Dissecting mRNA-ribosome interaction in AU-rich transcriptome of Plasmodium falciparum

解析恶性疟原虫富含 AU 的转录组中 mRNA-核糖体相互作用

• 批准号: 10798664
• 负责人: Sergej Djuranovic
• 金额: $ 11.14万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798664
• 关键词: Binding; Binding Proteins; Biological; Cells; Drug Targeting; Eukaryota; Evolution; Foundations; Gene Expression; Genes; Genome; Genomics; Goals; Health; Human; Human Cell Line; Lysine; Malaria; Messenger RNA; Organism; Parasites; Plasmodium; Plasmodium falciparum; Plasmodium falciparum genome; Polylysine; Process; Protein Biosynthesis; Proteins; Quality Control; RNA-Binding Proteins; Regulator Genes; Ribosomal Interaction; Ribosomal RNA; Ribosomes; Stretching; System; Translating; Translations; Variant; activation-induced cytidine deaminase; fighting; genome sequencing; in vivo; mRNA Decay; mRNA Surveillance; mRNA Translation; new therapeutic target; novel strategies; polyadenosine; single-molecule FRET; transcriptome

Project Summary/Abstract – from original application Genome sequencing of P. falciparum, the causative agent of malaria, has laid the foundation for significant biological advances by exposing surprising genomic information. The P. falciparum genome is extremely AT- rich (~80%) and comprised of a large number of genes encoding polyadenosine (polyA) tracks. In most eukaryotes, including humans, polyA tracks act as negative regulators of gene expression. Our recent studies have shown that the translation of mRNAs containing polyA track motifs results in ribosomal stalling and frameshifting in the majority of eukaryotic and bacterial organisms. In contrast to most organisms, P. falciparum can efficiently and accurately translate polyA tracks. Therefore, we want to understand how P. falciparum can effectively translate these genes. We hypothesize that potential contributors to P. falciparum’s unique translation mechanism are RNA-binding proteins, variations in the translation quality control machinery, and adaptations to the ribosomal RNA (rRNA) itself. P. falciparum evolutionary adaptation towards an AT-rich genome and polyA encoded lysine stretches remains to be explored. We first want to identify proteins in P. falciparum that bind to mRNAs containing polyA tracks or stalling sequences and determine the components of the no-go decay mRNA surveillance mechanism within the parasite (Aim 1). By understanding this process, we will begin to understand the fundamental differences between Plasmodium translation and all other characterized eukaryotes. We will use an adapted mRNA tagged system to pull-down mRNAs and examine the proteins binding to these mRNAs in P. falciparum cells (Aim 1a). We also hypothesize that to have an efficient translation of polyA track genes; there must be a unique relationship between mRNA-containing polyA tracks, ribosomes, and mRNA surveillance mechanisms in malaria parasites (Aim 1b). We will analyze features of P. falciparum rRNA involved in polyA translational fidelity and poly-lysine synthesis in vivo (Aim 2). We will use the MS2-tagged ribosome system adapted for P. falciparum rRNAs and ribosome isolation (Aim 2a). Finally, we believe that PfRACK1 protein aids in polyA track translation in Plasmodium cells. We will determine if PfRACK1 assists in polyA translation in both plasmodium and human cell lines and will also examine the differential, stage-dependent ribosomal binding of PfRACK1 within the parasite (Aim 2b). Association of PfRACK1 protein with P. falciparum and human ribosomes and their interaction with polyA mRNAs will be further characterized using single-molecule fluorescence resonance energy transfer (smFRET) The goals of this project are to characterize P. falciparum mRNA surveillance system fully, and we will be among the first to study the translational complexities in the P. falciparum genome. We believe that this information will be crucial to fighting malaria and that these unique features of the parasite can be exploited into new therapeutic targets, thus furthering the battle against malaria.

项目摘要/摘要-来自原始申请 恶性疟原虫是疟疾的病原体，其基因组测序为重大研究奠定了基础。 通过揭示令人惊讶的基因组信息来实现生物学进步。恶性疟原虫的基因组是非常AT- 丰富（约80%），并由大量编码聚腺苷（polyA）轨道的基因组成。在大多数 在包括人类在内的真核生物中，polyA轨道充当基因表达的负调节剂。我们最近的研究 已经表明，含有polyA轨道基序的mRNA的翻译导致核糖体停滞， 在大多数真核生物和细菌生物中的移码。与大多数生物相比，恶性疟原虫 可以高效准确地翻译polyA轨迹。因此，我们想了解恶性疟原虫是如何 有效地翻译这些基因。我们假设恶性疟原虫独特翻译的潜在贡献者 机制是RNA结合蛋白质，翻译质量控制机制的变化，以及适应 核糖体RNA（rRNA）恶性疟原虫向富含AT基因组和polyA的进化适应 编码的赖氨酸段仍有待探索。我们首先想确定恶性疟原虫中的蛋白质， 含有polyA轨道或停滞序列的mRNA，并决定了不去衰变mRNA的组分 寄生虫内的监测机制（目标1）。通过理解这个过程，我们将开始理解 疟原虫翻译与所有其他特征性真核生物之间的根本差异。我们将 使用一个适应的mRNA标记系统来拉下mRNA，并检查与这些mRNA结合的蛋白质 在恶性疟原虫细胞中（Aim 1a）。我们还假设，要有一个有效的翻译polyA轨道基因; 在含有mRNA的polyA轨道、核糖体和mRNA监视之间必须有一种独特的关系 疟疾寄生虫的机制（目标1b）。我们将分析参与polyA的恶性疟原虫rRNA的特征 翻译保真度和体内多聚赖氨酸合成（Aim 2）。我们将使用MS 2标记的核糖体系统 适用于恶性疟原虫rRNA和核糖体分离（Aim 2a）。最后，我们认为PfRACK 1蛋白有助于 跟踪疟原虫细胞中的翻译。我们将确定PfRACK 1是否在两种细胞中协助polyA翻译， 疟原虫和人类细胞系，并将检查差异，阶段依赖性的核糖体结合， PfRACK 1在寄生虫内（目标2b）。PfRACK 1蛋白与恶性疟原虫和人核糖体的结合 并且它们与polyA mRNA的相互作用将使用单分子荧光进一步表征 共振能量转移（smFRET）本项目的目标是表征恶性疟原虫mRNA 我们将成为第一个研究恶性疟原虫翻译复杂性的人。 基因组我们认为，这些信息对防治疟疾至关重要， 寄生虫可以被开发成新的治疗目标，从而进一步推动防治疟疾的斗争。