Fundamental Aspects of Kinetoplastid Mitochondrial RNA Stability

描述(申请人提供)：由昆虫传播的动体寄生虫引起的疾病的特点是它们在贫困人口中普遍存在，并且缺乏安全和有效的治疗策略。对布氏锥虫最好的研究是对动质体内不寻常的基因调控过程的理解，可能会发现针对这些病原体的更好的药物靶点。有证据表明，18个线粒体mRNAs的周转在基因调控中起着重要作用。试图确定和描述外切核酸酶识别的顺式元件的调控在布鲁氏锥虫线粒体中取得的成功有限，部分原因是已知的外切核酸酶的线粒体同源物不包括主要的RNA转换机制。未编码的3‘核苷酸尾巴影响所有细胞间的RNA稳定性，在锥虫线粒体中观察到的3’尾巴也可能是顺式作用的稳定因子。到目前为止获得的有限数据表明，布氏毛滴虫线粒体mRNAs的3‘末端在不同的转录本之间存在显著差异，但在特定转录本的组成和长度上是一致的。我们的假设是，3‘非编码RNA尾巴的长度、核苷酸组成和/或复杂性组成了一个由3’-5‘外切核酸酶读取的“稳定密码”。此外，这些3‘端延伸可能是寄生虫在昆虫和哺乳动物生活史阶段观察到不同线粒体转录丰度的原因。在目标1中，我们将首次通过减弱转录并利用qRT-PCR测量RNA随时间的衰减率来直接确定选定的一组线粒体转录本在生命周期阶段内和之间的相对稳定性。目的2通过对每个转录本的20-40个环状RT-PCR产物进行测序，同样是在两个生命周期阶段，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，尾部特征将与目标1中获得的稳定性进行比较，以具体确定3‘末端与转录本稳定性在生命周期阶段内和之间的关系。与上述方法并行，我们的最终目标是通过顺序层析步骤纯化线粒体蛋白质提取物中观察到的强劲衰退活性的主要线粒体外切核酸酶，通过LC-MS/MS确定其身份，并通过检测耗尽该酶的细胞中线粒体RNA的丰度来显示其对体内RNA周转的影响。该项目的长期目标是在更大的转录后调控范围内了解RNA稳定性在锥虫线粒体基因表达中的作用。综上所述，本文提出的研究将为医学上和经济上相关的动体寄生虫线粒体RNA周转的机制提供深入的见解。这些研究可能会揭示动质体生物学的独特方面，这些方面可以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、布氏锥虫和利什曼原虫，它们在世界热带和亚热带地区共同造成高发病率和死亡率，并威胁到美国军事和南部边境的人口。动态体RNA的稳定性被认为在基因调控中发挥着增强的作用，因此RNA稳定性调节被认为是一个潜在的和迫切需要的新药靶点。在这里，我们建议使用布氏锥虫作为实验模型系统来研究动质粒线粒体RNA的稳定性。

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.