Comparison of the maxicircle (mitochondrial) genomes of Leishmania tarentolae and Trypanosoma brucei at the level of nucleotide sequence

The entire 16.7-kilobase (kb) transcribed region of the Leishmania tarentolae maxicircle was compared to the entire 15-kb transcribed region of the Trypanosoma brucei maxicircle at the nucleotide sequence level by dot matrix analysis and by alignments of individual genes. The L. tarentolae NADH dehydrogenase subunit 1 (ND1) gene was identified in a newly obtained 2.9-kb sequence. All but two regions which flank the cytochrome b gene are highly conserved in both species. One 3.1-kb region in L. tarentolae that contains the cytochrome oxidase subunit III (COIII) gene and several open reading frames corresponds to a 2-kb sequence in T. brucei with limited sequence homology that lacks the COIII gene. Another 0.6-kb region that comprises an unidentified open reading frame (open reading frame 12) in L. tarentolae is substituted by a nonhomologous 0.4-kb open reading frame in T. brucei. A short intergenic region between the ND1 gene and the maxicircle unidentified reading frame 1 gene shows limited sequence homology, and the regions between the ND4 and ND5 genes and between the COI and ND4 genes are not conserved. All of the intergenic regions share G + C richness and a similar pattern of G versus C strand bias. 1.8 kb of the L. tarentolae divergent region (DV) and around 3 kb of the T. brucei DV were also obtained. The T. brucei DV sequences were not homologous to the L. tarentolae DV sequence but were organized in a similar fashion with tandem repeats of varying complexity.     

Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences.

Small guide RNAs (gRNAs) may direct RNA editing in kinetoplastid mitochondria. We have characterized multiple gRNA genes from Trypanosoma brucei (EATRO 164), that can specify up to 30% of the editing of the COIII, ND7, ND8, and A6 mRNAs and we have also found that the non-translated region of edited COIII mRNA of strain (EATRO 164) differs from that of another strain. Several of the gRNAs specify overlapping regions of the same mRNA often specifying sequence beyond that required for an anchor duplex with the next gRNA. Some gRNAs have different sequence but specify identical editing of the same region of mRNA. These data indicate a complex gRNA population and consequent complex pattern of editing in T. brucei.     

TbDSS-1, an essential Trypanosoma brucei exoribonuclease homolog that has pleiotropic effects on mitochondrial RNA metabolism

Mitochondrial gene expression in trypanosomes is controlled primarily at the levels of RNA processing and RNA stability. This regulation undoubtedly involves numerous ribonucleases. Here we characterize the Trypanosoma brucei homolog of the yeast DSS-1 mitochondrial exoribonuclease, which we term TbDSS-1. Biochemical fractionation indicates that TbDSS-1 is mitochondrially localized, as predicted by its N-terminal sequence. In contrast to its yeast homolog, TbDSS-1 does not appear to be associated with mitochondrial ribosomes. Targeted downregulation of TbDSS-1 by RNA interference in procyclic-form T. brucei results in a severe growth defect. In addition, TbDSS-1 depletion leads to a decrease in the levels of never edited cytochrome oxidase subunit I (COI) mRNA and both unedited and edited COIII mRNAs, indicating this enzyme functions in the control of mitochondrial RNA abundance. We also observe a considerable reduction in the level of edited apocytochrome b (CYb) mRNA and a corresponding increase in unedited CYb mRNA, suggesting that TbDSS-1 functions, either directly or indirectly, in the control of RNA editing. The abundance of both gCYb[560] and gA6[149] guide RNAs is reduced upon TbDSS-1 depletion, although the reduction in gCYb[560] is much more dramatic. The significant reduction in gCYb levels could potentially account for the observed decrease in CYb RNA editing. Western blot analyses of mitochondrial RNA editing and stability factors indicate that the perturbations of RNA levels observed in TbDSS-1 knock-downs do not result from secondary effects on other mitochondrial proteins. In all, these data demonstrate that TbDSS-1 is an essential protein that plays a role in mitochondrial RNA stability and RNA editing.     

Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing

We present evidence that addition of the 35 nucleotide spliced leader (SL) to the 5' end of T. brucei mRNAs occurs via trans RNA splicing. A 100 nucleotide fragment of the 135 base SL RNA (100-mer) is revealed by S1 nuclease analysis of total and poly(A)+ RNA. This 100-mer is not detected by Northern hybridization analysis, indicating that it does not exist free in the cell. The 5' end of the 100-mer maps precisely to the conserved splice junction sequence of the SL RNA. Purified debranching enzyme releases this 100-mer RNA as a free, 100 nucleotide species. This indicates that the 100-mer is covalently linked to poly(A)+ RNA by a 2'-5' phosphodiester bond, that the branched intermediate has a discontinuous intron or Y structure (rather than a lariat), which is expected of a trans-spliced mRNA, and that the SL RNA is indeed the donor of the SL sequence to trypanosome mRNAs.