Ng Kai-on. / "July 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 162-180). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-05-26Bitstream added on 2014-06-13T19:19:51Z : No. of bitstreams: 1 silva_mta_dr_araiq.pdf: 3269290 bytes, checksum: 226805a8de91ee25f88e83234f61efa0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A família Trypanosomatidae inclui diversos parasitas protozoários responsáveis por diferentes doenças humanas. Várias evidências sugerem importantes diferenças entre a maquinária de tradução e processamento de mRNA (trans-splicing) em Tripanosomatídeos quando comparados com eucariótos superiores. Neste contexto, alguns fatores importantes para o funcionamento da célula eucarióticas são os pequenos complexos constituídos de proteínas e RNA, chamados de ribonucleoproteínas (U snRNPs). Esta partículas possuem papel essencial no processamento de RNA mensageiros e durante a reação de splicing apresenta um core comum composto por proteínas (proteínas Sm) and RNAs estruturais (U snRNAs) e um conjunto de proteínas específicas de cada complexo. Embora bem definidas em mamíferos, snRNPs permanecem pouco caracterizadas em Tripanosomatídeos. Ferramentos de bioinformática identificaram quatro possíveis proteínas específicas do complexo U5 snRNP (U5-15K, U5-40K, U5-102K e U5-116K), e importantes parâmetros foram determinados, como peso molecular estimado, domínios e motivos conservados. Este trabalho demonstrou que U5-15K e 45-102K são altamente conservadas entre o Tripanosomatídeos e os domínios Dim1 and Prp1 foram identificados, respectivamente. Técnicas de purificação de complexos (PTP-tag) revelaram que estas proteínas interagem com o U5 snRNA, sugerindo que participem do complexo U5 snRNP. Análises funcionais demonstraram que U5-15K é essencial para viabilidade celular e que de alguma forma esta asssociada tanta a reação de cis quanto de tras-splcing. Experimentos de imunolocalização de U5-15K and U5-102K corroboram este dados, uma vez que as protínas em questão possuem localização nuclear. / There are several protozoan parasites in Trypanosomatidae family, including different agents responsible for human diseases. Several evidences suggest important differences in the translational system and mRNA processing (trans-splicing) in Trypanosomatids when compared to higher eukaryotes. In this context, some important factors for the functioning of eukaryotic cells are the small complexes of RNA and proteins; these particles of ribonucleoproteins (UsnRNPs) have an essential role in the pre-mRNA processing, mainly during splicing. UsnRNP presents a common protein core associated between itself and with the snRNA, named Sm proteins and specific proteins of each snRNP. Even though they are well defined in mammals, snRNPs are still not well characterized in certain Trypanosomatids. Bioinformatics analysis identified four possible U5 snRNP specific proteins (U5-15K, U5-40K, U5-102K and U5-116K), and important parameters were determinated, as estimated molecular weight, motifs and conserved domains. This work shows that the U5-15K and U5-102K proteins are highly conserved among different Tryponosomatids species and Dim1 and Prp1 domains were identified, respectively. Tandem affinity pull-down assay revealed that these proteins interact with U5snRNA, suggesting its participation in U5snRNP particle, and functional analysis showed that U5-15K is essential for cell viability and it is associated in some way to trans and cis-splicing machinery. Immunolocalization experiments corroborated those data, showed U5-15K and U5-102K in the nucleus of the cell.

