Conversion of aspartate and asparagine residues to isoaspartate is a prevalent covalent protein modification in cells. The accumulation of these altered residues can lead to the loss of protein function and the consequent loss of cellular function. The L-ISOASPARTATE METHYLTRANSFERASE (EC 2.1.1.77) (PIMT) iteratively methylates abnormal isoaspartyl residues leading to conversion to L-aspartate, thereby mitigating the injurious effects of aging. Arabidopsis thaliana is unique among eukaryotes studied to date in that it possesses two genes (At3g48330 (PIMT1) and At5g50240 (PIMT2)) encoding PIMT. The PIMT2 gene exhibits a complex transcriptional control involving different transcriptional initiation sites and 5'- and 3'- alternative splice site selection in the first intron. Varying the transcriptional initiation site results in alternative targeting of the PIMT2 proteins thus produced to: 1) the nucleus, or 2) the cytoplasm, while PIMT1 is cytosolic. Inclusion of a 51 nucleotide 5 alternatively spliced sequence with or without a nine nucleotide 3 alternatively spliced sequence dramatically alteres the subcellular protein localization from the cytoplasm and around the chloroplast to inside the chloroplast. All recombinant PIMT2 isoform tested exhibit PIMT activity, although solubility varied among them. Multiplex RT-PCR was used to establish PIMT1 and PIMT2 transcript presence and abundance, relative to -TUBULIN, in various tissues and under a variety of stresses imposed on seeds and seedlings. PIMT1 transcript is constitutively present but can increase, along with PIMT2, in developing seeds presumably in response to increasing endogenous ABA. Transcript from PIMT2 also increases in establishing seedlings due to exogenous ABA application or applied stress presumably through an ABA-dependent pathway. Furthermore, Cleaved Amplified Polymorphic Sequence analysis of the PIMT2 amplicons has shown that the ratio among the splicing variants alters upon ABA application, implicating a role for the spliceosome or differential RNA stability in orchestrating the plant's response to stress. T-DNA insertional mutants of both genes were isolated but no obvious phenotype has been identified. The double mutant has been generated and will be evaluated.

Calcium is a component of signal transduction pathways that allow plants to respond to numerous endogenous and environmental signals during growth and development. Calcium-mediated signaling involves multiple components including: 1) channels, pumps, and exchangers that act in concert to generate a change in cytosolic calcium, 2) calcium-binding proteins that sense the calcium change, and 3) downstream target proteins that modify enzyme activity and gene expression needed for the subsequent response. One method for achieving specificity during calcium signaling is through regulation of the calcium-binding proteins that perceive changes in cytosolic calcium. These proteins can be regulated through differences in expression in response to stimuli, localization within the cell or plant, affinity for calcium, and interaction with downstream target proteins; all of which can result in specific cellular responses. My projects have focused on the Arabidopsis thaliana (Arabidopsis) CALCINEURIN B-LIKE10 (CBL10) calcium-binding protein, and specifically on understanding: 1) how post-transcriptional regulation of the CBL10 gene is used to modulate seedling growth in saline conditions (salinity), and 2) CBL10’s function in the flower during growth in salinity. In addition, 3) I have examined the roles of two putative CBL10-interacting proteins in plant growth and development. CBL10 is alternatively spliced into two transcripts; CBL10 encoding the characterized, full-length protein and CBL10 LONG A (CBL10LA) encoding a putative truncated protein due to a pre-mature termination codon within a retained intron. When seedlings are grown in the absence of salinity, both alternatively spliced transcripts are detected; however, in response to salinity, levels of the CBL10LA transcript are reduced. My data suggest a model in which the relative abundance of the two transcripts regulates the SALT-OVERLY-SENSITIVE (SOS) pathway involved in maintaining cellular sodium ion homeostasis. The presence of CBL10LA in the absence of salinity ensures that the SOS pathway is inactive. The removal of CBL10LA in response to saline conditions results in CBL10 activation of the SOS pathway to prevent sodium ions from accumulating to toxic levels in the cytosol. Successful fertilization during flowering requires the coordinated development of stamens and pistils. Stamens must elongate and anthers dehisce to release pollen onto the stigma while the pistil prepares to receive the pollen and promote growth and targeting of the female gametophyte. When the cbl10 mutant is grown in salinity, flowers are sterile due to decreased stamen elongation, reduced anther dehiscence, and abnormal pistil development. My studies demonstrated that the SOS pathway is not involved in maintaining flower development in salinity and indicate that CBL10 functions in different pathways to regulate vegetative and reproductive development during growth in saline conditions. An in silico search for Arabidopsis proteins that might interact with CBL10 resulted in the identification of two components of the Mediator complex involved in the regulation of transcription in eukaryotes. While additional studies I carried out suggest that interaction with CBL10 is unlikely, I have shown that these proteins are important for plant growth in high levels of chloride and in maintenance of growth in short-day conditions.

