Dissecting RNA Regulation During Malaria Parasite Sexual Development

解析疟原虫性发育过程中的 RNA 调控

• 批准号: 9375224
• 负责人: Manuel Llinas
• 金额: $ 19.11万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9375224
• 关键词: 4-thiouracil; Address; Affect; Affinity; Amino Acid Sequence; Anopheles Genus; Antimalarials; Atlases; Biological Assay; Biology; Blood; Cessation of life; Child; Complex; Culicidae; DNA-Binding Proteins; Development; Developmental Process; Disease; Drug resistance; Elements; Epitopes; Family; Female; Foundations; Frequencies; Future; Gene Expression Regulation; Genes; Genetic Engineering; Genetic Transcription; Genome; Goals; Hour; Human; Immunoprecipitation; Infection; Insecticide Resistance; Intervention; Investigation; Label; Life; Life Cycle Stages; Malaria; Measures; Mediating; Messenger RNA; Metabolism; Methodology; Methods; Parasites; Phase; Physiologic pulse; Plasmodium; Plasmodium falciparum; Play; Population; Post-Transcriptional Regulation; Pregnant Women; Proteins; Proteomics; Pyrimidine; RNA; RNA Recognition Motif; RNA Stability; RNA-Binding Proteins; RNA-Protein Interaction; Regulation; Regulator Genes; Research; Ribonucleosides; Role; Scanning; Sexual Development; Sexual Maturation; Sexual Transmission; Sporozoites; System; Therapeutic; Time; Tissues; Transcript; Transcription Process; Transcriptional Regulation; Translational Repression; asexual; base; cell type; combat; cost effective; crosslink; design; disease transmission; disorder control; effective therapy; experimental study; falls; feeding; gene repression; global health; killings; mRNA Stability; malaria transmission; male; new therapeutic target; novel; novel strategies; oligo (dT); pathogen; programs; sex; tool; transcriptome; transmission process; vector mosquito

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. !

项目摘要/摘要： 人类病原体恶性疟原虫是疟疾的病原体，具有 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 向量。在人类感染期间，寄生虫主要每 48 小时进行一次无性复制， 每个周期产生 1 到 24 种新寄生虫。然而，在低频（1-3%）下，寄生虫 致力于性别分化计划，以产生至关重要的雄性和雌性配子细胞 用于传播到蚊子媒介并完成寄生虫的整个生命周期。发展 疟原虫寄生虫在各种细胞类型中的作用涉及新生 mRNA 的调节 转录以及影响 mRNA 稳定性的转录后机制。在大多数 真核系统转录后调控是由新生 mRNA 的相互作用介导的 具有特定的 RNA 结合蛋白 (RNABP)。在本提案中，我们重点关注 mRNA 的调控 寄生虫发育的有性阶段的动态，这个阶段相对而言 直到最近才进行此类研究。通过基因改造恶性疟原虫寄生虫 使它们能够清除嘧啶前体，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。这些硫醇化 RNA 使我们能够 通过捕获和识别这些新生RNA来专门解决有关RNA代谢的问题 RNA 及其 RNABP。我们的中心假设是转录后机制 由寄生虫特异性RNA结合蛋白介导的配子细胞- 特定的基因调控，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 稳定性，并将 鉴定参与疟原虫性阶段转录后控制的RNABP 恶性疟原虫的发展。目标 1 将使用我们的 4-TU 标记方法来捕获特定性阶段 通过测量新生转录和 mRNA 来了解整个配子细胞发生过程中的 mRNA 动态 稳定性始于对配子细胞发生的承诺。在目标 2 中，4-TU 标记的 RNA 将是 与其同源 RNABP 交联，并通过蛋白质组学进行分析，以确定所有性别的图谱。 阶段 RNA 结合蛋白并定义促进这种相互作用的 RNA 识别基序。 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 PfPuf2 在内的几种性阶段 RNABP 的蛋白质相互作用。这项研究将为 未来使用这些工具和方法进行 RNA 调控研究，并将能够设计 对抗疟疾传播的新抗疟策略。 ！