysis toolkit that automates the assembly and circularization of the mitochondrial genomes of Kinetoplastid parasites. While this tool overcomes the limitation of extracting mitochondrial assemblies from Next-Generation Sequencing datasets, interpreting and visualizing the genetic (dis)similarity within and between samples remains a time-consuming process. Results Here, we present a new analysis toolkit—rKOMICS—to streamline the analyses of minicircle sequence diversity in population-scale genome projects. rKOMICS is a user-friendly R package that has simple installation requirements and that is applicable to all 27 trypanosomatid genera. Once minicircle sequence alignments are generated, rKOMICS allows to examine, summarize and visualize minicircle sequence diversity within and between samples through the analyses of minicircle sequence clusters. We showcase the functionalities of the (r)KOMICS tool suite using a whole-genome sequencing dataset from a recently published study on the history of diversification of the Leishmania braziliensis species complex in Peru. Analyses of population diversity and structure highlighted differences in minicircle sequence richness and composition between Leishmania subspecies, and between subpopulations within subspecies. Conclusion The rKOMICS package establishes a critical framework to manipulate, explore and extract biologically relevant information from mitochondrial minicircle assemblies in tens to hundreds of samples simultaneously and efficiently. This should facilitate research that aims to develop new molecular markers for identifying species-specific minicircles, or to study the ancestry of parasites for complementary insights into their evolutionary history

Identification of novel components of Trypanosoma brucei editosomes

The editosome is a multiprotein complex that catalyzes the insertion and deletion of uridylates that occurs during RNA editing in trypanosomatids. We report the identification of nine novel editosome proteins in Trypanosoma brucei. They were identified by mass spectrometric analysis of functional editosomes that were purified by serial ion exchange/gel permeation chromatography, immunoaffinity chromatography specific to the TbMP63 editosome protein, or tandem affinity purification based on a tagged RNA editing ligase. The newly identified proteins have ribonuclease and/or RNA binding motifs suggesting nuclease function for at least some of these. Five of the proteins are interrelated, as are two others, and one is related to four previously identified editosome proteins. The implications of these findings are discussed

Parallel monitoring of RNA abundance, localisation and compactness with correlative single molecule FISH on LR White embedded samples

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans

Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes

Viability of the tsetse fly-transmitted African trypanosome Trypanosoma brucei depends on maintenance and expression of its kinetoplast (kDNA), the mitochondrial genome of this parasite and a putative target for veterinary and human antitrypanosomatid drugs. However, the closely related animal pathogens T. evansi and T. equiperdum are transmitted independently of tsetse flies and survive without a functional kinetoplast for reasons that have remained unclear. Here, we provide definitive evidence that single amino acid changes in the nuclearly encoded F(1)F(O)–ATPase subunit γ can compensate for complete physical loss of kDNA in these parasites. Our results provide insight into the molecular mechanism of compensation for kDNA loss by showing F(O)-independent generation of the mitochondrial membrane potential with increased dependence on the ADP/ATP carrier. Our findings also suggest that, in the pathogenic bloodstream stage of T. brucei, the huge and energetically demanding apparatus required for kDNA maintenance and expression serves the production of a single F(1)F(O)–ATPase subunit. These results have important implications for drug discovery and our understanding of the evolution of these parasites

A novel high-content phenotypic screen to identify inhibitors of mitochondrial DNA maintenance in trypanosomes

Kinetoplastid parasites cause diverse neglected diseases in humans and livestock, with an urgent need for new treatments. The survival of kinetoplastids depends on their uniquely structured mitochondrial genome (kDNA), the eponymous kinetoplast. Here, we report the development of a high-content screen for pharmacologically induced kDNA loss, based on specific staining of parasites and automated image analysis. As proof of concept, we screened a diverse set of ∼14,000 small molecules and exemplify a validated hit as a novel kDNA-targeting compound