Date of Award

4-30-2018

Publication Type

Master Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

Keywords

DEAD-Box RNA Helicase, Dicer, miRNA, RNAi, TgHODI, Toxoplasma gondii

Supervisor

Ananvoranich, Sirinart

Rights

info:eu-repo/semantics/openAccess

Abstract

The RNA-interference pathway is commonly found in eukaryotes, and plays an important role in the inhibition of gene expression. This study focuses on two aspects of the RNA-interference pathway of Toxoplasma gondii. The first aspect was to investigate the function of a putative Dicer, an enzyme responsible for the generation of small RNAs (siRNA and miRNA). A parasite strain, whose functional Tg-Dicer expression was abolished, was generated and used in this study in comparison to the parental strain. It was detected that the replication and invasion of the mutated Tg-Dicer strain (TgDicermut) is slower than that of the parental strain (TgDicer-wt). Using the dual luciferase assay I detected that TgDicer-mut lacks the ability to silence the expression of Renilla (RN) transcript containing Tg-miRNA binding sites. To determine if the inability to silence the RN expression was due to its incapability to process long dsRNA into short dsRNA, TgDicer-mut strain was electroporated with long or short dsRNA complementary to RN transcript. TgDicer-mut can use short dsRNA and cause a decrease in Renilla activity, but not long dsRNA. The study thus confirms that TgDicer is responsible for processing long dsRNA into short dsRNA. The other aspect of this investigation was to verify whether a predicted target of miRNAs named TgHODI (a DEAD-box RNA helicase) could be a target of the two most abundant miRNAs, miR4a and miR60a. A clonal transgenic, flag-tagged TgHODI was used for the study to determine its expression in the presence of miRNA inhibitor. When the inhibitors specific to miR4a and miR60a was used, the expression of TgHODI expression level increased to ~1.8 and ~1.6 times respectively. This indicates that TgHODI may be regulated by miR4a and miR60a.

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