Biochemistry and Pharmacology - Theses
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ItemNo Preview AvailableInvestigation of alternative splicing in apicomplexan parasites( 2021)Alternative splicing is the phenomenon by which coding and non-coding regions of pre-mRNA molecules can be differentially spliced to yield multiple mRNA isoforms from a single gene. In metazoans, alternative splicing occurs to a substantial degree, contributing to protein diversity and the post-transcriptional regulation of gene expression. However, to what extent this occurs in apicomplexan parasites is much less understood. This thesis examines the landscape, regulators and function of alternative splicing in two apicomplexan parasites, T. gondii and P. falciparum. Technological advances in the short read sequencing of nucleic acids at unprecedented depths have enabled deep profiling of the transcriptome. However, the short reads present a limitation in the analysis of complex splicing events that span beyond the length of the reads. We evaluated the capability of a third generation long read sequencing technology, Oxford Nanopore Technologies (ONT) sequencing, in sequencing full-length native mRNA from T. gondii and P. falciparum, and established a method to analyse the alternative splicing landscape from the long reads. We successfully identified full-length transcripts spanning annotated and non-annotated junctions, implying a suitability in exploring complex splicing events. The analysis reveals an unusually high level of intron retained transcripts with premature terminating codons (PTCs). This suggests that most alternative splicing events in T. gondii and P. falciparum are unlikely to be productive. Alternative splicing in metazoans is modulated by alterative splicing factors, most notably the SR (serine-arginine–rich) protein family. We characterised the suite of SR proteins and two putative kinases/regulators of SR proteins in T. gondii. The proteins were found localised to sub-nuclear compartments characteristic of splicing factors. We demonstrated through genetic ablation and whole-transcriptome sequencing that the SR proteins modulate distinct but overlapping subsets of mostly non-productive alternative splicing events, as well as impacting transcript abundance. Alternatively spliced junctions were also enriched in characteristic SR binding motifs. The putative kinases of SR proteins were found to be essential to parasite survival and modulate extensive splicing events, but the events poorly mirrored that modulated by the SR proteins. This suggests a complex system of splicing regulation that do not conform to other eukaryotic models. The targeting of non-productive alternatively spliced transcripts for degradation through the nonsense mediated decay (NMD) pathway is one mechanism by which metazoans post-transcriptionally regulate gene expression. To explore if this was the case for T. gondii, we characterised the three core NDM proteins- UPF1, UPF2 and UPF3. The three proteins were found to co-immunoprecipitate with one another, implying a conservation of the core NMD complex. However, when we conditionally ablated the UPF proteins, parasite growth and survival was not impacted. We sequenced the parasite mRNA and found that only UPF2 impacted global intron retention rates. Moreover, a link between intron retention and gene expression regulation could not be established. Our results show that the fitness cost of mis- splicing determines intron retention rates, rather than targeted regulation. Hence, this thesis has shown that although non-productive alternative splicing is widespread and regulated in T. gondii, it is not a mechanism for post-transcriptional regulation of gene expression through the NMD pathway.
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ItemTranscriptional regulation and functional differences driven by STAT3 isoforms, STAT3α and STAT3β( 2018)The Signal transducer and activator of transcription 3 (STAT3) protein, a member of the STAT family of transcription factors, plays important roles in the regulation of critical biological processes. STAT3 is best known for its role in the JAK-STAT pathway, mediating the transcriptional regulation of target gene expression following its activation by extracellular signal, and continues to be the subject of intense research efforts due to its essential roles in crucial physiological processes, such as embryonic development and inflammation, as well as its involvement in numerous types of cancer. While the genome-wide DNA-binding and transcriptional regulation activities of the full-length STAT3α have been widely studied, those of a naturally occurring shorter STAT3β spliceform are less understood. Despite having identical DNA-binding domains, the STAT3 spliceforms display significant differences in their transcriptional regulation activities. Furthermore, STAT3α and STAT3β can drive opposing oncogenic and tumour suppression outcomes, respectively. Thus, the major focus of this study is to explore STAT3α- and STAT3β-specific genome-wide DNA-binding, transcriptional regulation of non-coding micro-RNAs (miRNAs), and functional outcomes in cancer cell biology. By employing ChIP-seq and miRNA expression profiling assays, this study presents results revealing that STAT3α and STAT3β display clear differences in genome-wide DNA-binding and regulation of miRNA expression. Bioinformatic analyses of transcription factor binding sites also uncovered the possible roles of co-transcription factors in the distinct STAT3 spliceform-specific transcriptional regulation activities. In addition, a Morpholino-directed splicing modulation approach to drive STAT3 knockdown and STAT3α-to-β splicing switch in cancer cells showed that the STAT3 spliceforms can differentially alter the tumourigenic properties of cancer cells, with STAT3β expression being associated with tumour suppression outcomes by driving the suppression of cell proliferation, survival and migration. Taken together, this study highlights the distinct properties of the STAT3 spliceforms in genome-wide DNA-binding which possibly underlie their different gene transcriptional regulation activities in both protein-coding and non-coding genes, and further provides evidence that STAT3β can drive distinct tumour suppression outcomes.
