No description available.

ADAR (Adenosine Deaminases acting on RNA) family proteins are double-strand RNA binding proteins that deaminate specific adenosines into inosines. This A-to-I conversion is called A-to-I RNA editing and is well conserved in the animal kingdom from nematodes to humans. RNA editing is a pre-splicing event on nascent RNA that may affect alternative splicing when the editing occurs in the exon-intron junction or in the intron. Also, editing may change biological function of small RNAs by editing the premicroRNAs or other noncoding RNAs. Editing also alters protein amino acid sequences because inosine in the mRNA base pairs with cytosine and is therefore read as guanosine. In mammals, there are three ADAR family proteins, ADAR1, ADAR2, and ADAR3, encoded by three different genes. So far, no enzymatic activity of ADAR3 is detected. The most frequently edited targets of ADAR1 and ADAR2 are regions covering copies of Alu transposable elements in primates. In addition, loss of some specific editing events leads to profound phenotypes when the editing does not occur correctly. For example, some human neural disorders – such as epilepsy, forebrain ischemia, and Amyotrophic Lateral Sclerosis – are known to be associated with abnormally edited ion channel transcripts. Drosophila has a single ADAR protein (encoded by the Adar gene) that is highly conserved with human ADAR2 (encoded by the ADARB1 gene). To date, 972 editing sites have been identified in 597 transcripts in Drosophila, and approximately 20% of AGO2-associated esiRNAs are edited. Similar to mammals, many ion channel-encoding mRNA transcripts undergo ADAR-mediated A-to-I editing in Drosophila. While Adar1 null mice die at the embryonic stage and Adar2 null mice die shortly after birth due to seizures, Adar null flies are morphologically normal and have normal life span under ideal conditions. However, Adar null flies exhibit severe neurodegeneration and locomotion defects from eclosion, whilst Adar overexpression (OE) is lethal. To better understand the physiological role of RNA editing and ADAR, and to shed light on ADAR-related human disease, I used Drosophila Adar mutant flies as a model organism to investigate phenotypes, and to find chromosomal deletions and specific mutations that rescue the neural-behavioural phenotype of the Adar null mutant flies. Using the publicly available chromosomal deletions collectively covering more than 80% of the euchromatic genome of Chromsome III, I performed a genetic screen to find rescuers of the lethality caused by Adar overexpression. I confirmed that mutation in Rdl (Resistant to dieldrin, the gene encoding GABAA receptor main subunit) rescues. This rescue was not likely caused by effects on Adar expression level or activity. Driven by the hypothesis that the rescue may be due to reduction in GABAergic input to neurons, I recorded spontaneous firing activity of Drosophila larval aCC motor neurons using in vivo extracellular current recording technique. As expected, the neurons overexpressing Adar had much less activities compared with wild type neurons. Also, I found that Adar null fly neurons fired much more and showed epilepsy-like increased excitability. Although feeding PTX (Picrotoxin), a GABAA receptor antagonist, failed to rescue the lethality, reducing the expression of GAD1 to reduce synthesis of GABA was able to rescue the ADAR overexpression lethality. These results suggest that ADAR may finetune neuron activity synergistically with the GABAergic inhibitory signal pathway. I used MARCM (mosaic analysis using a repressible cell marker) to detect cellautonomous phenotypes in Adar null cells in otherwise wild type flies. Although neurodegeneration, observed as enlarged vacuoles formation in neurophils, was detected both in histological staining and EM images, the Adar null neurons marked with GFP from early developmental stages were not lost with age. Nevertheless, swelling in the axons or fragmentation of the axon branches of Adar null neurons was sometimes observed in the midbrain. By comparing the Poly-A RNA sequencing data from Adar null and wild type fly heads, we detected significant upregulation of innate immune genes. I confirmed this by qRT PCR and found that inactive ADAR reduces the innate immune gene transcript levels almost as much as active ADAR does. Further, using the locomotion assay, I confirmed that reintroducing inactive ADAR into Adar null flies can improve the flies’ climbing ability. Based on the Adar null flies having comparatively low viability, I performed a second deficiency screen to find rescuers of Adar null low viability using the same set of deficiencies as in the lethality rescue screen described above. I found seven deletions removing 1 to 37 genes that significantly increased the relative viability of the Adar null flies. However, not all the rescuing deficiencies also improved the Adar null locomotion. One rescuing gene, CG11357 was mapped from one of the rescuing deficiencies, and some mutant alleles of cry, JIL-1 and Gem3 also showed significant effects on the Adar null fly viability. The single gene viability rescuers were also not necessarily locomotion or neurodegeneration rescuers. Although the initial aim was to find neural-behavioural rescuing genes from the viability screen, the viability rescuers found in the screen are more likely to play a role in different aspects of stress response for survival.

