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: 8490509
• 负责人: Sara Lana Zimmer
• 金额: $ 5.39万
• 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/8490509
• 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.

描述（由申请人提供）：由昆虫传播的动质寄生虫引起的疾病以贫困人口的流行和缺乏安全有效的治疗策略为特征。在Brucei锥虫瘤中最好的研究，对动作质体中基因调节的异常过程的理解可能会发现这些病原体的较高药物靶标。有证据表明，18个线粒体mRNA的营业额在基因调节中起着重要作用。试图识别和描述通过驱虫核酸酶识别的顺式元件的调节，在布鲁氏菌线粒体中取得了有限的成功，部分是因为已知的驱虫核酸酶的线粒体同源物不包含主要RNA转换机械。未编码的3'核苷酸尾巴会影响所有细胞室的RNA稳定性，而在锥虫线粒体中观察到的3'尾巴也可能是顺式作用稳定性因子。迄今为止获得的有限数据表明，在转录本之间，布鲁氏猪笼草线粒体mRNA的3'尾巴差异很大，但对于特定的转录本的组成和长度是一致的。我们的假设是，长度，核苷酸组成和/或3'非编码的RNA尾巴的复杂性包括一个“稳定性代码”，由3'-5'Exoribonibonecleases读取。此外，这3'延伸可能是寄生虫的昆虫和哺乳动物生命周期阶段之间观察到的线粒体转录物丰度的原因。在AIM 1中，我们将首次直接确定所选线粒体转录本和生命周期阶段之间的相对稳定性，通过使用QRT-PCR来衰减转录和测量RNA衰减速率，利用QRT-PCR。 AIM 2通过在两个生命周期阶段再次对每个转录物进行20-40个圆形RT-PCR产物测序，对相同线粒体RNA的3'非编码尾部进行基本表征。将尾部特征与在配对和回归分析中在AIM 1中获得的稳定性进行比较，以确定3'尾巴与生命周期阶段之间和转录本稳定性之间的关系。与上述方法并联，我们的最终目的是确定主要的线粒体尖齿核酸酶通过在线粒体蛋白提取物中观察到的可靠衰变活性来确定通过顺序色谱步骤纯化该蛋白质的稳健衰变活性，从而通过LC-MS/MS确定其身份，并通过LC-MS/MS确定其对RNA的影响，并显示出对RNA的影响，并显示了RNA的影响。酶。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中较大的转录后调节范围中的作用。总而言之，此处提出的研究将洞悉医学和经济相关的动力质体寄生虫中线粒体RNA的机制。这些研究可能会揭示动作塑料生物学的独特方面，这些方面可能会在未来开发新的化学治疗剂。