Penschow Jonelle L, Sleve Daniel A, Ryan Christopher M, Read Laurie K
Department of Microbiology and Immunology, 138 Farber Hall, SUNY Buffalo School of Medicine, Buffalo, NY 14214, USA.
Eukaryot Cell. 2004 Oct;3(5):1206-16. doi: 10.1128/EC.3.5.1206-1216.2004.
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.
锥虫线粒体基因表达主要在RNA加工和RNA稳定性水平受到调控。这种调控无疑涉及众多核糖核酸酶。在此，我们鉴定了酵母DSS - 1线粒体外切核糖核酸酶在布氏锥虫中的同源物，我们将其命名为TbDSS - 1。生化分级分离表明，TbDSS - 1定位于线粒体，这与其N端序列预测的结果一致。与酵母同源物不同，TbDSS - 1似乎不与线粒体核糖体相关。通过RNA干扰在布氏锥虫前循环形式中靶向下调TbDSS - 1会导致严重的生长缺陷。此外，TbDSS - 1的缺失导致从未编辑的细胞色素氧化酶亚基I（COI）mRNA以及未编辑和编辑的COIII mRNA水平降低，表明该酶在控制线粒体RNA丰度中发挥作用。我们还观察到编辑后的脱辅基细胞色素b（CYb）mRNA水平显著降低，未编辑的CYb mRNA相应增加，这表明TbDSS - 1直接或间接在RNA编辑控制中发挥作用。TbDSS - 1缺失后，gCYb[560]和gA6[149]引导RNA的丰度均降低，尽管gCYb[560]的降低更为显著。gCYb水平的显著降低可能是观察到的CYb RNA编辑减少的原因。对线粒体RNA编辑和稳定性因子的蛋白质免疫印迹分析表明，在TbDSS - 1敲低中观察到的RNA水平扰动并非由对其他线粒体蛋白质的次级效应引起。总之，这些数据表明TbDSS - 1是一种在控制线粒体RNA稳定性和RNA编辑中发挥作用的必需蛋白质。
