Authors

Discussion

Function of the single mitochondrion of the sleeping sickness parasite Trypanosoma brucei shows striking regulation during the complex life cycle, with oxidative phosphorylation only active in the insect vector, the tsetse fly. Four out of the five mitochondrial respiratory chain protein complexes (complex I, NADH:ubiquinone oxidoreductase; complex III, cytochrome bc₁ complex; complex IV, cytochrome c oxidase; complex V, F₁F_O ATP synthase) are composed of both nuclearly and mitochondrially encoded subunits. Thus, while the slender bloodstream stage of T. brucei requires only two mitochondrially encoded proteins (F₁F_O ATP synthase subunit a and subunit RPS12 of the mitochondrial ribosome), the procyclic insect stage in thought to require at least five.

Trypanosome mitochondrial DNA (the kinetoplast) is organised as a massive network of intercalated ‘maxicircles’ and ‘minicircles’ - the most complex mitochondrial genome known. Maxicircles are the equivalent of mtDNA in other eukaryotes, while minicircles encode a vast number of short guide RNA (gRNA) molecules, typically of 40-60 nt length, that specify extensive post-transcriptional RNA editing of maxicircle-encoded mRNAs. The exact number of different types of minicircles per T. brucei kinetoplast was unknown, but most authors have predicted 100-300 distinct classes. As the procyclic insect stage requires more mitochondrially encoded gene products than the bloodstream stage, it is also expected to depend on a larger number of gRNA species, and therefore a more complex mitochondrial genome, but this has not been formally tested.

Using next-generation sequencing we have now determined the complete mitochondrial genome content of pleomorphic strain T. brucei EATRO 1125 (AnTat 1.1 90:13) and annotated the encoded genes using RNA-seq. We have identified 391 unique minicircles of varying copy numbers, assembled into a network of ~10,000 individual minicircle molecules per cell. Encoded within these minicircles we have identified 1035 distinct species of gRNA that cover virtually all known editing events in this organism (‘canonical’ gRNAs), at considerable redundancy. In addition, we have identified 225 expressed gRNA-like molecules (‘non-canonical’ gRNAs) of unknown function. We also have obtained conclusive evidence that T. brucei loses minicircle classes when grown as bloodstream stage parasite (i.e. independent of oxidative phosphorylation) and that during sexual recombination of the organism in the tsetse fly a new hybrid mtDNA network is generated that is composed of the parental networks in equal parts. Thus, highly unusual in nature, mtDNA inheritance in T. brucei is biparental.