Les introns sont des portions de gènes transcrites dans l’ARN messager, mais retirées pendant l’épissage avant la synthèse des produits du gène. Chez les eucaryotes, on rencontre les introns splicéosomaux, qui sont retirés de l’ARN messager par des splicéosomes. Les introns permettent plusieurs processus importants, tels que l'épissage alternatif, la dégradation des ARNs messagers non-sens, et l'encodage d'ARNs fonctionnels. Leurs rôles nous interrogent sur l'influence de la sélection naturelle sur leur évolution. Nous nous intéressons aux mutations qui peuvent modifier les produits d'un gène en changeant les sites d'épissage des introns. Ces mutations peuvent influencer le fonctionnement d'un organisme, et constituent donc un sujet d'étude intéressant, mais il n'existe actuellement pas de logiciels permettant de les étudier convenablement. Le but de notre projet était donc de concevoir une méthode pour détecter et analyser les changements des sites d'épissage des introns splicéosomaux. Nous avons finalement développé une méthode qui repère les évènements évolutifs qui affectent les introns splicéosomaux dans un jeu d'espèces données. La méthode a été exécutée sur un ensemble d'espèces d'oomycètes. Plusieurs évènements détectés ont changé les sites d’épissage et les protéines, mais de nombreux évènements trouvés ont modifié les introns sans affecter les produits des gènes. Il manque à notre méthode une étape finale d'analyse approfondie des données récoltées. Cependant, la méthode actuelle est facilement reproductible et automatise l'analyse des génomes pour la détection des évènements. Les fichiers produits peuvent ensuite être analysés dans chaque étude pour répondre à des questions spécifiques. / Introns are portions of genes transcribed into messenger RNA, but removed during RNA splicing. In eukaryotes, they are called spliceosomal introns as they are removed by spliceosomes. Introns allow many important processes such as alternative splicing, nonsense-mediated decay and functional-RNA coding. These roles leads to the question of the influence of natural selection on evolution of introns. We focus on mutations that are able to change gene products by modifing introns splice sites. These mutations seems to be an interesting topic as they can affect proteins, but there is currently no software to study them properly. The aim of our project was to design a method to detect and analyze changes in splice sites of spliceosomal introns. We finally developed a method that locates the evolutionary events on splice sites of spliceosomal introns in a given species set. The method was performed on a set of oomycetes. Several detected events change splice sites and proteins, but there is also many events that seems to modify introns without affecting gene products. Our method lacks a final step for thorough analysis of the collected events. However, the current method is easily reusable and automates genome analysis for the detection of events. The resulting files can then be analyzed in each study to answer specific questions.