Adenosine (A) to inosine (I) RNA editing is a widespread post-transcriptional mechanism in mammals that contributes to increase the protein diversity. Adenosine deaminases that act on RNA (ADARs) are the enzymes catalyzing RNA editing. ADARs are particularly active within the brain where they act on transcripts involved in neurotransmission. In this work the editing efficiency of all known site-selectively edited substrates have been analyzed during development of the mouse brain. We show that there is a global regulation of RNA editing, where editing levels of sites increase as the brain matures. This increase in editing efficiency cannot be explained by an increase in ADAR protein expression. During differentiation of primary cells from the mouse brain, editing levels increases similar to what we observe in vivo. Interestingly, the subcellular localization of the ADAR enzymes of cultured neurons show a different distribution in immature compared mature neurons. An accumulation of the ADAR enzymes in the nucleus may explain elevated A-to-I editing during brain development. Furthermore, we find that certain adenosines work as principal sites where editing of the transcript is initiated. Presumably, these sites are kinetically favored and are hypothesized to recruit the ADAR enzymes to the RNA substrate. Editing is then coupled to sites located in multiples of 12 nucleotides from each other. Interestingly, these sites reside on the same side in the 3D helix structure. The Gabra-3 transcript is site-selectively edited at a single position changing an isoleucine codon for a methionine upon editing. Gabra-3 encodes the a3 subunit of the GABAA receptor. We show that receptors assembled with edited a3 are less stable at the cell surface than the non-edited a3. We propose that the amino acid change upon editing, could affect protein interactions important for trafficking and stability of the GABAA receptors. Further, the editing event in a3 may have the function to reduce the number of a3 subunits in favor of other a subunits. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

The SARS-CoV2 pandemic has demonstrated on a global scale that viral infections can be highly contagious, can evolve rapidly, and are challenging to treat. The immune system provides cells with various control mechanisms to detect and prevent the spread of viral infection and further damage to the host. However, viruses have evolved methods to evade immunity, resulting in persevered viral replication and proliferation. Chronic viral infections occur when a virus evades immunity and persists in the body for an extended period, which can lead to increasingly harmful damage to the host, including increased risk of cancer. When immunity proves insufficient, alternative methods to sense virally infected cells can allow for detection and targeted elimination of the virus, which is especially necessary in cases of chronic viral infection. In this thesis, the development and characterization of RNA-editing enzymes based on adenosine deaminase acting on RNA (ADAR) that have been engineered to activate in response to viral protease is discussed. Specifically, methods for targeting ADAR editing to specific mRNA transcripts and strategies in which the editing activity of engineered ADARs has been made conditional upon viral proteolytic activity are explored. The development of fluorescent and quantitative assays to characterize systems are described and the implementation of the system to control downstream transcriptional activity is discussed. This thesis explores establishing the viability of a viral protease sensor able to be self-contained in an RNA circuit, which in the future may provide a treatment method for patients with severe symptoms or chronic viral infection. The ability to sense virally infected cells and create a functional output in specific response to viral protease presence as a potential future treatment of chronic viral infection is explored through viral protease activation of engineered ADAR enzymes to enable editing of specific mRNA transcripts. / 2025-05-24T00:00:00Z

