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

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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.