Le cycle cellulaire eucaryote se divise en une série de phases ordonnées qui ont pour finalité la division cellulaire. Ce processus est primordial dans la prolifération des cellules normales et le développement, mais il est aussi très fortement dérégulé dans les cellules cancéreuses. Les phases de cycle cellulaire sont différenciées par les tâches effectuées au cours de celles-ci et requièrent l’expression de gènes spécifiques à chacune des phases. Chez l’humain, il existe environ 1000 gènes dont l’expression est dépendante de la phase du cycle cellulaire. Les mécanismes impliqués dans le contrôle de l’expression périodique de ces gènes ont principalement été étudié aux niveaux transcriptionnels et post-traductionnels. Cependant, la régulation post-transcriptionnelle demeure encore peu étudiée dans le contexte du cycle cellulaire, malgré son importance dans le contrôle de l’expression génique. Afin d’identifier des régulateurs post-transcriptionnels du cycle cellulaire, nous avons analysé la corrélation existante entre l’expression des gènes périodiques du cycle cellulaire et celle de 687 protéines liant l’ARN (RNA-binding protein; RBP) sur plus de 1000 spécimens de cancer du sein. Cette analyse nous a permis d’identifier 39 RBP dont les protéines Nuclear Factor 45 (NF45) et Nuclear Factor 90 (NF90). NF45 et NF90 forment un hétérodimère qui lie des structures d’ARN double brin et qui contrôle l’expression génique à différents niveaux de l’épissage à la stabilisation des ARNm. La déplétion de NF45 ou NF90 inhibe la prolifération des cellules en induisant de nombreux défauts mitotiques qui résultent d’une baisse d’expression de plusieurs gènes essentiels à la mitose. D’autre part, à l’aide d’une méthode de protéomique nous avons réalisé l’interactome du complexe NF45-NF90 afin de déterminer à quels niveaux ce mécanisme de régulation prend place et avons identifié une interaction avec le complexe Staufen1/2-UPF1 responsable de la dégradation des ARNm. Ainsi, les niveaux d’expression de certains ARNm importants à la mitose sont conditionnés par une compétition entre NF45-NF90 et Staufen1/2 pour la liaison à ces ARNm. Dans une seconde étude, nous avons recherché les régulations potentielles sur NF45 et NF90 au cours du cycle cellulaire et avons i découvert des évènements de phosphorylation sur NF90 prenant place en phase G2/M. Nous avons montré que cette phosphorylation est médiée par CDK1, et l’activation de CDK1 provoque la translocation de NF90 du noyau vers le cytoplasme. Enfin, au vu de l’implication du complexe NF45-NF90 dans la prolifération des cellules cancéreuses, nous avons réalisé un essai de criblage à haut débit de 120 000 molécules sur l’interaction entre NF45 et NF90. Cet essai nous as permis d’identifier plus de plus 1000 molécules pouvant potentiellement interférer avec le complexe NF45-NF90. Parmi celles-ci, nous avons retrouvé 14 molécules de la famille des glycosides cardiaques, qui sont des composés antiarythmiques mais qui par ailleurs possèdent des effets anticancéreux décrits depuis plusieurs décennies. De façon intéressante, le traitement des cellules à ces composés mène à un phénotype mitotique très similaire à la déplétion de NF45 ou NF90, suggérant une implication du complexe NF45-NF90 dans les effets antimitotiques induits par les glycosides cardiaques. En conclusion, ces études nous ont permis d’éclairer le rôle du complexe NF45-NF90 dans la prolifération cellulaire, mais aussi d’approfondir la compréhension des différents mécanismes impliqués dans le contrôle du cycle cellulaire. / The eukaryotic cell cycle is divided into a series of ordered phases leading to cell division. This process is essential in normal cell proliferation and development, but it is also largely deregulated in cancer cells. Cell cycle phases are differentiated by the different molecular processes performed and expression of specific genes at each phase is determinant. In humans, there are approximately 1000 genes that are periodically expressed throughout the cell cycle. Control of this periodic expression has been well characterized at the transcriptional and post-translational levels. However, post-transcriptional regulation remains little studied in the context of the cell cycle, despite its importance in the