Microtubules (MT) are regulated by multiple categories of proteins, including proteins responsible for severing MTs that are therefore called MT-severing proteins. Studies of katanin, spastin, and fidgetin in animal systems have clarified that these proteins are MT-severing. However, studies in plants have been limited to katanin p60, and little is known about spastin or fidgetin and their function in plants. I looked at plant genomes to identify MT-severing protein homologues to clarify which severing proteins exist in plants. I obtained data from a variety of eukaryotic species to look for MT-severing proteins using homology to human proteins and analyzed these protein sequences to obtain information on the evolution of MT-severing proteins in different species. I focused this analysis on MT-severing proteins in the maize and Arabidopsis thaliana genomes. I created evolutionary phylogenetic trees for katanin-p60, katanin-p80, spastin, and fidgetin using sequences from animal, plant, and fungal genomes. I focused on Arabidopsis spastin and worked to understand its functionality by identifying protein interaction partners. The yeast two-hybrid technique was used to screen an Arabidopsis cDNA library to identify putative spastin interactors. I sought to confirm the putative protein interactions by using molecular tools for protein localization such as the YFP system. Finally, a Biomolecular Fluorescence Complementation (BiFC) assay was initiated as a proof of concept for confirmation of in vivo protein-protein interaction.

Die vorliegende Arbeit beschäftigt sich mit verschiedenen Aspekten der Assoziation von Saccharose-Synthase (SUS) mit subzellulären Strukturen. Durch cDNA-Durchmusterungen konnten proteinogene Bindepartner von SUS sowie Aktin identifiziert und zum Teil verifiziert werden. Die dritte Isoform SuS3 aus Mais wurde auf molekularer Ebene identifiziert und das rekombinante Protein biochemisch charakterisiert. Trotz signifikanter Sequenzunterschiede zwischen den SUS-Isoformen, wurden ähnliche katalytische Eigenschaften und mögliche posttranslationale Modifikationen der Enzyme nachgewiesen, darunter die Redox-Modifikation der Enzymaktivität und das Potential zur reversiblen Phosphorylierung. Der Einfluss der Phoshorylierung von SUS auf dessen enzymatische und assoziative Aktivität wurde mittels mutagenisiertem Protein untersucht und zeigte kein stark verändertes Verhalten infolge der Mutationen. Eine metabolische Regulation der SUS-Aktin-Wechselwirkung durch Zucker konnte bestätigt und die katalytische Aktivität von SUS in Gegenwart von Aktin gezeigt werden. Assoziationsstudien von Aktin mit synthetischen Peptiden sowie immunologische Untersuchungen lieferten Hinweise für die Aktinbindedomäne in SUS. Co-Pelletierungsexperimente zeigten die Assoziation von SUS mit Mikrotubuli aus Rinderhirn. In vitro konkurriert SUS mit Aldolase um die Bindung an Miktotubuli. Als proteinogene Bindepartner von SUS wurden einige im Kohlenhydratstoffwechsel sowie im 26S-Proteasom-Komplex involvierte Proteine identifiziert. Ebenso wurde eine Glutathion-Peroxidase identifiziert, die ubiquitäre Transkriptakkumulation dokumentiert und die katalytische Aktivität des rekombinanten Proteins gezeigt. Eine weitere cDNA-Durchmusterung führte zur Identifikation verschiedener glykolytischer Enzyme als potentielle Interaktionspartner von Mais-Aktin sowie zu Bindepartnern, die nach Sequenzanalyse Domänen mit Homologien zu bekannten ABPs aus tierischen Organismen zeigten.

