Pseudomonas aeruginosa RsmA belongs to the CsrA family of RNA binding proteins. CsrA family members are post-transcriptional regulators of global gene expression and usually function to inhibit translation of target genes, but in some cases can also exert positive regulatory effects. Previous work from our lab determined that RsmA is required for maximal T3SS gene expression in P. aeruginosa strain PA103. Nevertheless, the molecular mechanism underlying the RsmA-mediated control of T3SS gene expression was unknown. Expression of the T3SS is under the direct control of ExsA, a transcriptional activator. Previous microarray analyses showed that exsA transcript levels were reduced two-fold in an rsmA mutant. In chapter II I examine the role of RsmA in regulating ExsA expression. I demonstrate that expression of a ExsA-LacZ translational fusion was reduced two-fold in an rsmA mutant suggesting a specific effect of RsmA on ExsA expression. The effect of RsmA on ExsA expression occurs at a post-transcriptional level and is independent of mRNA and protein stabilization mechanisms. RsmA directly interacts with the exsCEBA transcript at multiple sites. Truncation analyses indicate that the -37 to +85 region (relative to the ATG start codon) is necessary and sufficient for RsmA-dependent control. I identified two binding sites, BS1 (-25 bp) and BS2 (+84), involved in the interaction of RsmA with the exsA transcript using sequence analysis, site-directed mutagenesis, EMSA assays, RNase footprints, and RNaseH cleavage assays. Mutagenesis of both binding sites results in an RsmA-independent phenotype. I further demonstrate that RsmA is able to activate ExsA expression. I propose a model wherein RsmA relieves a block on ExsA translation. Collectively, this work shows that RsmA directly binds and activates ExsA expression at the post-transcriptional level. Most Pseudomonas species carry at least two homologs of CsrA on the chromosome, but only one copy had been identified in P. aeruginosa. Through the course of other projects in the lab, we observed several phenotypes that could not be accounted for by a single copy of RsmA. In collaboration with the Wolfgang lab, we identified a second CsrA homolog, RsmF in P. aeruginosa. RsmF is dimeric in solution. The structure of RsmF differs substantially from other CsrA homologs by having alpha-helices located between the beta-2 and beta-3 strands. In chapter III I examine the role of RsmF in regulating RsmA-controlled processes associated with acute (T3SS) and chronic (T6SS and biofilm formation) infection. I discovered that while an rsmF mutant alone does not exhibit a phenotype, simultaneous deletion of both rsmA and rsmF significantly accentuates the phenotypes exhibited by an rsmA mutant alone. I show that RsmA directly binds and represses RsmF translation and that the small regulatory RNAs RsmZ and RsmY do not significantly modulate RsmF activity. Site-directed mutagenesis revealed that Arg 62, located in the beta-1 and beta-5 fold, is essential for biological activity in vivo and RNA-binding in vitro suggesting a conserved mechanism of RNA recognition maintained across all CsrA family members. Finally, I show that RsmF binds to only a subset of RsmA targets and is not involved in the regulation of all RsmA-controlled processes. In chapter IV I identified high-affinity RNA ligands from a chemically synthesized oligonucleotide library using systematic evolution of ligands by exponential enrichment (SELEX) and high-througput sequencing. From preliminary analyses of high-throughput sequencing data, the RsmF-binding consensus was determined as 5'-RUACARGGAC-3', with the ARGGA motif being 95% conserved. Collectively, this work shows that Rsm homologs play important roles in regulating virulence gene expression in P. aeruginosa.