Les éléments Alu sont les retrotransposons les plus prolifiques chez l’humain avec plus d’1 million de copies occupant plus de 10% du génome. Afin de contrecarrer l’expansion des rétro-éléments, les organismes ont développés différents mécanismes pour préserver l’intégrité de leurs génomes. Le plus proéminent, également utilisé pour lutter contre la réinsertion d’ADN viral dans le génome hôte, est l’édition de l’ARN. Chez les mammifères, la plus courante est la déamination de l’adénine en inosine catalysée par la famille de protéine ADAR dont Les principales cibles sont les éléments Alu chez l’humain. L’édition des éléments Alu conduit à leur séquestration dans le noyau des cellules, mute leurs promoteurs internes, cible de l’ARN polymérase III (POLIII), et leurs queues poly-A, prévenant ainsi leur future rétrotransposition. Dans la première partie de cette étude, l’analyse de données de séquençage haut-débit révèle que ~40% des éléments Alu sont reconnus par POLIII, qu’ils sont présents en tant que petits ARN dans le cytoplasme et le noyau des cellules, que certain d’entre eux sont associés à la chromatine, et que la transcription des éléments Alu est un phénomène courant dans les tissus somatiques qui concorde avec l’expression d’éléments LINE1 fonctionnels. Ceci suggère que la rétrotransposition peut être un mécanisme normal dans la plupart des tissus humains. Enfin, l’analyse de l’expression des éléments Alu et LINE1 chez la souris montre que la transcription de rétrotransposons n’est pas spécifique de l’humain. Dans la seconde partie de cette étude, une nouvelle méthode a été développée pour explorer l’impact de l’édition de l’ARN sur le transcriptome en identifiant les ARN édités par séquençage haut-débit. Dans un premier temps, un anticorps ciblant ADAR a été utilisé pour extraire les ARN associés aux protéines de l’édition. Cette méthode n’étant pas suffisamment efficace, une autre stratégie, qui extrait directement les ARN contenant de l’inosine, a été développée : dans un premier temps, l’ARN est fixé à des billes magnétiques par leurs extrémités 3’, ensuite, les billes sont traitées au glyoxal/acide borique et à la RNAse T1 pour libérer la région 5’ des ARN contenant une ou plusieurs inosines, et enfin, les ARN libérés sont séquencés par séquençage haut débit. En utilisant cette méthode, 1822 sites d’éditions ont été identifiés dans l’ARN de cerveau de souris, incluant 28 nouveaux sites présents dans des séquences codantes qui conduisent à des mutations non-synonymes des futures protéines. Des sites d’éditions ont aussi été observés pour la première fois dans les ARN ribosomaux, les snoRNA et les snRNA. / The Alu repeats comprise more than 10% of the human genome. They spread in the genome by retrotransposition. As a response to this invasion, organisms developed mechanisms to preserve the integrity of their genome, such as RNA editing. The most abundant type of editing in mammals is A-to-I editing where the ADAR proteins transform adenosine into inosine and targets mainly Alu elements in human. Editing of the Alu elements leads to their sequestration in the nucleus and mutates their internal POLIII promoter and their poly-A tail, thus preventing their subsequent transposition. In the first part of this study, we challenged the view that Alu elements are dormant occupant of the genome by characterizing their activity. Deep-sequencing data analyses revealed that ~40% of Alu elements can bind POLIII, they present a definite localization in the cell and associate with chromatin and polysomes, and that Alu elements transcription is a widespread phenomenon in normal tissues which correlates with functional LINE1 elements expression. This suggested that Alu element retrotransposition may be a natural mechanism in most normal human tissues. Further analyses showed that SINE and LINE expression in somatic tissues was not exclusive to human but also occurs in mouse. Finally, attempts were made to identify tissue specific insertions in the human genome resulting from retrotransposition events. In the second part of this study, a new method was developed to understand the full impact of RNA editing on transcriptomes by characterizing the edited RNA in a high-throughput fashion. First, immunoprecipitation was attempted to pull-down RNA associated with the editing enzymes ADARs. Since this method was inefficient, another approach purifying directly the edited RNA was developed. First, the RNA was sequestered on magnetic beads. Then an inosine specific cleavage based on RNAseT1 treatment of RNA protected with glyoxal and borate allowed the separation of the edited RNA from the total RNA. Finally, deep sequencing was used to identify edited RNA. 1,822 editing sites were found in mouse brain RNA by this method, including 28 new editing sites modifying the coding sequences of genes and editing in rRNA, snoRNA and snRNA which were never observed before.

The transcriptome of the mammalian brain is extensively modified by adenosine to inosine (A-to-I) nucleotide conversion by two adenosine deaminases (ADAR1 and ADAR2). As adenosine and inosine have different base pairing properties, A-to-I RNA editing shapes the functional output of both coding and non-coding RNAs (ncRNAs) in the brain. The aim of this thesis was to identify editing events in small regulatory ncRNAs (miRNAs) and to determine their temporal and spatial editing status in the developing and adult mouse brain. To do this, we initially analyzed the editing status of miRNAs from different developmental time points of the mouse brain. We detected novel miRNA substrates subjected to A-to-I editing and found a general increase in miRNA editing during brain development, implicating a more stringent control of miRNAs as the brain matures. Most of the edited miRNAs were found to be transcribed as a single long consecutive transcript from a large gene cluster. However, maturation from this primary miRNA (pri-miRNA) transcript into functional forms of miRNAs is regulated individually, and might be influenced by the ADAR proteins in an editing independent matter. We also found that edited miRNAs were highly expressed at the synapse, implicating a role as local regulators of synaptic translation. We further show that the increase in editing during development is explained by a gradual accumulation of the ADAR enzymes in the nucleus. Specifically for ADAR2, we found a developmentally increasing interaction with two factors, importin-α4 and Pin1, that facilitate nuclear localization of the editing enzyme. We have also found that selectively edited stem loops often are flanked by other long stem loop structures that induce editing in cis. This may explain why multiple pri-miRNAs are edited within the same cluster. In conclusion, this thesis has significantly increased the understanding of the dynamics of both editing substrates and enzymes in the developing and mature brain. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Extreme acid resistance contributes to the successful transmission of enterohemorrhagic E. coli (EHEC) through acidic food matrices and the stomach, allowing it to gain access to the intestine and elicit disease in humans. Alternative sigma factor N (σN, encoded by rpoN) was previously identified as a novel regulator of extreme acid resistance in EHEC. This study investigated the role for σN and co-expressed products of the rpoN operon in the acid resistance phenotype of EHEC. The results revealed that σN primarily controls acid resistance through repression of the glutamate-dependent acid resistance (GDAR) system through control of the σS-directed GadXW pathway. σN was also determined to repress additional acid resistance systems, including arginine-dependent acid resistance, and an anaerobic acid resistance mechanism. Two gene products of the rpoN operon, hpf and ptsN, were also determined to negatively affect GDAR, as well as expression of the σN dependent genes glnA, astA, and pspA. Mutation of hpf and ptsN did not however alter the transcription of rpoN. Transcript levels of rpoN operon genes were observed to be differential, and inconsistent with the hypothesis of expression as a single transcriptional unit. Together this data signifies the importance of rpoN operon genes in the negative regulation of extreme acid resistance systems, and suggests that the products of hpf and ptsN control the activity of σN at its promoters.

