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： 人类的病原体恶性疟原虫是疟疾的病原体，它有一种 复杂的生命周期，包括在人类宿主和蚊子体内的多个组织中发育 向量。在人类感染期间，寄生虫主要每48小时无性复制一次， 每个周期有一种到多达24种新的寄生虫。然而，在低频率(1-3%)，寄生虫 致力于性别分化计划以产生关键的雄配子体和雌配子体 以传播到蚊子媒介，并完成寄生虫的整个生命周期。发展 疟原虫在不同细胞类型中的作用涉及新生mRNA的调节 转录以及转录后影响信使核糖核酸稳定性的机制。在大多数 真核系统转录后调控是由新生mRNAs相互作用介导的 与特定的RNA结合蛋白(RNABP)结合。在这项建议中，我们关注的是信使核糖核酸的调节。 寄生虫有性发育阶段的动态，这一阶段相对 直到最近才能接触到这样的研究。通过对恶性疟原虫进行基因工程来 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。这些硫基化的RNA使我们能够 通过捕获和识别这些新生细胞来专门解决有关RNA新陈代谢的问题 RNAs及其RNABP。我们的中心假设是转录后机制 由寄生虫介导的特异RNA结合蛋白在配子体中发挥着重要作用。 是寄生虫传播阶段的基本决定因素。 发展。这项研究将直接描述实时转录和RNA稳定，并将 鉴定参与疟原虫有性期转录后调控的RNABP 恶性疟原虫发育。目标1将使用我们的4-TU标记方法来捕捉特定的性阶段 通过检测新生转录和mRNA在配子发生过程中的mRNA动态 稳定始于对配子体发生的承诺。在AIM 2中，4-TU标记的RNA将 与它们的同源RNABP交叉连接，并通过蛋白质组学检测来确定所有性别- 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，我们将使用基于定向免疫沉淀的方法来定义特定的RNA- 包括PfPuf2在内的几种有性阶段RNABP的蛋白质相互作用。这项研究将为 用于未来使用这些工具和方法进行RNA调节的研究，并将使设计 防治疟疾传播的新抗疟疾战略。 好了！