Authors

Z Chen¹; L Tavernier²; L Vermeiren²; J Van Den Abbeele²; N Savill¹; A Schnaufer¹; F Van den Broeck²; F Van den Broeck³;  ¹ University of Edinburgh, UK;  ² Institute of Tropical Medicine, Antwerp, Belgium;  ³ Department of Microbiology, Immunology and Transplantation, Rega Institute, Belgium

Discussion

Nick Savill, Achim Schnaufer and Frederik Van den Broeck contributed equally to this work and wish to be regarded as co-senior authors Trypanosoma congolense is a causative agent of African animal trypanosomiasis, also known as Nagana. These kinetoplastid protists adopt different metabolic pathways and physiology in mammalian bloodstream and insect host, partially due to the difference in nutrient availability {Read, 1994 #17}. These pathways involve components of respiratory chain complexes and the mitoribosome that are encoded on the mitochondrial genome {Read, 1993 Aug 25 #1} . The mitochondrial DNA of T. congolense, known as kinetoplast (k) DNA, consists of multiple copies of a 28-kb circular DNA called ‘maxicircle’ and thousands of approximately 1-kb circular DNA molecules called ‘minicircles’. The maxicircle is homologous to the mitochondrial genomes of other eukaryotes, whereas the minicircles encode guide RNAs (gRNAs) that direct essential post-transcriptional editing of the pre-mRNA products from encrypted maxicircle genes by virtue of their complementarity to the fully edited version. Multiprotein complexes called editosomes resolve initial mismatches between pre-mRNA and gRNA by insertion and deletion of uridine residues until complementarity is achieved. The extent of uridine insertion/deletion editing varies between species; in the model Salivarian species Trypanosoma brucei this mechanism edits transcripts of 12 maxicircle protein coding genes, nine of which are edited through their entire lengths. While decades of research have provided a detailed description of kDNA composition and editing events for T. brucei {Cooper, 2019 #21}, data for T. congolense are scant. An in-depth comparison of kDNA structure and expression between these two species is expected to offer valuable insight into the evolution and function of this defining feature of kinetoplastid biology. This project used next-generation sequencing of purified kDNA (Illumina MiSeq platform) and the KOMICS pipeline {Van den Broeck, 2020 Oct 6 #5}to assemble the minicircle genome of the T. congolense reference strain IL3000. Sequences of fully edited mRNAs were obtained by a combination of Illumina (DNBSeq) and PacBio RNA sequencing of bloodstream stage parasites and careful manual curation, considering sequence information from the maxicircle-encoded genes and from published T. brucei edited mRNAs. Minicircle-encoded gRNA genes were identified by alignment to edited sequences and mapping of short RNAs isolated from bloodstream form cells. The endeavour resulted in a nearly complete set of 184 minicircles, encoding 410 canonical gRNA genes. These gRNAs provide extensive coverage for the predicted fully edited mRNA sequences for cytochrome oxidase subunit (COX) 2, C-rich region 3, apocytochrome b, NADH dehydrogenase (ND) subunits 3, 7, 8, and 9, and ribosomal protein S12. Edited sequences for ATP synthase subunit 6, COX3, and maxicircle unidentified reading frame 2 contain considerable gaps in gRNA coverage, indicating the need for further refinement. The analysis also revealed potential alternative editing patterns in RSP12. In total, the canonical gRNAs identified so far explain > 93% of the editing events for the predicted mRNAs. Similar to T. brucei, the vast majority of gRNAs are encoded in ‘cassettes’ defined by inverted repeats. Analysis of nucleotide bias and the short RNA transcriptomes also identified 129 putative non-canonical gRNA genes potentially responsible for alterative editing
events or non-functional transcripts. Our ongoing comparative analysis of the minicircle genomes of T. congolense and T. brucei is identifying interesting similarities and differences between the two organisms, which will be presented at the meeting. . References Cooper, S., E. S. Wadsworth, T. Ochsenreiter, A. Ivens, N. J. Savill and A. Schnaufer (2019). "Assembly and annotation of the mitochondrial minicircle genome of a differentiation-competent strain of Trypanosoma brucei." Nucleic Acids Res 47(21): 11304-11325. Read, L. K., W. R. Fish, A. M. Muthiani and K. Stuart (1993 Aug 25). "Maxicircle DNA and edited mRNA sequences of closely related trypanosome species: implications of kRNA editing for evolution of maxicircle genomes." Nucleic Acids Res 21(17): 4073-4078. Read, L. K., K. A. Stankey, W. R. Fish, A. M. Muthiani and K. Stuart (1994). "Developmental regulation of RNA editing and polyadenylation in four life cycle stages of Trypanosoma congolense." Mol Biochem Parasitol 68(2): 297-306. Van den Broeck, F., N. J. Savill, H. Imamura, M. Sanders, I. Maes, S. Cooper, D. Mateus, A. Jara M, V, , J. Arevalo, A. Llanos-Cuentas, L. Garcia, E. Cupolillo, M. Miles, M. Berriman, A. Schnaufer, J. A. Cotton and J. C. Dujardin (2020 Oct 6). "Ecological divergence and hybridization of Neotropical Leishmania parasites . doi: 10.1073/pnas.1920136117. ." Proc Natl Acad Sci U S A. 117(40): 25159-25168.