Date of Award
Chemistry and Biochemistry
Argonaute, gene regulation, microRNA, RNA interference
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Toxoplasma gondii is an intracellular obligate parasite of phylum Apicomplexa. Toxoplasma is able to infect any nucleated cell including up to one third of the world’s population. Within its hosts, the asexual life cycle of Toxoplasma consists of two distinct forms; a rapidly growing form called tachyzoite and a latent cyst encapsulated form called bradyzoite. Although tachyzoites can be removed by the host’s immune system, parasites can convert to bradyzoites thereby evading the host’s immune response. If the host’s immune system becomes weakened, bradyzoites are able to reconvert to tachyzoites. Taken together, these observations suggest Toxoplasma contains intricate gene regulation mechanisms that could be shared by other intracellular parasites. MicroRNAs (miRNAs) are crucial genetic effectors involved in numerous gene regulation mechanisms in eukaryotes. Although a post-transcriptional gene silencing can be observed in Toxoplasma the roles and functions of Tg-miRNAs are still elusive. In this work an engineered dual luciferase reporter system was used to examine and standardize the ability of long and short double-stranded RNA to control gene expression in Toxoplasma. The effects of endogenous Tg-miRNAs were also evaluated based on (i) their abundance and (ii) the number of binding sites within a transcript. Tg-miRNA effectiveness can be altered by use of miRNA mimics and inhibitors. Also a genetic knockdown system that exploits and directs Tg-miRNAs for loss-of-function analysis of essential and non-essential genes was developed. To further our understanding of miRNA induced gene silencing on parasite cell biology, a model gene target was selected for the most abundant Tg-miR-60a. The locus of the Ubiquitin-like protease 1 (Ulp1) contains the highest number of predicted Tg-miR-60a binding sites. The antisense RNA expresses a likely precursor of Tg-miR-60a suggesting TgUlp1 expression could be self-regulating. Ulp1 activity is required for the first and last step in the SUMOylation pathway. TgUlp1 is able to compliment the function of yeast Ulp1 and exhibits cleavage activity in vitro. Knockout of TgUlp1 is detrimental to parasite egress and survival. Since a miR-60a inhibitor or antisense RNA can alter TgUlp1 expression and activity, a TgUlp1-SF line was created to study the effects of miR-60a and its effects on total protein SUMOylation to demonstrate the importance of proper gene regulation by miRNAs. Furthermore, Toxoplasma Argonaute, the core protein of RNA-induced gene silencing that is required for a gene silencing effect, is expressed as two isoforms from the same locus, suggesting alternative translational starts sites. Overall, this dissertation reveals the importance of proper gene regulation directed by non-coding RNAs in Toxoplasma that could lead to the development of anti-Toxoplasma therapeutic.
Crater-Potter, Anna, "RNA Directed Gene Regulation in Toxoplasma gondii" (2017). Electronic Theses and Dissertations. 5933.