Microsporidia are spore-forming, obligate intracellular pathogens typically associated with opportunistic infections in immunocompromised individuals. Treatment options for microsporidia infections in humans are limited and additional research is necessary to create better therapeutic agents. For many pathogenic organisms, adhesion to the host cell surface is a prerequisite for tissue colonization and invasion. Our previous research has demonstrated a direct relationship between adherence of microsporidia spores to the surface of host cells and infectivity in vitro. In an effort to better understand adherence, we have turned our attention to determining what proteins may be involved in this process. Examination of the Encephalitozoon cuniculi genome database revealed a gene encoding a protein with sequence homology to members of the ADAM (a disintegrin and metalloprotease) family of type I transmembrane glycoproteins. The microsporidia ADAM (MADAM) protein is of interest because ADAMs are known to be involved in a variety of biological processes including cell adhesion, proteolysis, cell fusion, and signaling. The objectives for this study were to examine the localization of MADAM, analyze its potential involvement during adherence and/or host cell infection, and to identify potential binding partners or substrates. Through the use of immunoelectron transmission microscopy, we demonstrated that MADAM is localized to the surface exposed exospore, plasma membrane, and the polar sac-anchoring disk complex (a bell-shaped structure at the spore apex involved in the infection process). Location of MADAM within the exospore and polar sac-anchoring disk suggests that MADAM is in a position to facilitate spore adherence or host cell infection. Thus far, we have been unable to conclusively demonstrate that MADAM is involved in either event. Through the use of a yeast two-hybrid system, we were able to identify polar tube protein 3 (PTP3) as a potential binding partner or substrate for the MADAM protein. The interaction between MADAM and PTP3 was confirmed by in vitro co-immunoprecipitation. PTP3 is hypothesized to be involved in the process of polar tube extrusion by stabilizing the interaction between PTP1-PTP2 polymers. Further analysis of the interaction between MADAM and PTP3 may lead to a better understanding of the events that occur during polar tube extrusion.

No description available.

No description available.

Pluripotenz wird durch viele Stammzell-spezifische Transkriptionsfaktoren wie Oct3/4, Nanog und Sox2 sowie deren Funktion in ihrem regulatorischen Netzwerk etabliert und aufrechterhalten. Viele Studien haben gezeigt, wie diese Pluripotenz-assoziierten Faktoren ihre Zielgene regulieren. Dies geschieht durch die Interaktion mit bekannten und unbekannten Interaktionspartnern. In der vorliegenden Arbeit haben wir Zfp819 als einen neuen Pluripotenz-assoziierten Faktor beschrieben und dessen Funktion in pluripotenten Stammzellen untersucht. Im ersten Teil der vorliegenden Arbeit haben wir zwei cDNA-Banken für Yeast two Hybdrid (Y2H)-Assays aus unterschiedlichen pluripotenten Stammzelltypen generiert. Dies hatte zum Ziel, potentielle Interaktionspartner eines Kandidatenproteins zu identifizieren um dadurch Eindrücke über die Funktion des Proteins zu gewinnen. Für die Identifizierung von potentiellen Interaktionspartnern von Zfp819 haben wir die cDNA-Bank aus embryonalen Stammzellen benutzt. Wir konnten 17 putative Interaktionspartner identifizieren und daraus ein hypothetisches „Interaktom“ von Zfp819 generieren. Die Einordnung der putativen Interaktionspartner nach ihrer Gen-Ontologie (GO) ließ vermuten, dass Zfp819 eine Rolle in der Regulation der Transkription, der Aufrechterhaltung der genetischen Integrität und im Zellzyklus bzw. bei der Apoptose spielt. Im zweiten Teil der vorliegenden Arbeit wurde die sehr intensive Expression von Zfp819 in undifferenzierten pluripotenten Zelllinien gezeigt. Desweiteren konnte die Promotorregion von Zfp819 identifiziert werden, und es wurde gezeigt, dass diese mit epigenetischen Mustern ausgestattet ist. Zusätzlich konnten wir Regionen im Zfp819-Gen identifizieren, die für die nukleäre Lokalisation von Zfp819 verantwortlich sind. Desweiteren konnten wir zeigen, dass Zfp819 in der transkriptionellen Repression von spezifischen endogenen, retroviralen Elementen (ERVs) in pluripotenten Zellen eine Rolle spielt. Durch zelluläre und biochemische Studien konnten wir zeigen, dass Zfp819 mit vielen Proteinen interagiert (z.B. Kap1 und Chd4), welche für die Aufrechterhaltung der genomischen Integrität von Bedeutung sind. Tatsächlich resultierte der Verlust von Zfp819 in embryonalen Stammzellen in einer erhöhten Anfälligkeit für DNA-Schäden und in einer verminderten DNA-Reparatur. Zusammenfassend lassen die Identifizierung der Interaktionspartner sowie die Ergebnisse der molekularen und der funktionellen Studien vermuten, dass Zfp819 durch die Unterdrückung von ausgewählten ERVs eine Rolle in der Regulation der genomischen Stabilität von pluripotenten Zellen spielt.