Chez les organismes multicellulaires la diversité protéique dans chaque cellule et chaque tissu est obtenue initialement en régulant l’expression d’une partie des gènes d’un génome. Ces gènes sélectionnés peuvent ensuite être soumis à un épissage alternatif de sorte que certains exons sont retenus ou exclus dans l’ARNm final. Nous étudions les détails moléculaires de la protéine MEC-8, un facteur d’épissage tissu spécifique chez Caenorhabditis elegans. Les mutants MEC-8 sont responsables d’un phénotype insensible au touché chez Caenorhabditis elegans. Plus précisément, MEC-8 lie le pré-ARNm de mec-2 un composant des récepteurs mécanosensoriels afin de réguler la production d’un isoforme particulier nécessaire pour la transduction du signal mécanosensoriel. Des études portant sur le motif conservé de reconnaissance à l’ARN (RRM) chez des orthologues des vertébrés (RBPMS) et des insectes (couch potato, CPO) ont démontré la présence d’un motif d’homodimérisation dans le domaine RRM1 de MEC-8. Cependant MEC-8 contient aussi un second domaine RRM dans sa partie C-terminale, domaine qui n’est pas retrouvé dans les protéines RBPMS et CPO. Nous avons donc exprimé chaque domaine RRM de MEC-8 indépendamment ainsi que la protéine entière et ces constructions ont été utilisées pour diverses expériences biophysiques. Nous avons ainsi identifié la séquence de liaison optimale pour les deux domaines RRM1 et RRM2. Ces analyses ont aussi été menées sur les domaines homologues issus des protéines RBPMS et CPO qui présentent une forte affinité pour la même séquence d’ARN. Nous avons donc découvert que malgré des différences de fonction et de localisation les membres de la famille RBPMS lient tous le même motif d’ARN. Les détails atomiques des deux RRM en complexe avec leur motif de liaison ont été obtenus en utilisant de la spectroscopie RMN et de la cristallographique des rayons X. Les deux complexes RRM-ligand de MEC-8 présentent de surprenantes similarités dans leur architecture. / In multicellular organisms, proteomic diversity in each cell and tissue is provided initially by selective expression of gene subsets from the total genome, which are further subjected to alternative splicing, such that a different pattern of exons can be retained or excluded in the final protein coding mRNA. We are investigating the molecular details of the tissue-specific splicing factor protein MEC-8 from the worm Caenorhabditis elegans. The MEC-8 mutant protein is responsible for a touch insensitive phenotype in Caenorhabditis elegans, relating to its role as an alternative splicing factor. More precisely, MEC-8 can bind to the mec-2 pre-mRNA, a component of mechanosensory receptor, to regulate the production of a certain isoform required for transducing the touch signal. Previous studies of the conserved RNA Recognition Motif (RRM) domain in orthologues from vertebrate (RBPMS) and insect (couch potato; CPO) have demonstrated a homodimerization motif in MEC-8 RRM1. However, MEC-8 also contains a second RRM domain in the C-terminus that is not found in the characterized RBPMS and CPO proteins. We have therefore expressed the independent RNA-binding domains of MEC-8 as well as the full-length protein and have used these constructs in a variety of biophysical assays. We identified the optimal RNA binding sequence for both the RRM1 and RRM2, and quantified the penalty of sequence variations. The investigation has also been extended to the homologous domains from human RBPMS and Drosophila CPO, which show a high affinity to the same RNA sequence. We therefore find that despite differences in function and localization, the members of the RBPMS protein family all bind to the same RNA motif. Atomic details of binding have also been obtained by using a combination of NMR spectroscopy and X-ray crystallography. The ligand-bound complexes reveal a surprising similarity in the architecture of the bound ligand for the first and second RRM domains from MEC-8.

