Somatic embryogenesis can provide researchers with an important tool to study the physiological and molecular mechanisms involved in embryo development. In spruce, few lines are able to produce fully developed embryos due to the presence of malformed meristems. Genes from two families known as KNOX (knotted-like homeobox) and ARGONAUTE (AGO) have previously been found to be involved in meristem development and maintenance. This work documents the discovery of a new member of the AGO family of proteins designated as PgAGO and the further study of a KNOX gene known as HBK2. The complete coding sequence of both PgAGO and HBK2 was obtained through screening of cDNA libraries generated from white spruce (Picea glauca) somatic embryos. RNA in-situ hybridization studies showed that PgAGO mRNAs accumulate preferentially within cells of the shoot and root apical meristems in developing spruce embryos. In addition, the expression of PgAGO was low in white spruce lines unable to produce embryos in culture. Norway spruce (Picea abies) embryogenic tissue was transformed via microprojectile bombardment with an antisense construct of PgAGO. Down-regulation of PgAGO altered proper development of the apical meristems and reduced embryo regeneration. RNA in-situ hybridization studies showed that HBK2 is specifically expressed in the sub-apical and cortical regions of developing embryos. Like PgAGO, HBK2 expression was diminished in white spruce lines unable to produce embryos in culture. Transformation experiments with antisense constructs of HBK2 completely arrested somatic embryo development. This study reveals the importance of a functional meristem during embryo development. / October 2006

Somatic embryogenesis can provide researchers with an important tool to study the physiological and molecular mechanisms involved in embryo development. In spruce, few lines are able to produce fully developed embryos due to the presence of malformed meristems. Genes from two families known as KNOX (knotted-like homeobox) and ARGONAUTE (AGO) have previously been found to be involved in meristem development and maintenance. This work documents the discovery of a new member of the AGO family of proteins designated as PgAGO and the further study of a KNOX gene known as HBK2. The complete coding sequence of both PgAGO and HBK2 was obtained through screening of cDNA libraries generated from white spruce (Picea glauca) somatic embryos. RNA in-situ hybridization studies showed that PgAGO mRNAs accumulate preferentially within cells of the shoot and root apical meristems in developing spruce embryos. In addition, the expression of PgAGO was low in white spruce lines unable to produce embryos in culture. Norway spruce (Picea abies) embryogenic tissue was transformed via microprojectile bombardment with an antisense construct of PgAGO. Down-regulation of PgAGO altered proper development of the apical meristems and reduced embryo regeneration. RNA in-situ hybridization studies showed that HBK2 is specifically expressed in the sub-apical and cortical regions of developing embryos. Like PgAGO, HBK2 expression was diminished in white spruce lines unable to produce embryos in culture. Transformation experiments with antisense constructs of HBK2 completely arrested somatic embryo development. This study reveals the importance of a functional meristem during embryo development.

Somatic embryogenesis can provide researchers with an important tool to study the physiological and molecular mechanisms involved in embryo development. In spruce, few lines are able to produce fully developed embryos due to the presence of malformed meristems. Genes from two families known as KNOX (knotted-like homeobox) and ARGONAUTE (AGO) have previously been found to be involved in meristem development and maintenance. This work documents the discovery of a new member of the AGO family of proteins designated as PgAGO and the further study of a KNOX gene known as HBK2. The complete coding sequence of both PgAGO and HBK2 was obtained through screening of cDNA libraries generated from white spruce (Picea glauca) somatic embryos. RNA in-situ hybridization studies showed that PgAGO mRNAs accumulate preferentially within cells of the shoot and root apical meristems in developing spruce embryos. In addition, the expression of PgAGO was low in white spruce lines unable to produce embryos in culture. Norway spruce (Picea abies) embryogenic tissue was transformed via microprojectile bombardment with an antisense construct of PgAGO. Down-regulation of PgAGO altered proper development of the apical meristems and reduced embryo regeneration. RNA in-situ hybridization studies showed that HBK2 is specifically expressed in the sub-apical and cortical regions of developing embryos. Like PgAGO, HBK2 expression was diminished in white spruce lines unable to produce embryos in culture. Transformation experiments with antisense constructs of HBK2 completely arrested somatic embryo development. This study reveals the importance of a functional meristem during embryo development.