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) occurs when the cardiomyocytes in the left ventricle become enlarged by increasing in mass in response to haemodynamic pressure overload. This can either be attributed to a normal physiological response to exercise or can be the result of a maladaptive process or disease state, such as chronic hypertension. Hypertrophic cardiomyopathy (HCM) is the most common form of Mendelian-inherited cardiac disease. A defining characteristic thereof is primary LVH that occurs when there are no other hypertrophy-predisposing conditions present. Therefore, HCM provides a unique opportunity to study the molecular determinants of LVH in the context of a Mendelian disorder, instead of in more complex disorders such as hypertension. Over 1000 HCM-causing mutations in 19 genes have been identified thus far, most of them encoding sarcomeric proteins residing in the sarcomeric C-zone. However, for many HCM patients no disease-causing genes have been identified. Moreover, studies have shown phenotypic variation in presentation of disease in, as well as between, families in which the same HCM-causing mutation segregates. This has led many investigators to conclude that genetic modifiers of hypertrophy exist. The aim of the study was to identify novel plausible HCM-causing or modifier genes by searching for interactors of a known HCM-causing protein, namely titin. The hypothesis was that genes encoding proteins, which interact with proteins that are encoded by known HCM-causative genes, may also be considered HCM-causing or may modify the HCM phenotype. To this end, the aim was to identify novel interactors of the 11-domain super-repeat region of titin, which resides within the sarcomeric C-zone, using yeast two-hybrid analysis. Five putative interactors of the 11-domain super-repeat region of titin were identified in this study. These interactions were subsequently verified by colocalisation in H9C2 rat cardiomyocytes, providing further evidence for possible interactions between titin and these proteins. The putative interactor proteins of titin determined from the Y2H library screen were: filamin C (FLNC), phosphatidylethanolamine-binding protein 4 (PEBP4), heart-type fatty acid binding protein 3 (H-FABP3), myomesin 2 (MYOM2) and myomesin 1 (MYOM1). The FLNC gene could be a candidate for cardiac diseases, especially cardiomyopathies that are associated with hypertrophy or developmental defects. The putative interaction of titin and PEBP4 is speculated to be indicative of the formation of the interstitial fibrosis and myocyte disarray seen in HCM. Heart-type fatty acid-binding protein 3 has prognostic value to predict recurrent cardiac events. Its suggested interaction with titin is speculated to play a role in inhibiting its functional abilities. Myomesin 2 is jointly responsible, with MYOM1, for the formation of a head structure on one end of the titin string that connects the Z and M bands of the sarcomere. This is speculated to be linked to a developmental error with the result being a defect in sarcomeric structure formation, which could result in pathologies such as HCM. Therefore, these identified proteins could likely play a functional role in HCM due to their interactions with titin. This research could thus help with new insights into the further understanding of HCM patho-aetiology. / AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) ontstaan wanneer die kardiomyosiete in die linkerventrikel vergroot as gevolg van 'n verhoging in massa in reaksie op hemodinamiese drukoorlading. Dit kan toegeskryf word aan 'n normale fisiologiese respons op oefening of kan die gevolg wees van 'n wanaangepaste of siektetoestand, soos chroniese hipertensie. Hipertrofiese kardiomiopatie (HKM) is die mees algemene vorm van Mendeliese oorerflike hartsiekte. 