The spliceosomal U5 snRNP contains several proteins with well characterised functions in splicing, including: Brr2, an ATPase/RNA helicase that disrupts U4/U6 and U2/U6 snRNA base pairing during activation of the spliceosome; Snu114, a GTPase that controls the action of Brr2; and Prp8, the largest and most conserved protein considered to have a central role in the spliceosome, which interacts directly with Snu114 and Brr2. Yeast Cwc21 is one of twelve Bact complex proteins that associate with spliceosomes shortly before the first step of splicing catalysis. Cwc21 interacts directly with Prp8 and Snu114, as does its human orthologue, the SR protein SRm300/SRRM2. Although, Cwc21 is not essential for yeast cell viability, it is required for sporulation. This work aims to identify the function of Cwc21 during meiosis. PP1 is a protein phosphatase required for both steps of splicing. Multiple sequence alignments of Snu114 and Prp8 revealed the presence of putative PP1 binding motifs that are well conserved among different species. This led me to hypothesize that PP1 may interact with Snu114 and/or Prp8 to regulate these or other interacting proteins. By screening intron-containing genes that are expressed in meiosis, I found that Cwc21 is required for splicing HRB1 transcripts. In addition, I show that HRB1 is also required during meiosis. The HRB1 intron contains an unusual branchsite sequence, TACTAATG, which when changed to the consensus branchsite sequence restores sporulation in the absence of Cwc21. Therefore, it is likely that Cwc21 promotes the expression of HRB1 during an early stage of meiosis by stabilising its pre-mRNA in the catalytic centre of the spliceosome. This study demonstrates a novel function for Cwc21 during meiosis. Using yeast two hybrid assay I have identified the interacting regions of Cwc21, PP1 and Brr2 in Snu114. Through biochemical studies I provide evidence for mutually exclusive interaction of Cwc21 and PP1 in the putative PP1 binding motif situated in Snu114 domain ‘IVa’. In the case of yeast Snu114, the PP1 binding motif has a novel sequence ‘YGVQYK’. I also show that the affinity of Cwc21 and PP1 for Snu114 is influenced by the different nucleotide-bound states of Snu114. Furthermore, I show that mutations in Snu114 domain ‘IVa’ restrict Snu114 function during meiosis and affect the MER1 splicing regulatory network. Therefore, Snu114 may play a role in modulating the conformational state of the catalytic spliceosome through its interactions with Cwc21/PP1 in regulating subsets of genes during meiosis. Finally, I show that PP1 is a putative regulator of Prp8.

This study seeks to address one of the simplest questions that can be asked about an interconnected system; what happens to one process in the absence of the other process? This is a more difficult task than it would appear at first, due to the absence of small molecule inhibitors that can inhibit splicing globally in yeast cells. The first results chapter describes the adaptation of a system called the auxin induced degron (AID) to the task of inhibiting pre-mRNA splicing. This system appears to have several advantages over previous methods of inhibiting splicing and has many potential applications. Another hurdle to understanding what happens to transcription in the absence of splicing is the differential stability of pre-mRNA versus mRNA. At steady state the vast majority of transcripts of a specified gene will be mRNA transcripts. This means that even if you could rapidly inhibit splicing it would be a long time before all the pre-existing mRNA would turn-over. If you waited until specified mRNAs turned over it is likely that the cells would be very sick making it difficult to separate primary and secondary effects. The second results chapter shows the use of a metabolic labelling technique using a uracil analogue called 4-thiouracil (4SU). 4SU is added for an extremely short amount of time (1.5 min, 2.5 min, and 5 min) and the RNA produced during the labelling time is isolated by affinity purification. This allows us to study the kinetics of pre-mRNA splicing in wild-type cells and to seek correlations between splicing kinetics and gene architecture. The third results chapter combines the methods used in the previous two chapters to give a new technique called AID4U-seq. AID4U-seq allows for rapid inhibition of splicing and then the ability to isolate only the transcripts that were created after this inhibition came into effect. This should allow for examination of the primary consequences of blocking pre-mRNA splicing at multiple stages during spliceosome assembly. Additionally AID4U-seq is immediately applicable to the study of other areas of RNA processing. Defining the effects on the transcriptome of inhibiting splicing at multiple stages of assembly is an ambitious aim likely to require many more years of research. Therefore this thesis chiefly seeks to illustrate a novel strategy to begin dissecting a complex issue in which splicing, transcription, degradation and the post-transcriptional modification of histones are all likely to have roles.

