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.

b Institute of Parasitology Biology Center and Faculty of Sciences University of South Bohemia České Budějovice Czech Republic

c Canadian Institute for Advanced Research Toronto Canada

Department of Biochemistry and Robert Cedergren Center for Bioinformatics and Genomics Université de Montréal Montreal Canada

Zobrazit více v PubMed

Burger G, Moreira S, Valach M. Genes in hiding. Trends Genet 2016; 32:553-65; PMID:27460648; http://dx.doi.org/10.1016/j.tig.2016.06.005 PubMed DOI

Knoop V. When you can't trust the DNA: RNA editing changes transcript sequences. Cell Mol Life Sci 2010; 68:567-86; PMID:20938709; http://dx.doi.org/10.1007/s00018-010-0538-9 PubMed DOI PMC

Maslov DA, Yasuhira S, Simpson L. Phylogenetic affinities of Diplonema within the Euglenozoa as inferred from the SSU rRNA gene and partial COI protein sequences. Protist 1999; 150:33-42; PMID:10724517; http://dx.doi.org/10.1016/S1434-4610(99)70007-6 PubMed DOI

Simpson AGB, Roger AJ. Protein phylogenies robustly resolve the deep-level relationships within Euglenozoa. Mol Phylogenet Evol 2004; 30:201-12; PMID:15022770; http://dx.doi.org/10.1016/S1055-7903(03)00177-5 PubMed DOI

Lara E, Moreira D, Vereshchaka A, López-García P. Pan-oceanic distribution of new highly diverse clades of deep-sea diplonemids. Environ Microbiol 2009; 11:47-55; PMID:18803646; http://dx.doi.org/10.1111/j.1462-2920.2008.01737.x PubMed DOI

de Vargas C, Audic S, Henry N, Decelle J, Mahé F, Logares R, Lara E, Berney C, Le Bescot N, Probert I, et al.. Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean. Science 2015; 348:1261605; PMID:25999516; http://dx.doi.org/10.1126/science.1261605 PubMed DOI

Lukeš J, Flegontova O, Horák A. Diplonemids. Curr Biol 2015; 25:R702-4; http://dx.doi.org/10.1016/j.cub.2015.04.052 PubMed DOI

Flegontova O, Flegontov P, Malviya S, Audic S, Wincker P, de Vargas C, Bowler C, Lukeš J, Horák A. (2016) Unexpected diversity and abundance of planktonic diplonemids in the world ocean. Curr. Biol; http://dx.doi.org/10.1016/j.cub.2016.09.031 PubMed DOI

Gawryluk RMR, del Campo J, Okamoto N, Strassert JFH, Lukeš J, Richards TA, Worden AZ, Santoro AE, Keeling PJ. (2016) Morphological identification and single-cell genomics of marine diplonemids. Curr. Biol; http://dx.doi.org/10.1016/j.cub.2016.09.013. PubMed DOI

Vlcek C, Marande W, Teijeiro S, Lukeš J, Burger G. Systematically fragmented genes in a multipartite mitochondrial genome. Nucleic Acids Res 2011; 39:979-88; PMID:20935050; http://dx.doi.org/10.1093/nar/gkq883 PubMed DOI PMC

Moreira S, Valach M, Aoulad-Aissa M, Otto C, Burger G. Novel modes of RNA editing in mitochondria. Nucleic Acids Res 2016; 44:4907-19; PMID:27001515; http://dx.doi.org/10.1093/nar/gkw188 PubMed DOI PMC

Marande W, Lukeš J, Burger G. Unique mitochondrial genome structure in diplonemids, the sister group of kinetoplastids. Eukaryot Cell 2005; 4:1137-46; PMID:15947205; http://dx.doi.org/10.1128/EC.4.6.1137-1146.2005 PubMed DOI PMC

Marande W, Burger G. Mitochondrial DNA as a genomic jigsaw puzzle. Science 2007; 318:415; PMID:17947575; http://dx.doi.org/10.1126/science.1148033 PubMed DOI

Kiethega GN, Yan Y, Turcotte M, Burger G. RNA-level unscrambling of fragmented genes in Diplonema mitochondria. RNA Biol 2013; 10:301-13; PMID:23324603; http://dx.doi.org/10.4161/rna.23340 PubMed DOI PMC

Kiethega GN, Turcotte M, Burger G. Evolutionarily conserved cox1 trans-splicing without cis-motifs. Mol Biol Evol 2011; 28:2425-8; PMID:21436119; http://dx.doi.org/10.1093/molbev/msr075 PubMed DOI

Moreira S, Breton S, Burger G. Unscrambling genetic information at the RNA level. Wiley Interdiscip Rev RNA 2012; 3:213-28; PMID:22275292; http://dx.doi.org/10.1002/wrna.1106 PubMed DOI

Valach M, Moreira S, Kiethega GN, Burger G. Trans-splicing and RNA editing of LSU rRNA in Diplonema mitochondria. Nucleic Acids Res 2014; 42:2660-72; PMID:24259427; http://dx.doi.org/10.1093/nar/gkt1152 PubMed DOI PMC

