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

摘要 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，P。 恶性疟原虫可以有效和准确地翻译polyA轨道。因此，我们想知道P。 恶性疟原虫可以有效地翻译这些基因。我们假设恶性疟原虫的潜在致病因素 独特的翻译机制是RNA结合蛋白，翻译质量控制机制的变化， 以及对核糖体RNA（rRNA）本身的适应。恶性疟原虫进化适应AT丰富 基因组和polyA编码的赖氨酸段仍有待探索。我们首先要鉴定P. 与含有polyA轨道或stalling序列的mRNA结合，并确定恶性疟原虫的组分， 寄生虫内的非去衰变mRNA监视机制（Aim 1）。通过了解这个过程， 我们将开始了解疟原虫翻译和所有其他翻译之间的根本区别。 以真核生物为特征。我们将使用一个适应的mRNA标记系统来拉下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， polyA跟踪基因的有效翻译;含有mRNA的polyA之间必须存在独特的关系 疟疾寄生虫中的追踪、核糖体和mRNA监测机制（目标1b）。我们将分析 恶性疟原虫rRNA的特征涉及体内polyA翻译保真度和多聚赖氨酸合成（Aim 2）。 我们将使用适用于恶性疟原虫rRNA和核糖体分离的MS 2标记的核糖体系统（Aim 2a）。最后，我们认为PfRACK 1蛋白有助于疟原虫细胞中polyA轨道的翻译。我们将 确定PfRACK 1是否在疟原虫和人类细胞系中协助polyA翻译， 检查PfRACK 1在寄生虫内的差异、阶段依赖性核糖体结合（Aim 2b）。 PfRACK 1蛋白与恶性疟原虫和人核糖体的结合及其与polyA的相互作用 将使用单分子荧光共振能量转移（smFRET）进一步表征mRNA 本项目的目标是充分表征恶性疟原虫mRNA监测系统， 是最早研究恶性疟原虫基因组翻译复杂性的人之一。我们认为这 这些信息对抗击疟疾至关重要，而且可以利用寄生虫的这些独特特征， 转化为新的治疗目标，从而进一步推进与疟疾的斗争。

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.