RAPs-mediated post-transcriptional control in Apicomplexan parasites

项目摘要/摘要 在疟疾寄生虫中，基因调控严重依赖于作用于 转录后水平和翻译水平。更重要的是，疟原虫拥有许多 与人类宿主相比，这两种途径都具有非典型特征。这些不同寻常的 因此，这些特征延长了我们对这种致命寄生虫的治疗窗口，但这些 在分子水平上，人们对这些特征的探索很少。这个项目的主要目标是 鉴定新发现的apicomplexan特异性RNA结合蛋白(RAPs)在 寄生虫的发展和毒力，并验证其作为新的药物靶标的潜力。 疟原虫在转录后调节部分His基因表达的事实 Level的灵感来自许多早期研究，这些研究表明全球蛋白质组图谱通常 落后于转录档案。我们最新出版的大规模生物信息学 当我们确定至少有18%的编码基因被预测时，加强了这个猜想 是一种潜在的RNA结合域蛋白(RBP)。此外，这些寄生虫中的许多 实验表明，限制性商业惯例可与mRNAs进行原位相互作用。在所有真核生物中， 限制性商业惯例在包括剪接在内的多个水平上调节mRNA的加工是必不可少的， 运输、信使核糖核酸的稳定性和周转，以及信使核糖核酸的定位和翻译 效率。在人类疟疾寄生虫中，很少有限制性商业惯例被描述出来。我们建议 通过检查寄生虫特异性RNA结合的功能来解决这一知识差距 在Apicomplexans或RAP中丰富的结构域蛋白。更具体地说，我们将使用 最新的基因组学、分子和细胞方法来确定21 确定了RAP在寄生虫发育和生存中的作用。在目标1中，我们将描述 恶性疟原虫RAP的重要性、亚细胞定位和表型 使用新的CRISPR/CAS9方法研究不同发育阶段。在目标2中，我们将定义 跨多种寄生虫的全球RAP相关核糖核蛋白相互作用网络 通过开发高通量测序技术和下拉菜单来实现发展阶段 策略后进行质谱分析。我们互补的方法不仅将 确定有助于寄生虫发展的关键的特定限制性商业惯例，但也发现独特的 真核人类病原体中的转录后网络。通过提供 对疟原虫翻译调控机制的基本见解，该项目将 提高我们设计抗击疟疾和许多其他疾病的新药和新防线的能力 传染病病原体。

Project Summary/Abstract In the malaria parasite, gene regulation is heavily dependent on mechanisms acting at the post- transcriptional and translational levels. More importantly, Plasmodium possesses many atypical characteristics in both pathways when compared to its human host. These unusual features hence extend our therapeutic window against this deadly parasite, yet these characteristics have been poorly explored at the molecular level. The main goal of this project is to characterize the role of newly identified apicomplexan-specific RNA- binding proteins {RAPs) in parasite development and virulence, and to validate their potential as novel drug targets. The fact that Plasmodium parasites modulate part of his gene expression at the post-transcriptional level was inspired by many early studies that demonstrated that the global proteomic profiles often lagged behind the transcriptional profiles. Our newly published large-scale bioinformatics reinforce this conjecture as we determined that at least 18% of all coding-gene are predicted to be potential RNA binding domain-containing protein (RBP). Furthermore, many of these parasite RBPs were experimentally shown to interact in situ with mRNAs. In all eukaryotic organisms, RBPs are essential to regulate mRNA processing at multiple levels including splicing, transport, mRNA stability and turnover, as well as mRNA localization and translational efficiency. In the human malaria parasite, very few RBPs have been characterized. We propose to address this knowledge gap by examining the function of the parasite specific RNA-binding domain proteins abundant in Apicomplexans, or RAPs. More specifically, we will use state-of-the-art genomics, molecular, and cellular approaches to determine the role of the 21 identified RAPs in parasite development and survival. In Aim 1, we will characterize the essentiality, subcellular localization and phenotypes of RAPs in P. falciparum across different developmental stage using novel CRISPR/Cas9 approaches. In Aim 2, we will define the global RAP- related ribonucleoprotein interaction network across various parasite developmental stages by developing high-throughput sequencing technologies and pull-down strategies followed by mass spectrometry analysis. Our complementary approaches will not only identify key specific RBPs contributing to parasite development, but also uncover unique post-transcriptional networks in a eukaryotic human pathogen. By providing fundamental insights into mechanisms regulating translation in Plasmodium, this project will improve our ability to design new drugs and novel lines of defense against malaria and many other infectious disease agents.