miRNAs are short, non-coding RNA molecules that regulate gene expression posttranscriptionally through inhibition of translation and/or mRNA degradation. Mammalian development is a complex series of developmental transitions, which relies on accurate spatial and temporal regulation of gene expression and we are interested in the role that miRNAs may play in these developmental transitions. An initial objective was to establish which, if any, miRNAs were dynamically regulated in a cell model of an early developmental transition, and to establish whether differential expression of any particular miRNA played a functional role in this developmental process. Having established a role for specific miRNAs, further objectives were to assess the reliability of current miRNA-mRNA target identification procedures and to assess the general role of miRNAs in cellular differentiation. In order to explore the roles of miRNAs during an early developmental transition, an embryonic stem (ES) cell model of trophectoderm differentiation was used. In this model system the expression of the key ES cell regulatory gene, Oct4, can be conditionally repressed, which induces the ES cells to differentiate down the trophectoderm lineage. The expression of microRNAs was profiled in this model system by cloning and sequencing of small RNAs. This approach identified miRNAs that were dynamically regulated during differentiation. The expression patterns of differentially regulated miRNAs were confirmed by miRNA northern analysis. The miRNA profiling data showed that mmu-miR-294 and mmu-mir-295 are expressed at similar levels in ES cells and differentiated cells, which disagrees with previous reports that these miRNAs are ES cell specific. Several of the miRNAs with higher expression levels in differentiated cells are encoded within a placental-enriched polycomb group gene, Sfmbt2, suggesting an important role for these miRNAs in extraembryonic development. One of the miRNAs that was expressed at higher levels in ES cells than in differentiated cells, mmu-miR-92a, was shown to play a role in regulation of cell proliferation. Three current methods of identifying miRNA targets were assessed. A sequencebased method using the web-based utility miRecords, which amalgamates results from numerous target prediction databases, was used to generate lists of potential targets of the Sfmbt2 miRNA cluster and of mmu-miR-92a. Amalgamating results from multiple target prediction programs may improve the likelihood that the predicted targets are real. Exemplifying this, the single mmu-miR-92a target that was predicted by six different target prediction programs had been previously experimentally verified. An experimental method of identifying direct miRNA targets, PAR-CLIP, was investigated but proved technically limiting for routine use in the laboratory. A proteome-based experimental method for identifying potential miRNA targets, called SILAC, was successfully used to identify proteins that were differentially expressed in the cell model of trophectoderm differentiation. Differential expression of two of these proteins, CTBP2 and CKB, was confirmed by western analysis. miRecords was then used to assess whether the differentially expressed proteins were likely to be targets of the differentially expressed miRNAs that had been identified in the miRNA profiling analysis. The general role of miRNAs in cell differentiation was investigated using a cell line that does not express miRNAs. This ES cell line is deficient for the miRNAprocessing enzyme DGCR8, which results in loss of expression of mature miRNAs in these cells. Compared to wild type ES cells, miRNA-deficient ES cells expressed normal levels of the ES cell marker genes Oct4 and Sox2 but elevated levels of Nanog. In contrast to wild type ES cells, miRNA-deficient ES cells did not upregulate the mesoderm marker gene Brachyury during embryoid body differentiation and showed reduced upregulation of the endoderm marker gene Gata6. These findings suggest that miRNAs are not required for maintenance of pluripotency, but are essential for proper ES cell differentiation. The results presented in this thesis show that miRNAs are dynamically expressed during a mammalian developmental transition and are involved in regulating early developmental processes. We believe that miRNAs act as an additional level of genetic regulation to ensure canalisation during embryonic development.

This thesis describes a molecular-based study undertaken to analyse the expression of the RAMOSUS1 (RMS1) and RAMOSUS5 (RMS5) genes in pea (Pisum sativum). Both genes encode carotenoid cleavage dioxygenase (CCD) enzymes that are together proposed to control the synthesis of an inhibitor of bud outgrowth termed SMS (Shoot Multiplication Signal). SMS was recently identified as strigolactone. Expression analyses of RMS1 presented here have built upon earlier experiments which demonstrate it to be a highly regulated transcript. RMS1 mRNA levels are known to be rapidly decreased following removal of the shoot apex but are subsequently restored to that of intact plants by auxin (indole-3-acetic acid or IAA). This regulatory mechanism is retained in all five ramosus mutants tested to date. Together with physiological data, this indicates RMS1, and therefore SMS, are required in IAA-mediated suppression of bud outgrowth. Another significant aspect of RMS1 regulation identified in previous studies involves a graft-transmissible, long-distance feedback signal that moves from shoot to root. This feedback regulation is dependent on the RMS2 gene and enhances RMS1 expression levels. Prior to the cloning of RMS5 and its discovery as a second CCD enzyme in the RMS network, reciprocal grafting studies with the rms mutants indicated RMS5 may act in the same pathway as RMS1 to produce SMS. Multiple studies presented here demonstrate that these two CCD genes are expressed in similar tissues and are regulated by the same signals, specifically IAA and the RMS2-dependent feedback signal. Like RMS1, the RMS5 gene also retains its