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.

Belozersky Institute of Physico Chemical Biology M 5 Lomonosov Moscow State University Moscow 119991 Russia

Central European Institute of Technology Masaryk University Brno 625 00 Czech Republic

Department of Biomedical Sciences University of Minnesota Medical School Duluth MN 55812 3031 USA

Faculty of Biology M 5 Lomonosov Moscow State University Moscow 119991 Russia

Faculty of Science University of South Bohemia České Budějovice 370 05 Czech Republic

Institute for Information Transmission Problems Russian Academy of Sciences Moscow 127051 Russia

Institute of Environmental Technologies Faculty of Science University of Ostrava Ostrava 710 00 Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice 370 05 Czech Republic

Life Science Research Centre Faculty of Science University of Ostrava Ostrava 710 00 Czech Republic

Russia Extreme Biology Laboratory Institute of Fundamental Medicine and Biology Kazan Federal University Kazan 420008 Russia

Skolkovo Institute of Science and Technology Moscow 14326 Russia

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Gray M.W., Lukeš J., Archibald J.M., Keeling P.J., Doolittle W.F.. Cell biology. Irremediable complexity?. Science. 2010; 330:920–921. PubMed

Koonin E.V. Splendor and misery of adaptation, or the importance of neutral null for understanding evolution. BMC Biol. 2016; 14:114. PubMed PMC

Lukeš J., Archibald J.M., Keeling P.J., Doolittle W.F., Gray M.W.. How a neutral evolutionary ratchet can build cellular complexity. IUBMB Life. 2011; 63:528–537. PubMed

Stoltzfus A. Constructive neutral evolution: exploring evolutionary theory's curious disconnect. Biol Direct. 2012; 7:35. PubMed PMC

Smith D.R., Keeling P.J.. Protists and the Wild, Wild West of gene expression: new frontiers, Lawlessness, and Misfits. Annu. Rev. Microbiol. 2016; 70:161–178. PubMed

Burger G., Moreira S., Valach M.. Genes in Hiding. Trends Genet. 2016; 32:553–565. PubMed

Faktorová D., Dobáková E., Peña-Diaz P., Lukeš J.. From simple to supercomplex: mitochondrial genomes of euglenozoan protists. F1000Research. 2016; 5:392. PubMed PMC

Valach M., Moreira S., Faktorová D., Lukeš J., Burger G.. Post-transcriptional mending of gene sequences: looking under the hood of mitochondrial gene expression in diplonemids. RNA Biol. 2016; 13:1204–1211. PubMed PMC

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; 8:2870–2878. PubMed PMC

Aphasizheva I., Aphasizhev R.. U-insertion/deletion mRNA-editing holoenzyme: definition in sight. Trends Parasitol. 2016; 32:144–156. PubMed PMC

Read L.K., 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. PubMed PMC

David V., Flegontov P., Gerasimov E., Tanifuji G., Hashimi H., Logacheva M.D., Maruyama S., Onodera N.T., Gray M.W., Archibald J.M. et al. . Gene loss and error-prone RNA editing in the mitochondrion of Perkinsela, an endosymbiotic kinetoplastid. mBio. 2015; 6:e01498–15. PubMed PMC

Simpson L., Thiemann O.H., Savill N.J., Alfonzo J.D., Maslov D.A.. Evolution of RNA editing in trypanosome mitochondria. Proc. Natl. Acad. Sci. U.S.A. 2000; 97:6986–6993. PubMed PMC

Simpson L., Maslov D.A.. Evolution of the U-insertion/deletion RNA editing in mitochondria of kinetoplastid protozoa. Ann. N. Y. Acad. Sci. 1999; 870:190–205. PubMed

Aravin A.A., Yurchenko V., Merzlyak E.M., Kolesnikov A.A.. The mitochondrial ND8 gene from Crithidia oncopelti is not pan-edited. FEBS Lett. 1998; 431:457–460. PubMed

Gerasimov E.S., Kostygov A.Y., Yan S., Kolesnikov A.A.. From cryptogene to gene? ND8 editing domain reduction in insect trypanosomatids. Eur. J. Protistol. 2012; 48:185–193. PubMed

McDermott S.M., Luo J., Carnes J., Ranish J.A., Stuart K.. The architecture of Trypanosoma brucei editosomes. Proc. Natl. Acad. Sci. U.S.A. 2016; 113:e6476–e6485. PubMed PMC