The RNA interference (RNAi) mechanism is a conserved system of nucleic acid manipulation, based on the interaction between small RNA guide molecules and Argonaute effector proteins. RNAi pathways are found in the vast majority of eukaryotes, and have diversified into a broad array of functions including gene regulation, antiviral immunity and transposable element (TE) suppression. Many of these functional innovations coincide with duplication of Argonaute genes, suggesting that gene duplication may be a key driving force in the diversification of RNAi. However, few studies have explicitly investigated Argonaute evolution after duplication. In this thesis, I focused on the impact of gene duplication on the evolution of Argonaute genes. Argonaute genes in different species exhibit a broad array of functions; however, most of our knowledge of Argonaute function in the arthropods is based on studies in D. melanogaster. To compare the rate of duplication and its evolutionary effect between different Argonaute subclades, I quantified gene turnover rates and evolutionary rate change in Argonaute genes from 86 Dipteran species (Chapter 2). I find that duplication rate varies widely between subclades and lineages, and that duplication drives an increase in evolutionary rate, suggesting that functional divergence after Argonaute duplication is prevalent throughout the Diptera. In the obscura group of Drosophila I identified a series of recent duplications of Argonaute2 (Ago2), which has antiviral and anti-TE functions in D. melanogaster. To quantify the extent of functional divergence between these paralogues, I measured the expression of paralogues from three species (D. subobscura, D. obscura and D. pseudoobscura), in different tissues and under viral challenge (Chapter 3). I find that the majority of Ago2 paralogues have specialised to a derived testis-specific role, potentially to suppress TE activity or meiotic drive. While CRISPR-Cas9 mediated knockout of these genes ultimately proved unsuccessful (Chapter 5), the selective importance of their derived function is suggested by its multiple independent origins. Functional novelty, as appears to have evolved in the obscura group Ago2 paralogues, is often driven by strong selection. To quantify the evolutionary rate and positive selection on these paralogues, I gathered intraspecies polymorphism data for all paralogues in D. subobscura, D. obscura and D. pseudoobscura, combining this with publicly-available population genomic data for D. pseudoobscura (Chapter 4). I find that the majority of paralogues in all species have extremely low diversity, indicative of recent selection, and identify recent selective sweeps on three paralogues in D. pseudoobscura. This suggests that the majority of Ago2 paralogues in the obscura group are evolving under strong positive selection. In this thesis I have aimed to quantify the effect of gene duplication on Argonaute evolution. I find that Argonaute genes duplicate frequently in some lineages, resulting in the evolution of derived functions that may be driven by positive selection. This suggests that functional diversification is prevalent in eukaryotic RNAi, and is likely to coincide with expansion of the Argonaute gene family.

It has previously been demonstrated that lactic acid bacteria are able to influence the innate immune response of host cells. One way this can be achieved is through modulation of inflammatory cascades initiated by pattern recognition elements such as toll-like receptors. Micro RNA can also have an effect on innate immunity, and has been shown to have an influence in regulation of these pathways in immune responsive cells. However, it is yet to be determined if the interaction between lactic acid bacteria and host cells involves regulation of the RNA interference machinery involved in micro RNA biogenesis. Three of the key proteins responsible for miRNA production and activation are Argonaute 2, Dicer and Drosha. Together, these are responsible for the processing and activation of miRNA to enable post-transcriptional gene regulation. In this study we have used quantitative PCR to evaluate changes in gene expression of these enzymes in HT29 and VK2/E6E7 mucosal epithelial cells after treatment with Lactobacillus and uropathogenic bacteria. We have found that bacterial treatment downregulates gene expression of elements responsible for miRNA biogenesis, and our results showed different responses dependent on the cell line. In addition to this we have also determined stable reference genes for use in further studies involving this model. Our findings indicate that modulation of the RNAi machinery might be an important element of immune regulation by bacterial colonists.

No description available.