Productive HIV-1 transcription yields a single ∼­9.2kb RNA. From this ∼9.2kb genomic RNA, greater than 40 different subgenomic mRNAs can be produced through alternative splicing using four 5' splice sites (ss) and seven 3'ss. Splice site utilization is governed by the inherent strength of the splice sites and by several identified cis acting elements. The HIV-1 Vif protein, required to overcome the cellular antiviral factor APOBEC3G, is encoded by a singly-spliced mRNA coupling 5'ss D1 to 3'ss A1. Alternatively, mRNAs spliced at A1 can utilize a downstream 5'ss, D2, resulting in inclusion of non-coding exon 2 in a small percentage of mRNAs. Expression of vif mRNA within infected cells is required but maintained at low levels. The purpose of studies described in this thesis was to identify and characterize elements within the HIV-1 genome regulating vif mRNA splicing. We identified an exonic splicing enhancer (ESE) within the 18nt downstream of HIV-1 3'ss A1, ESEVif. Mutation of ESEVif within the HIV-1 proviral clone pNL4-3 resulted in a dramatic decrease in vif mRNA, Vif protein, and undetectable levels of non-coding exon 2 inclusion. The cellular splicing factor SRp75 was found to selectively bind ESEVif in vitro. ESEVif mutant virus replicated in APOBEC3G-deficient T-cell lines as efficiently as wild-type virus. In APOBEC3G-producing T-cell lines, ESEVif mutant virus replicated to lower levels than wild-type virus. Other studies have identified additional mRNA splicing elements regulating splicing at A1: the downstream 5'ss D2 can promote or repress splicing, a G4 motif downstream of D2 represses splicing, and two ASF/SF2 dependent splicing enhancers, ESEM1 and ESEM2, promote splicing. Mutational analysis described in this thesis determined that loss of both ESEVif and the G4 motif resulted in wild-type levels of splicing at A1. Mutations of each identified ESE influenced vif mRNA splicing in the order ESEVif>ESEM2>ESEM1. The data presented in this thesis support a model of vif mRNA splicing regulation in which exon 2 ESE act to overcome the negative G4 motif insuring sufficient levels of Vif production for efficient replication in the presence of APOBEC3G.

Mitochondria are important cellular organelles whose functions include generation of ATP, sequestration and release of pro-apoptotic molecules and calcium buffering. Mitochondria function is tightly linked to organelle morphology, which exits in a dynamic spectrum between a highly interconnected/fused mitochondria network to a punctate/fragmented scattering of individual mitochondrion. A family of large GTPase enzymes modulates this spectrum, with fusion catalyzed through the actions of mitofusin 1 and 2 (Mfn1/2) on the outer mitochondria membrane (OMM) and optic atrophy 1 (Opa1) causing fusion of the inner mitochondria membrane (IMM). On the other end of the spectrum, fragmentation is catalyzed through the actions of dynamin-related protein 1 (Drp1). Drp1 is recruited from the cytosol to binding partners at the OMM, organizes into concentric spiral rings, undergoes GTP hydrolysis to constrict the ring and pinches mitochondrion in two. While fragmentation is achieved through the action of only one GTPase enzyme, the mechanisms behind the complex regulation of Drp1 remain relatively obscure. In order to expand upon known Drp1 regulatory mechanisms, an examination of how both Drp1 splicing and Drp1 recruitment to the OMM contributes to protein regulation is necessary. Drp1 contains three alternatively spliced exons, resulting in the potential generation of eight protein isoforms. Each of these isoforms is capable of inducing mitochondrial fragmentation, however one exon arrangement (termed Drp1-x01) can also bind to microtubules within the cell. Characterization of the Drp1-x01 isoform at both the RNA and protein level indicate an important, yet incompletely characterized, role in immune system biology. Drp1 is capable of interacting with several proteins localized at the OMM. Among these, mitochondria fission factor (Mff) has been implicated in the formation of Drp1 spirals and the eventual fragmentation process. Mff contains four alternatively spliced exons as well as several phosphorylation sites identified through nonbiased phosphoproteomic screens. Inclusion of alternative exons to the Mff structure decreases its ability to recruit Drp1 from the cytosol, while phosphomimetic substitutions to conserved serine residues enhances the Drp1::Mff interaction. Taken together, this suggests that regulation of mitochondrial fragmentation occurs at the pretranslational (alternative splicing) and the posttranslational (phosphorylation) level is critical for maintaining the complex, yet essential, balance between mitochondrial fission and fragmentation.