'n Bepalende eienskap daarvan is primêre LVH, wat plaasvind wanneer daar geen ander hipertrofie-predisponerende voorwaardes teenwoordig is nie. Gevolglik bied HKM 'n unieke geleentheid om die molekulêre derterminante van LVH te bestudeer, in die konteks van 'n Mendeliese oorerflike siekte, in plaas van om dit in die meer komplekse siektes soos hoë bloeddruk te bestudeer. Meer as 1000 HKM-veroorsakende mutasies is tot dusver in 19 gene geïdentifiseer. Die meeste van hulle kodeer vir sarkomeriese proteïene wat in die C-sone voorkom. Egter, vir baie HKM-pasiënte is geen siekte-veroorsakende gene al geïdentifiseer nie. Daarbenewens het studies getoon dat variasie in fenotipiese aanbieding van die siekte in, sowel as tussen, families voorkom wat dieselfde HKM-veroorsakende mutasie het. Dit het daartoe gelei dat baie navorsers tot die gevolgtrekking gekom het dat genetiese wysigers van hipertrofie wel bestaan. Die doel van die studie was om nuwe moontlike HKM-veroorsakende of wysiger-gene te identifiseer deur te soek vir interaktors van 'n bekende HKM-veroorsakende proteïen, naamlik titin. Die hipotese was dat gene wat vir proteïene kodeer, wat in wisselwerking is met proteïene wat geïnkripteer word deur bekende HKM-veroorsakende gene, ook oorweeg kan word om HKM te veroorsaak. Dit kan ook die HKM fenotipe verander. Dus was die doel om nuwe interaktors van die 11-domein super-herhaalstreek van titin, soos gevind binne die sarkomeriese C-sone, te identifiseer deur middel van gis-twee-hibried-analise. Vyf vermeende interaktors van die 11-domein super-herhaalstreek van titin is in hierdie studie geïdentifiseer. Hierdie interaksies is later geverifieer met behulp van ko-lokalisering in H9C2-rotkardiomyosiete, wat verdere bewyse vir moontlike interaksies tussen titin en hierdie proteïene verskaf. Die vermeende interaktor-proteïene van titin wat bepaal is vanaf die gis-twee-hibried-biblioteeksifting was as volg: filamin C (FLNC), phosphatidylethanolamine-bindingsproteïen 4 (PEBP4), hart-tipe-vetsuur bindingsproteïen 3 (H-FABP3), myomesin 2 (MYOM2) en myomesin 1 (MYOM1). Die FLNC-geen kan 'n kandidaat vir kardiale siektes, veral kardiomiopatieë, wees wat geassosieer word met hipertrofie of ontwikkelingsafwykings. Die vermeende interaksie van titin en PEBP4 dui daarop om 'n aanduiding te wees vir die vorming van die interstisiële fibrose en miokardiale wanorde, soos gesien in HKM. Hart-tipe-vetsuur bindingsproteïen 3 het prognostiese waarde om herhalende kardiale gebeure te voorspel. Verder dui sy voorgestelde interaksie met titin moontlik daarop dat dit 'n rol kan speel in die inhibering van sy funksionele vermoëns. Myomesin 2 tesame met MYOM1 is verantwoordelik vir die vorming van 'n kopstruktuur aan die een kant van die titinstring wat dan die Z- en M-bande van die sarkomeer verbind. Daar word vermoed dat dit gekoppel is aan 'n ontwikkelingsfout, met die gevolg dat daar 'n defek is in sarkomeriese struktuurvorming, wat weer kan lei tot patologieë soos HKM. / Mrs Wendy Ackerman / Prof Paul van Helden / National Research Foundation (NRF) / Stellenbosch University

A biologia da reprodução de plantas é um campo de grande interesse, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes). O pistilo é o órgão reprodutivo feminino, composto de estigma, estilete e ovário. Devido à importância central do pistilo no sucesso da reprodução de plantas, faz-se necessário um melhor conhecimento dos genes e processos que regulam seu desenvolvimento e funcionamento. Estudos comparativos da expressão gênica nos órgãos vegetativos e reprodutivos de Nicotiana tabacum revelaram genes de expressão preferencial nos órgãos reprodutivos, entre eles alguns codificando proteínas de função ainda desconhecida. Um destes genes foi caracterizado e denominado SCI1 (Stigma/style Cell-cycle Inhibitor 1), por apresentar um papel importante no desenvolvimento do estigma/estilete, atuando como um inibidor de ciclo celular tecido-específico (DePaoli et al., 2011). O presente trabalho teve como objetivo estudar os mecanismos moleculares pelos quais NtSCI1 regula o ciclo celular, investigando seus parceiros de interação. Em um ensaio de pull-down, utilizando-se extrato proteico nuclear de estigmas/estiletes de N. tabacum, vários putativos reguladores de ciclo celular foram identificados, sendo a interação entre NtSCI1 e NtCDKG;2 confirmada por BiFC e localizada no nucléolo. Uma biblioteca de cDNAs de estigmas/estiletes de N. tabacum, no sistema de duplo-híbrido de levedura, foi construída com sucesso. O screening desta biblioteca, utilizando BD-NtSCI1 como \"isca\", permitiu a identificação de vários parceiros de interação com NtSCI1, entre eles: uma helicase de RNA DEAD-BOX, a proteína 14-3-3D2, dois fatores de transcrição (HOMEOBOX-22 e