Feagin JE, Abraham JM, Stuart K. Extensive editing of the cytochrome c oxidase III transcript in Trypanosoma brucei. Cell 1988; 53:413-22; PMID:2452697; http://dx.doi.org/10.1016/0092-8674(88)90161-4 PubMed DOI

Read LK, Lukeš J, Hashimi H. Trypanosome RNA editing: the complexity of getting U in and taking U out. Wiley Interdiscip Rev RNA 2016; 7:33-51; PMID:26522170; http://dx.doi.org/10.1002/wrna.1313 PubMed DOI PMC

Roy J, Faktorová D, Benada O, Lukeš J, Burger G. Description of Rhynchopus euleeides n. sp. (Diplonemea), a free-living marine euglenozoan. J Eukaryot Microbiol 2007; 54:137-45; PMID:17403154; http://dx.doi.org/10.1111/j.1550-7408.2007.00244.x PubMed DOI

Roy J, Faktorová D, Lukeš J, Burger G. Unusual mitochondrial genome structures throughout the Euglenozoa. Protist 2007; 158:385-96; PMID:17499547; http://dx.doi.org/10.1016/j.protis.2007.03.002 PubMed DOI

Flegontov P, Gray MW, Burger G, Lukeš J. Gene fragmentation: a key to mitochondrial genome evolution in Euglenozoa? Curr Genet 2011; 57:225-32; PMID:21544620; http://dx.doi.org/10.1007/s00294-011-0340-8 PubMed DOI

Yabuki A, Tanifuji G, Kusaka C, Takishita K, Fujikura K. Hyper-eccentric structural genes in the mitochondrial genome of the algal parasite Hemistasia phaeocysticola. Genome Biol Evol 2016; evw207; http://dx.doi.org/10.1093/gbe/evw207 PubMed DOI PMC

Yabuki A, Tame A. Phylogeny and reclassification of Hemistasia phaeocysticola (Scherffel) Elbrächter & Schnepf, 1996. J Eukaryot Microbiol 2015; 62:426-9; PMID:25377132; http://dx.doi.org/10.1111/jeu.12191 PubMed DOI

Wallach DFH, Kamat VB. Plasma and cytoplasmic membrane fragments from Ehrlich ascites carcinoma. Proc Natl Acad Sci USA 1964; 52:721-8; PMID:14212548; http://dx.doi.org/10.1073/pnas.52.3.721 PubMed DOI PMC

Hauser R, Pypaert M, Häusler T, Horn EK, Schneider A. In vitro import of proteins into mitochondria of Trypanosoma brucei and Leishmania tarentolae. J Cell Sci 1996; 109:517-23; PMID:8838675 PubMed

Schneider A, Charrière F, Pusnik M, Horn EK. Isolation of mitochondria from procyclic Trypanosoma brucei. Methods Mol Biol 2007; 372:67-80; PMID:18314718; http://dx.doi.org/10.1007/978-1-59745-365-3_5 PubMed DOI

Burger G, Lavrov DV, Forget L, Lang BF. Sequencing complete mitochondrial and plastid genomes. Nat Protoc 2007; 2:603-14; PMID:17406621; http://dx.doi.org/10.1038/nprot.2007.59 PubMed DOI

Xie Y, Wu G, Tang J, Luo R, Patterson J, Liu S, Huang W, He G, Gu S, Li S, et al.. SOAPdenovo-Trans: de novo transcriptome assembly with short RNA-Seq reads. Bioinformatics 2014; 30:1660-6; PMID:24532719; http://dx.doi.org/10.1093/bioinformatics/btu077 PubMed DOI

Schulz MH, Zerbino DR, Vingron M, Birney E. Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels. Bioinformatics 2012; 28:1086-92; PMID:22368243; http://dx.doi.org/10.1093/bioinformatics/bts094 PubMed DOI PMC

Eddy SR. Accelerated profile HMM searches. PLoS Comput Biol 2011; 7:e1002195; PMID:22039361; http://dx.doi.org/10.1371/journal.pcbi.1002195 PubMed DOI PMC

Gautheret D, Lambert A. Direct RNA motif definition and identification from multiple sequence alignments using secondary structure profiles. J Mol Biol 2001; 313:1003-11; PMID:11700055; http://dx.doi.org/10.1006/jmbi.2001.5102 PubMed DOI

Nawrocki EP, Eddy SR. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 2013; 29:2933-5; PMID:24008419; http://dx.doi.org/10.1093/bioinformatics/btt509 PubMed DOI PMC

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012; 9:357-9; PMID:22388286; http://dx.doi.org/10.1038/nmeth.1923 PubMed DOI PMC

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 2013; 29:15-21; PMID:23104886; http://dx.doi.org/10.1093/bioinformatics/bts635 PubMed DOI PMC

Otto C, Stadler PF, Hoffmann S. Fast and sensitive mapping of bisulfite-treated sequencing data. Bioinformatics 2012; 28:1698-704; PMID:22581174; http://dx.doi.org/10.1093/bioinformatics/bts254 PubMed DOI

Neupert W, Herrmann JM. Translocation of proteins into mitochondria. Annu Rev Biochem 2007; 76:723-49; PMID:17263664; http://dx.doi.org/10.1146/annurev.biochem.76.052705.163409 PubMed DOI

