Cis- and trans-acting factors in mitochondrial RNA decay

描述(申请人提供)：在布氏锥虫线粒体中，RNA是以多顺反子方式合成的。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，稳定状态的RNA丰度，从而基因表达，是由转录后过程控制的，这些过程是发育调节的。我们的假设是，在这个系统中，RNA衰变途径是基因调控的关键决定因素。我们以前的研究发现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。在未经编辑的mRNA上，Poly(A)尾巴的存在显著刺激了RNA的周转。与之形成直接对比的是，多聚腺苷可以稳定编辑过的RNA。此外，与未编辑的对应元件相比，将聚(A)尾部从不稳定元件切换到稳定元件的相同的小编辑元件足以促进缺少聚(A)尾部的编辑RNA的快速衰变。这些数据揭示了两种与线粒体膜外围相关的外切核糖核酸酶活性的存在：(I)快速降解聚(A+)未编辑RNA的活性和(Ii)特定降解缺少聚(A)尾巴的编辑RNA的活性。在布氏锥虫基因组中鉴定出两种候选外切核糖核酸酶，其中一种被证明是最适生长所必需的。第三种参与线粒体RNA周转的反式作用酶是RET1末端尿苷酰转移酶(TUTase)。在细胞器数据中，RET1涉及促进聚(A+)RNA子集的周转，可能是通过修改它们的3‘尾部序列。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。其具体目的是：1)定义调节编辑的RNA稳定性的顺式作用元件。我们将确定被多聚腺苷化稳定的编辑RNA的范围，并精确地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研究编辑的顺式元件稳定多腺化RNA的机制。2)鉴定与线粒体RNA转换有关的外切核糖核酸酶。我们将生化分离Poly(A)RNA选择性外切核酸酶，并利用RNAi分析其在线粒体RNA代谢中的作用。使用双RNAi策略，我们将识别快速降解非腺苷化编辑RNA的外切核糖核酸酶。在这里，候选外切核糖核酸酶将在工程细胞中被去除，以产生更高水平的聚(A-)编辑的RNA。3)确定RET1 TUTase在mRNA3‘末端形成和稳定性中的作用。通过对RET1敲除细胞的分析，我们将确定其在体内的周转受到RET1影响的RNA，并评估其3‘端的修饰。这些研究将极大地增加我们对这种医学和经济上重要的寄生虫基因调控的理解，并将对高等生物中线粒体基因调控的机制提供重要的见解。公共卫生相关性：该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鲁氏锥虫线粒体RNA衰退中的作用，并了解这些因素调节线粒体基因表达的机制。这些研究将极大地增加我们对这种医学和经济上重要的寄生虫的基因调控的了解，并将为高等生物中线粒体基因调控的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.