Résumé non transmis / SOX10 is a transcription factor with well-known functions in neural crest and oligodendrocyte development. Mutations in SOX10 were first associated with Waardenburg-Hirschsprung disease (WS4; deafness, pigmentation defects and intestinal aganglionosis). However, variable phenotypes that extend beyond the WS4 definition are now reported. The neurological phenotypes associated with some truncating mutations are suggested to be the result of escape from the nonsense-mediated mRNA decay pathway; but, to date, no mechanism has been suggested for missense mutations, of which approximately 20 have now been reported, and about half of which are redistributed in vitro to nuclear bodies of undetermined nature and function. Here, we reported that the paraspeckle protein p54NRB, which plays a crucial role in the regulation of gene expression during many cellular processes including differentiation, and is a member of the Drosophila behavior Human Splicing (DBHS) protein family, interacts and acts synergistically with SOX10 to regulate several target genes. Interestingly, this multifunctional protein, as well as two other members of the DBHS protein family, co-localized with SOX10 mutants in nuclear bodies, suggesting the possible paraspeckle nature of these foci or re-localization of the DBHS members to other subnuclear compartments. Remarkably, the co-transfection of wild-type and mutant SOX10 constructs led to the sequestration of wild-type SOX10 in mutant-induced foci. However, only foci forming mutants exclusively found in the nucleus altered synergistic activity between SOX10 and p54NRB. We proposed that such a dominant negative effect may contribute to or be at the origin of the progressive neurological phenotype observed in affected patients.One of the roles of p54NRB is the regulation of gene expression via nuclear retention, by binding to hyperedited IRAlu sequences this protein blocks their efficient export to the cytoplasm (Zhang and Carmichael., 2001), we then decided to get into the world of editing. Editing, is a molecular mechanism characterized by the deaminase conversion of adenosines into inosines (A-to-I). In mammals, this molecular modification, is performed by a cluster of three enzymes named Adenosine deaminases acting on RNA (ADARs 1-3) (Wagner RW et al., 1989).In order to evaluate the role of ADAR1 in NC development, we decided to conditionally invalidate the expression of this enzyme using the NC specific HtPA-Cre line. Two main crossing strategies were followed, one including the Rosa26R-LacZ marker (RADR crossing) to track the NCCs and one not (CADR crossing). Globally, the Adar1 deficient pups harvested from the CADR crossing presented with 100% mortality within the first three days after birth. The survival rate of the mutants generated using the second strategy (RADR) was higher, however, none of the mutants survived up to P30. In general, the mutants of the latest crossing, presented with pleiotropic NC phenotype: abnormal melanocyte, ENS and sciatic nerve defects were observed.

mRNA molecules form an intermediate in the transfer of sequences from DNA to ribosomes in order to guide protein production. Errors can be introduced into mRNA, producing aberrant proteins which place a strain on cellular regulatory machinery, causing increased risks of apoptosis, cancer, and decreased fitness. These errors may be introduced due to decreased transcriptional proofreading capabilities, exposure to chemicals, or mistakes in RNA editing machinery. It is important to investigate these causes of transcription errors to better understand the long-neglected area of mRNA fidelity which has such significant impacts on our cellular functions. In this paper, it was determined that addition of adenine opposite from abasic sites, not genomic uracil pairing with adenine, are a probable cause of G-to-A transcription errors. That exposure to Roundup causes increased levels of transcription errors, potentially due to oxidative stress. And finally, that off-target ADAR gene editing of transcripts occurs at high levels.

No description available.