control of gene expression. In order to identify post-transcriptional cell cycle regulators, we have correlated the expression of cell cycle genes with the expression of 687 RNA-binding proteins (RBP) in more than 1000 breast cancer specimens. This analysis allowed us to identify 39 RBPs, including Nuclear Factor 45 (NF45) and Nuclear Factor 90 (NF90). NF45 and NF90 form a heterodimer that binds double-stranded RNA structures and controls gene expression at various levels, from splicing to stabilization of mRNAs. Depletion of NF45 or NF90 inhibits cell proliferation by inducing several mitotic defects resulting from decreased expression of many genes essential for mitosis. In order to determine at which levels this regulatory mechanism takes place, we performed a proteomic method to identifyNF45-NF90 proximal interactors and identified an interaction with the Staufen1/2-UPF1 complex responsible for the degradation of mRNAs. Thus, it appears that the expression of some mitotic mRNAs is controlled by a competition between NF45-NF90 and Staufen1/2 for binding to these mRNAs. In a second study, we looked for potential regulations on NF45 and NF90 during the cell cycle and found phosphorylation events on NF90 taking place in the G2/M phase of the cell cycle. We have shown that this phosphorylation is CDK1-dependent, and that CDK1 activation leads to the translocation of NF90 from the nucleus to the cytoplasm. Finally, based on the involvement of the NF45-NF90 complex in cancer cell proliferation, we carried out a high-throughput screening assay of 120,000 ii. Abstract ii molecules on the interaction between NF45 and NF90. This assay allowed us to identify more than 1000 molecules that could potentially interfere with the NF45-NF90 complex. Amongst them, we found 14 molecules belonging to the cardiac glycoside family, which are antiarrhythmic drugs that also display anticancer effects. Interestingly, treatment with cardiac glycosides leads to a mitotic phenotype very similar to the depletion of NF45 or NF90, suggesting an involvement of the NF45-NF90 complex in the antimitotic effects induced by cardiac glycosides. In conclusion, these studies have shed light on the role of the NF45-NF90 complex in cell proliferation, but also deepened our understanding of the different mechanisms involved in cell cycle control.

No description available.

No description available.

RNA-bindande proteiner (RBP:er) har framträtt som kritiska regulatorer i post-transkriptionell genreglering, och deras dysreglering bidrar till cancerpatogenes. CAPRIN1, ett cytoplasmiskt protein, har identifierats som ett onkofetalt RBP med potentiell betydelse i hepatocellulärt karcinom (HCC), en mycket malign form av levercancer med dålig prognos. Detta examensarbete syftade till att använda gensredigeringsteknologier som CRISPR-interferens (CRISPRi) och RNA-interferens (RNAi) för att störa CAPRIN1-uttryck i HCC-cellinjer och analysera dess effekter på cellproliferation och mängden av CAPRIN1:s främsta RNA-mål. CRISPRi-konstruktioner som riktade sig mot transkriptionsstartstället (TSS) av CAPRIN1 designades och validerades, men trots lyckad kloning och uttryck av guideRNA:er (gRNA:er) resulterade CRISPRi-medierad gensläckning (KD) inte i någon signifikant minskning av CAPRIN1-uttryck. Ineffektiviteten hos CRISPRi tillskrevs låg transfektionseffektivitet, vilket bekräftades av fluorescensmikroskopi med EGFP-expressande plasmider i HepG2- och Huh7-celler. Detta kräver ytterligare optimering av elektroporationsparametrar eller utforskning av alternativa leveransmetoder. I kontrast uppnådde RNAi-medierad KD en betydande minskning av CAPRIN1-uttryck, vilket visade hög KD-effektivitet (90%) enligt RT-qPCR. Denna nivå av gensläckning gav en robust modell för vidare analyser. Efterföljande RT-qPCR av sex topp-RNA-mål identifierade av RAPseq visade varierande svar, med oväntad uppreglering av MYC och Cyclin D2, vilket går emot litteratur som antyder att deras stabilitet beror på CAPRIN1. Denna diskrepans kan förklaras av betydande standardavvikelse i uttrycksnivåerna, vilket understryker behovet av att upprepa experimenten samt genomföra en transkriptomanalys med RNA-seq för att identifiera meningsfulla mål såsom långa icke-kodande RNA (lncRNA:er). Ytterligare, MTT-proliferationstester, visade ökad cellproliferation efter CAPRIN1-KD, även om experimentella inkonsekvenser såsom ojämna cellantal begränsade slutsatsernas pålitlighet. För att erhålla tillförlitliga resultat behöver dock MTT-testet upprepas för att säkerställa konsistens med den positiva kontrollen och samla in statistiskt signifikanta data.Sammanfattningsvis, trots att CRISPRi-försöket stötte på betydande utmaningar, kan gRNA-designen och klonade plasmider användas i framtida studier när elektroporationsprotokollet har optimerats. Dessutom visade den framgångsrika RNAi-medierade KD av CAPRIN1 en alternativ metod som kan antas för vidare studier med andra RBP-kandidater. Framtida studier bör syfta till att optimera elektroporationssystemet, validera KD på proteinnivå och använda RNA-seq för en omfattande analys av CAPRIN1:s inverkan på transkriptomet i HCC-cellinjer. Dessa ansträngningar kommer att förbättra förståelsen av CAPRIN1:s regulatoriska mekanismer och dess potential som terapeutiskt mål vid HCC. / RNA-binding proteins (RBPs) play a crucial role in post-transcriptional gene regulation, and their dysregulation is implicated in cancer pathogenesis. CAPRIN1, a cytoplasmic activation/proliferation-associated protein, identified as an oncofetal RBP with potential significance in hepatocellular carcinoma (HCC), is a worthy candidate to be studied. Objective: This project aimed to establish a protocol for silencing RBPs gene expression via CRISPR interference (CRISPRi) technology using CAPRIN1 as a case study, as the roles of CAPRIN1 in tumorigenesis remains unknown. Results: Despite successful cloning of the designed gRNAs, CRISPRi-mediated knockdown did not reduce CAPRIN1 expression, attributed to low transfection efficiency as indicated by fluorescence microscopy. RNAi-mediated knockdown, however, achieved substantial reduction in CAPRIN1 expression (90%). Subsequent analysis of CAPRIN1 top RNA interactors showed substantial standard deviations, highlighting the need for repeating the experiments. The MTT assay showed uneven initial seeding densities, necessitating further repetitions of the assay. Conclusions: The study highlights the challenges associated with CRISPi-mediated knockdown, especially in transfecting HepG2 cells with large plasmids using electroporation. However, the designed gRNAs and cloned plasmids have the potential to be used in future studies, provided that the transfection process is further optimized. RNAi proved to be an effective alternative for gene silencing, achieving high knockdown efficiency and enabling subsequent analysis of CAPRIN1 target RNAs. Prioritizing further optimization of CRISPRi transfection protocols in upcoming projects will facilitate the adoption of this workflow for investigating other RBP candidates. Additionally, performing a comprehensive RNA-seq analysis could provide a deeper understanding of CAPRIN1’s role in HCC.

Die chloroplastidäre und mitochondriale Genexpression ist abhängig von einer großen Anzahl an RNA-Bindeproteinen (RBPs). Eine besonders abundante Familie sind die chloroplastidären Ribonukleoproteine (cpRNPs). Während Ziel-RNAs mehrerer cpRNPs und die Phänotypen entsprechender Mutanten beschrieben wurden, bleibt ihre molekulare Funktion weitgehend ungeklärt. In dieser Arbeit wurden Studien der cp29a Mutante durch genomweite Analysen erweitert. Diese legen nahe, dass die eigentliche Rolle von CP29A in phänotypisch erkennbarem Mutanten-Gewebe durch sekundäre Defekte maskiert wird. Um primäre Defekte zu identifizieren, wurden in vivo Bindestellen von CP29A mit einer neuen Chloroplasten-adaptierten Methode, die UV-Licht zur Quervernetzungen nutzt, bestimmt. Transkripte, die für Untereinheiten des Photosystem II und des Cytochrom-b6f-Komplexes kodieren, waren unter den Zielen von CP29A überrepräsentiert. Weiterhin wurden mehrere Bindestellen in Nachbarschaft zu Bindestellen von PPR-Proteinen identifiziert. Mit einer alternativen Methode, die chemische Quervernetzung nutzt, wurden Ziel-RNAs eines weiteren cpRNP, CP31A, identifiziert. Transkripte, die für Untereinheiten des NADH-Dehydrogenase Komplexes kodieren, waren überrepräsentiert. Diese Daten führten zu einer neuen Hypothese, die die Funktion von cpRNPs im Zusammenspiel mit PPR-Proteinen