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-来自原始应用程序 疟疾病原体恶性疟原虫的基因组测序为有意义的 通过揭示令人惊讶的基因组信息而取得的生物学进展。恶性疟原虫基因组极不稳定- 富含(~80%)，由大量编码多腺苷(PolyA)轨迹的基因组成。在大多数 真核生物，包括人类，Polya Track扮演着基因表达的负调控者的角色。我们最近的研究 已经表明，含有PolA Track基序的mRNAs的翻译会导致核糖体停滞和 在大多数真核生物和细菌生物体中的移码。与大多数生物不同，恶性疟原虫 可以高效、准确地翻译Polya曲目。因此，我们想了解恶性疟原虫是如何 有效地翻译这些基因。我们推测恶性疟原虫独特翻译的潜在贡献者 机制是RNA结合蛋白，翻译质量控制机制的变异，以及适应 核糖体RNA(RRNA)本身。恶性疟原虫对富含AT的基因组和PolA的进化适应 编码的赖氨酸延伸仍有待探索。我们首先想要鉴定恶性疟原虫结合蛋白 含有Polya轨道或失速序列的mRNAs，并确定no-go Decay mRNAs的组成 寄生虫内的监测机制(目标1)。通过了解这一过程，我们将开始理解 疟原虫翻译与所有其他特化的真核生物之间的根本区别。我们会 使用经过调整的信使核糖核酸标签系统下拉mRNAs并检测与这些mRNAs结合的蛋白质 恶性疟原虫细胞(目标1a)。我们还假设Polya Track基因有一个有效的翻译； 在含有mRNA的PolyA轨迹、核糖体和mRNA监测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Polya涉及的恶性疟原虫rRNA的特征 翻译保真度与体内多赖氨酸合成(目标2)。我们将使用MS2标记的核糖体系统 适用于恶性疟原虫rRNA和核糖体分离(目标2a)。最后，我们认为PfRACK1蛋白有助于 在Polya中，疟原虫细胞中的轨道翻译。我们将确定PfRACK1是否在两者中协助Polya翻译 并将研究不同阶段依赖于核糖体的差异结合。 寄生虫内的PfRACK1(目标2b)。PfRACK1蛋白与恶性疟原虫及人核糖体的关系 并将利用单分子荧光进一步表征它们与Polya mRNAs的相互作用。 共振能量转移(SmFRET)这个项目的目标是鉴定恶性疟原虫的mRNA 我们将成为第一批研究恶性疟原虫翻译复杂性的人之一。 基因组。我们认为，这些信息将对防治疟疾至关重要，而这些独特的 寄生虫可以被开发成新的治疗目标，从而进一步抗击疟疾。