Kinetoplastids are protists defined by one of the most complex mitochondrial genomes in nature, the kinetoplast. In the sleeping sickness parasite Trypanosoma brucei, the kinetoplast is a chain mail-like network of two types of interlocked DNA molecules: a few dozen ∼23-kb maxicircles (homologs of the mitochondrial genome of other eukaryotes) and thousands of ∼1-kb minicircles. Maxicircles encode components of respiratory chain complexes and the mitoribosome. Several maxicircle-encoded mRNAs undergo extensive post-transcriptional RNA editing via addition and deletion of uridines. The process is mediated by hundreds of species of minicircle-encoded guide RNAs (gRNAs), but the precise number of minicircle classes and gRNA genes was unknown. Here we present the first essentially complete assembly and annotation of the kinetoplast genome of T. brucei. We have identified 391 minicircles, encoding not only ∼930 predicted ‘canonical’ gRNA genes that cover nearly all known editing events (accessible via the web at http://hank.bio.ed.ac.uk), but also ∼370 ‘non-canonical’ gRNA genes of unknown function. Small RNA transcriptome data confirmed expression of the majority of both categories of gRNAs. Finally, we have used our data set to refine definitions for minicircle structure and to explore dynamics of minicircle copy numbers.

No abstract

Mitochondrial DNA of protists of order Kinetoplastida comprises thousands of interlinked circular molecules arranged in a disk-like network. It composes of two types of molecules called maxicircles and minicircles. Minicircles encode so called guide RNA (gRNA) genes that mediate post-transcriptional editing of maxicircle encoded genes, many extensively so along their full length. There are thousands of minicircles per network. They are diverse and vary in copy number. The human sleeping sickness parasite Trypanosoma brucei has one of the most diverse sets of minicircle classes of all studied trypanosomatids with hundreds of different classes, each encoding one to four gRNA genes within cassettes defined by 18 bp imperfect inverted repeats. About a third of cassettes in T. brucei have no identifiable gRNA genes even though their sequence structures are similar to cassettes with identifiable gRNAs. These so called non-canonical gRNA genes have remained a mystery for many years. It is only until recently that the sequences of almost all minicircle classes for some subspecies and isolates of T. brucei have been sequenced and annotated with corresponding verification of gRNA expression by small-RNA transcriptome data. These datasets provide a rich resource for understanding the structure of minicircle classes, cassettes and gRNA genes and their transcription, and for studying the evolution and maintenance of the diverse, and often redundant, classes and genes. Here we provide a statistical description of the functionality, expression, structure and sequence of gRNA genes in a differentiation-competent, laboratory-adapted strain of T. brucei. We obtain a clearer definition of what is a gRNA gene in this species. Our analysis supports the idea that many, if not all, of the cassettes without an identifiable gRNA gene contain decaying remnants of once functional gRNA genes. We also report several new, unexplained discoveries such as the association between cassette position on the minicircle and gene expression and functionality, and the association between a gene's initiation sequence and the position of its anchor sequence.

No abstract