in der Prozessierung funktionell verwandter RNAs postuliert. Ein weiterer für die Genexpression relevanter Mechanismus ist die Bildung membranloser Kompartimente durch flüssig-flüssig Phasentrennung. Es wurde eine in silico Analyse durchgeführt, um organelläre Proteine mit Domänen, die auf flüssig-flüssig Phasentrennung hindeuten, zu identifizieren. Funktionen mit Bezug zu Genexpression, insbesondere RNA-Edierung, waren bei diesen Proteinen mit Prionen-ähnlichen Domänen (PLDs) überrepräsentiert. Zwei Kandidaten wurden auf ihre Neigung zur flüssig-flüssig Phasentrennung durch in vitro Experimente und in vivo Mikroskopie untersucht. / Gene expression in chloroplasts and mitochondria relies on a large number of RNA-binding proteins (RBPs), which are involved in the processing of polycistronic precursor transcripts. A particular abundant family are the chloroplast ribonucleoproteins (cpRNPs). While target RNAs and mutant phenotypes of several cpRNPs were described, insights on their molecular function remained sparse. In this thesis, analyses of cp29a mutants were extended by genome-wide transcriptome data, which suggest that in phenotypically noticeable mutant tissue the actual role of CP29A might be masked by secondary effects. To identify primary defects, in vivo binding sites of CP29A on its target transcripts were determined using a novel chloroplast-adapted approach using crosslinking by UV-light. Identified targets of CP29A are functionally enriched in mRNAs encoding subunits of the photosystem II and the cytochrome b6f complex. Moreover, several binding sites were identified in close proximity to characterized binding sites of PPR proteins. Using an alternative approach, employing chemical crosslinking, targets of another cpRNP, CP31A, were identified. Targets are enriched in genes encoding subunits of the NADH-like dehydrogenase complex. In combination, these data led to a novel hypothesis on the molecular function of cpRNPs working together with PPR proteins in the processing of functionally related RNAs. Another increasingly recognized mechanism in gene expression is the formation of membraneless organelles by liquid-liquid phase separation. An in silico screen for organellar proteins containing domains indicative of phase separation was performed. The identified set of proteins with prion-like domains (PLDs) is enriched in functions related to gene expression, particularly RNA-editing. Two selected candidate proteins were characterized for their propensity to undergo phase separation by in vitro phase separation assays and in vivo microscopy.

RNA-bindende Proteine spielen eine zentrale Rolle in der posttranskriptionellen Kontrolle von mRNAs, die zwischen Transkription und Abbau von mRNAs stattfindet. RNA-bindende Proteine beeinflussen Spleißen, Export, Stabilität, Lokalisierung und Translation von mRNAs. FUS, EWSR1 und TAF15 gehören zu der Familie der FET Proteine. Diese wirken an verschiedenen zellulären Prozessen wie Transkription, Spleißen und der Prozessierung von miRNAs mit. Translokationen und Mutationen der FET Proteine führen zu verschiedenen Krankheiten. FUS spielt eine Rolle bei den neurodegenerativen Krankheiten frontotemporale Lobärdegeneration (FTLD) und amyotrophe Lateralsklerose (ALS). In dieser Arbeit wurde die mithilfe von photoaktivierbaren Ribonukleotiden UV-Licht induzierte Quervernetzung und Immunpräzipitation (PAR-CLIP) Methode genutzt, um die RNA-Bindestellen von FUS, EWSR1 und TAF15, einer ALS-verursachenden FUS Mutante und einem anderen, mit ALS in Verbindung stehenden Protein, TARDBP, zu bestimmen. Die RNA-Bindestellen der FET-Proteine lagen größtenteils in Introns. Passend dazu konnte durch knockdown der FET Proteine eine Rolle von FUS und EWSR1 im Spleißen von mRNAs validiert werden. Dem Ubiquitin-Proteasom-System zugehörige RNAs waren unter den sowohl von FUS als auch TARDBP gebundenen mRNAs überrepräsentiert. Dies bestätigt die Annahme, dass Störungen in der Proteindegradation die ALS-Pathogenese beeinflussen. Zusätzlich konnte gezeigt werden, dass FUS und TAF15 bevorzugt UAC-reiche, einzelsträngige RNA-Sequenzen binden. Sequenzierung von mRNAs nach Depletion von FUS, EWSR1 und TAF15 in HEK293-Zellen zeigte einen stabilisierenden Effekt der FET-Proteine auf gebundene