Fundamental Aspects of Kinetoplastid Mitochondrial RNA Stability

描述（由申请人提供）：由昆虫传播的着丝体寄生虫引起的疾病，其特点是在贫困人口中流行，缺乏安全有效的治疗策略。对动胞体中基因调控的不寻常过程的理解，在布鲁氏锥虫中得到了最好的研究，可能会发现这些病原体的优越药物靶点。有证据表明，18种线粒体mrna的周转在基因调控中起着重要作用。鉴定和描述外核糖核酸酶识别的顺式元件调控的尝试在布氏体线粒体中取得了有限的成功，部分原因是已知的外核糖核酸酶的线粒体同源物不包括主要的RNA转换机制。非编码的3‘核苷酸尾部影响所有细胞区室中RNA的稳定性，在锥虫线粒体中观察到的3’尾部也可能是顺式作用的稳定因子。迄今为止获得的有限数据表明，布鲁氏体线粒体mrna的3'尾在转录本中存在显著差异，但在特定转录本的组成和长度上是一致的。我们的假设是，长度、核苷酸组成和/或3‘非编码RNA尾部的复杂性组成了一个由3’-5'外核糖核酸酶读取的“稳定性代码”。此外，这些3'延伸可能是在昆虫和哺乳动物生命周期阶段观察到的不同线粒体转录物丰度的原因。在Aim 1中，我们将首次通过利用qRT-PCR减弱转录和测量RNA衰减率，直接确定一组选定的线粒体转录物在生命周期阶段内和之间的相对稳定性。Aim 2通过对每个转录物进行20-40个环状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，同样在两个生命周期阶段。在配对和回归分析中，将尾部特征与Aim 1中获得的稳定性进行比较，以确定3'尾与生命周期阶段内和之间的转录本稳定性之间的具体关系。与上述方法平行，我们的最终目标是通过顺序色谱步骤纯化线粒体外核糖核酸酶，通过LC-MS/MS确定其身份，并通过检测线粒体RNA丰度来显示其对体内RNA转换的影响，从而确定线粒体外核糖核酸酶对线粒体蛋白质提取物中观察到的强大衰变活性负责。该项目的长期目标是在转录后调控的更大范围内了解RNA稳定性在锥虫线粒体基因表达中的作用。总之，本文提出的研究将为线粒体RNA周转机制在医学和经济相关的着丝体寄生虫中提供见解。这些研究可能揭示着丝体生物学的独特方面，可以在未来开发新的化疗药物。

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.