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ItemUnderstanding the role of Maurer’s clefts in virulence protein trafficking( 2017)The malaria parasite Plasmodium falciparum modifies the host red blood cell to establish virulence protein-trafficking pathways. The major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1) is exported from the parasite to the red blood cell surface, where it mediates attachment of the infected cell to ligands on the host vascular endothelium. This process of sequestration enables infected red blood cells to avoid immune detection in the spleen and contributes to the development of severe malaria. The Maurer’s clefts are organelles formed by the parasite and are present in the red blood cell cytoplasm. The primary function of Maurer’s clefts is thought to be the transport of PfEMP1 to the red blood cell membrane. We investigate a Maurer’s clefts protein, ring-exported protein-1 (REX1) and its role in PfEMP1 trafficking. We show that Maurer’s clefts morphology is disrupted by knocking down REX1 and that PfEMP1 surface display is decreased. Using transfectant parasites expressing truncated forms of the protein, we identify a repeat region of REX1 that mediates Maurer’s clefts morphology and is required for efficient PfEMP1 trafficking. We have developed a method to enrich the Maurer’s clefts from infected red blood cells and define their protein composition by tandem mass spectrometry. We epitope-tag a number of putative and established Maurer’s clefts proteins and confirm the location of several novel Maurer’s clefts proteins. Using super-resolution microscopy, we localise Maurer’s clefts proteins to subcompartments within these organelles. Finally, we use co- precipitation to describe a protein interaction network at the Maurer’s clefts.
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ItemAlternative splicing and stage differentiation in apicomplexan parasites( 2017)Alternative splicing is the phenomenon by which single genes code for multiple mRNA isoforms. This is common in metazoans, with alternative splicing observed in over 90% of human genes (Wang et al., 2008). However, the full extent of alternative splicing in apicomplexans has been previously under-reported. Here, I address this deficiency by transcriptomic analysis of two apicomplexan parasites: Toxoplasma gondii, which causes toxoplasmosis; and Plasmodium berghei, which is a murine model for human malaria. I identified apicomplexan homologues to SR (serine-arginine–rich) proteins, which are alternative-splicing factors in humans. I then localised a homologue, which I named TgSR3, to a subnuclear compartment in T. gondii. Conditional overexpression of TgSR3 was deleterious to growth. I detected perturbation of alternative splicing by qRT-PCR. Parasites were sequenced with RNA-seq, and 2000 genes were identified as constitutively alternatively spliced. Overexpression of TgSR3 perturbed alternative splicing in over 1000 genes. Previously, computational tools were poorly suited to compacted parasite genomes, making these analyses difficult. I alleviated this by writing a program, GeneGuillotine, which deconvolutes RNA-seq reads mapped to these genomes. I wrote another program, JunctionJuror, which estimates the amount of constitutive alternative splicing in single samples. Most alternative splicing in humans is tissue specific (Wang et al., 2008; Pan et al., 2008). However, unicellular parasites including Apicomplexa lack tissue. Nevertheless, I have shown that alternative splicing can still be common. I hypothesised that the tissue-specific alternative splicing of metazoans is analogous to stage-specific alternative splicing in unicellular organisms. I purified female and male gametocytes of P. berghei and sequenced these stages, with the aim of investigating alternative splicing and its relationship to stage differentiation. As a reference point, I first established the wild-type differences between female and male gametocytes. I detected a trend towards downregulation of transcripts in gametocytes compared to asexual erythrocytic stages, with this phenomenon more marked in female gametocytes. I was also able to identify many female- and male-specific genes, some previously-characterised, and some novel. My findings were further placed in an evolutionary context. Sex-specific genes were well conserved within the Plasmodium genus, but relatively poorly conserved outside this clade, suggesting that many Plasmodium sex-related genes evolved within this genus. This trend is least pronounced in male-specific genes, which suggests that sexual development of male gametocytes may have preferentially evolved from genes already present in organisms outside this genus. I then analysed these transcriptomes, now focusing on changes in alternative splicing. While non-gendered gametocyte differentiation is modulated by known transcription factors such as AP2-G (Sinha et al., 2014), I provide evidence that alternative splicing adds another level of regulation, which is required for differentiation into specific genders. I ablated a Plasmodium SR-protein homologue, which I named PbSR-MG. By transcriptomic analysis, I show that it regulates alternative splicing, predominantly in male gametocytes. Ablation was also associated with a drastic reduction in the viability of male gametocytes. Hence, I have shown that alternative splicing is common in apicomplexan parasites, is regulated by specific genes, and acts on specific targets. Alternative splicing is important for parasite viability and fundamental to stage differentiation in Plasmodium.
