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.
Penschow
Jonelle L
JL
Department of Microbiology and Immunology, 138 Farber Hall, SUNY Buffalo School of Medicine, Buffalo, NY 14214, USA.
Sleve
Daniel A
DA
Ryan
Christopher M
CM
Read
Laurie K
LK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D045303:Cytochromes b / Q000235:genetics / Q000378:metabolism; D016054:DNA, Protozoan / Q000235:genetics; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D017393:RNA Editing; D034622:RNA Interference; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D012313:RNA; D045303:Cytochromes b; D005095:Exoribonucleases