Simpson R.M., Bruno A.E., Chen R., Lott K., Tylec B.L., Bard J.E., Sun Y., Buck M.J., Read L.K.. Trypanosome RNA Editing Mediator Complex proteins have distinct functions in gRNA utilization. Nucleic Acids Res. 2017; 45:7965–7983. PubMed PMC

Kirby L.E., Sun Y., Judah D., Nowak S., Koslowsky D.. Analysis of the Trypanosoma brucei EATRO 164 bloodstream guide RNA transcriptome. PLoS Negl. Trop. Dis. 2016; 10:e0004793. PubMed PMC

Koslowsky D., Sun Y., Hindenach J., Theisen T., Lucas J.. The insect-phase gRNA transcriptome in Trypanosoma brucei. Nucleic Acids Res. 2014; 42:1873–1886. PubMed PMC

Simpson L., Douglass S.M., Lake J.A., Pellegrini M., Li F.. Comparison of the mitochondrial genomes and steady state transcriptomes of two strains of the trypanosomatid parasite, Leishmania tarentolae. PLoS Negl. Trop. Dis. 2015; 9:e0003841. PubMed PMC

Ammerman M.L., Presnyak V., Fisk J.C., Foda B.M., Read L.K.. TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites. RNA. 2010; 16:2239–2251. PubMed PMC

Koslowsky D.J., Bhat G.J., Read L.K., Stuart K.. Cycles of progressive realignment of gRNA with mRNA in RNA editing. Cell. 1991; 67:537–546. PubMed

Simpson R.M., Bruno A.E., Bard J.E., Buck M.J., Read L.K.. High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing. RNA. 2016; 22:677–695. PubMed PMC

Arts G.J., van der Spek H., Speijer D., van den Burg J., van Steeg H., Sloof P., Benne R.. Implications of novel guide RNA features for the mechanism of RNA editing in Crithidia fasciculata. EMBO J. 1993; 12:1523–1532. PubMed PMC

Maslov D.A., Simpson L.. The polarity of editing within a multiple gRNA-mediated domain is due to formation of anchors for upstream gRNAs by downstream editing. Cell. 1992; 70:459–467. PubMed

Maslov D.A., Thiemann O., Simpson L.. Editing and misediting of transcripts of the kinetoplast maxicircle G5 (ND3) cryptogene in an old laboratory strain of Leishmania tarentolae. Mol. Biochem. Parasitol. 1994; 68:155–159. PubMed

Sturm N.R., Maslov D.A., Blum B., Simpson L.. Generation of unexpected editing patterns in Leishmania tarentolae mitochondrial mRNAs: misediting produced by misguiding. Cell. 1992; 70:469–476. PubMed

Ochsenreiter T., Hajduk S.L.. Alternative editing of cytochrome c oxidase III mRNA in trypanosome mitochondria generates protein diversity. EMBO Rep. 2006; 7:1128–1133. PubMed PMC

Ochsenreiter T., Anderson S., Wood Z.A., Hajduk S.L.. Alternative RNA editing produces a novel protein involved in mitochondrial DNA maintenance in trypanosomes. Mol. Cell. Biol. 2008; 28:5595–5604. PubMed PMC

Ochsenreiter T., Cipriano M., Hajduk S.L.. Alternative mRNA editing in trypanosomes is extensive and may contribute to mitochondrial protein diversity. PLoS One. 2008; 3:e1566. PubMed PMC

Madina B.R., Kumar V., Metz R., Mooers B.H., Bundschuh R., Cruz-Reyes J.. Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition. RNA. 2014; 20:1142–1152. PubMed PMC

Flegontov P., Gray M.W., Burger G., Lukeš J.. Gene fragmentation: a key to mitochondrial genome evolution in Euglenozoa?. Curr. Genet. 2011; 57:225–232. PubMed

Gray M.W. Evolutionary origin of RNA editing. Biochemistry. 2012; 51:5235–5242. PubMed

Speijer D. Is kinetoplastid pan-editing the result of an evolutionary balancing act?. IUBMB Life. 2006; 58:91–96. PubMed

Speijer D. Does constructive neutral evolution play an important role in the origin of cellular complexity? Making sense of the origins and uses of biological complexity. Bioessays. 2011; 33:344–349. PubMed

Kirby L.E., Koslowsky D.. Mitochondrial dual-coding genes in Trypanosome brucei. PLoS Negl. Trop. Dis. 2017; 11:e0005989. PubMed PMC