Gerber AP, Keller W. RNA editing by base deamination: more enzymes, more targets, new mysteries. Trends Biochem Sci 2001; 26:376-84; PMID:11406411; http://dx.doi.org/10.1016/S0968-0004(01)01827-8 PubMed DOI

Kwak JE, Wickens M. A family of poly(U) polymerases. RNA 2007; 13:860-7; PMID:17449726; http://dx.doi.org/10.1261/rna.514007 PubMed DOI PMC

Tanaka N, Meineke B, Shuman S. RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo. J Biol Chem 2011; 286:30253-7; PMID:21757685; http://dx.doi.org/10.1074/jbc.C111.274597 PubMed DOI PMC

Sun T, Bentolila S, Hanson MR. The unexpected diversity of plant organelle RNA editosomes. Trends Plant Sci 2016; S1360-1385; http://dx.doi.org/10.1016/j.tplants.2016.07.005 PubMed DOI

Moreira S, Noutahi E, Lamoureux G, Burger G. Three-dimensional structure model and predicted ATP interaction rewiring of a deviant RNA ligase 2. BMC Struct Biol 2015; 15:20. PubMed PMC

Wittig I, Braun HP, Schägger H. Blue native PAGE. Nat Protoc 2006; 1:418-28; PMID:17406264; http://dx.doi.org/10.1038/nprot.2006.62 PubMed DOI

Petrov A, Wu T, Puglisi EV, Puglisi JD. RNA purification by preparative polyacrylamide gel electrophoresis. Meth Enzymol 2013; 530:315-30; PMID:24034329; http://dx.doi.org/10.1016/B978-0-12-420037-1.00017-8 PubMed DOI

Greber BJ, Ban N. Structure and function of the mitochondrial ribosome. Annu Rev Biochem 2016; 85:103-32; PMID:27023846; http://dx.doi.org/10.1146/annurev-biochem-060815-014343 PubMed DOI

Göringer HU. “Gestalt,” composition and function of the Trypanosoma brucei editosome. Annu Rev Microbiol 2012; 66:65-82; PMID:22994488; http://dx.doi.org/10.1146/annurev-micro-092611-150150 PubMed DOI

Papasaikas P, Valcárcel J. The spliceosome: the ultimate RNA chaperone and sculptor. Trends Biochem Sci 2016; 41:33-45; PMID:26682498; http://dx.doi.org/10.1016/j.tibs.2015.11.003 PubMed DOI

Engreitz JM, Sirokman K, McDonel P, Shishkin AA, Surka C, Russell P, Grossman SR, Chow AY, Guttman M, Lander ES. RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent pre-mRNAs and chromatin sites. Cell 2014; 159:188-99; PMID:25259926; http://dx.doi.org/10.1016/j.cell.2014.08.018 PubMed DOI PMC

McHugh CA, Chen CK, Chow A, Surka CF, Tran C, McDonel P, Pandya-Jones A, Blanco M, Burghard C, Moradian A, et al.. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 2015; 521:232-6; PMID:25915022; http://dx.doi.org/10.1038/nature14443 PubMed DOI PMC

Chu C, Zhang QC, da Rocha ST, Flynn RA, Bharadwaj M, Calabrese JM, Magnuson T, Heard E, Chang HY. Systematic discovery of Xist RNA binding proteins. Cell 2015; 161:404-16; PMID:25843628; http://dx.doi.org/10.1016/j.cell.2015.03.025 PubMed DOI PMC

Ho CK, Wang LK, Lima CD, Shuman S. Structure and mechanism of RNA ligase. Structure 2004; 12:327-39; PMID:14962393; http://dx.doi.org/10.1016/j.str.2004.01.011 PubMed DOI

Tanaka N, Chakravarty AK, Maughan B, Shuman S. Novel mechanism of RNA repair by RtcB via sequential 2′,3′-cyclic phosphodiesterase and 3′-phosphate/5′-hydroxyl ligation reactions. J Biol Chem 2011; 286:43134-43; PMID:22045815; http://dx.doi.org/10.1074/jbc.M111.302133 PubMed DOI PMC

Popow J, Schleiffer A, Martinez J. Diversity and roles of (t)RNA ligases. Cell Mol Life Sci 2012; 69:2657-70; PMID:22426497; http://dx.doi.org/10.1007/s00018-012-0944-2 PubMed DOI PMC

Aphasizheva I, Aphasizhev R. U-insertion/deletion mRNA-editing holoenzyme: definition in sight. Trends Parasitol 2016; 32:144-56; PMID:26572691; http://dx.doi.org/10.1016/j.pt.2015.10.004 PubMed DOI PMC

Gene fragmentation and RNA editing without borders: eccentric mitochondrial genomes of diplonemids

Evolution of metabolic capabilities and molecular features of diplonemids, kinetoplastids, and euglenids

Massive mitochondrial DNA content in diplonemid and kinetoplastid protists

Trypanosomatid mitochondrial RNA editing: dramatically complex transcript repertoires revealed with a dedicated mapping tool