<p>The six-layered neuron structure in the cerebral cortex is the foundation for human mental abilities. In the developing cerebral cortex, neural stem cells undergo proliferation and differentiate into intermediate progenitors and neurons, a process known as embryonic neurogenesis. Disrupted embryonic neurogenesis is the root cause of a wide range of neurodevelopmental disorders, including microcephaly and intellectual disabilities. Multiple layers of regulatory networks have been identified and extensively studied over the past decades to understand this complex but extremely crucial process of brain development. In recent years, post-transcriptional RNA regulation through RNA binding proteins has emerged as a critical regulatory nexus in embryonic neurogenesis. The exon junction complex (EJC) is a highly conserved RNA binding complex composed of four core proteins, Magoh, Rbm8a, Eif4a3, and Casc3. The EJC plays a major role in regulating RNA splicing, nuclear export, subcellular localization, translation, and nonsense mediated RNA decay. Human genetic studies have associated individual EJC components with various developmental disorders. We showed previously that haploinsufficiency of Magoh causes microcephaly and disrupted neural stem cell differentiation in mouse. However, it is unclear if other EJC core components are also required for embryonic neurogenesis. More importantly, the molecular mechanism through which the EJC regulates embryonic neurogenesis remains largely unknown. Here, we demonstrated with genetically modified mouse models that both Rbm8a and Eif4a3 are required for proper embryonic neurogenesis and the formation of a normal brain. Using transcriptome and proteomic analysis, we showed that the EJC posttranscriptionally regulates genes involved in the p53 pathway, splicing and translation regulation, as well as ribosomal biogenesis. This is the first in vivo evidence suggesting that the etiology of EJC associated neurodevelopmental diseases can be ribosomopathies. We also showed that, different from other EJC core components, depletion of Casc3 only led to mild neurogenesis defects in the mouse model. However, our data suggested that Casc3 is required for embryo viability, development progression, and is potentially a regulator of cardiac development. Together, data presented in this thesis suggests that the EJC is crucial for embryonic neurogenesis and that the EJC and its peripheral factors may regulate development in a tissue-specific manner.</p> / Dissertation

The aim of this thesis was to identify genetic factors involved in frontotemporal lobar degeneration (FTLD), a neurodegenerative disorder clinically characterised by a progressive change in personality, behaviour and language. FTLD is a genetically complex disorder and a positive family history is found in up to 40% of the cases. In 10-20% of the familial cases the disease can be explained by mutations in the gene encoding the microtubule associated protein tau (MAPT). In the first study we describe the clinical and neuropathological features of a Finnish family with FTLD caused by a mutation in MAPT. We also provide evidence that the pathogenic mechanism of this mutation is through altered splicing of MAPT transcripts. Recently, mutations in the gene encoding progranulin (PGRN) were identified as a major cause of FTLD. In the second study we describe a Swedish family with FTLD caused by a frameshift mutation in PGRN. We provide a clinical and neuropathological description of the family, as well as evidence that the pathogenicity of this mutation is through nonsense-mediated decay of the mutant mRNA transcripts and PGRN haploinsufficiency. In the third study we describe a novel PGRN splice site mutation and a previously described PGRN frameshift mutation, found in a mutation screen of 51 FTLD patients. We describe the clinical and neuropathological characteristics of the mutation carriers and demonstrate that haploinsufficiency is the pathogenic mechanism of the two mutations. In the fourth study we investigate the prevalence of PGRN and MAPT gene dosage alterations in 39 patients with FTLD. No gene dosage alterations were identified, indicating that variations in copy number of the PGRN and MAPT genes are not a common cause of disease, at least not in this FTLD patient collection.

OBJECTIVES: The role of heterogeneous nuclear ribonucleoproteins in controlling hormone production is not well-understood. This study aims to determine the regulatory roles of hnRNP L and LL in hormone production. RESULTS: HnRNP LL knockdown dramatically reduced the mRNA level of growth hormone (GH) and prolactin (PRL) in GH3 pituitary cells, which was efficiently rescued by a Myc-hnRNP LL. In contrast, hnRNP L knockdown caused a 93-nucleotide intron retention with a decrease in the full-length Prl, which was rescued by a FLAG-hnRNP L. Double knockdown of hnRNPs indicates the interactions of hnRNPs for particular effects. The essential role of hnRNP LL for the secretion of GH and PRL was verified in primary pituitary cultures. Using RNA sequencing, a group of genes in hormone production that were differentially regulated by hnRNP L or LL were identified. CONCLUSION: These findings demonstrate the differential functions of two paralogous RNA processing factors in hormone production. / February 2016

