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. In the mitochondria of the protozoan parasite Trypanosoma brucei, posttranscriptional mechanisms of gene regulation are of major importance. Transcription of both the maxicircle (30, 48) and minicircle (22) genomes is polycistronic. This suggests that regulation of processing and stability of rRNA-, mRNA-, and guide RNA (gRNA)-containing transcripts are likely to be critical steps in gene expression. For example, both endonuclease cleavage and exonuclease trimming are presumably required for the production of mature RNAs from polycistronic precursors. Processing is a particularly critical step in the formation of those mRNAs whose genes overlap such that it is impossible to produce two mature monocistronic RNAs from the same precursor molecule (30, 48). Moreover, this overlapping gene arrangement coupled with polycistronic transcription indicates that a system must exist for the rapid degradation of nonfunctional by-products of processing reactions. The majority of mitochondrial RNAs in T. brucei also require an extensive RNA editing process involving uridine insertion and deletion to form translatable RNAs (55, 56). The steady-state RNA pool contains large amounts of improperly edited RNAs (12, 29). These RNAs may be intermediates destined to become properly edited or they may represent aberrantly processed RNAs that need to be removed. The latter scenario would require the presence of an RNA surveillance system to identify and degrade improperly edited RNAs. In addition to