Biochemistry and Pharmacology - Theses

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Start date: 2018 × Subject: alternative splicing × Subject: cell biology ×

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    Investigation of alternative splicing in apicomplexan parasites
    Lee, V Vern ( 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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    Transcriptional regulation and functional differences driven by STAT3 isoforms, STAT3α and STAT3β
    Tano, Vincent ( 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.