STOREKEEPER), um fator de splicing portador do domínio SWAP, uma quinase de adenosina e uma transposase. As interações entre NtSCI1 e os três primeiros parceiros citados já foram confirmadas por BiFC (observadas no núcleo e nucléolo) e a interação entre NtSCI1 e Nt14-3-3D2 foi confirmada também por co-imunoprecipitação. O envolvimento de NtSCI1 com a regulação do ciclo celular foi corroborado pela interação entre NtSCI1 e a proteína NtCICLINA-L1 (subunidade regulatória de CDKG;2), confirmada por duplo-híbrido e por BiFC, no nucléolo. A interação entre NtSCI1 e NtCICLINA-RELATED também foi confirmada por BiFC. Para entender a dinâmica de NtSCI1 no nucléolo, foi estudada a localização subcelular da proteína de fusão NtSCI1-GFP durante as fases do ciclo celular. NtSCI1-GFP foi observada no nucléolo de células BY-2 em interfase e prófase, desaparecendo na metáfase e anáfase e reaparecendo no nucléolo no final da telófase, mostrando que a presença de NtSCI1 na célula é controlada pelo ciclo celular. A construção de uma primeira versão do interactoma de NtSCI1 mostrou seu envolvimento direto e indireto com proteínas relacionadas ao metabolismo de RNAs, controle da transcrição e regulação do ciclo celular. Estes resultados sugerem que NtSCI1 possa atuar no controle do ciclo celular de forma não canônica, por meio de múltiplos processos paralelos que interconectam aspectos da regulação da transcrição e o processamento de RNAs com o controle do ciclo celular. / The biology of plant reproduction is a field of great interest, since most of the food consumed by humans is composed of reproductive parts of plants (fruits and seeds). The pistil is the female reproductive organ, composed of stigma, style and ovary. Due to the central importance of the pistil in the success of plant reproduction, a better knowledge of the genes and processes that regulate pistil development and function is necessary. Comparative studies of gene expression in vegetative and reproductive organs of Nicotiana tabacum have revealed genes preferentially expressed in the reproductive organs, among them some encoding proteins of unknown function. One of these genes was characterized and denominated SCI1 (Stigma/style Cell-cycle Inhibitor 1), since it has an important role in stigma/style development, acting as a tissue-specific cell-cycle inhibitor (DePaoli et al., 2011). The objective of the present work was to study the molecular mechanisms through which NtSCI1 regulates the cell cycle investigating its interaction partners. In a pull-down assay, using nuclear protein extracts from N. tabacum stigmas/styles, several putative cell cycle regulators were identified. Among them, the interaction between NtSCI1 and NtCDKG;2 was confirmed by BiFC and localized in the nucleolus. A N. tabacum stigma/style cDNA library in the yeast two-hybrid system was successfully constructed. The screening of this library, using BD-NtSCI1 as bait, allowed the identification of several NtSCI1 interaction partners, among them: a DEAD-BOX RNA helicase; the 14-3-3D2 protein; two transcription factors (HOMEOBOX-22 and STOREKEEPER); a splicing factor containing a SWAP domain; an adenosine kinase; and a transposase. The interactions between NtSCI1 and the first three mentioned partners have already been confirmed by BiFC (observed in the nucleus and nucleolus) and the interaction between NtSCI1 and Nt14-3-3D2 was also wconfirmed by co-immunoprecipitation. The NtSCI1 involvement in cell cycle regulation was corroborated by the interaction between NtSCI1 and the NtCYCLIN-L1 (a regulatory subunit of CDKG;2), which was confirmed by two-hybrid and BiFC in the nucleolus. The interaction between NtSCI1 and NtCYCLIN-RELATED was also confirmed by BiFC. To understand the dynamics of NtSCI1 in the nucleolus, the subcellular localization of the fusion protein NtSCI1-GFP was studied during the different cell cycle phases. NtSCI1-GFP was observed in the nucleolus of BY-2 cells at interphase and prophase, disappearing at metaphase and anaphase and reappearing in the nucleolus at the end of telophase, showing that NtSCI1 presence in the cell is controlled by the cell cycle. The construction of the first version of NtSCI1 interactome showed its direct and indirect involvement with proteins related to RNA metabolism, transcription control and cell cycle regulation. These results suggest that NtSCI1 may act in cell cycle control in a non-canonical way, through multiple parallel processes interconnecting aspects of transcription regulation, RNA processing and cell cycle control.

