Abstract
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.
Highlights
Single-cellular flagellates of the order Kinetoplastida include many important parasites of humans and their livestock [1,2] and are characterized by an eponymous structure within their single mitochondrion, the kinetoplast [3,4]
The precise sites of U insertion and deletion are specified by ‘guide RNAs’, short transcripts of typically 40– 60 nt in lengths that, from 5 to 3, are characterized by the following features: (i) a 5 triphosphate, marking them as primary transcripts, (ii) a semi-conserved 5 transcript initiation sequence that has been reported to conform to a 5 -RYAYA consensus [9], (iii) an ‘anchor’ sequence that is crucial for recognising the cognate pre-edited mRNA via Watson– Crick base-pairing, (iv) the ‘information region’, which is complementary to a particular stretch of edited mRNA and (v) a 3 oligo(U) tail that is added post-transcriptionally and that has been suggested to stabilise the gRNA-mRNA interaction [10]
Notes: aNote that editing sites only matched by an anchor sequence are not considered as being covered. bSee below for discussion and definition of the high confidence set. cThe mRNA encoding COX2 is not included here as its single gRNA is present in cis at its 3 end. dCR3 and CR4 were suggested by Duarte and Tomas (2014) to represent subunits ND4L and ND6, respectively, of respiratory complex I. eNumbers for mitochondrial unidentified open reading frame 2 (MURF2) include the maxicircle-encoded gRNA. fThe total numbers given take into account editing events and gRNAs that are shared between the two versions of A6 and ND8. gn/a = not applicable
Summary
Single-cellular flagellates of the order Kinetoplastida include many important parasites of humans and their livestock [1,2] and are characterized by an eponymous structure within their single mitochondrion, the kinetoplast [3,4]. In Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT, or sleeping sickness), kDNA is complex. It consists of two types of molecules: dozens of essentially identical copies of a ∼23kb maxicircle, and 5000–10 000 minicircle molecules, which are ∼1-kb in size and highly diverse. 12 of the protein-coding genes are ‘cryptogenes’––their mRNAs require post-transcriptional RNA editing by insertion and, less frequently, deletion of uridylate (U) residues [7,8]. Nine of these mRNAs require extensive ‘pan-editing’ via the insertion of hundreds and the deletion of dozens of Us per transcript. The precise sites of U insertion and deletion are specified by ‘guide RNAs’ (gRNAs), short transcripts of typically 40– 60 nt in lengths that, from 5 to 3 , are characterized by the following features: (i) a 5 triphosphate, marking them as primary transcripts, (ii) a semi-conserved 5 transcript initiation sequence that has been reported to conform to a 5 -RYAYA consensus [9], (iii) an ‘anchor’ sequence (which can overlap with the initiation sequence) that is crucial for recognising the cognate pre-edited mRNA via Watson– Crick base-pairing, (iv) the ‘information region’, which is complementary to a particular stretch of edited mRNA (but involves non-Watson-Crick G:U base pairs) and (v) a 3 oligo(U) tail that is added post-transcriptionally and that has been suggested to stabilise the gRNA-mRNA interaction [10]
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