Genetic variation is the major mechanism behind adaptation and evolutionary change. As most proteins operate through interactions with other proteins, changes in protein complex composition and subunit sequence provide potentially new functions. Comparative genomics can reveal expansions, losses and sequence divergence within protein-coding genes, but in silico analysis cannot detect subunit substitutions or replacements of entire protein complexes. Insights into these fundamental evolutionary processes require broad and extensive comparative analyses, from both in silico and experimental evidence. Here, we combine data from both approaches and consider the gamut of possible protein complex compositional changes that arise during evolution, citing examples of complete conservation to partial and total replacement by functional analogues. We focus in part on complexes in trypanosomes as they represent one of the better studied non-animal/non-fungal lineages, but extend insights across the eukaryotes by extensive comparative genomic analysis. We argue that gene loss plays an important role in diversification of protein complexes and hence enhancement of eukaryotic diversity.

• Klíčová slova
• constructive neutral evolution, evolutionary divergence, evolutionary mechanisms, gene replacement, molecular evolution, protein complexes,
• MeSH
• Eukaryota * genetika   MeSH
• fylogeneze MeSH
• genomika MeSH
• molekulární evoluce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• editace RNA * MeSH
• fylogeneze MeSH
• genom protozoální * MeSH
• messenger RNA metabolismus   MeSH
• RNA mitochondriální metabolismus   MeSH
• transkriptom MeSH
• Trypanosomatina genetika   metabolismus   MeSH
• vodící RNA, systémy CRISPR-Cas MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• RNA mitochondriální MeSH

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.

• Klíčová slova
• Euglena gracilis, Euglenozoa, mitochondria, proteome, protist,
• MeSH
• Euglena gracilis metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• proteom * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteom * 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
• chromozomy genetika   MeSH
• editace RNA genetika   MeSH
• Euglenozoa genetika   MeSH
• fylogeneze MeSH
• genom mitochondriální * MeSH
• geny * MeSH
• konzervovaná sekvence MeSH
• mitochondriální DNA genetika   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH

The mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans-splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper-inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4',6-diamidino-2-phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein-coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet-like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 IUBMB Life, 70(12):1267-1274, 2018.

• Klíčová slova
• DNA content, kinetoplast DNA, mitochondrial DNA, protist,
• MeSH
• Euglenozoa genetika   MeSH
• fylogeneze MeSH
• Kinetoplastida genetika   MeSH
• mitochondriální DNA genetika   izolace a purifikace   ultrastruktura   MeSH
• mitochondrie genetika   MeSH
• trans-splicing genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA 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
• editace RNA * MeSH
• genom mitochondriální genetika   MeSH
• genom protozoální genetika   MeSH
• izoformy RNA genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• RNA mitochondriální genetika   metabolismus   MeSH
• RNA protozoální genetika   metabolismus   MeSH
• sestřih RNA MeSH
• stanovení celkové genové exprese metody   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Trypanosomatina genetika   metabolismus   MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• izoformy RNA MeSH
• RNA mitochondriální MeSH
• RNA protozoální MeSH

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.

• MeSH
• DNA vazebné proteiny genetika   metabolismus   ultrastruktura   MeSH
• elektronová mikroskopie MeSH
• fluorescenční polarizace MeSH
• fylogeneze MeSH
• genetická variace MeSH
• lidé MeSH
• molekulární evoluce MeSH
• oligonukleotidy genetika   metabolismus   MeSH
• proteiny vázající telomery klasifikace   genetika   metabolismus   ultrastruktura   MeSH
• retardační test MeSH
• Schizosaccharomyces pombe - proteiny genetika   metabolismus   ultrastruktura   MeSH
• Schizosaccharomyces genetika   MeSH
• sekvence nukleotidů MeSH
• telomery genetika   metabolismus   ultrastruktura   MeSH
• transkripční faktory genetika   metabolismus   ultrastruktura   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• oligonukleotidy MeSH
• proteiny vázající telomery MeSH
• Schizosaccharomyces pombe - proteiny MeSH
• taz1 protein, S pombe MeSH Prohlížeč
• Teb1 protein, S pombe MeSH Prohlížeč
• transkripční faktory 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.

• Klíčová slova
• euglenozoa, mitochondria, mitochondrial genome,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

UNLABELLED: Perkinsela is an enigmatic early-branching kinetoplastid protist that lives as an obligate endosymbiont inside Paramoeba (Amoebozoa). We have sequenced the highly reduced mitochondrial genome of Perkinsela, which possesses only six protein-coding genes (cox1, cox2, cox3, cob, atp6, and rps12), despite the fact that the organelle itself contains more DNA than is present in either the host or endosymbiont nuclear genomes. An in silico analysis of two Perkinsela strains showed that mitochondrial RNA editing and processing machineries typical of kinetoplastid flagellates are generally conserved, and all mitochondrial transcripts undergo U-insertion/deletion editing. Canonical kinetoplastid mitochondrial ribosomes are also present. We have developed software tools for accurate and exhaustive mapping of transcriptome sequencing (RNA-seq) reads with extensive U-insertions/deletions, which allows detailed investigation of RNA editing via deep sequencing. With these methods, we show that up to 50% of reads for a given edited region contain errors of the editing system or, less likely, correspond to alternatively edited transcripts. IMPORTANCE: Uridine insertion/deletion-type RNA editing, which occurs in the mitochondrion of kinetoplastid protists, has been well-studied in the model parasite genera Trypanosoma, Leishmania, and Crithidia. Perkinsela provides a unique opportunity to broaden our knowledge of RNA editing machinery from an evolutionary perspective, as it represents the earliest kinetoplastid branch and is an obligatory endosymbiont with extensive reductive trends. Interestingly, up to 50% of mitochondrial transcripts in Perkinsela contain errors. Our study was complemented by use of newly developed software designed for accurate mapping of extensively edited RNA-seq reads obtained by deep sequencing.

• MeSH
• Amoebozoa parazitologie   MeSH
• delece genu * MeSH
• editace RNA * MeSH
• Kinetoplastida genetika   růst a vývoj   MeSH
• mitochondriální DNA chemie   genetika   MeSH
• mitochondrie genetika   MeSH
• sekvenční analýza DNA MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA 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.

• Klíčová slova
• Euglena gracilis, RNA editing, mitochondrial genome, transcription,
• MeSH
• editace RNA MeSH
• elektronový transportní řetězec genetika   MeSH
• Euglena gracilis genetika   MeSH
• genom mitochondriální * MeSH
• molekulární evoluce * MeSH
• RNA ribozomální genetika   MeSH
• sestřih RNA MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• elektronový transportní řetězec MeSH
• RNA ribozomální MeSH