IAA response in the rms mutants. However, RMS5 is generally less responsive to changes in IAA and RMS2-dependent feedback, as it exhibits smaller fluctuations than RMS1 in its expression levels. Together these findings support a general view that RMS1 is more likely to control a rate-limiting step in SMS synthesis. A previous study indicated that RMS1 expression may be up-regulated by IAA through a posttranscriptional mechanism. This thesis sought to more closely examine the RMS1 and RMS5 IAA response by separately observing the effect of IAA on subsequent transcription. New transcripts, termed heterogenous nuclear RNAs (hnRNAs), were relatively quantified in parallel with existing mRNAs in the steady-state cytoplasmic pool. The experiments conducted here provide further evidence that IAA may act post-transcriptionally to stabilise RMS1 mRNA because the changes in hnRNA are not proportional to the changes in mRNA following IAA-modifying treatments. IAA may still function to induce transcription of RMS1, but this does not appear to be a significant mechanism by which IAA regulates RMS1 expression. In contrast, the IAA induction of RMS5 occurs predominantly via new transcription and RMS5 either lacks or is not as strongly subjected to the IAA-mediated mRNA stabilisation mechanism proposed for RMS1. Initial studies described in this thesis also suggest that IAA could act to regulate the expression of the Arabidopsis orthologues MORE AXILLARY BRANCHING (MAX) genes via a post-transcriptional mechanism. Analyses of MAX hnRNA and mRNA levels in Arabidopsis to date indicate it is the RMS5 orthologue MAX3 which exhibits an IAA response most like RMS1. Additional studies into the regulation of RMS1 and RMS5 presented in this thesis provide further insights into the molecular mechanisms controlling their expression levels. In vitro experiments with the translation inhibitor cycloheximide demonstrate that RMS5 expression levels are increased when protein synthesis is reduced, as previously shown for RMS1. Relative quantification of RMS1 and RMS5 hnRNA levels further demonstrate that the induction by cycloheximide is due primarily to an increase in new transcription, indicating that RMS1 and RMS5 are negatively regulated by a rapidly turned-over transcriptional repressor. Tissue specific effects on RMS1 expression were also observed which are consistent with a protein degradation function of the RMS4 F-box in the shoot. This thesis provides further evidence to suggest that SMS acts in concert with IAA to inhibit the sustained outgrowth of axillary buds. RMS1 and RMS5 expression levels are not regulated by a hypothetical fast decapitation signal which is proposed to cause the initial bud outgrowth occurring prior to decapitation-induced IAA depletion. RMS1, RMS5 and SMS are therefore unlikely to control the initial exit of buds from dormancy to an intermediate transition state. Studies here also suggest that enhanced shoot auxin transport and cytokinin biosynthesis are associated with axillary bud outgrowth because the rms mutants contain elevated shoot expression levels of a gene encoding the auxin efflux carrier PIN1 and two genes controlling cytokinin biosynthesis. Several approaches described in this study were used to characterise the RMS1 and RMS5 proteins. Anti-peptide antibodies were generated against both proteins and the results obtained show that although the antibodies are likely to recognise the full-length proteins, further work is required to effectively detect RMS1 and RMS5 in plant tissues via western blotting. Preliminary in situ immunolocalisation results indicate the RMS1 and RMS5 proteins are localised to the vasculature, consistent with gene expression analyses.

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Parkinson's disease (PD) is the second most common neurodegenerative disease, yet it has no cure. It is characterized by the loss of dopaminergic neurons and accumulation of dense aggregates, primarily composed of α-synuclein protein. Many causative genes have been identified including SNCA, encoding α-synuclein, and Leucine-rich-repeat kinase 2 (LRRK2). The LRRK2 G2019S mutation is known to cause hyperactive kinase activity, but its cellular functions, including its kinase substrates, remain poorly understood. PD has many risk factors including environmental and genetic modifiers. Polymorphisms in the Embryonic lethal-abnormal vision-like 4 (ELAVL4) gene modify PD age-of-onset or susceptibility. Incidentally, a genetic screen in Drosophila identified an ELAVL homologue as required for LRRK2-induced pathology. Therefore, we hypothesized that LRRK2 phosphorylates ELAVL4 to control phenotypes relevant to PD. We discovered that three neuronal ELAVLs including ELAVL4 (also known as HuD) bind to, and post-transcriptionally regulate mRNA encoding α-synuclein and LRRK2. We also show that LRRK2 phosphorylates HuD and its homologues HuB and HuC. This controls binding of nELAVLs (i.e., HuB, HuC, and HuD) to mRNA and post-transcriptionally regulates mRNA abundance and splicing in the mouse midbrain. In mice, the complex interaction between HuD and Lrrk2 G2019S is associated with motor deficits, dopaminergic neuron loss, and accumulated α-synuclein protein levels. Targets of nELAVLs are also selectively misregulated in iPSC-derived neurons and tissues from PD patients. In a model of PD-relevant inflammation, we also show that the ubiquitously expressed ELAVL homologue, HuR, controls LRRK2 protein levels. We show that mice lacking Lrrk2 are more susceptible to an acute model of dextran sodium sulfate (DSS) chemical-induced colitis. Lrrk2-deficient mice treated with DSS also show accumulated α-synuclein in brain tissue. Using in vitro models and mouse tissue we show that LRRK2 controls HuR binding to RNA probes and to the proinflammatory cytokine Tnfa in colon tissue, and this has implications for intestinal pathology relevant to PD. Together, this suggests that misregulation of ELAVLs may be implicated in neurodegeneration and inflammation observed in Parkinson's disease.