mRNAs. Desweiteren scheinen die FET Proteine durch Interaktion mit Promotor-assoziierten, nicht-kodierenden RNAs die Transkription zu beeinflussen. / Post-transcriptional regulation of gene expression takes place at multiple levels between transcription and decay of the mRNA. RNA-binding proteins play a key role in orchestrating splicing, export, stability, localization and translation of mRNAs. FUS, EWSR1 and TAF15 constitute the FET protein family which participates in multiple levels of cellular function. FET proteins have been implicated to function in various cellular processes including transcription, pre-mRNA splicing and miRNA processing. Translocations and mutations in FET proteins lead to diverse pathologies. FUS is involved in neurodegenerative diseases like frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). In this study, Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) was used to determine RNA-targets and binding sites of FUS, EWSR1 and TAF15, an ALS-causing FUS mutant and another ALS-related protein, TARDBP. The identified binding sites of FET proteins were mainly intronic, supporting the involvement of FUS and EWSR1 in splicing, which was validated by FET protein knockdown. Comparison of FUS and TARDBP RNA targets revealed that ubiquitin-proteasome related gene categories were overrepresented, further illustrating that aberrations in protein degradation are implicated in the pathogenesis of ALS. In addition, it was shown that FUS and TAF15 proteins preferentially bind UAC rich, single-stranded RNA sequences. mRNA sequencing after FUS, EWSR1 and TAF15 depletion in HEK293 cells revealed a stabilizing effect on their targets. Interestingly, FET proteins also seem to influence transcription by interaction with promoter-associated noncoding RNAs. In summary, we identified the RNA-targets and binding sites of all human FET proteins in comparison with an ALS-causing FUS mutant and TARDBP. Functional studies revealed an involvement of FET proteins in mRNA stabilization, splicing and transcriptional regulation.

Salmonella enterica gehört zu den gramnegativen Bakterien und ist der Erreger von verschiedenen Darmerkrankungen, von Gastroenteritis bis systemische Infektionen, und jährlich die Ursache für hunderttausende Todesfälle. In den letzten 30 Jahren wurden grundlegende Mechanismen der Invasion und des intrazellulären Wachstums von Salmonella gelöst: Salmonella verfügt über eine Reihe von Virulenzfaktoren (Effektorproteine), die mittels Typ-III-Sekretionssystemen (T3SS) zur molekularen Manipulation der Wirtszelle ausgeschüttet werden. Allerdings sind die Funktionen einiger dieser Effektorproteine nur unzureichend charakterisiert. Darüber hinaus hat die Rolle von RNA-Protein-Wechselwirkungen in bakteriellen Prozessen, einschließlich Infektionen, an Bedeutung gewonnen. Eine der am häufigsten verwendeten Techniken zur Untersuchung von Effektorproteinen ist der Gentamicin Protection Assay (GPA), eine einfache Methode, die den Salmonella-Infektionsprozess in vitro nachbildet. Da der GPA jedoch starken Schwankungen unterliegt und eine Endpunktmessungen darstellt, ist dieser unzureichend, wenn gleichzeitig mehrere Bedingungen oder zeitabhängige Dynamiken untersucht werden müssen. Um diese Einschränkungen zu umgeben, wurde InFluence entwickelt, eine Hochdurchsatz-Fluoreszenz-Mikroskopiemethode. Diese Methode ermöglicht Einblicke in die von Salmonellen besiedelten intrazellulären Nischen, und erwies sich als nützliches Instrument zur Charakterisierung von nicht nur von Effektorproteinen, sondern auch von Wirtsproteinen im Infektionsprozess. Darüber hinaus haben wir zur Entwicklung von TriPepSVM, einer Support Vector Machine (SVM), beigetragen. Dieser Algorithmus, der zusammen mit der AG Marsico (ICB - Helmholtz Zentrum München) entwickelt wurde, sagt RNA-Bindeproteine voraus, mithilfe von Tripeptiden in intrinsisch ungefalteten Regionen (IDR). 