Organization of minicircle cassettes and guide RNA genes in Trypanosoma brucei.

Mitochondrial DNA of protists of order Kinetoplastida comprises thousands of interlinked circular molecules arranged in a network. There are two types of molecules called minicircles and maxicircles. Minicircles encode guide RNA (gRNA) genes whose transcripts mediate post-transcriptional editing of maxicircle encoded genes. Minicircles are diverse. 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 genes mainly within cassettes framed by 18 bp inverted repeats. A third of cassettes have no identifiable gRNA genes even though their sequence structures are similar to cassettes with identifiable genes. Only recently have almost all minicircle classes for some subspecies and isolates of T. brucei been sequenced and annotated with corresponding verification of gRNA expression by small-RNA transcriptome data. These data sets provide a rich resource for understanding the structure of minicircle classes, cassettes and gRNA genes and their transcription. Here, we provide a statistical description of the functionality, expression status, 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. Our analysis supports the idea that many, if not all, cassettes without an identifiable gRNA gene contain decaying remnants of once functional gRNA genes. Finally, we report several new, unexplained discoveries such as the association between cassette position on the minicircle and gene expression and functionality, and the association between gene initiation sequence and anchor position.

Assembly and annotation of the mitochondrial minicircle genome of a differentiation-competent strain of Trypanosoma brucei.

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.