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

摘要 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。与大多数生物相比，P. 恶性疟原虫可以高效、准确地翻译Polya轨迹。因此，我们想要了解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贡献者 独特的翻译机制是RNA结合蛋白，翻译质量控制机制中的变异， 以及对核糖体RNA(RRNA)本身的适应。恶性疟原虫对富含AT基因的进化适应 基因组和Polya编码的赖氨酸片段仍有待探索。我们首先想要鉴定P。 恶性疟原虫结合含有PolA轨道或失速序列的mRNAs，并确定其组成 寄生虫内非GO衰变信使核糖核酸的监视机制(目标1)。通过了解这一过程， 我们将开始理解疟原虫翻译和所有其他 以真核生物为特征的。我们将使用一种改编的mrna标签系统来下拉mrna并检查 恶性疟原虫细胞中与这些mRNAs结合的蛋白质(目标1a)。我们还假设有一个 Polya Track基因的高效翻译；含mRNA的PolyA之间必须存在唯一的关系 疟疾寄生虫的跟踪、核糖体和信使核糖核酸监测机制(目标1b)。我们将分析 恶性疟原虫rRNA在体内参与PolA翻译保真度和多赖氨酸合成的特征(目标2)。 我们将使用MS2标记的核糖体系统来适应恶性疟原虫rRNA和核糖体分离(AIM 2a)。最后，我们认为PfRACK1蛋白有助于在疟原虫细胞中进行PolA Track翻译。我们会 确定PfRACK1是否有助于原虫和人类细胞系中的PolyA翻译，并将 检查PfRACK1在寄生虫内的差异、阶段相关的核糖体结合(目标2b)。 PfRACK1蛋白与恶性疟原虫和人核糖体的关系及其与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mRNAs 这个项目的目标是全面描述恶性疟原虫mrna监测系统的特征，我们将 是最早研究恶性疟原虫基因组翻译复杂性的人之一。我们相信这一点 信息将对防治疟疾至关重要，这种寄生虫的这些独特特征可以被利用 转变为新的治疗目标，从而进一步加强与疟疾的斗争。

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.