The adenosine deaminase acting on RNA (ADAR) enzymes catalyse the hydrolytic deamination of adenosine (A) to inosine (I) in double stranded (ds) RNA. Mutations in ADAR1 underlie the autoimmune disorder Aicardi Goutiѐres syndrome (AGS). Patients with AGS display heightened levels of type I interferon (IFN) and IFN stimulated genes (ISGs). The first aim of my thesis was to determine whether the mutations found in the human ADAR1 gene affected RNA editing. I found that the ADAR1 mutants identified in the AGS patients have reduced editing activity. Interestingly, the mutations have a greater effect on the IFN-inducible cytoplasmic isoform, ADAR1p150 than on the constitutive ADAR1p110 isoform. These results imply that A-to-I editing plays a role in regulating the type I IFN response. The Adar1 null mouse dies by E12.5 with a type I IFN signature similar to that observed in the AGS patients. The second aim of my thesis was to characterize the immune signalling pathway aberrantly activated in the absence of Adar1. A colleague in our research group rescued the Adar1 null mouse to birth by blocking the cellular response to cytoplasmic dsRNA by generating a double mutant with the mitochondrial antiviral signalling adaptor, Mavs. In the Adar1-/-; Mavs-/- mutant I found that the aberrant immune response is rescued at E11.5. This indicates that MAVS is the downstream adaptor in the aberrant immune response that underlies the embryonic lethality in the Adar1-/- mouse. The third aim of my thesis was to determine if the lack of inosine modification within cellular RNA was triggering the aberrant immune response in the Adar1-/- mouse. To study this, Adar1-/-; p53 -/- mouse embryonic fibroblasts (MEFs) were generated. By reintroducing various ADAR isoforms into the Adar1-/-; p53 -/- MEFs I found that to rescue the aberrant immune response requires both catalytic activity and the location of an ADAR protein within the cytoplasm. Moreover, I demonstrated that transfecting inosine-containing dsRNA oligonucleotides into Adar1-/-; p53 -/- MEFs suppresses the aberrant immune response. Overall my results suggest that A-to-I editing by ADAR1 is an essential RNA modification that is required by the cell to distinguish between ‘self’ and ‘non-self’ RNA. Editing of cellular RNAs prevents an autoimmune response whereas editing of viral RNA may act to suppress a heightened antiviral immune response and prevent long-term damage to the cell.

RNA editing had been considered as a rare exception to the central dogma of molecular biology in which the mRNA truthfully carries genetic code from nucleus to the ribosome for translation. However, researches in the last five years have revealed numerous, widespread RNA A-to-I editing sites in human genome. Although the effects of these editing events require further study, this finding strongly suggests RNA editing occurs frequently, and affects large number of genes. By selectively modifying a few sequences of a gene, RNA editing allows a cell to produce a population of proteins with different properties from a single gene. The major question of this thesis study is whether such editing event is actually dynamically regulated when the cellular physiological processes have to be adjusted in response to changing environment. A previous study screening for Drosophila mutants defected in hypoxia and heat tolerance discovered a hypnos-2 mutant strain which was later found to be defective in dADAR, the drosophila gene encoding the A-to-I editing enzyme, supporting the hypothesis that cells/organisms response to stressful environment by dADAR-mediated RNA editing. Two directions are used to approach how Drosophila uses A-to-I editing to adapt ¡§heat¡¨ environment stress. First, whether the expression pattern of dADAR changes after heat shock was investegated. The result showed the dADAR gene exon 7 self-editing frequency was decreased by heat shock, thus possibly enhances dADAR activity after heat shock processing. Moreover it is worth noting that the isoform without -1 exon transcript were obviously up-regulated, and transcript with -1 sequence is relatively down-regulated. On the other hand, no significant changes in the dADAR mRNA expression levels and in the degrees of two dADAR promoters activity were observed. Second, the changes of editing frequency of 30 known A-to-I editing sites were investigated. Generally the editing frequency of majority editing sites changed after heat shock. Therefore, the dADAR activity, the dADAR gene transcript expression alternations, and A-to-I editing frequency of dADAR target genes did change after heat shock, supporting the notion that change of RNA editing pattern is a mechanism for organism to adapt to drastic environmental change. However, how the edited protein isoforms contribute to heat resistance requires further investigation.