Veränderungen in phosphorylierungsabhängigen Signalwegen, Akkumulation von Proteinaggregaten im Gehirn und neuronaler Zelltod sind Neurodegenerationskennzeichen und Indikatoren für überlappende molekulare Mechanismen. Um Einblicke in die involvierten Signalwege zu erhalten, wurde mit Hilfe eines modifizierten Hefe-Zwei-Hybrid (Y2H)-Systems für 71 Proteine, die mit neurologischen Erkrankungen assoziiert sind, proteomweit nach Protein-Protein Interaktionen (PPIs) gesucht. Für 21 dieser Proteine wurden PPIs identifiziert. Das Gesamtnetzwerk besteht aus 79 Proteinen und 90 PPIs von denen 5 phosphorylierungsabhängig sind. Ein Teil dieser PPIs wurde in unabhängigen Interaktionsassays mit einer Validierungsrate von 66 % getestet. Der netzwerkbasierte Versuch verbindet erfolgreich neurologische Erkrankungen untereinander aber auch mit zellulären Prozessen. Ser/Thr-Kinase abhängige PPIs verknüpfen zum Beispiel das Parkinson Protein 7 (PARK7, DJ1) mit den E3 Ligase Komponenten ASB3 und RNF31 (HOIP). Die Funktion dieser Proteine bekräftigt den Zusammenhang zwischen dem Ubiquitin-Proteasom-System und der Parkinson Krankheit (PD). Neurofibromin 2 (NF2, merlin) Isoformen und PARK7 interagieren mit der regulatorischen PI3K Untereinheit p55-gamma (PIK3R3). Diese PPIs basieren auf Tyr-Kinase Aktivität im modifizierten Y2H System und funktionellen PIK3R3 pTyr-Erkennungsmodulen (SH2 Domänen) in co-IP und Venus PCA Versuchen. Dies verknüpft den PI3K/AKT Überlebenssignalweg mit zwei unterschiedlichen neurologischen Erkrankungsphenotypen: dem PD assoziierten neuronalen Zelltod und der Neurofibromatose Typ 2-assoziierten Tumorentstehung. Die vergleichende Beobachtung von PIK3R3, AOF2 (KDM1A, LSD1) Interaktionen auf NF2 Isoformlevel offenbart eine Bevorzugung von Isoform 7 bei zytoplasmatischer Lokalisation, wohingegen Isoform 1 PPIs an der Membran lokalisiert sind. Das modifizierungsabhängige und isoformspezifische PPI Netzwerk ermöglichte neue Hypothesen zu molekularen Pathomechanismen. / Alterations in phosphorylation-dependent signalling pathways, accumulation of aggregated proteins in the brain and neuronal apoptosis are common to neurodegeneration and implicate overlapping molecular mechanism. To gain insight into involved pathways, a modified yeast-two hybrid (Y2H) system was applied to screen 71 proteins associated with neurological disorders in a proteome-wide manner. For 21 of these proteins interactions were identified including 5 phosphorylation-dependent ones. In total, the network connected 79 proteins through 90 protein-protein interactions (PPIs). A fraction of these Y2H PPIs was tested in secondary interaction assays with a validation rate of 66 %. The described network-based approach successfully identified proteins associated with more than one disorder and cellular functions connected to specific disorders. In particular, the network revealed Ser/Thr kinase-dependent PPIs between the Parkinson protein 7 (PARK7, DJ1) and the E3 ligase components ASB3 and RNF31 (HOIP). The function of these proteins further substantiates the established connection between Parkinson’s disease (PD) and ubiquitination-mediated proteasome (dis)functions. Neurofibromin 2 (NF2, merlin) isoforms and PARK7 were identified as PI3K regulatory subunit p55-gamma (PIK3R3) interactors. These PPIs required Tyr kinase coexpression in the modified Y2H system and functional PIK3R3 pTyr-recognition modules (SH2 domains) in co-IP and Venus PCA experiments. This finding implicates the PI3K/AKT survival pathway in PD-associated neuronal apoptosis and Neurofibromatosis type 2-associated tumour formation. Investigation of PIK3R3, AOF2 (KDM1A, LSD1) and EMILIN1 PPIs on NF2 isoform level revealed preferential isoform 7 binding and cytoplasmic or membrane localisation of these PPIs for isoform 7 or 1, respectively. The generated modification-dependent and isoform-specific PPI network triggered many hypotheses on the molecular mechanisms implicated in neurological disorders.

