Kinetoplast (k) DNA, a specialized form of mitochondrial DNA in trypanosomes, is composed of catenated maxicircles and minicircles, and is essential for the survival of these parasitic protists. In this study, we provide a detailed description of the near-complete minicircle complement of Trypanosoma musculi, a globally distributed, mouse-specific haemoflagellate. We have identified 39 minicircle classes, which can be grouped via their structural organization into five distinct categories. Using RNA-seq, we have reconstructed mitochondrial transcripts of ATP6, RPS12, CYTB, COX2 and COX3, all of which undergo canonical editing that renders them translatable, as well as abnormally edited transcripts of four respiratory Complex I subunits. Additionally, through a combined analysis of the kDNA minicircles, edited mRNAs and small RNAs, we identified 108 guide (g)RNA genes and further categorized their conservation in comparison with Trypanosoma lewisi, an opportunistic human parasite. Moreover, we demonstrate a small respiratory Complex I in T. musculi, highlighting the functional consequences of missing gRNAs. Finally, we propose a model for the evolutionary maintenance of gRNAs, contributing to our understanding of kDNA dynamics in trypanosomes and other kinetoplastid flagellates.

Ecosystems and Environment Research Centre and Biomedical Research Centre School of Science Engineering and Environment University of Salford Salford UK

Faculty of Science University of South Bohemia České Budějovice Czech Republic

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

MOE Key Laboratory of Gene Function and Regulation State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Aquatic Economic Animals School of Life Sciences Sun Yat Sen University Guangzhou P R China

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