Most human genes undergo alternative splicing, and many abnormal splicing processes are associated with human diseases. However, the molecular relationship between alternative splicing and tumorigenesis is not well understood. Here, we identified novel Krüppel-like factor 4 (KLF4) splicing variants produced by exon skipping in human cancer cell lines as well as colon tumor tissues. To elucidate the mechanism involved in KLF4 alternative splicing, we developed KLF4 minigene system and found that RNA binding motif protein 5 (RBM5) plays an important role in KLF4 splicing, as assessed by gain and loss of functional studies. Several anti-tumorigenic compounds were also tested for KLF4 splicing. Interestingly, sulindac sulfide restored wild type KLF4 (KLF4L) expression and this is mediated by dephosphorylation of RBM5. Another splicing variant, small KLF4 (KLF4S), localizes in the cytoplasm and nucleus, and antagonizes transcriptional activity of wild type KLF4. Our data suggest that RBM5 plays a pivotal role in the alternative splicing of KLF4, and these splicing variant forms may impact tumorigenesis.

Le processus d'excision-épissage des RNAs pré-messagers (pré-mRNAs) est une étape essentielle dans l'expression de la majorité des gènes eucaryotiques. Lépissage se déroule dans le noyau au sein dun complexe macromoléculaire appelé spliceosome, ou particule dépissage, qui sassemble sur des sites précis le long des pré-mRNAs. Il consiste en cinq petites particules nucléaires ribonucléoprotéiques dénommées snRNPs (small nuclear Ribonucleoproteins) constituées de snRNAs (small nuclear RNAs) riches en uridines (U1, U2, U4/U6 et U5) et denviron 150 protéines associées (Patel et Steitz, 2003; Jurica et al., 2004). Lépissage requiert également de nombreuses protéines non constitutives des snRNPs appelées de manière générique facteurs essentiels dépissage. Parmi ceux-ci, les protéines SR constituent une famille de facteurs dépissage conservés chez les Eucaryotes (Barta et al., 2008; Long et Caceres, 2009). Ces protéines possèdent toutes un ou deux domaines de liaison au RNA appelé RRM (RNA Recognition Motif) en N-terminal et un domaine riche en dipeptides sérine et arginine (SR ou RS) en C-terminal. Elles jouent un rôle crucial dans lépissage constitutif et alternatif et ce, par un jeu complexe d'interactions protéine-protéine et protéine-RNA (Bourgeois et al., 2004; Reddy, 2007). Le mécanisme dépissage alternatif permet de produire différents mRNAs à partir dun seul pré-mRNA et de ce fait peut amener à la synthèse de plusieurs isoformes protéiques. Chez Arabidopsis de ~20 à 40% des pré-mRNAs sont épissés alternativement (Campbell et al., 2006; Wang et Brendel, 2006; Severing et al., 2009; Filichkin et al., 2010). Le séquençage complet du génome dArabidopsis a révélé que ~80% des régions codantes des gènes nucléaires sont interrompues par des introns (Iida et al., 2004) et a également permis lidentification de 19 protéines SR (Kalyna et Barta, 2004). Ces protéines sont classées en 7 sous-familles, certaines ayant leur homologue chez lhomme, dautres étant spécifiques aux végétaux (Kalyna et Barta, 2004). Le nombre de protéines SR est plus élevé chez Arabidopsis comparé à lhomme, chez qui on en dénombre seulement 11. Cela soulignerait des différences entre ces deux règnes au niveau des mécanismes dépissage et des facteurs impliqués dans ce processus. Les travaux antérieurs réalisés par immunofluorescence (Docquier et al., 2004) et par fusion traductionnelle avec la GFP (Green Fluorescent Protein) ont permis de montrer que les protéines SR dArabidopsis se localisent dans le noyau et présentent une organisation nucléaire en speckles (ou Splicing Factors Compartments, SFCs) et ce dans différents types cellulaires (Ali et al., 2003; Docquier et al., 2004; Fang et al., 2004; Tillemans et al., 2005). Les speckles sont considérés comme des sites de stockage et/ou dassemblage des complexes dépissage (Lamond et Spector, 2003). La phosphorylation des protéines SR joue un rôle important dans la régulation de leur localisation et de leurs fonctions. Une hyper- ou hypophosphorylation réduit leur activité générale suggérant que leur niveau de phosphorylation est strictement régulé in vivo (Misteli et Spector, 1996; Lai et al., 2003; Lin et al., 2005). Ainsi, la relocalisation des protéines SR au sein des speckles est activement dépendante de leur état de phosphorylation (Misteli, 2000; Docquier et al., 2004; Huang et Steitz, 2005; Tillemans et al., 2005). Le flux des facteurs