Cis- and trans-acting factors in mitochondrial RNA decay

线粒体 RNA 衰减中的顺式和反式作用因子

• 批准号: 7617872
• 负责人: Laurie K. Read
• 金额: $ 38.81万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7617872
• 关键词: Address; Affect; Be++ element; Beryllium; Biochemical; Biochemical Genetics; Biological Assay; Cell Line; Cells; Cis-Acting Sequence; Critical Pathways; Data; Digestion; Down-Regulation; Electrophoretic Mobility Shift Assay; Elements; Enzymes; Event; Evolution; Exoribonucleases; Gene Expression; Gene Expression Regulation; Genome; Goals; Growth; In Vitro; Length; Life Cycle Stages; Mediating; Messenger RNA; Metabolism; Mitochondria; Mitochondrial RNA; Modeling; Modification; Northern Blotting; Organism; Parasites; Pathway interactions; Poly A; Poly(A) Tail; Poly(A)+ RNA; Polyadenylation; Polynucleotide Adenylyltransferase; Population; Positioning Attribute; Process; RNA; RNA Decay; RNA Editing; RNA Interference; RNA Stability; RNA analysis; RNA-Binding Proteins; RNA-Protein Interaction; Race; Reverse Transcriptase Polymerase Chain Reaction; Ribonuclease H; Ribonucleases; Role; Sequence Analysis; Structure; System; Tail; Trans-Activators; Transcriptional Regulation; Trypanosoma; Trypanosoma brucei brucei; UTP-RNA uridylyltransferase; Uridine; cellular engineering; cis acting element; in vivo; insight; knock-down; mRNA Decay; mitochondrial membrane; mitochondrial messenger RNA; novel; public health relevance; research study

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.

描述（由申请人提供）：在布氏锥虫线粒体中，RNA 是多顺反子合成的。然而，成熟的单顺反子 RNA 的水平在不同的生命周期阶段差异很大。这表明稳态 RNA 丰度以及基因表达受转录后过程控制，并且这些过程受到发育调节。我们的假设是 RNA 衰减途径是该系统中基因调控的关键决定因素。我们之前的研究确定了顺式和反式作用因子，它们定义了锥虫基因调控的新途径，并表明未编辑和编辑的线粒体 RNA 存在不同的衰减途径。在未经编辑的 mRNA 上，poly(A) 尾的存在会显着刺激 RNA 周转。与此相反，聚腺苷酸化可以稳定编辑后的 ​​RNA。此外，与未编辑的对应物相比，将poly(A)尾从不稳定元件切换为稳定元件的相同的小编辑元件足以促进缺乏poly(A)尾的编辑RNA的快速衰减。这些数据揭示了两种外核糖核酸酶活性的存在，我们已经证明它们与线粒体膜外周相关：（i）一种快速降解聚（A+）未编辑RNA的活性和（ii）一种特异性降解缺乏聚（A+）的编辑RNA的活性A）尾巴。在布氏锥虫基因组中鉴定出两种候选核糖核酸外切酶，其中一种被证明对于最佳生长至关重要。参与线粒体 RNA 周转的第三种反式作用酶是 RET1 末端尿苷酰转移酶 (TUTase)。在细胞器数据中，RET1 可能通过修饰其 3' 尾序列来促进 Poly(A+) RNA 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。具体目标是： 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-蛋白质相互作用测定来解决通过编辑顺式元件稳定聚腺苷酸化 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。我们将通过生化方法分离出 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，我们将鉴定可快速降解非腺苷酸化编辑 RNA 的核糖核酸外切酶。在这里，候选核糖核酸外切酶将在经过改造的细胞中被消除，以产生更高水平的多聚腺苷酸编辑RNA。 3)确定RET1 TUTase在mRNA 3'末端形成和稳定性中的作用。通过对 RET1 敲低细胞的分析，我们将鉴定其周转率在体内受 RET1 影响的 RNA，并评估其 3' 末端修饰。这些研究将极大地增加我们对具有医学和经济重要性的寄生虫的基因调控的理解，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。公共健康相关性：该项目的长期目标是阐明特定顺式作用元件和反式作用因子在布氏锥虫线粒体 RNA 衰变中的作用，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医学和经济上重要的寄生虫基因调控的理解，并将为高等生物线粒体基因调控机制提供重要见解。