Thesis (M.A.) -- Central Connecticut State University, 2008. / Thesis advisor: Barry Hoopengardner. "... in partial fulfillment of the requirements for the degree of Master of Arts in Biomolecular Sciences." Includes bibliographical references (leaves 34-36). Also available via the World Wide Web.

Post-transcriptional regulation of gene expression involves a diverse set of mechanisms such as RNA splicing, RNA localization, and RNA turn-over. Adenosine to Inosine (A-to-I) RNA editing is an additional post-transcriptional regulatory mechanism. Temporally, it occurs after transcription and before RNA splicing and has been shown in some instances to possibly modulate alternative splicing events. This is the case for example, with the pre-mRNA encoding the GluR- 2 subunit of AMPA receptor, a glutamate-activated ion channel. ADAR (Adenosine deaminase acting on RNA) proteins bind to double-stranded regions in pre-messenger RNAs. They deaminate specific adenosines, generating inosines; if the editing event occurs within the coding region, inosine is then interpreted as guanosine by the ribosomal translational machinery, changing codon meaning. These editing events can increase the repertoire of translated proteins, generating molecular diversity and modifying protein function. In mammals there are four ADAR genes: ADAR1, ADAR2, ADAR3 and TENR. ADAR3 and TENR are enzymatically inactive. All the proteins have two types of functional domains: (i) the catalytic deaminase domain at the carboxyl-terminus and (ii) the double stranded RNA binding domains, dsRBDs, at the amino terminus. ADAR1 and ADAR2 differ significantly at the amino terminus, by the number of the dsRNA binding domains (three and two dsRBDs for ADAR1 and ADAR2 protein, respectively). The differences observed between ADAR1 and ADAR2 are likely to reflect the different repertoires of substrates edited by these two enzymes. Data concerning the conservation of Adar genes throughout evolution suggest that Drosophila melanogaster has a unique Adar gene which is a true ortholog of human ADAR2 rather than an invertebrate gene ancestral for both vertebrate genes. Flies that are null mutants for Adar (Adar5G1 mutants) display profound behavioral and locomotive deficits. Impairment in motor activity of the mutants is succeeded by age-dependent neurodegeneration, characterized by swelling within the Adar-null mutant fly brain. The initial focus of my thesis was to elucidate what causes Adar mutant phenotypes or, whether it is possible, to suppress them. I took advantage of Drosophila genetics to establish a forward genetic screen for suppressors of reduced Adar5G1 viability which is approximately 20-30% in comparison to control flies at eclosion. The results from an interaction screen on Chromosome 2L were further confirmed using Exelixis P-element insertion lines. The screen revealed that decreasing Tor (Target of rapamycin) expression suppresses Adar mutant phenotypes. TOR plays a role in maintaining cellular homeostasis by balancing the metabolic processes. It controls anabolic events by phosphorylating eukaryotic translation initiation factor 4E-binding protein (4E-BP) and p70 S6 kinase (S6K) and inducing cap-mediated translation. However, different types of stress, signals or increased demand in catabolic processes, converge to reduce TOR enzymatic activity. This results in long-lived proteins and organelles being engulfed in double-membrane vesicles and degraded; this bulk degradation process is called (macro)autophagy. The second aim of my thesis was to clarify which pathway, downstream to TOR, was responsible for the suppression of Adar-null phenotypes. I mimicked the effect of reduced Tor expression by manipulating genetically the cap-dependent translation and the autophagy pathways. Interestingly, boosting the expression of Atg (autophagy specific genes) genes, such as, Atg1 and Atg5, thereby increasing the activation rate of the autophagy pathway, suppresses Adar5G1 phenotypes. Finally, I found that Adar5G1 mutant flies have an increased level of autophagy that is observable from the larval stage. I investigated possible stresses affecting our mutants; Adar-mutant larval fat cells show ER stress triggering an unfolded protein response as indicated by expression of XbpI-eGFP reporter. Thus, ER stress might induce increased autophagy and it can lead to locomotive impairments and neurodegeneration in Adar-null mutants. These results suggest a function for the Adar gene in regulating cellular stress.

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.

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.

Thesis (Ph. D.)--Colorado State University, 2004. / Includes bibliographical references.

Thesis (M.S.)--Villanova University, 2010. / Chemistry Dept. Includes bibliographical references.