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, and dysregulation of caspase-9 splice variant ratio or expression of caspase-9b isoform has been linked to augmentation of the anchorage-independent growth and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. This study focuses on cell signaling pathway(s) regulating the alternative splicing of caspase-9 pre-mRNA and mechanistic roles of caspase-9b in a certain oncogenic/survival pathway. In regards to the former, we have identified hnRNP U as a novel splice-enhancer associated with exon 3 of caspase-9 (C9/E3). Moreover, hnRNP U binds specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interferes with hnRNP U for binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Overall, a mechanistic model has been revealed where hnRNP U competes with hnRNP L for C9/E3 binding to enhance the inclusion of the four-exon cassette, and this splice-enhancing effect is blocked by the AKT pathway via phosphorylation of hnRNP L. As to the latter aim, it is unknown about the mechanistic roles of caspase-9b besides the inhibitory effect on caspase-9a processing. In this study, caspase-9b has been demonstrated to have a dual function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway, which is independent from modulating caspase-9a activation. In particular, caspase-9b has been shown to activate the canonical arm and inhibit the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for caspase-9b contributes to the enhanced survival and anchorage-independent growth of NSCLC cells conferred by caspase-9b expression. Further mechanistic studies have demonstrated a direct association of caspase-9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif. Through this interaction, caspase-9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the survival, anchorage-independent growth and tumorigenicity of NSCLC cells. Overall, a novel tumorigenic mechanism has been identified, by which alternative mRNA processing regulates the NF-κB signaling independent of external agonist.

In the presented study, we demonstrate that the alternative splicing of caspase 9 was dysregulated in a large percentage of NSCLC tumors and cell lines. These findings led to the hypothesis that survival pathways activated by oncogenic mutation regulated this mechanism. Indeed, the oncogenic PI3-Kinase/Akt pathway was demonstrated to regulate the alternative splicing of caspase 9. Further mechanistic studies demonstrate that multiple Akt isoforms can regulate the alternative splicing of caspase 9 in NSCLC. Akt was additionally shown to mediate the exclusion of the exon 3,4,5,6 cassette of caspase 9 via the phospho-state of the RNA trans-factor, SRp30a. Mutagenesis studies identified serine 199, serine 201, serine 227, and serine 234 as critical residues regulating the alternative splicing of caspase 9, as well as playing a role in the anchorage-independent growth of A549 cells. Since dysregulation of this splicing mechanism correlated with NSCLC tumors/cell lines and constitutively active Akt, oncogenic factors for NSCLC known to activate the PI3-Kinase/Akt pathway were examined in HBEC-3KT cells. In contrast to k-ras V12 expression, the overexpression/mutation of EGFR affected the alternative splicing of caspase 9 in a pro-oncogenic manner, dramatically lowering the caspase 9a/9b mRNA ratio. Stable downregulation of caspase 9b by shRNA blocked the ability of E746-A750 del EGFR expressing HBEC-3KTs to induce anchorage-independent growth, suggesting a role for caspase 9b as a cooperative oncogenic factor. These findings were further corroborated by the ability of caspase 9b expression to completely block the inhibition of clonogenic colony formation by erlotinib. Therefore, this study demonstrates that oncogenic factors activating the PI3-Kinase/Akt pathway regulate the alternative splicing of caspase 9, to produce caspase 9b, via a coordinated mechanism involving the phosphorylation of SRp30a. In additional studies, we demonstrate that the PI3-Kinase/PKCι pathway, a pathway important for cancer cell survival and transformation of lung epithelial cells, regulates the alternative splicing of Bcl-x pre-mRNA via modulation of SAP155 expression to produce an anti-apoptotic phenotype in NSCLC. Therefore, these studies link oncogenic mechanisms in NSCLC to the therapeutically relevant and distal target mechanisms of caspase 9 and Bcl-x pre-mRNA splicing.