Maslov D.A., Sturm N.R., Niner B.M., Gruszynski E.S., Peris M., Simpson L.. An intergenic G-rich region in Leishmania tarentolae kinetoplast maxicircle DNA is a pan-edited cryptogene encoding ribosomal protein S12. Mol. Cell. Biol. 1992; 12:56–67. PubMed PMC

Feagin J.E., Abraham J.M., Stuart K.. Extensive editing of the cytochrome c oxidase III transcript in Trypanosoma brucei. Cell. 1988; 53:413–422. PubMed

Shaw J.M., Feagin J.E., Stuart K., Simpson L.. Editing of kinetoplastid mitochondrial mRNAs by uridine addition and deletion generates conserved amino acid sequences and AUG initiation codons. Cell. 1988; 53:401–411. PubMed

Feagin J.E., Shaw J.M., Simpson L., Stuart K.. Creation of AUG initiation codons by addition of uridines within cytochrome b transcripts of kinetoplastids. Proc. Natl. Acad. Sci. U.S.A. 1988; 85:539–543. PubMed PMC

Carnes J., McDermott S., Anupama A., Oliver B.G., Sather D.N., Stuart K.. In vivo cleavage specificity of Trypanosoma brucei editosome endonucleases. Nucleic Acids Res. 2017; 45:4667–4686. PubMed PMC

del Campo J., Sieracki M.E., Molestina R., Keeling P., Massana R., Ruiz-Trillo I.. The others: our biased perspective of eukaryotic genomes. Trends Ecol. Evol. 2014; 29:252–259. PubMed PMC

Votýpka J., Klepetková H., Yurchenko V.Y., Horák A., Lukeš J., Maslov D.A.. Cosmopolitan distribution of a trypanosomatid Leptomonas pyrrhocoris. Protist. 2012; 163:616–631. PubMed

Maslov D.A., Votýpka J., Yurchenko V., Lukeš J.. Diversity and phylogeny of insect trypanosomatids: all that is hidden shall be revealed. Trends Parasitol. 2013; 29:43–52. PubMed

Flegontov P., Butenko A., Firsov S., Kraeva N., Eliáš M., Field M.C., Filatov D., Flegontova O., Gerasimov E.S., Hlaváčová J. et al. . Genome of Leptomonas pyrrhocoris: a high-quality reference for monoxenous trypanosomatids and new insights into evolution of Leishmania. Sci. Rep. 2016; 6:23704. PubMed PMC

Castellani O., Ribeiro L.V., Fernandes J.F.. Differentiation of Trypanosoma cruzi in culture. J. Protozool. 1967; 14:447–451. PubMed

Pelletier M., Read L.K., Aphasizhev R.. Isolation of RNA binding proteins involved in insertion/deletion editing. Methods Enzymol. 2007; 424:75–105. PubMed

Horváth A., Horáková E., Dunajcíková P., Verner Z., Pravdová E., Slapetová I., Cuninková L., Lukes J.. Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei. Mol. Microbiol. 2005; 58:116–130. PubMed

Záhonová K., Hadariová L., Vacula R., Yurchenko V., Eliáš M., Krajčovič J., Vesteg M.. A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis. FEBS Lett. 2014; 588:783–788. PubMed

Zhang L., Sement F.M., Suematsu T., Yu T., Monti S., Huang L., Aphasizhev R., Aphasizheva I.. PPR polyadenylation factor defines mitochondrial mRNA identity and stability in trypanosomes. EMBO J. 2017; 36:2435–2454. PubMed PMC

Gazestani V.H., Hampton M., Shaw A.K., Liggett C., Salavati R., Zimmer S.L.. Tail characteristics of Trypanosoma brucei mitochondrial transcripts are developmentally altered in a transcript-specific manner. Int. J. Parasitol. 2017; doi:10.1016/j.ijpara.2017.08.012. PubMed

Aslett M., Aurrecoechea C., Berriman M., Brestelli J., Brunk B.P., Carrington M., Depledge D.P., Fischer S., Gajria B., Gao X. et al. . TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010; 38:D457–D462. PubMed PMC

Souza A.E., Myler P.J., Stuart K.. Maxicircle CR1 transcripts of Trypanosoma brucei are edited and developmentally regulated and encode a putative iron-sulfur protein homologous to an NADH dehydrogenase subunit. Mol. Cell. Biol. 1992; 12:2100–2107. PubMed PMC

