Ebola virus (EBOV) is an enveloped, negative-sense single-stranded RNA virus that causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes multiple transcripts due to RNA editing at a conserved editing site (ES) (a hepta-uridine stretche). The majority of GP gene transcript is unedited and encodes for a soluble glycoprotein (sGP); a defined function has not been assigned for sGP. In contrast, the transmembrane glycoprotein (GP1,2) dictates viral tropism and is expressed through RNA editing by insertion of a nontemplate adenosine (A) residue. Hypothetically, the insertion/deletion of a different number of A residues through RNA editing would result in another yet unidentified GP gene product, the small soluble glycoprotein (ssGP). I have shown that ssGP specific transcripts were indeed produced during EBOV infection. Detection of ssGP during infection was challenging due to the abundance of sGP over ssGP and the absence of distinguishing antibodies for ssGP. Optimized two- dimensional (2-D) gel electrophoresis verified the expression of ssGP during infection. Biophysical characterization revealed ssGP is a disulfide-linked homodimer that is exclusively N-glycosylated. Although ssGP appears to share similar structural properties with sGP, it does not have the same anti-inflammatory function. Using a new rapid transcript quantification assay (RTQA), I was able to demonstrate that RNA editing is an inherent feature of the genus Ebolavirus and all species of EBOV produce multiple GP gene products. A newly developed dual-reporter minigenome system was utilized to characterize EBOV RNA editing and determined the conserved ES sequence and cis-acting sequences as primary and secondary requirements for RNA editing, respectively. Viral protein (VP) 30, a transcription activator, was identified as a contributing factor of RNA editing— a proposed novel function for this largely uncharacterized viral protein. Finally, I could show that EBOV RNA editing is GP gene-specific because a similar sequence located in L gene did not serve as an ES, most likely due to the lack of the necessary cis-acting sequences. In conclusion, I identified a novel soluble protein of EBOV whose function needs further characterization. I also shed light into the mechanism of EBOV RNA editing, a potential novel target for intervention.

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Metallothioneins (MTs) have a major role in metal metabolism and may also protect DNA against oxidants. MT protein has been found localized in the nucleus during S-phase. The mRNA encoding for the MT-1 isoform is found localized around the nucleus and associated with the cytoskeleton; this is due to targeting signals within the 3'untranslated region (3'UTR). Using cells transfected with gene constructs differing in their 3'UTRs, the role of perinuclear mRNA localization in facilitating MT synthesis close to its site of function and subsequent import of protein into the nucleus has been investigated, as well as the role of MT protein in the nucleus. We transfected CHO cells, which have a low constitutive level of MT expression, with either the full MT-gene (MTMT) or with MR 5'UTR and coding region linked to the 3'UTR of glutathione peroxidase (MTGSH). Immunocytochemistry showed that MT protein was localized in the perinuclear cytoplasm in the MTMT cells whereas no distinct localization was found in the MTGSH cells. The cells were then synchronised in S-phase by serum depletion/repletion. After serum repletion, MT was found in the nucleus of MTMT cells but not in the MTGSH cells. This suggests that perinuclear localization of MT-1 mRNA and its association with the cytoskeleton is necessary for MT protein localization, particularly for the shuttling of MT protein into the nucleus during S-phase. Functional studies demonstrated that the extent of oxidative stress and DNA damage was lower in the MTMT than the MTGSH, showing that a loss of MT protein localization led to a reduced protection of the cell. Therefore, it seems that perinuclear localization of mRNAs coding for MT is necessary for subsequent transport and targeting of proteins into the nucleus and that the localization of the protein within the cell is important for its function.

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The precise location of pre-messenger RNA (pre-mRNA) was determined within the cell nucleus of the amphibian N. maculosus (mudpuppy) oocyte. Pre-messenger RNA or heterogeneous nuclear RNA (hnRNA) is directly transcribed from the gene in the cell nucleus (Jelinek et al., 1983). This highly unstable, high molecular weight complex is then processed into messenger RNA (mRNA). Again this transformation is thought to take place in the nucleus.By employing the techniques of in situ hybridization along with light and high resolution autoradiography an effort was made to localize, via a radioactive probe, ( 3H) poly (U), pre-mRNA within the nucleus. Additionally, various inhibitors were employed in this study to analyze changes in the amount and distribution of radioactive material within the cell.Light microscopy observations of autoradiographs reveal a consistent pattern of probe localization over the nucleoli with a broader dispersal across the nuclei. Ultrastructural studies reveal the presence of granules (perichromatin granules) localized over both the nucleolar-associated chromatin and at the border of condensed chromatin. These structures are contained in the cell nucleus and are presumed to function in the transport and storage of mRNA. / Department of Physiology and Health Science

Frequent subgraph mining is a useful method for extracting biologically relevant patterns from a set of graphs or a single large graph. Here, the graph represents all possible RNA structures and interactions. Patterns that are significantly more frequent in this graph over a random graph are extracted. We hypothesize that these patterns are most likely to represent a biological mechanisms. The graph representation used is a directed dual graph, extended to handle intermolecular interactions. The graph is sampled for subgraphs, which are labeled using a canonical labeling method and counted. The resulting patterns are compared to those created from a randomized dataset and scored. The algorithm was applied to the mitochondrial genome of the kinetoplastid species Trypanosoma brucei. This species has a unique RNA editing mechanism that has been well studied, making it a good model organism to test RiboFSM. The most significant patterns contain two stem-loops, indicative of gRNA, and represent interactions of these structures with target mRNA.

RNA interference (RNAi) is a gene silencing mechanism induced by double-stranded RNA (dsRNA). RNAi has been used extensively to create loss-of-function mutants in many species to identify the functions of genes, but it also has the potential to be used as a species-specific pesticide if the dsRNA can silence essential genes in pests. The mosquito Aedes aegypti is a vector of numerous viruses including Dengue and West Nile virus, and is frequently controlled by chemical insecticides. With growing concerns about the extensive use of broad-spectrum pesticides, new control methods are eagerly sought. In this study, I examined the efficacy of feeding pesticidal dsRNAs to mosquito larvae. A dose-dependent RNAi response and mortality was observed when larvae were fed dsRNA targeting several different genes. Unlike RNAi in the related dipteran Drosophila melanogaster, RNAi in A. aegypti also appeared to be systemic, spreading beyond the gut to other tissues. A degree of species-specificity was also observed, as dsRNA specific to the D. melanogaster β-tubulin gene killed D. melanogaster larvae but did not kill mosquito larvae. RNAi was also used to determine the function of a newly-identified A. aegypti cytochrome P450 (CYP) gene, Aacyp. This gene showed male-biased expression in the mosquitoes, and was expressed primarily in the male abdomen and/or thorax, but unlike some other insect male-biased CYPs, Aacyp was not highly expressed in the reproductive structures. While dsRNA injections successfully knocked down expression of Aacyp, no discernable change in reproductive or male-specific behaviours were noted. Nevertheless, RNAi is still considered a highly versatile tool for both gene function studies and has promising potential to be developed into a novel class of pesticides.