nucléaires au sein de ces compartiments et leurs interactions transitoires et rapides soulignent une organisation spatiale et temporelle très dynamique (Eils et al., 2000; Phair et Misteli, 2000; Dundr et Misteli, 2001). Le but de cette thèse de doctorat est détablir un profil dexpression précis de RSZp22 au cours du développement de la plante, de caractériser les propriétés nucléocytoplasmiques de RSZp22 et enfin définir les rôles des domaines de liaison au RNA dans sa dynamique. RSZp22 est un homologue de la protéine 9G8 humaine. Cette protéine SR possède un domaine Zn-knuckle à motif CCHC localisé entre un domaine RRM unique et le domaine RS. Le domaine RRM reconnait les séquences activatrices dépissage présentes sur les exons (ESEs Exonic Splicing Enhancers) alors que le domaine RS est impliqué dans les interactions protéine-protéine et protéine-RNA (Shen et al., 2004). Le rôle du Zn-knuckle, qui se retrouve dans deux sous-familles de protéines SR dArabidopsis (RSZ et RS2Z), nest pas encore bien caractérisé et les spécificités dinteractions des domaines RRM et Zn-knuckle nont pas encore été étudiées. Parmi les protéines SR étudiées, RSZp22 semble être la seule à se localiser et se concentrer au sein du nucléole selon les conditions physiologiques dans lesquelles la cellule se trouve (Tillemans et al., 2005). Les travaux réalisés récemment au laboratoire montrent que RSZp22 fusionnée à la GFP et surexprimée transitoirement dans des cellules foliaires est une protéine hautement dynamique. Sa mobilité dépend du niveau de phosphorylation et de la concentration en ATP de la cellule. Le développement puis lutilisation des approches de FRAP (Fluorescence Recovery After Photobleaching) et de FLIP (Fluorescence Loss In Photobleaching) ont permis de suggérer que RSZp22 est une protéine SR dynamique et quelle peut transiter entre le noyau et le cytoplasme. RSZp22 ferait ainsi partie des protéines SR dites navettes comme, entre autres, son homologue humain 9G8 (Tillemans et al., 2006). Basés sur la délétion de domaines entiers de la protéine, nos travaux montrent également que le domaine RS est impliqué dans lorganisation en speckles des protéines SR dArabidopsis au sein du nucléoplasme et quil jouerait le rôle de signal de localisation nucléaire (NLS Nuclear Localisation Signal). Cette étude suggère aussi que le domaine RRM nest pas indispensable pour lorganisation en speckles de RSZp22 et que le domaine Zn-knuckle interviendrait dans lexportation de la protéine (Tillemans et al., 2006). Les études antérieures de la dynamique des protéines SR d'Arabidopsis et en particulier de RSZp22 ont été réalisées après surexpression ectopique -et quelquefois hétérologue- des facteurs d'épissage. Dans cette étude, nous avons exprimé la protéine RSZp22-GFP sous le contrôle du promoteur endogène RSZp22 après transformation stable dArabidopsis. Parallèlement, une analyse de RT-PCR quantitative et lutilisation du gène rapporteur -glucuronidase (GUS), nous ont permis d'établir un profil dexpression précis de RSZp22 et de complémenter -et valider- la localisation tissulaire de la protéine de fusion. Nous avons ensuite étudié en plantes transgéniques, la dynamique de la protéine RSZp22-GFP dans des types cellulaires spécifiques. Par analyses de FLIP cytoplasmique (dénommé FLIP-shuttling), nous avons démontré que RSZp22 est bien une protéine SR navette nucléocytoplasmique et établi une cinétique d'exportation. Nous avons également confirmé que son exportation vers le cytoplasme est partiellement dépendante de la voie du récepteur CRM1/XPO1. Notre travail a ainsi permis de mettre en évidence les avantages complémentaires des techniques de surexpression en transformation transitoire et de lexpression stable et spécifique pour létude de la dynamique des protéines nucléaires. Enfin, nous avons montré par mutagenèse dirigée que les motifs RNP1 et Zn-knuckle ne sont pas nécessaires pour la localisation nucléaire de la protéine RSZp22 ni pour sa concentration en speckles. Ces motifs de liaison au RNA sont cependant impliqués dans lexportation de RSZp22 par la voie CRM1/XPO1. De plus, les expériences de FRET (Fluorescence Resonance Energy Transfert) indiquent que ces motifs interviennent dans les interactions moléculaires impliquant RSZp22. Ainsi, ce travail a permis de caractériser la dynamique nucléocytoplasmique de RSZp22 dans des tissus spécifiques dArabidopsis et de mettre en évidence limportance de son interaction avec le mRNA pour son exportation par la voie CRM1/XPO1.