La mitochondrie est une organite ayant un rôle central dans le métabolisme énergétique de la cellule. Bien que la mitochondrie exprime son propre génome, plusieurs protéines et ARN non-codants issus du génome nucléaire sont nécessaires à la biogenèse et aux fonctions mitochondriales. Les microARNs (miRNAs) sont de petits ARN non-codants qui s'associent à la protéine Argonaute 2 (Ago2) pour moduler l'expression génique au niveau post-transcriptionnelle par ARN interférence. Classiquement, les miRNAs s’apparient à des sites de liaison complémentaires situés dans le 3’-UTR de l’ARNm cible. Nous faisons l'hypothèse que les miRNAs seraient impliqués dans la communication entre le noyau et la mitochondrie. Notre travail a donc porté sur l’étude du rôle des miRNAs dans le contexte de maladies génétiques caractérisées par une dysfonction mitochondriale. Nous avons choisi d’étudier l’ataxie de Friedreich, la plus fréquente des ataxies héréditaires, qui est causée par un déficit d’expression de la protéine mitochondriale frataxine (FXN). Nous avons montré qu'environ 90% de patients étaient homozygotes pour un haplotype spécifique des variants génétiques du 3'-UTR du gène FXN. Ce résultat a été retrouvé dans deux cohortes de patients indépendantes. Par une combinaison d’approche bioinformatique et d’expériences de co-transfections, nous avons montré que les miRNAs, et en particulier miR-124, ciblent les variants du 3’-UTR. En parallèle, nous avons évalué la possibilité que les miRNAs ciblent directement la mitochondrie. Pour cela, nous avons analysé l’expression des miRNAs dans des fractions d'ARN mitochondriale et cytosolique isolées à partir de mêmes cellules HeLa. Nous avons identifié une signature de 13 miRNAs spécifiquement enrichis dans la fraction d'ARN mitochondriale que nous avons appelé «mitomiRs». Nos prédictions ont révèle des fonctions spécifiques de ces mitomiRs à la mitochondrie, y compris la modulation de l'activité de la chaîne respiratoire. Nous avons également montré une localisation de la protéine Ago2 à l’espace inter-membranaire mitochondriale.Notre travail définit ainsi les miRNAs et Ago2 comme un nouveau niveau de communication entre le noyau et les mitochondries. Nous discutons de la possibilité que la mitochondrie agisse comme acteur du ARN interférence ou plutôt comme organite cible. Notre travail ouvre la voie à un nouveau domaine de recherche, qui pourrait avoir une utilité thérapeutique pour palier les dysfonctions mitochondriales. / Mitochondria are organelles that have a central role in the energetic metabolism of the cell. Although mitochondria express their own genome, they rely on the expression of the nuclear genome for their biogenesis and function. microRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute 2 (Ago2) protein to regulate gene expression post-transcriptionallythrough RNA interference. The ‘classic’ view of RNA interference describes the pairing of miRNAs with complementary binding sites within the 3’untranslated region (3’-UTR) of the target mRNA. We hypothesized that miRNAs might be instrumental to the cross-talk between the nucleus and the mitochondria. In the first part, we assessed the role of miRNAs in the context of a rare genetic disease involving mitochondrial dysfunction. We focused on Friedreich's ataxia, the most frequent of inherited ataxia in Europe, which is caused by reduced expression of the mitochondrial protein frataxin (FXN). Intwo independent cohorts of patients, we discovered that about 90% of patients were homozygous forone specific haplotype of genetic variants of the FXN3'-UTR. By a combination of computational target prediction analysis and co-transfection experiments, we showed that miRNAs, and specifically miR-124, are involved in the regulation of the FXN.We then challenged further the relationship between the miRNAs and mitochondria through questioning their localization at mitochondria. To this end, we studied miRNAs from mitochondrial and cytosolic RNA fractions isolated from the same HeLa cells. We identified a signature of 13 miRNAs specifically enriched in the mitochondrial RNA fraction that we termed ‘mitomiRs’. Through pathway-enrichement analysis, we observed a specific mitochondrial role for mitomiRs, including regulation of ATP synthesis coupled electron trasport. We also provided the evidence of Ago2 protein location inside human mitochondria at the inter-membrane space. Our work sketches miRNAs and Ago2 as a novel layer of the interplay between the nucleus and the mitochondria. We discuss whether mitochondria may be instrumental to RNA interference or a target per se. Our work paves the way to a new field of research, which may unravel therapeutic outcomes to rescue mitochondrial dysfunction.

