Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

• Keywords
• Euglena gracilis, Euglenozoa, mitochondria, proteome, protist,
• MeSH
• Euglena gracilis metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Proteome * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Proteome * MeSH

Diplonemids are highly abundant heterotrophic marine protists. Previous studies showed that their strikingly bloated mitochondrial genome is unique because of systematic gene fragmentation and manifold RNA editing. Here we report a comparative study of mitochondrial genome architecture, gene structure and RNA editing of six recently isolated, phylogenetically diverse diplonemid species. Mitochondrial gene fragmentation and modes of RNA editing, which include cytidine-to-uridine (C-to-U) and adenosine-to-inosine (A-to-I) substitutions and 3' uridine additions (U-appendage), are conserved across diplonemids. Yet as we show here, all these features have been pushed to their extremes in the Hemistasiidae lineage. For example, Namystynia karyoxenos has its genes fragmented into more than twice as many modules than other diplonemids, with modules as short as four nucleotides. Furthermore, we detected in this group multiple A-appendage and guanosine-to-adenosine (G-to-A) substitution editing events not observed before in diplonemids and found very rarely elsewhere. With >1,000 sites, C-to-U and A-to-I editing in Namystynia is nearly 10 times more frequent than in other diplonemids. The editing density of 12% in coding regions makes Namystynia's the most extensively edited transcriptome described so far. Diplonemid mitochondrial genome architecture, gene structure and post-transcriptional processes display such high complexity that they challenge all other currently known systems.

• MeSH
• Chromosomes genetics   MeSH
• RNA Editing genetics   MeSH
• Euglenozoa genetics   MeSH
• Phylogeny MeSH
• Genome, Mitochondrial * MeSH
• Genes * MeSH
• Conserved Sequence MeSH
• DNA, Mitochondrial genetics   MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Mitochondrial MeSH

BACKGROUND: Comparative analyses have indicated that the mitochondrion of the last eukaryotic common ancestor likely possessed all the key core structures and functions that are widely conserved throughout the domain Eucarya. To date, such studies have largely focused on animals, fungi, and land plants (primarily multicellular eukaryotes); relatively few mitochondrial proteomes from protists (primarily unicellular eukaryotic microbes) have been examined. To gauge the full extent of mitochondrial structural and functional complexity and to identify potential evolutionary trends in mitochondrial proteomes, more comprehensive explorations of phylogenetically diverse mitochondrial proteomes are required. In this regard, a key group is the jakobids, a clade of protists belonging to the eukaryotic supergroup Discoba, distinguished by having the most gene-rich and most bacteria-like mitochondrial genomes discovered to date. RESULTS: In this study, we assembled the draft nuclear genome sequence for the jakobid Andalucia godoyi and used a comprehensive in silico approach to infer the nucleus-encoded portion of the mitochondrial proteome of this protist, identifying 864 candidate mitochondrial proteins. The A. godoyi mitochondrial proteome has a complexity that parallels that of other eukaryotes, while exhibiting an unusually large number of ancestral features that have been lost particularly in opisthokont (animal and fungal) mitochondria. Notably, we find no evidence that the A. godoyi nuclear genome has or had a gene encoding a single-subunit, T3/T7 bacteriophage-like RNA polymerase, which functions as the mitochondrial transcriptase in all eukaryotes except the jakobids. CONCLUSIONS: As genome and mitochondrial proteome data have become more widely available, a strikingly punctuate phylogenetic distribution of different mitochondrial components has been revealed, emphasizing that the pathways of mitochondrial proteome evolution are likely complex and lineage-specific. Unraveling this complexity will require comprehensive comparative analyses of mitochondrial proteomes from a phylogenetically broad range of eukaryotes, especially protists. The systematic in silico approach described here offers a valuable adjunct to direct proteomic analysis (e.g., via mass spectrometry), particularly in cases where the latter approach is constrained by sample limitation or other practical considerations.

• Keywords
• Andalucia godoyi, Jakobids, Mitochondrial evolution, Mitochondrial genome, Mitochondrial proteome, Mitochondrion, Protist,
• MeSH
• Cell Nucleus genetics   MeSH
• Eukaryota genetics   MeSH
• Genome, Mitochondrial * MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Proteome * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Proteome * MeSH

RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3' to 5' on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. We also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.

