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Eukaryotic Cell, October 2004, p. 1206-1216, Vol. 3, No. 5
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.5.1206-1216.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

TbDSS-1, an Essential Trypanosoma brucei Exoribonuclease Homolog That Has Pleiotropic Effects on Mitochondrial RNA Metabolism

Jonelle L. Penschow,{dagger} Daniel A. Sleve,{dagger} Christopher M. Ryan, and Laurie K. Read*

Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, SUNY Buffalo School of Medicine, Buffalo, New York

Received 13 April 2004/ Accepted 21 June 2004

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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, 138 Farber Hall, SUNY Buffalo School of Medicine, Buffalo, NY 14214. Phone: (716) 829-3307. Fax: (716) 829-2158. E-mail: lread{at}acsu.buffalo.edu.

{dagger} J.L.P. and D.A.S. contributed equally to this work.

Eukaryotic Cell, October 2004, p. 1206-1216, Vol. 3, No. 5
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.5.1206-1216.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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