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（疟疾的病原体）的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。恶性疟原虫基因组极其 AT- 丰富（~80%），由大量编码多聚腺苷（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 轨迹充当基因表达的负调节因子。我们最近的研究 已经表明，含有多聚腺苷酸轨道基序的 mRNA 的翻译会导致核糖体停滞和 大多数真核生物和细菌生物体中的移码。与大多数生物体相比，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 机制包括RNA结合蛋白、翻译质量控制机制的变化以及对 核糖体 RNA (rRNA) 本身。恶性疟原虫对富含 AT 的基因组和多聚腺苷酸的进化适应 编码的赖氨酸片段仍有待探索。我们首先要鉴定恶性疟原虫中与 含有 PolyA 的 mRNA 跟踪或停滞序列并确定不可行衰变 mRNA 的成分 寄生虫内部的监视机制（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有特征的真核生物之间的根本区别。我们将 使用适应的 mRNA 标记系统来下拉 mRNA 并检查与这些 mRNA 结合的蛋白质 恶性疟原虫细胞（目标 1a）。我们还假设对多聚腺苷酸轨道基因进行有效的翻译； 含有 mRNA 的 PolyA 轨迹、核糖体和 mRNA 监视之间必定存在独特的关系 疟疾寄生虫的机制（目标 1b）。我们将分析参与多聚A的恶性疟原虫rRNA的特征 翻译保真度和体内聚赖氨酸合成（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，我们相信 PfRACK1 蛋白有助于 疟原虫细胞中的多聚A轨道翻译。我们将确定 PfRACK1 是否有助于多聚腺苷酸翻译 疟原虫和人类细胞系，还将检查不同阶段依赖的核糖体结合 寄生虫内的 PfRACK1（目标 2b）。 PfRACK1 蛋白与恶性疟原虫和人核糖体的关联 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) 该项目的目标是表征恶性疟原虫 mRNA 全面的监测系统，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。我们相信，这些信息对于抗击疟疾至关重要，而且疟疾的这些独特特征 寄生虫可以被开发成新的治疗靶点，从而进一步推进抗击疟疾的斗争。