• MeSH
• RNA Editing * MeSH
• Genome, Mitochondrial genetics   MeSH
• Genome, Protozoan genetics   MeSH
• RNA Isoforms genetics   metabolism   MeSH
• Mitochondria genetics   metabolism   MeSH
• RNA, Mitochondrial genetics   metabolism   MeSH
• RNA, Protozoan genetics   metabolism   MeSH
• RNA Splicing MeSH
• Gene Expression Profiling methods   MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Trypanosomatina genetics   metabolism   MeSH
• Computational Biology methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA Isoforms MeSH
• RNA, Mitochondrial MeSH
• RNA, Protozoan MeSH

The instructions to make proteins and structural RNAs are laid down in gene sequences. Yet, in certain instances, these primary instructions need to be modified considerably during gene expression, most often at the transcript level. Here we review a case of massive post-transcriptional revisions via trans-splicing and RNA editing, a phenomenon occurring in mitochondria of a recently recognized protist group, the diplonemids. As of now, the various post-transcriptional steps have been cataloged in detail, but how these processes function is still unknown. Since genetic manipulation techniques such as gene replacement and RNA interference have not yet been established for these organisms, alternative strategies have to be deployed. Here, we discuss the experimental and bioinformatics approaches that promise to unravel the molecular machineries of trans-splicing and RNA editing in Diplonema mitochondria.

• Keywords
• Cryptic genes, RNA editing, diplonemids, gene fragmentation, multipartite mtDNA, trans-splicing,
• MeSH
• RNA Editing MeSH
• Euglenozoa genetics   MeSH
• Mitochondrial Proteins genetics   MeSH
• Mitochondria genetics   MeSH
• RNA Processing, Post-Transcriptional MeSH
• Protozoan Proteins genetics   MeSH
• Gene Expression Regulation MeSH
• RNA, Transfer metabolism   MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Mitochondrial Proteins MeSH
• Protozoan Proteins MeSH
• RNA, Transfer MeSH

Mitochondria are double membrane organelles of endosymbiotic origin, best known for constituting the centre of energetics of a eukaryotic cell. They contain their own mitochondrial genome, which as a consequence of gradual reduction during evolution typically contains less than two dozens of genes. In this review, we highlight the extremely diverse architecture of mitochondrial genomes and mechanisms of gene expression between the three sister groups constituting the phylum Euglenozoa - Euglenida, Diplonemea and Kinetoplastea. The earliest diverging euglenids possess a simplified mitochondrial genome and a conventional gene expression, whereas both are highly complex in the two other groups. The expression of their mitochondrial-encoded proteins requires extensive post-transcriptional modifications guided by complex protein machineries and multiple small RNA molecules. Moreover, the least studied diplonemids, which have been recently discovered as a highly abundant component of the world ocean plankton, possess one of the most complicated mitochondrial genome organisations known to date.

• Keywords
• euglenozoa, mitochondria, mitochondrial genome,
• Publication type
• Journal Article MeSH
• Review MeSH

In this study, we describe the mitochondrial genome of the excavate flagellate Euglena gracilis. Its gene complement is reduced as compared with the well-studied sister groups Diplonemea and Kinetoplastea. We have identified seven protein-coding genes: Three subunits of respiratory complex I (nad1, nad4, and nad5), one subunit of complex III (cob), and three subunits of complex IV (cox1, cox2, and a highly divergent cox3). Moreover, fragments of ribosomal RNA genes have also been identified. Genes encoding subunits of complex V, ribosomal proteins and tRNAs were missing, and are likely located in the nuclear genome. Although mitochondrial genomes of diplonemids and kinetoplastids possess the most complex RNA processing machineries known, including trans-splicing and editing of the uridine insertion/deletion type, respectively, our transcriptomic data suggest their total absence in E. gracilis. This finding supports a scenario in which the complex mitochondrial processing machineries of both sister groups evolved relatively late in evolution from a streamlined genome and transcriptome of their common predecessor.

• Keywords
• Euglena gracilis, RNA editing, mitochondrial genome, transcription,
• MeSH
• RNA Editing MeSH
• Electron Transport Chain Complex Proteins genetics   MeSH
• Euglena gracilis genetics   MeSH
• Genome, Mitochondrial * MeSH
• Evolution, Molecular * MeSH
• RNA, Ribosomal genetics   MeSH
• RNA Splicing MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Electron Transport Chain Complex Proteins MeSH
• RNA, Ribosomal MeSH