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: 10415144
• 负责人: Sergej Djuranovic
• 金额: $ 31.5万
• 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/10415144
• 关键词: Adenosine; Affect; Amino Acids; Binding; Binding Proteins; Biological; Cell Line; Cells; Chimera organism; Codon Nucleotides; Development; Drug Targeting; Ensure; Eukaryota; Evolution; Exhibits; Foundations; Gene Expression; Genes; Genetic Translation; Genome; Genomics; Goals; Health; Human; Human Cell Line; Knowledge; Lysine; Malaria; Mass Spectrum Analysis; Messenger RNA; Modification; Nonsense-Mediated Decay; Organism; Outcome; Parasites; Pathway interactions; Plasmodium; Plasmodium falciparum; Plasmodium falciparum genome; Polylysine; Process; Protein Biosynthesis; Proteins; Proteome; Protocols documentation; Quality Control; RNA; RNA Decay; RNA-Binding Proteins; Regulator Genes; Reporter; Ribosomal Interaction; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Running; Starvation; Stretching; Structure; System; Technology; Testing; Therapeutic; Tobramycin; Translating; Translations; Variant; Work; activation-induced cytidine deaminase; aptamer; experimental study; fight against; fighting; genome sequencing; in vivo; mRNA Decay; mRNA Surveillance; mRNA tagging; new therapeutic target; novel strategies; polyadenosine; recruit; single-molecule FRET; stem; transcriptome

Abstract 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.

抽象的 恶性疟原虫的基因组测序是疟疾的致病药物，为重要的基础奠定了基础 通过暴露令人惊讶的基因组信息来进步。恶性疟原虫基因组极为 丰富（约80％），由大量编码多腺苷（Polya）轨道的基因组成。大多数 真核生物，包括人类，Polya Tracks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的翻译导致核糖体失速和 大多数真核生物和细菌生物的帧。与大多数生物相反，P。 恶性菌可以有效，准确地翻译Polya轨道。因此，我们想了解P. 恶性菌可以有效地翻译这些基因。我们假设对恶性疟原虫的潜在贡献者 独特的翻译机制是RNA结合蛋白，翻译质量控制机制的变化， 并适应核糖体RNA（rRNA）本身。恶性疟原虫的进化适应性 基因组和Polya编码的赖氨酸拉伸仍有待探索。我们首先想鉴定P中的蛋白质。 恶性与包含多拉轨道或失速序列的mRNA结合并确定的成分 寄生虫内的无衰减mRNA监测机制（AIM 1）。通过了解这个过程， 我们将开始了解疟原虫翻译与所有其他所有其他之间的根本差异 真核生物的特征。我们将使用改编的mRNA标记系统进行下拉mRNA并检查 在恶性疟原虫细胞中与这些mRNA结合的蛋白质（AIM 1A）。我们还假设有一个 Polya轨道基因的有效翻译；含mRNA的polya之间必须存在独特的关系 疟原虫中的轨道，核糖体和mRNA监测机制（AIM 1B）。我们将分析 恶性疟原虫rRNA的特征参与体内Polya转化保真度和聚赖氨酸合成的特征（AIM 2）。 我们将使用适用于恶性疟原虫RRNA和核糖体隔离的MS2标记的核糖体系统（AIM 2a）。最后，我们认为pfrack1蛋白在疟原虫细胞中Polya Track翻译中有助于。我们将 确定pfrack1是否有助于质子和人类细胞系中的polya翻译，也将 检查寄生虫中PFRACK1的差异，依赖性核糖体结合（AIM 2B）。 PFRACK1蛋白与恶性疟原虫和人核糖体的关联及其与Polya的相互作用 MRNA将进一步表征使用单分子荧光共振能传递（SMFRET） 该项目的目标是完全表征恶性疟原虫mRNA监视系统，我们将是 在第一个研究恶性疟原虫基因组中的翻译复杂性的其中之一。我们相信这个 信息对于与疟疾作斗争至关重要，并且可以利用寄生虫的这些独特特征 进入新的治疗靶标，从而促进与疟疾的战斗。