Thesis (Ph. D. in Pharmacology)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

archives@tulane.edu / A-to-I RNA editing, catalyzed by a family of enzymes called adenosine deaminases acting on RNA (ADARs), brings broad significance in various biological processes. To date, the roles of ADARs and its associated RNA editing in Epstein-Barr virus (EBV)’s life cycle and pathogenesis are still largely unknown. To fill this significant knowledge gap, we utilized our well-established next-generation RNA sequencing-based computational approaches and traditional molecular biology methodologies to elucidate the triangle relationship between ADARs, RNA-editing, and EBV infection. The expression of ADARs was first evaluated in a cohort of EBV-associated lymphoma cells. A constitutive expression of ADAR1, the predominant form of ADARs, was observed in the examined cells. In synchronous EBV reactivation cell models, we found that EBV reactivation led to a decreased expression of ADAR1 as well as a global suppression of A-to-I RNA editing. Further, we found that expression of the key viral trans-activator Zta inhibited ADAR1 expression in EBV-associated lymphoma cells. Analyses of the ADARs-mediated RNA editing events revealed novel editing sites on viral lytic transcripts. Knockdown of ADAR1 led to a global suppression of RNA-editing accompanied by a more robust EBV reactivation. Meanwhile, the enhanced expression of ADAR1 inhibited Zta’s expression and transactivation function. Together, our findings reveal a novel mechanism controlling the balance of EBV life cycle, in which ADAR1 and associated RNA editing events help maintain the viral latency by silencing Zta; whereas a bona fide lytic signal leads to high-level Zta expression by inhibiting ADAR1 and ADARs-mediated RNA editing. / 1 / Yi Yu

Unlike mammals, adult salamanders possess an intrinsic ability to regenerate complex organs and tissue types, making them an exciting and useful model to study tissue regeneration. The aims of this thesis are two fold, (1) to develop and characterize a reproducible cardiac regeneration model system in the newt, and (2) to decipher the cellular and molecular underpinnings involved in regeneration. In Paper I of this thesis we developed a novel and reproducible heart regeneration model system in the red-spotted newt and demonstrated for the first time the newt’s ability to regenerate functional myocardial muscle, following resection injury, without scarring. The observed findings coincide with an increase in several developmental cardiac transcription factors, wide-spread cellular proliferation of cardiomyocytes and non-cardiomyocyte populations in the ventricle and reverse-remodeling at later time points during regeneration. Of further interest was the identification of functionally active Islet1+ve and GATA4+ve cardiac precursor cells in regenerating areas. The observation of such cell types further compels the similarity between mammalian cardiac development and newt cardiac regeneration and justifies these animals as suitable model organisms for studying heart regeneration. In Paper II we wanted to decipher the molecular cues possibly driving cardiac regeneration in newts. Here we used qualitative and quantitative methods to delineate the function microRNAs (miRNAs) have in this process. One interesting candidate, miR-128, a known tumor suppressor miRNA and regulator of myogenesis, was found to have a regulatory role in controlling non-cardiomyocyte hyperplasia during newt cardiac regeneration. Of further interest was the discovery of a novel binding site of miR-128 in the 3’UTR of Islet1. We speculate that the natural increase in miR-128 expression levels during cardiac regeneration functions as a fine-tuning mechanism to control cellular proliferation of precursor cells. In Paper III of my thesis we sought to explore if a link exists between RNA editing, a wide-spread post-transcriptional process and regeneration. We observed that A-to-I editing enzymes (ADARs) are present in regenerating newt tissues and the localization of ADAR1 alternates between nuclear and cytoplasmic compartments during regeneration. This activity of ADAR1 during regeneration may be partly responsible for driving the cellular plasticity that is needed during multiple phases of tissue regeneration in the red-spotted newt. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

RNA editing by adenosine deamination is catalyzed by members of an enzyme family known as adenosine deaminases that act on RNA (ADARs). ADARs can change the structure of RNA by changing an AU base-pair to an IU mismatch. This frequently modifies the function of the encoded protein, and an emerging theme associated with A-to-I mRNA editing is that tissues often regulate the ratio of proteins expressed from edited and unedited mRNAs to fine-tune cellular responses and functions. In mammals, pre-mRNA of receptor proteins involved in neurotransmission, including serotonin receptors and glutamate receptors, are edited. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. To identify RNAs containing inosine residues, this study used a multi step approach; including (1) inosine-specific base cleavage and RNase T1 digestion, (2) purification of polyA-tailed mRNA, (3) RT w/ T7-polydT primer, (4) probe synthesis and microarray analysis. Using this method it is possible to identify novel targets of A to I editing. Approximately 100 genes showed a significant decrease in two arrays. Future analysis of these targets should reveal the biomedical significance of A-to-I editing.