In Trypanosoma brucei mitochondria, RNAs are synthesized polycistronically. Nevertheless, levels of mature monocistronic RNAs vary dramatically between life cycle stages. This indicates that steady-state RNA abundance, and thus gene expression, is controlled by posttranscriptional processes and these processes are developmentally regulated. Our hypothesis is that RNA decay pathways are critical determinants of gene regulation in this system. Our previous studies identified both cis- and trans-acting factors that define novel pathways of trypanosome gene regulation, and indicate that distinct decay pathways exist for unedited and edited mitochondrial RNAs. On unedited mRNA, the presence of a poly(A) tail significantly stimulates RNA turnover. In direct contrast, polyadenylation stabilizes edited RNAs. Moreover, the same small edited element that switches the poly(A) tail from a destabilizing to a stabilizing element is sufficient to facilitate rapid decay of edited RNA lacking a poly(A) tail compared to its unedited counterpart. These data reveal the presence of two exoribonuclease activities that we have shown are peripherally associated with mitochondrial membranes: (i) an activity that rapidly degrades poly(A+) unedited RNAs and (ii) an activity that specifically degrades edited RNAs that lack a poly(A) tail. Two candidate exoribonucleases were identified in the T. brucei genome, and one was shown to be essential for optimal growth. A third trans-acting enzyme involved in mitochondrial RNA turnover is the RET1 terminal uridylyltransferase (TUTase). In organello data implicate RET1 in facilitating turnover of a subset of poly(A+) RNAs, potentially through modification of their 3'tail sequences. The long-term goal of this project is to understand the roles of RNA turnover in trypanosome mitochondrial gene expression, and to elucidate the underlying biochemical mechanisms that regulate these events.
The Specific Aims are: 1) Define cis-acting elements that regulate edited RNA stability. We will determine the range of edited RNAs that are stabilized by polyadenylation, and precisely define cis-acting stabilization sequences. We will use RNA structure determination and RNA-protein interaction assays to address the mechanism by edited cis-elements stabilize polyadenylated RNAs. 2) Identify and characterize exoribonucleases involved in mitochondrial RNA turnover. We will biochemically isolate the poly(A) RNA selective exoribonuclease and analyze its role in mitochondrial RNA metabolism using RNAi. Using a dual-RNAi strategy, we will identify the exoribonuclease that rapidly degrades non-adenylated edited RNAs. Here, candidate exoribonucleases will be ablated in cells engineered to produce increased levels of poly(A-) edited RNAs. 3) Determine the role of RET1 TUTase in mRNA 3'end formation and stability. Through analysis of RET1 knock-down cells, we will identify RNAs whose turnover is impacted by RET1 in vivo and assess their 3'end modifications. These studies will greatly increase our understanding of gene regulation in a medically and economically important parasite, and will provide important insights in to the mechanisms of mitochondrial gene regulation in higher organisms.

Public Health Relevance

The long term goal of this project is to elucidate the roles of specific cis-acting elements and trans-acting factors in mitochondrial RNA decay in Trypanosoma brucei, and to understand the mechanisms by which these factors regulate mitochondrial gene expression. These studies will greatly increase our understanding of gene regulation in a medically and economically important parasite, and will provide important insights into the mechanisms of mitochondrial gene regulation in higher organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI077520-05
Application #
8260373
Study Section
Special Emphasis Panel (ZRG1-IDM-R (02))
Program Officer
Mcgugan, Glen C
Project Start
2008-05-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2012
Total Cost
$380,376
Indirect Cost
$135,351
Name
State University of New York at Buffalo
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Sakyiama, Joseph; Zimmer, Sara L; Ciganda, Martin et al. (2013) Ribosome biogenesis requires a highly diverged XRN family 5'->3' exoribonuclease for rRNA processing in Trypanosoma brucei. RNA 19:1419-31
Ammerman, Michelle L; Downey, Kurtis M; Hashimi, Hassan et al. (2012) Architecture of the trypanosome RNA editing accessory complex, MRB1. Nucleic Acids Res 40:5637-50
Zimmer, Sara L; McEvoy, Sarah M; Li, Jun et al. (2011) A novel member of the RNase D exoribonuclease family functions in mitochondrial guide RNA metabolism in Trypanosoma brucei. J Biol Chem 286:10329-40
Fisk, John C; Presnyak, Vladimir; Ammerman, Michelle L et al. (2009) Distinct and overlapping functions of MRP1/2 and RBP16 in mitochondrial RNA metabolism. Mol Cell Biol 29:5214-25
Mattiacio, Jonelle L; Read, Laurie K (2009) Evidence for a degradosome-like complex in the mitochondria of Trypanosoma brucei. FEBS Lett 583:2333-8