Les petites particules ribonucléoprotéiques nucléaires (snRNPs) sont les composants majeurs du splicéosome, la machinerie responsable de l'épissage des pré-messagers. La biogenèse des snRNPs est un processus complexe qui fait intervenir de nombreux facteurs comme les protéines SMN et ICln. Au cours de ma thèse, je me suis intéressé à l'étude du rôle de ces deux protéines dans la maturation et la production des snRNPs du splicéosome.Dans la première partie de mon travail, les modifications internes des snRNAs ont été caractérisées dans des cellules dont les corpuscules de Cajal sont dispersés à cause d'une déficience de la protéine SMN. En effet, en plus de son rôle dans les étapes précoces de formation des snRNPs, la protéine SMN est également requise pour la formation des corpuscules de Cajal, structures nucléaires qui concentrent les scaRNAs impliqués dans le processus de modification post-transcriptionnelle des ARNs. Nous avons pu ainsi montrer que la protéine SMN et les corps de Cajal ne sont pas essentiels à la production des résidus 2'-O-methyl et pseudouridine dans les snRNAs majeurs et mineurs.La deuxième partie de mon travail a porté sur l'étude des relations fonctionnelles entre les protéines ICln et SMN in vivo en utilisant l'organisme modèle S. pombe. Après avoir caractérisé un homologue de la protéine humaine dans la levure fissipare, nous avons montré que la protéine ICln n'est pas essentielle mais est importante pour une croissance optimale des cellules de levure. Notre étude montre aussi que la modulation de l'activité de la protéine ICln ne permet pas de compenser les défauts dans la production de snRNPs observés dans les cellules portant un allèle muté de SMN. Finalement, l'utilisation d'une approche génomique montre que la délétion du gène ICln entraine des défauts différentiels d'épissage, indiquant que le choix des sites et la cinétique d'épissage sont fortement dépendants de la concentration des composants de base du splicéosome. / Small nuclear ribonucleoproteins (snRNPs) are the major components of the spliceosome, the machinery responsible for the splicing of pre-messenger RNAs. The biogenesis of snRNPs is a complex process that involves many factors such as the SMN and ICln proteins. During my thesis, I studied the role of these two proteins in the maturation and the production of the spliceosome snRNPs.The goal of the first part of my work was to characterize the internal modifications of snRNAs in SMN-deficient cells carrying disrupted Cajal bodies. Indeed, in addition to its role in the early stages of snRNPs assembly, the SMN protein is also required for the formation of Cajal bodies which are nuclear structures carrying the scaRNAs involved in the post-transcriptional modification process of RNAs. We could show that the SMN protein and Cajal bodies are not essential for the formation of 2'-O-methyl and pseudouridine residues in the major and minor snRNAs.In the second part of my work, the functional relationships between the ICln and SMN proteins were examined in vivo using the S. pombe model organism. We first identified a fission yeast homologue of the human ICln protein and found that the ICln protein is not essential but important for optimal growth of yeast cells. Our study also showed that the modulation of the activity of the ICln protein does not compensate for defects in the production of snRNPs observed in yeast cells carrying a SMN mutated allele. Finally, the use of a genome-wide approach allowed us to show that deletion of the ICln gene resulted in differential splicing defects, indicating that the choice of splice sites and the kinetics of splicing are strongly dependent on the concentration of the basic components of the spliceosome.

Alternative splicing is an important cellular mechanism. It allows to produce multiple protein isoforms from a limited number of genes. Regulation of alternative splicing involves cis-acting elements on pre-mRNA and trans-acting splicing factors (SR and hnRNP proteins). Because splicing occurs co-transcriptionaly, chromatin structure appears to have a role in the regulation of alternative splicing. We have studied the effect of histone acetylation on alternative splicing. We have prepared splicing reporter for alternative EDB exon, which is part of the fibronectin gene. We have shown, that the inhibition of histone deacetylases affects splicing pattern of EDB exon from the reporter in the same way as the splicing of the endogenous EDB exon. Furthermore, we have shown, that the structure of the promoter affects splicing of alternative EDB exon from splicing reporter. Currently we have found out, that the structure of the promoter influences the degree of histone H4 acetylation. Inclusion of alternative EDB exon in mRNA was inversely proportional to histon acetylation on the reporter. This work might explain why various promoters have different splicing patterns of alternative exons.