Fundamental Aspects of Kinetoplastid Mitochondrial RNA Stability

动质体线粒体 RNA 稳定性的基本方面

• 批准号: 8201858
• 负责人: Sara Lana Zimmer
• 金额: $ 5.13万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-05 至 2014-08-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8201858
• 关键词: 5' -exoribonuclease; Address; Attenuated; Biological Assay; Biological Models; Biology; Cells; Characteristics; Chromatography; Code; Dactinomycin; Data; Development; Disease; Drug Delivery Systems; Elements; Enzymes; Exoribonucleases; Experimental Models; Future; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; Homidium Bromide; Homologous Gene; Human; Insecta; Leishmania; Length; Life Cycle Stages; Measurement; Measures; Messenger RNA; Military Personnel; Mitochondria; Mitochondrial Proteins; Mitochondrial RNA; Morbidity - disease rate; Nucleotides; Oligonucleotides; Organism; Parasites; Pharmaceutical Preparations; Play; Population; Post-Transcriptional Regulation; Poverty; Prevalence; Process; Proteins; RNA; RNA Decay; RNA Degradation; RNA Editing; RNA Interference; RNA Sequences; RNA Stability; RNA, ribosomal, 12S; Reading; Regression Analysis; Regulation; Relative (related person); Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Sequence Analysis; Staging; System; Tail; Time; Transcript; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; cis acting element; effective therapy; in vivo; insight; mRNA Decay; meetings; mitochondrial genome; mitochondrial messenger RNA; mortality; new technology; novel; pathogen; success; treatment strategy

DESCRIPTION (provided by applicant): Diseases caused by insect-borne kinetoplastid parasites are marked by their prevalence in poverty-stricken populations and their lack of safe and effective treatment strategies. An understanding of the unusual processes of gene regulation in kinetoplastids, best studied in Trypanosoma brucei, may uncover superior drug targets for these pathogens. Evidence suggests that turnover of the 18 mitochondrial mRNAs plays a significant role in gene regulation. Attempts to identify and describe the regulation of cis elements recognized by exoribonucleases have met with limited success in T. brucei mitochondria, partly because mitochondrial homologues of known exoribonucleases do not comprise the primary RNA turnover machinery. Non-encoded 3' nucleotide tails impact RNA stability in all cellular compartments, and the 3' tails observed in trypanosome mitochondria may be cis-acting stability factors as well. Limited data obtained to date suggest that 3' tails of T. brucei mitochondrial mRNAs are strikingly varied among transcripts but are consistent in composition and length for a particular transcript. Our hypothesis is that the length, nucleotide composition, and/or the complexity of 3' non-encoded RNA tails comprise a "stability code" that is read by 3'-5' exoribonucleases. Furthermore, these 3' extensions may be the reason for the varied mitochondrial transcript abundances observed between the insect and mammalian life cycle stages of the parasite. In Aim 1, we will for the first time directly determine relative stabilities of a selected group of mitochondrial transcripts within and between life cycle stages by attenuating transcription and measuring RNA decay rates over time utilizing qRT-PCR. Aim 2 undertakes the fundamental characterization of 3' non-encoded tails of the same mitochondrial RNAs by sequencing 20-40 circular RT-PCR products for each transcript, again in two life cycle stages. Tail characteristics will be compared with the stabilities obtained in Aim 1 in paired and regression analyses to determine specifically the relationship between 3' tails and transcript stability within and between life cycle stages. In parallel with the above approaches, our final aim is to identify the primary mitochondrial exoribonuclease responsible for a robust decay activity observed in mitochondrial protein extracts by purifying this protein through sequential chromatography steps, determining its identity by LC-MS/MS, and showing its impact on RNA turnover in vivo by examining mitochondrial RNA abundance in cells depleted for this enzyme. The long-term goal of this project is to understand the role of RNA stability within the larger scope of post-transcriptional regulation in trypanosome mitochondrial gene expression. In summary, the research proposed here will provide insight into the mechanisms of mitochondrial RNA turnover in the medically and economically relevant kinetoplastid parasites. These studies may uncover unique aspects of kinetoplastid biology that can be exploited in the future for development of new chemotherapeutics. PUBLIC HEALTH RELEVANCE: Kinetoplastid parasites are a group of important human pathogens including Trypanosoma cruzi, T. brucei, and Leishmania spp., which collectively cause high morbidity and mortality in tropical and subtropical regions of the world, and also threaten military and southern border U.S. populations. Kinetoplastid RNA stability is hypothesized to play an enhanced role in gene regulation, and thus RNA stability regulation has been identified as a potential and urgently needed new drug target. Here, we propose the use of T. brucei as an experimental model system to study kinetoplastid mitochondrial RNA stability.

描述（由申请人提供）：由虫媒动质体寄生虫引起的疾病的特点是在贫困人口中流行，缺乏安全有效的治疗策略。对动质体中基因调控的不寻常过程的理解，最好是在布氏锥虫中进行研究，可能会发现这些病原体的上级药物靶点。有证据表明，18个线粒体mRNA的周转在基因调控中起着重要作用。试图鉴定和描述由核糖核酸外切酶识别的顺式元件的调节在T.这是因为已知核糖核酸外切酶的线粒体同源物不包含主要的RNA周转机制。非编码的3'核苷酸尾影响所有细胞区室中的RNA稳定性，并且在锥虫线粒体中观察到的3'尾也可能是顺式作用稳定因子。目前获得的有限数据表明，T.布氏杆菌线粒体mRNA在转录物中显著变化，但对于特定转录物在组成和长度上是一致的。我们的假设是3'非编码RNA尾的长度、核苷酸组成和/或复杂性包含由3'-5 '核糖核酸外切酶读取的“稳定性密码”。此外，这些3'延伸可能是在寄生虫的昆虫和哺乳动物生命周期阶段之间观察到的不同线粒体转录本丰度的原因。在目标1中，我们将首次通过衰减转录并利用qRT-PCR测量RNA随时间的衰减率来直接确定生命周期阶段内和之间的选定组线粒体转录物的相对稳定性。目的2通过对每个转录物的20-40个环状RT-PCR产物进行测序，再次在两个生命周期阶段中对相同线粒体RNA的3'非编码尾进行基本表征。在配对和回归分析中将尾特征与目标1中获得的稳定性进行比较，以具体确定3'尾与生命周期阶段内和之间的转录物稳定性之间的关系。与上述方法平行，我们的最终目的是鉴定负责线粒体蛋白提取物中观察到的稳健衰变活性的主要线粒体核糖核酸外切酶，通过连续色谱步骤纯化该蛋白，通过LC-MS/MS确定其身份，并通过检查耗尽该酶的细胞中的线粒体RNA丰度来显示其对体内RNA周转的影响。该项目的长期目标是了解RNA稳定性在锥虫线粒体基因表达转录后调节的更大范围内的作用。总之，这里提出的研究将提供深入了解线粒体RNA周转的机制，在医学和经济相关的动质体寄生虫。这些研究可能揭示动质体生物学的独特方面，可在未来开发新的化疗药物。 公共卫生相关性：动体寄生虫是一组重要的人类病原体，包括克氏锥虫、T。布氏杆菌和利什曼原虫属，这些疾病共同导致世界热带和亚热带地区的高发病率和死亡率，并且还威胁到美国军队和南部边境的人口。动质体RNA稳定性被认为在基因调控中起着重要作用，因此RNA稳定性调控已被确定为一个潜在的和迫切需要的新的药物靶点。在这里，我们建议使用T。brucei作为实验模型系统研究动质体线粒体RNA的稳定性。