Monogenic diseases make excellent models for the study of gene functions and basal cellular mechanisms in humans. The aim of this thesis was to elucidate how genetic mutations affect the basal cellular mechanisms in the monogenic diseases Nerve growth factor (NGF) dependent pain insensitivity and Iron-Sulphur cluster assembly protein U (ISCU) myopathy. NGF dependent pain insensitivity is a rare genetic disorder with clinical manifestations that include insensitivity to deep pain, development of Charcot joints, and impaired temperature sensation but with no effect on mental abilities. The disease is caused by a missense mutation in the NGFβ gene causing a drastic amino acid substitution (R221W) in a well-conserved region of the protein. NGF is secreted in limited amounts by its target tissues and is important for the development and maintenance of the cholinergic forebrain neurons as well as the sensory and sympathetic neurons. To reveal the underlying mechanisms of disease we performed functional studies of the mutant NGF protein. We could show that mutant NGF was unable to induce differentiation of PC12 cells as a consequence of impaired secretion. Furthermore, mutant NGF had different intracellular localisation compared to normal NGF and resided mostly in its unprocessed form proNGF. Mature NGF and proNGF have different binding properties to the receptors TrkA and p75. Individuals with mutations in TRKA are, aside from pain insensitive mentally affected; therefore it has been proposed that the R221W mutation mainly affects the interaction with p75. In agreement with this, we could show that R221W NGF was able to bind and activate TrkA whereas the interaction with p75 was impaired as compared to normal NGF. ISCU myopathy is a monogenic disease where the affected patients suffer from severe exercise intolerance resulting in muscle cramps and sometimes severe lactic acidosis. The disease is caused by a point mutation in the last intron of the Iron sulphur cluster assembly gene, ISCU, resulting in the inclusion of a part of the intron in the mRNA. ISCU functions as a scaffold protein in the assembly of iron-sulphur (Fe-S) clusters important for electron transport in Kreb’s cycle and the respiratory chain. We have shown that ISCU is vital in mammals since complete knock-down of Iscu in mice results in early embryonic death. The deletion of ISCU homologous in lower organisms has also been shown fatal. In spite this central role in energy metabolism the disease is restricted to the patient’s skeletal muscles while other energy demanding organs seem unaffected. To address this contradiction we examined if tissue-specific differences in the splicing of mutant ISCU could explain the muscle-specific phenotype. We could show that the splicing pattern did, indeed, differ with more incorrectly spliced ISCU in muscle compared to other tissues. This was accompanied by a decrease in Fe-S containing proteins in muscle, while no decrease was observed in other tissues. Alternative splicing is more common then previously thought and may depend upon interacting factors and/or differences in the surrounding milieu. To reveal plausible mechanisms involved in the tissue-specific splicing we identified nuclear factors that interacted with the region where the mutation was located. Five interacting factors were identified, out of which three affected the splicing of ISCU. PTBP1 was shown to repress the incorrect splicing while IGF2BP1 and RBM39 repressed the formation of normal transcript and could also counteract the effect of PTBP1. IGF2BP1 was the only factor that showed higher affinity to the mutant sequence making it a possible key factor in the incorrect splicing of the mutant ISCU gene. Together, these results offer important insights into the cellular mechanisms causing these diseases. We found impaired secretion and inaccurate sorting of NGF to be cellular mechanisms contributing to NGF dependent pain insensitivity while tissue-specific splicing of ISCU was found to be the event contributing to the phenotype of ISCU myopathy.