Les particules ribonucléoprotéiques (RNP) à boîtes C/D et H/ACA sont impliquées dans la maturation des UsnRNA et des précurseurs des ARNr. L'assemblage de ces RNP dans les cellules est un processus complexe faisant intervenir de nombreux facteurs cellulaires dont NUFIP, commun aux deux RNP, et NAF1, spécifique aux RNP à boîtes H/ACA. Le complexe de Survie des Motoneurones (SMN) est essentiel à la survie cellulaire et est nécessaire à l'assemblage d'une autre RNP, les UsnRNP, composants des spliceosomes. Un déficit en protéine SMN conduit à une pathologie grave, l'amyotrophie spinale. Plusieurs études suggèrent que le complexe SMN puisse également jouer un rôle dans l'assemblage des RNP à boîtes C/D et H/ACA. Dans le but d'obtenir de plus amples informations, nous avons testé si des interactions existent entre les constituants du complexe SMN et i) les protéines associées aux RNP matures, ainsi que ii) les autres facteurs d'assemblage déjà connus. Ainsi, par une approche de double hybride chez la levure, nous avons observé des interactions fortes entre NAF1 et les protéines Gemin3 et Gemin8 du complexe SMN. Comme la protéine coeur GAR1 des RNP à boîte H/ACA interagit avec la protéine SMN, ces données suggèrent que le complexe SMN participe à l'échange de NAF1 par GAR1, qui est une étape clé de la biogenèse des RNP à boîtes H/ACA. De plus, nous avons mis en évidence des interactions entre Gemin3/NUFIP, Gemin4/NUFIP et Gemin6/NUFIP. L'étude de cette dernière interaction a été approfondie. Nous avons montré que l'interaction est directe, qu'elle existe dans les cellules de mammifères à la fois dans le cytoplasme et le noyau, et nous avons défini les domaines de chaque protéine nécessaires à l'interaction, en collaboration avec l'équipe d'E. Bertrand (IGM Montpellier). Ces résultats ouvrent de larges perspectives quant à un lien fonctionnel entre le complexe SMN et NUFIP dans l'assemblage des RNP à boîtes C/D et H/ACA, mais aussi dans l'assemblage de la snRNP U4 et dans le mécanisme de traduction localisée dans les cellules / Box C/D and H/ACA ribonucleoparticles (RNPs) are required for UsnRNA and ribosomal RNA maturation. Their assembly in cells is a complex process, which implicates numerous cellular factors, such as NUFIP, a common assembly factor, and NAF1, which is a specific factor for H/ACA box RNP assembly. The Survival of Motoneurons (SMN) complex is essential for cell survival and is required for the assembly of another class of RNPs, the UsnRNPs, which are essential components of the splicing machinery. Decreased levels of the SMN protein lead to a severe disease, the spinal muscular atrophy. Several studies led to the proposal that the SMN complex also plays a role in the assembly of box C/D and H/ACA RNPs. In order to obtain more information, we analyzed whether some interactions may exist between components of the SMN complex and i) core proteins of mature RNPs, or ii) factors already known to be involved in the assembly. Using a yeast two-hybrid approach, we observed strong interactions between NAF1 and the SMN complex components, Gemin3 and Gemin8. Since the core H/ACA protein GAR1 interacts with the SMN protein, our data suggest that the SMN complex participates to the exchange of NAF1 by GAR1, which is a crucial step of H/ACA box RNP biogenesis. Furthermore, we discovered strong interactions between Gemin3/NUFIP, Gemin4/NUFIP and Gemin6/NUFIP. Concerning the Gemin6/NUFIP interaction, we showed that is direct, that it exists in both compartments in mammalian cells and we defined domains of both proteins necessary for the interaction in collaboration with the E. Bertrand team (IGM Montpellier). These results open new perspectives concerning functional links between the SMN complex and NUFIP in box H/ACA and C/D RNP assembly, but also in U4 snRNP assembly and in the mechanism of localized translation