Dissecting RNA Regulation During Malaria Parasite Sexual Development

项目概要/摘要： 人类病原体恶性疟原虫是疟疾的病原体， 复杂的生命周期，包括人类宿主和蚊子体内多个组织的发育 vector.在人类感染期间，寄生虫主要每48小时无性复制一次， 从一个到多达24个新的寄生虫在每个周期。然而，在低频率（1-3%），寄生虫 致力于性别分化计划，以产生男性和女性配子体，这是至关重要的 以便传播给蚊子媒介并完成寄生虫的整个生命周期。发展 疟原虫在各种细胞类型中的表达涉及新生mRNA的调节 转录以及影响mRNA稳定性的转录后机制。在大多数 真核系统的转录后调节是由新生mRNA的相互作用介导的 RNA结合蛋白（RNABP）。在这个建议中，我们专注于mRNA的调控 在寄生虫发育的有性阶段的动态，一个阶段，这是相对 直到最近才有这样的研究。通过基因工程改造恶性疟原虫， 使它们能够合成嘧啶前体，我们现在可以给它们喂食修饰的4-硫氧嘧啶（4-TU）， 它们很容易将其整合到新转录的RNA中。这些巯基化的RNA使我们能够 通过捕获和鉴定这些新生的 RNA及其RNABP。我们的核心假设是转录后机制 由寄生虫特异性RNA结合蛋白介导， 特定的基因调节并且是寄生虫传播阶段的重要决定因素 发展这项研究将直接分析实时转录和RNA稳定化， 鉴定在疟原虫有性阶段参与转录后控制的RNABP 恶性疟原虫发育目的1将使用我们的4-TU标记方法来捕获性阶段特异性 通过测量新生转录和mRNA水平， 稳定性始于配子体发生。在目标2中，4-TU标记的RNA将是 与它们的同源RNABP交联，并通过蛋白质组学进行测定，以确定所有性- 阶段RNA结合蛋白和定义RNA识别基序，促进这种相互作用。 此外，我们将使用基于靶向免疫沉淀的方法来定义特定的RNA， 包括PfPuf 2在内的几种有性阶段RNABP的蛋白质相互作用。这项研究将为 为将来使用这些工具和方法进行RNA调控的研究奠定了基础，并将使设计 新的抗疟战略，以防止疟疾传播。 !

Project Summary/Abstract: The human pathogen, Plasmodium falciparum, the causative agent of the disease malaria, has a complex life cycle which includes development in multiple tissues within the human host and mosquito vector. During human infection, the parasite predominantly replicates asexually every 48 hours, going from one to up to 24 new parasites in every cycle. However, at a low frequency (1-3%), the parasite commits to a sexual differentiation program to produce male and female gametocytes that are critical for transmission to the mosquito vector and to complete the full lifecycle of the parasite. Development of the Plasmodium parasite within various cell types involves the regulation of nascent mRNA transcription as well as post-transcriptional mechanisms that impact mRNA stability. In most eukaryotic systems post-transcriptional regulation is mediated by the interaction of nascent mRNAs with specific RNA binding proteins (RNABPs). In this proposal we focus on the regulation of mRNA dynamics during the sexual stage of parasite development, a stage which has been relatively inaccessible to such studies until recently. By genetically engineering P. falciparum parasites to enable them to scavenge pyrimidine precursors, we can now feed them modified 4-thiouracil (4-TU), which they readily incorporate into newly transcribed RNA. These thiolated RNAs allow us to specifically address questions regarding RNA metabolism by capturing and identifying these nascent RNAs as well as their RNABPs. Our central hypothesis is that post-transcriptional mechanisms mediated by parasite specific RNA-binding proteins play a prominent role in gametocyte- specific gene regulation and are essential determinants of the parasite-transmissible stages of development. This study will directly profile real-time transcription and RNA stabilization, and will identify the RNABP involved in post-transcriptional control during the sexual stages of Plasmodium falciparum development. Aim 1 will use our 4-TU labeling method to capture sexual-stage specific mRNA dynamics throughout gametocytogenesis by measuring nascent transcription and mRNA stability beginning with commitment to gametocytogenesis. In Aim 2 4-TU labeled RNAs will be crosslinked to their cognate RNABPs and assayed by proteomics to determine an atlas of all sexual- stage RNA-binding proteins and define the RNA recognition motifs that promote this interaction. Additionally, we will use a targeted immunoprecipitation-based approach to define the specific RNA- protein interactions of several sexual-stage RNABPs including PfPuf2. This study will pave the way for future studies of RNA regulation using these tools and approaches and will enable the design of new antimalarial strategies to combat malaria transmission. !