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ItemProtein localisation in a divergent eukaryotic parasite( 2013)Malaria infects 200 million people every year, with more than half a million of those cases resulting in death. Understanding the cell biology of Plasmodium falciparum remains largely an unsolved and unexplored problem. This doctoral assertion furthers understanding by investigating the sub-cellular localisation of Plasmodium falciparum proteins. A comprehensive literature review was undertaken, and the sub-cellular localisations of hundreds of proteins has been recorded in a transparent, traceable, publicly accessible and tactile fashion, and this serves as the basis for the further studies undertaken. It has been named ApiLoc. Using this curated set of protein localisations, the first Plasmodium falciparum-specific algorithm to predict sub-cellular localisation globally was created. Amino-acid based protein features were useful for prediction, but the predictor leveraged other predictive data types, such as microarray information and evolutionary conservation of the protein. Predictions were rigorously validated using both computational and epitope tagging approaches. ApiLoc also served as the basis for studies into the evolutionary conservation of protein localisation itself. This showed a remarkable lack of conservation, with only ~50% of protein localisations conserved across Apicomplexa, and ~20% throughout Eukaryota. The nucleus was studied in particular detail, given my involvement in a project to analyse isolated nuclei using a proteomic approach. Biochemical and bioinformatic studies were undertaken, providing further evidence that a classical nuclear import system involving basic-rich nuclear localisation signals is functional in Plasmodium falciparum and the evolutionarily related parasite Toxoplasma gondii.
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ItemAnalysis of architectural rearrangements in Plasmodium falciparum gametocytes( 2013)Transmission of the most virulent human malaria parasite, Plasmodium falciparum, is dependent on the parasite’s ability to produce viable gametocytes. Over two weeks the parasite prepares itself for transmission into mosquitoes by undergoing a series of cellular rearrangements. Once the metamorphosis is completed, mature gametocytes release from their sequestration sites, enter the circulation and become accessible to feeding mosquitoes. Whilst the formation of mature gametocytes represents a bottleneck in the parasite’s lifecycle, and thus an attractive target for transmission blocking strategies, very little of the basic biology of this lifecycle stage has been described. As the intraerythrocytic asexual stage parasite develops, it modifies its host red blood cell (RBC) by forming an exomembrane network of parasite-derived protein sorting organelles that facilitate the delivery of proteins to the RBC cytoplasm and membrane. Despite early ultrastructural descriptions of the gametocyte exomembrane network, no molecular characterisation of this system has been performed. In this thesis, modern high-resolution microscopy and immuno labelling techniques were used to re-evaluate the fine structure and molecular identity of several key components of the exomembrane system in the gametocyte. Early ultrastructural studies identified a sub-pellicular membrane complex in gametocytes. This structure consists of a flattened cisternal membrane beneath the parasite plasma membrane, which is supported by a network of microtubules. We have further described the molecular composition and origin of the sub-pellicular membrane complex, by identifying the presence of glideosome-associated proteins in gametocytes. We show that the gametocyte pellicle is analogous to the inner membrane complex (IMC), an organelle with structural and motor functions that is conserved across the Apicomplexan phylum. Thus we have proposed that the sub-pellicular membrane complex be renamed the gametocyte IMC (gIMC). We have also shown that the coordinated assembly of the microtubule network alongside the gIMC is responsible for driving shape change in the P. falciparum gametocyte. Interestingly, changes in cellular deformability correspond with gametocyte shape shifting. An increase in deformability coincides with the time the banana-shaped stage V gametocytes reappear in circulation. It has therefore been postulated that modifying the deformability of the host cell enables the gametocyte to circulate in the blood stream without being detected and removed by the mechanical filtering mechanisms in the host's spleen. Further investigation of how these changes occur show that the loss of cellular deformability is not dependent of the tubulin cytoskeleton of the parasite. Instead, it is associated with an altered molecular organization of the host cell membrane, as indicated by a loss of lateral mobility of the major RBC membrane protein, Band 3. Our studies have shown that several parasite proteins, which modulate rigidity of the RBC membrane in asexual stage parasites, are not exported in gametocytes. It appears that changes in gametocyte deformability are due to the rearrangement of cytoskeletal components, including host cell actin. The work presented in this thesis demonstrates that changes in gametocyte morphology and cellular deformability are crucial for the development of the P. falciparum gametocyte and discusses their likely contribution to the survival and transmission of the parasite.