Master of Science, The Ohio State University, 2012, Plant Pathology

The interaction between Arabidopsis thaliana double stranded RNA binding protein 4 (DRB4) and dicer-like 4 (DCL4) has been shown to be necessary for RNA silencing. In addition, DRB proteins may also facilitate the loading of siRNAs into RISC. However a mechanistic understanding of their role in anti-viral silencing requires a detailed knowledge of interactions between the silencing pathway components, as well as interactions between different domains of the individual components. Here we report our efforts at resolving these issues using both the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Using both BiFC and Y2H assay we show that DRB4 interacts with itself, and that it's N-terminal half, consisting of two dsRNA binding motifs (dsRBMs), mediates this interaction. Splitting these two dsRBMs resulted in a complete loss of interactions in yeast. Conversely, the C-terminal half of DRB4 was unable to self-interact, and was completely dispensable for DRB4 dimerization. We further delineated the amino acid residues essential for this interaction by mutating the highly conserved amino acid residues within the two dsRBMs. Mutating both the histidine and lysine residues, at position 32 and 133 respectively, led to a complete loss of DRB4 self-interaction in yeast. Interestingly, these amino acids have been previously shown to be critical for the DRB4/DCL4 interaction. Our current data suggests that DRB4 likely exist as a dimer in cells and this dimerization is necessary for its interaction with DCL4 and hence anti-viral RNA silencing. Our findings provide a better understanding of the molecular events at the initiation step of anti-viral RNA silencing pathway in Arabidopsis thaliana.

Committee: Feng Qu Dr. (Advisor); Thomas Mitchell Dr. (Committee Member); Lucy Stewart Dr. (Committee Member) Subjects: Biology; Molecular Biology; Plant Biology; Plant Pathology; Plant Sciences

Doctor of Philosophy, University of Toledo, 2020, Biology (Cell-Molecular Biology)

Virus infection leads to activation of the interferon (IFN)-induced endoribonuclease RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRRs), like retinoic acid-inducible I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), to further amplify IFN production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how cells coordinate RNA sensing with signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce the formation of antiviral stress granules (avSGs) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) and recruit the antiviral proteins RIG-I, PKR, OAS, and RNase L to avSGs. Biochemical analysis of purified avSGs showed interaction of a key stress granule protein, G3BP1, with only PKR and RIG-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production, but not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection, demonstrating the importance of avSGs in RNase L-mediated host defense. We propose a role during viral infection for RNase L-cleaved RNAs in inducing avSGs containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to mount an effective antiviral response.

Committee: Malathi Krishnamurthy (Committee Chair); Travis Taylor (Committee Member); Saurabh Chattopadhyay (Committee Member); Scott Leisner (Committee Member); Deborah Chadee (Committee Member) Subjects: Biology; Virology

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2010, College of Science

Project I: Currently, chemotherapy is the only treatment for metastatic prostate cancer. However, due to toxicity and resistance, the currently available chemotherapeutic drugs are not good enough to control this disease. To find a novel and effective method to treat this disease, we studied the effect of interferons (IFNs) and double-stranded RNA (dsRNA) on the apoptosis of prostate cancer cells. Interestingly, pretreatment of PC3 cells, a human prostate adenocarcinoma cell line, with IFNs significantly sensitized these cells to dsRNA induced apoptosis, and cell apoptosis was confirmed by a variety of assays such as Annexin V, TUNEL, DNA fragmentation and the activity of caspase 3. In comparison with IFN-α or beta treatment, IFN-γ treatment remarkably augmented dsRNA-induced apoptosis in PC3 cells. By using mutant cell lines, we demonstrated that IFN-signaling is necessary for these effects. Silence of dsRNA-dependent protein kinase R (PKR) and RNAse L by siRNA did not have any significant impact on this event, suggesting that neither RNase L nor PKR is involved. Further investigation of the apoptotic pathway revealed that Bak, a pro-apoptotic member of the Bcl-2 family, is up-regulated by IFN-γ and dsRNA. Our findings may lead to the design of novel therapeutic strategies for prostate cancer. Project II: Transmembrane and coiled-coil domains 1 (TMCO1) is a membrane-associated protein with unknown function. Recently, a homozygous frame shift mutation, c.139_140delAG, has been identified in the TMCO1 gene in patients with TMCO1 defect syndrome (TDS). TDS is characterized by distinctive craniofacial dysmorphism, skeletal anomalies, and mental retardation. In order to study the biological function of this gene, human TMCO1 was expressed in both bacteria and mammalian cells. The recombinant TMCO1 expressed in bacteria was purified in order to prepare an antibody, and subcellular localization revealed that TMCO1 may be expressed in the mitochondria of cells. Further (open full item for complete abstract)

Committee: Aimin Zhou PhD (Advisor); David Anderson PhD (Committee Member); Xue-long Sun PhD (Committee Member); Crystal Weyman PhD (Committee Member); Sihe Wang PhD (Committee Member) Subjects: Molecular Biology