TbDSS-1 functions in mitochondrial RNA turnover and surveillance in Trypanosoma brucei
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The parasitic protozoan Trypanosoma brucei is the causative agent of African sleeping sickness and nagana in wildlife and livestock. T. brucei is a member of the Order Kinetoplastida, so termed because of the unusual organization of their mitochondrial DNA or kinetoplast DNA (kDNA), which is contained in the single large mitochondrion of the organism. The posttranscriptional mechanisms affecting mitochondrial gene expression, including RNA turnover, are of major importance because transcriptional control is relatively simple. In this work, proteins involved in RNA turnover and processing in T. brucei mitochondria were investigated. Here, a T. brucei homolog of the yeast mitochondrial degradosome component DSS-1, was investigated for a role in RNA turnover and surveillance. To begin to assess the function of TbDSS-1 in vivo , we constructed an RNAi cell line to selectively down-regulate TbDSS-1 mRNA levels in procyclic-form T. brucei . Targeted depletion of TbDSS-1 results in a decrease in the levels of various RNA species, including never edited, unedited and edited mRNAs as well as guide RNAs. Using this cell line, we also investigated the role of TbDSS-1 in RNA processing and surveillance by analyzing 12S rRNA processing intermediates. Multiple RNA fragments corresponding to leader sequence upstream of the 5' extremity of 12S rRNA accumulate in TbDSS-1 depleted cells. In addition, 12S rRNAs with mature 3' ends and unprocessed 5' ends also accumulate in TbDSS-1 depleted cells, suggesting that these RNAs represent non-functional 12S processing intermediates that are normally removed from the system by a RNA surveillance pathway involving TbDSS-1. TbDSS-1 degrades RNA originating upstream of the first gene on the minor strand of the mitochondrial maxicircle suggesting TbDSS-1 degrades RNAs generated from other regions of the genome. The results from these experiments suggest that TbDSS-1 plays multiple roles in RNA degradation in T. brucei mitochondria and analysis of accumulating 12S rRNA precursor transcripts suggests a major pathway for 12S rRNA processing. Finally, we describe a protein that shares homology with yeast SUV3 RNA helicase, from T. brucei , which we term TbSUV3. Biochemical fractionation of TbSUV3-PTP tagged cell line indicates TbSUV3 is mitochondrially localized and is present in a high-molecular weight complex that co-sediments with TbDSS-1. Furthermore, we demonstrate that TbDSS-1 associates with TbSUV3 in the first report of a mitochondrial degradosome-like complex in any organism other than yeast.