Cis- and trans-acting factors in mitochondrial RNA decay

描述（由申请人提供）：在布氏锥虫线粒体中，RNA以多顺反子方式合成。然而，成熟的单顺反子RNA的水平在生命周期阶段之间变化很大。这表明，稳态RNA丰度，从而基因表达，是由转录后过程控制，这些过程是发育调节。我们的假设是，RNA衰变途径是该系统中基因调控的关键决定因素。我们以前的研究确定了定义锥虫基因调控新途径的顺式和反式作用因子，并表明未编辑和编辑的线粒体RNA存在不同的衰变途径。在未编辑的mRNA上，poly（A）尾的存在显著刺激RNA周转。与此形成直接对比的是，多聚腺苷酸化使编辑的RNA稳定。此外，将poly（A）尾从去稳定化元件转换为稳定化元件的相同的小编辑元件足以促进缺乏poly（A）尾的编辑RNA与其未编辑的对应物相比的快速衰变。这些数据揭示了两种核糖核酸外切酶活性的存在，我们已经表明它们与线粒体膜外周相关：（i）快速降解聚（A+）未编辑RNA的活性和（ii）特异性降解缺乏聚（A）尾的编辑RNA的活性。在T.布氏杆菌基因组，其中一个被证明是必不可少的最佳增长。参与线粒体RNA周转的第三种反式作用酶是RET 1末端尿苷酰转移酶（TUTase）。在organello数据中，RET 1可能通过修饰它们的3'尾序列促进poly（A+）RNA的子集的周转。该项目的长期目标是了解RNA周转在锥虫线粒体基因表达中的作用，并阐明调节这些事件的潜在生化机制。具体目的是：1）定义调节编辑RNA稳定性的顺式作用元件。我们将确定通过聚腺苷酸化稳定的编辑RNA的范围，并精确定义顺式作用稳定序列。我们将使用RNA结构测定和RNA-蛋白质相互作用分析来解决编辑顺式元件稳定多聚腺苷酸化RNA的机制。2)鉴定和表征参与线粒体RNA周转的核糖核酸外切酶。我们将通过生物化学方法分离poly（A）RNA选择性核糖核酸外切酶，并使用RNAi分析其在线粒体RNA代谢中的作用。使用双RNAi策略，我们将鉴定快速降解非腺苷酸化编辑RNA的核糖核酸外切酶。在此，候选核糖核酸外切酶将在工程化以产生增加水平的聚（A-）编辑的RNA的细胞中被消融。3)确定RET 1 TUTase在mRNA 3'末端形成和稳定性中的作用。通过分析RET 1敲低细胞，我们将鉴定其周转受RET 1体内影响的RNA，并评估其3'端修饰。这些研究将大大增加我们对医学和经济上重要的寄生虫的基因调控的理解，并将为高等生物线粒体基因调控机制提供重要的见解。公共卫生相关性：本项目的长期目标是阐明特定的顺式作用元件和反式作用因子在布氏锥虫线粒体RNA降解中的作用，并了解这些因子调控线粒体基因表达的机制。这些研究将大大增加我们对医学和经济上重要的寄生虫的基因调控的理解，并将为高等生物线粒体基因调控机制提供重要的见解。

DESCRIPTION (provided by applicant): 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.