Die Lokalisierung von mRNA ist ein wichtiger regulativer Mechanismus in polarisierten Zellen und in frühen Embryonalstadien. Dort sind räumliche Muster maternaler mRNA für die korrekte Entwicklung der Körperachsen und die Spezifizierung der Keimzellen verantwortlich. Systematische Analysen dieser Prozesse wurden jedoch bisher limitiert durch einen Mangel an räumlicher und zeitlicher Auflösung von Einzelzell- Sequenzierungsdaten. Wir analysierten die Dynamik des räumlichen und zeitlichen Transkriptoms während frühen Embryonalstadien von Zebrafischen. Wir verbesserten Empfindlichkeit und Auflösung von tomo-seq und erfassten damit systematisch räumlich aufgelöste Transkriptome entlang der animal-vegetalen-Achse Embryonen im Einzell-Stadium und fanden 97 vegetal lokalisierte Gene. Außerdem etablierten wir eine Hochdurchsatz kompatible Variante der RNA-Markierungsmethode scSLAM-seq. Wir wendeten diese in Embryonen während der Gastrulation. Von den vegetal lokalisierten Genen waren 22 angereichert in Keimzellen, was eine funktionelle Rolle bei der Spezifizierung von Keimzellen nahelegt. Mit tomo-seq untersuchten wir die evolutionäre Konservierung der RNA-Lokalisierung zwischen Zebrafischen und gereiften Oozyten zweier Xenopus-Arten. Wir verglichen die lokalisierten Gene, suchten nach konservierten 3'UTR-Motiven, und fanden zum Teil überlappende Motive, was auf eine mögliche mechanistische Konservierung der Lokalisierungsmechanismen hinweist. Wir untersuchten auch RNA-Editierung von Adenin zu Inosin während der Embryonalentwicklung und in den Organen erwachsener Fische. In im Gehirn exprimierten Transkripten fanden wir 117 Editierstellen, die hauptsächlich für Ionentransporter kodieren und zum Teil zum Menschen konserviert sind. Die höchsten Editierraten konnten wir in Eierstöcken, Hoden und frühen Embryonen nachweisen, was auf eine mögliche Rolle bei der Regulierung der RNA-Stabilität hindeutet. / Subcellular localization of mRNA is an important regulatory mechanism in polarized cells. In early embryos of many species, spatial patterns of maternal mRNA are essential for the proper development of body axes and the specification of germ cells. These processes have been studied in zebrafish, but systematic analyses have been hindered by a lack of spatial and temporal information in single-cell RNA sequencing. We performed a spatial-temporal analysis of the zebrafish transcriptome during early embryonic development to systematically characterize localized mRNA and the fate of maternal transcripts until gastrulation stage. We enhanced sensitivity and resolution of the tomo-seq method and systematically acquired spatially-resolved transcriptomes along the animal-vegetal axis of one-cell stage zebrafish embryos, and found 97 genes to be localized vegetally. Furthermore, we established an in vivo and high-throughput compatible version of the single-cell RNA labeling method scSLAM-seq in gastrulation stage embryos. We followed localized transcripts until gastrulation and found transcripts of 22 of the vegetally localized genes enriched in primordial germ cells. We propose that these genes have a functional role in the early priming of the germ cell fate. To investigate the evolutionary conservation of vegetal RNA localization, we acquired tomo-seq datasets of mature oocytes of two xenopus species. We compared the pools of localized RNA and searched for conserved 3’UTR motifs. The resulting sets showed high similarity, possibly reflecting a mechanistic conservation of localization pathways. We also investigated RNA A-to-I editing during embryonic development and in organs of adult fish. Specifically, we identified 117 recoding editing sites in the brain that mainly encode for ion transporters and are partly conserved in humans. We detected the highest editing levels in ovary, testes and in early embryos, implicating a potential role in regulating RNA stability.