Read L.K., Myler P.J., Stuart K.. Extensive editing of both processed and preprocessed maxicircle CR6 transcripts in Trypanosoma brucei. J. Biol. Chem. 1992; 267:1123–1128. PubMed

Ruvalcaba-Trejo L.I., Sturm N.R.. The Trypanosoma cruzi Sylvio X10 strain maxicircle sequence: the third musketeer. BMC Genomics. 2011; 12:58. PubMed PMC

Langmead B., Salzberg S.. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012; 9:357–359. PubMed PMC

Gazestani V.H., Hampton M., Abrahante J.E., Salavati R., Zimmer S.L.. circTAIL-seq, a targeted method for deep analysis of RNA 3′ tails, reveals transcript-specific differences by multiple metrics. RNA. 2016; 22:477–486. PubMed PMC

Jirků M., Yurchenko V.Y., Lukeš J., Maslov D.A.. New species of insect trypanosomatids from Costa Rica and the proposal for a new subfamily within the Trypanosomatidae. J. Eukaryot. Microbiol. 2012; 59:537–547. PubMed

Landweber L.F., Gilbert W.. RNA editing as a source of genetic variation. Nature. 1993; 363:179–182. PubMed

Yasuhira S., Simpson L.. Minicircle-encoded guide RNAs from Crithidia fasciculata. RNA. 1995; 1:634–643. PubMed PMC

Nebohácová M., Kim C.E., Simpson L., Maslov D.A.. RNA editing and mitochondrial activity in promastigotes and amastigotes of Leishmania donovani. Int. J. Parasitol. 2009; 39:635–644. PubMed PMC

Read L.K., Wilson K.D., Myler P.J., Stuart K.. Editing of Trypanosoma brucei maxicircle CR5 mRNA generates variable carboxy terminal predicted protein sequences. Nucleic Acids Res. 1994; 22:1489–1495. PubMed PMC

Maslov D.A. Complete set of mitochondrial pan-edited mRNAs in Leishmania mexicana amazonensis LV78. Mol. Biochem. Parasitol. 2010; 173:107–114. PubMed PMC

Westenberger S.J., Cerqueira G.C., El-Sayed N.M., Zingales B., Campbell D.A., Sturm N.R.. Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region. BMC Genomics. 2006; 7:60. PubMed PMC

Thomas S., Martinez L.L., Westenberger S.J., Sturm N.R.. A population study of the minicircles in Trypanosoma cruzi: predicting guide RNAs in the absence of empirical RNA editing. BMC Genomics. 2007; 8:133. PubMed PMC

Greif G., Rodriguez M., Reyna-Bello A., Robello C., Alvarez-Valin F.. Kinetoplast adaptations in American strains from Trypanosoma vivax. Mutat. Res. 2015; 773:69–82. PubMed

Etheridge R.D., Aphasizheva I., Gershon P.D., Aphasizhev R.. 3′ adenylation determines mRNA abundance and monitors completion of RNA editing in T. brucei mitochondria. EMBO J. 2008; 27:1596–1608. PubMed PMC

Flegontova O., Flegontov P., Malviya S., Audic S., Wincker P., de Vargas C., Bowler C., Lukeš J., Horák A.. Extreme diversity of diplonemid eukaryotes in the ocean. Curr. Biol. 2016; 26:3060–3065. PubMed

Gawryluk R.M., Del Campo J., Okamoto N., Strassert J.F., Lukeš J., Richards T.A., Worden A.Z., Santoro A.E., Keeling P.J.. Morphological identification and single-cell genomics of marine diplonemids. Curr. Biol. 2016; 26:3053–3059. PubMed

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. PubMed

Lukeš J., Skalický T., Týč J., Votýpka J., Yurchenko V.. Evolution of parasitism in kinetoplastid flagellates. Mol. Biochem. Parasitol. 2014; 195:115–122. PubMed

Rodrigues J.C., Godinho J.L., de Souza W.. Biology of human pathogenic trypanosomatids: epidemiology, lifecycle and ultrastructure. Subcell. Biochem. 2014; 74:1–42. PubMed

Parsons M., Ramasamy G., Vasconcelos E.J., Jensen B.C., Myler P.J.. Advancing Trypanosoma brucei genome annotation through ribosome profiling and spliced leader mapping. Mol. Biochem. Parasitol. 2015; 202:1–10. PubMed PMC

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