<p>The cellular microRNA (miRNA) pathway has emerged as an important regulator of host-virus interactions. While miRNAs of viral and cellular origin have been demonstrated to modulate viral gene expression and host immune responses, reports detailing these activities in the context of mammalian retroviruses have been controversial. Using modern, high-throughput small RNA sequencing we provide evidence that the spumaretrovirus bovine foamy virus expresses high levels of viral miRNAs via noncanonical biogenesis mechanisms. In contrast, the lentivirus human immunodeficiency virus type 1 (HIV-1) does not express any viral miRNAs in a number of cellular contexts. Comprehensive analysis of miRNA binding sites in HIV-1 infected cells yielded several viral sequences that can be targeted by cellular miRNAs. However, this analysis indicated that HIV-1 transcripts are largely refractory to binding and inhibition by cellular miRNAs. In addition, we demonstrate that HIV-1 exerts minimal perturbations on cellular miRNA profiles and that viral replication is not affected by the ablation of mature cellular miRNAs. Together, these data demonstrate that the ability of retroviruses to encode miRNAs is not broadly conserved and that lentiviruses, particularly HIV-1, have evolved to avoid targeting by cellular miRNAs.</p> / Dissertation

AnalysisofshortArgonauteisoformsfrommouseoocytes Abstract: Argonaute proteins carrying small RNAs form the conserved core of RNA silencing mechanisms, which repress viruses, mobile genetic elements, and genes in a sequence specific manner. The microRNA (miRNA) pathway is a dominant mammalian RNA silencing mechanism in somatic cells, which post-transcriptionally regulates large fraction of genes and thereby adjusts protein levels. miRNA-guided Argonautes inhibit translation and induce deadenylation of complementary mRNAs, ultimately resulting in their decay. In contrast to RNA interference (RNAi), which employs Argonaute slicer activity to directly cleave perfectly complementary RNAs, an effective miRNA-mediated mRNA repression requires multiple Argonaute-associated protein factors and enzymes. The miRNA pathway has been implicated in many complex biological processes ranging from organogenesis, stress-response to haematopoiesis or cancer. Surprisingly, canonical miRNAs are not essential for oocytes and early embryonic development in mice. Even the most abundant miRNAs present in mouse oocytes are unable to effectively repress target genes. However, RNAi, which shares key enzymes with the miRNA pathway, is highly active in oocytes and early embryos. The cause of miRNA inactivity in mouse oocytes remains...

In recent years, LIM domains-containing protein 1 (LIMD1) has been identified as a critical component in microRNA (miRNA)-induced silencing complex (miRISC) to regulate miRNA-mediated gene silencing. Human Argonaute (AGO) 2 with its family members (AGO1-4) are critical for the biogenesis of miRNA and thus miRNA-mediated gene silencing. In this study, we have investigated the direct interaction interfaces between LIMD1 and AGO2. A distinct interface within LIMD1, amino acid (a.a) 140-166, is identified to be responsible for the binding to AGO2 and other members of AGO family. Furthermore, the Linker-2 (L2) domain within AGO2 is identified to be responsible for LIMD1 binding and its dependency on the phosphorylation at serine 387 (S387) residue within the L2 domain of AGO2. The phospho-mimic mutant (S387E) enhances the binding of AGO2 to LIMD1, whereas the phospho-deficient mutant (S387A) attenuates AGO2-LIMD1 interaction. In addition, the association of LIMD1 with other AGOs is also dependent on the phosphorylation at the equivalent conserved serine residue within the L2 domain on other AGOs. In addition to the above aspects, LIMD1 is a tumour suppressor gene frequently down-regulated in more than 75% human lung tumours. Because of their loss of expressions or functions, it is of the inherent difficulty in targeting tumour suppressor genes to treat cancers. In this study, the concept of synthetic lethality was used to identify possible protein kinases, the ablation of which are synthetically lethal to LIMD1 negative cancer cell lines. As a result, drugs that target these kinases may represent novel targeted therapies for LIMD1 negative lung tumours. ACVR2B and STK39 are validated to be synthetically lethal with LIMD1 loss. Additionally, the complete loss of LIMD1 expression causes a dramatic increase of STK39 expression due to miRNA-mediated gene silencing pathway. The inverse relationship between LIMD1 and STK39 may represent a conserved and fundamental signalling response and may be a predictive marker for STK39-targeted therapy.