Protéines difficiles à exprimer: un goulot d'étranglement pour la plupart des biologistes. J'ai choisi d'utiliser comme modèle d’étude Acanthamoeba polyphaga Mimivirus. Ce virus géant à ADN possède des protéines subissant des modifications post-traductionnelles, des structures multi-protéiques ou encore des voies enzymatiques jamais identifiées auparavant dans un virus, ce qui en font un modèle idéal pour l’étude de protéines récalcitrantes. Le but ultime de cette thèse, était de produire les protéines de capsides de Mimivirus. Le rôle de la protéine de capside dans l’assemblage de la particule virale, son infectivité et ses caractéristiques moléculaires sont d’une grande importance. Pour aller du gène à la protéine, J’ai participé à la compréhension de ce qui gouverne la terminaison de la transcription de Mimivirus et également participé à l'analyse globale du transcriptome au cours du cycle d'infection des amibes par Mimivirus. Nous avons montré que les transcrits de Mimivirus sont systématiquement polyadénylés dans des régions formant une structure secondaire en tige-boucle, même s’il n’existe pas de signal de polyadénylation canonique en amont. Nous en avons conclu que la polyadénylation de Mimivirus suit exclusivement une règle «épingle à cheveux». De plus, l’étude du transcriptome a révélé 3 phases temporelles distinctes dans le cycle infectieux: précoce, intermédiaire et tardive. Les transcrits de capsides sont tous exprimés durant la phase tardive mais leur profil d’expression ne sont pas superposables dans le temps. Les données de transcriptomique ont révélées la présence de plusieurs glycosyltransférases chez Mimivirus, dans la phase tardive du cycle, concomitant avec la production de la protéine de capside. Les informations recueillies sur l'expression des gènes à différents temps post-infection ont contribué à la conception de protocoles pour la production des protéines de capsides (la protéine majeure de capside (MCP) et ses paralogues) dans de systèmes eucaryote. / Difficult to express proteins: a bottleneck for most biologists. I have chosen to use Acanthamoeba polyphaga Mimivirus as my study model. This giant dsDNA virus possesses post-translationally modified proteins, multi-protein structures and enzyme pathways never before seen in a virus, which makes it ideal for refractory studies. The ultimate goal of my thesis was to produce the capsid proteins of Mimivirus. The role of the capsid protein in the assembly of the viral particle, its infectivity, and molecular features are of great importance. To go from gene to protein, I participated in the comprehension of what governs the post-transcriptional termination in Mimivirus and equally participated in the global analysis of the transcriptome during the infectious cycle of Acanthamoeba by Mimivirus. We have shown that the Mimivirus transcripts are systematically polyadenylated in the regions forming a stem-loop secondary structure; even when a canonical poyadenylation signal is absent We concluded that Mimivirus polyadenylation obeys a strict “Hairpin rule”. Moreover, the transcriptomic study revealed three distinct temporal phases: early, intermediate and late. The capsid transcripts are all expressed during the late phase but their expression profiles are not superimposable. The transcriptomic data also revealed the presence of several Mimivirus glycosyltransferases in the late temporal phase, concomitant with the capsid proteins. The expression data gathered throughout my thesis has contributed to the rational design of a protein production experiment to produce the major capsid protein and its three paralogs in eukaryotic systems.

BACKGROUND:Colony stimulating factor-1 (CSF-1) plays an important role in ovarian cancer biology and as a prognostic factor in ovarian cancer. Elevated levels of CSF-1 promote progression of ovarian cancer, by binding to CSF-1R (the tyrosine kinase receptor encoded by c-fms proto-oncogene).Post-transcriptional regulation of CSF-1 mRNA by its 3' untranslated region (3'UTR) has been studied previously. Several cis-acting elements in 3'UTR are involved in post-transcriptional regulation of CSF-1 mRNA. These include conserved protein-binding motifs as well as miRNA targets. miRNAs are 21-23nt single strand RNA which bind the complementary sequences in mRNAs, suppressing translation and enhancing mRNA degradation.RESULTS:In this report, we investigate the effect of miRNAs on post-transcriptional regulation of CSF-1 mRNA in human ovarian cancer. Bioinformatics analysis predicts at least 14 miRNAs targeting CSF-1 mRNA 3'UTR. By mutations in putative miRNA targets in CSF-1 mRNA 3'UTR, we identified a common target for both miR-128 and miR-152. We have also found that both miR-128 and miR-152 down-regulate CSF-1 mRNA and protein expression in ovarian cancer cells leading to decreased cell motility and adhesion in vitro, two major aspects of the metastatic potential of cancer cells.CONCLUSION:The major CSF-1 mRNA 3'UTR contains a common miRNA target which is involved in post-transcriptional regulation of CSF-1. Our results provide the evidence for a mechanism by which miR-128 and miR-152 down-regulate CSF-1, an important regulator of ovarian cancer.