66 RBPs hat TriPepSVM in Salmonella vorausgesagt, wovon drei im Rahmen dieser Arbeit experimentell validiert wurden. / Salmonella enterica is a species of Gram-negative bacteria and the causative agent of enteric diseases, ranging from gastroenteritis to systemic infections, causing hundreds of thousands of deaths every year. In the last 30 years the basic mechanisms underpinning invasion and intracellular growth have been unravelled: Salmonella produces tens of virulence factors - termed “effectors” - which are secreted by two distinct Type III Secretion Systems (T3SS) for the hijacking of the host cell molecular machinery via protein-protein interactions. Although the biochemical activities of many effectors have been characterised, the functions of some have remained elusive. In addition, post-transcriptional regulation has emerged to prominence in bacteria, with RNA-protein interactions playing a pivotal role in many bacterial functions, including infection. One of the most frequently used techniques to study Salmonella effectors is the Gentamicin Protection Assay (GPA), a simple method which replicates the infection process in vitro; however, as GPA is subject to high levels of variation and only generates end-point measurements, the method can be inadequate when studying multiple conditions at once or following time-dependent dynamics. InFluence, a high-throughput, fluorescence microscopy-based analysis pipeline, was designed to address these issues. InFluence offered insights into the intracellular niches occupied by Salmonella, and proved to be a useful tool in assessing not only the contribution to the infection process of effectors, but that of host proteins too. In addition, we contributed to the development of TriPepSVM, a support vector machine-based (SVM) method developed with the Marsico group (Institute of Computational Biology (ICB) - Helmholtz Zentrum Munchen) for predicting RNA-binding proteins (RBPs) using tripeptides that are frequent in Intrinsic Disordered Regions (IDRs). TriPepSVM predicted 66 new RBPs in Salmonella, and three were experimentally validated.

PPR-Proteine bilden die größte Familie von RNA-Bindeproteinen in Pflanzen und sie werden fast ausschließlich in die Mitochondrien oder Plastiden importiert, wo sie eine wesentliche Rolle im RNA-Metabolismus spielen (Lurin et al., 2004). Doch die Funktionsweise der Proteine ist noch weitgehend unbekannt. In dieser Arbeit wurde das plastidäre PPR-Protein PPR5 in Zea mays funktionell charakterisiert, dessen Ortholog in Arabidopsis thaliana essentiell für die Embryogenese ist (Cushing et al., 2005). Mittels PPR5-Immunopräzipitation und einer Analyse der kopräzipitierten RNA konnte in vivo eine spezifische Assoziation mit der ungespleißten tRNA-Glycin (UCC) nachgewiesen werden. Analysen von ppr5-Mais-Mutanten offenbarten einen Stabilitätsverlust dieser RNA. Es wurde gefolgert, dass PPR5 das Transkript vor einem endonukleolytischen Abbau schützt. Die weiteren Projekte der Arbeit widmeten sich der Evolution der Familie. Um Erkenntnisse zur Funktion und Spezifität nahe verwandter PPR-Proteine zu erhalten, wurden die drei nächsten Verwandten von PPR5 identifiziert und Mais-Mutanten isoliert. Weiterhin wurde PPR54 untersucht. Es konnte gezeigt werden, dass PPR54 in Mais – wie in Arabidopsis (Tillich, nicht publiziert) – für das Spleißen des ndhA-Introns benötigt wird. Damit wurden erstmalig orthologe PPR-Proteine in einer Mono- und einer Dikotylen funktionell analysiert. Die vorgelegten Analysen mündeten in drei allgemeingültigen Schlussfolgerungen zur Funktion der PPR-Proteine. 1) Plastidäre PPR-Proteine, die in Dikotylen wie Arabidopsis für die Embryogenese notwendig sind, üben eine Funktion in der plastidären Translation aus. 2) Die vorgeschlagene Funktionsweise von PPR5 erfordert nicht die Rekrutierung anderer, katalytisch aktiver Proteine, sondern ihr liegt ein passiver, auf der Bindung einer RNA beruhender Mechanismus zugrunde. 3) Die Funktion orthologer PPR-Proteine ist in Mono- und Dikotylen konserviert, wie am Beispiel von PPR54 experimentell nachgewiesen wurde. / PPR proteins are the largest family of RNA binding proteins in plants and the vast majority of them is localized to mitochondria or chloroplasts, where they are major players in the RNA metabolism of defined transcripts (Lurin et al., 2004). However, the mechanistic function of these proteins is still not clear. In this study, the plastid PPR protein PPR5, whose ortholog in Arabidopsis thaliana is embryo-essential (Cushing et al., 2005), was functionally characterized in Zea mays. By PPR5 immunoprecipitation and analyses of the coimmunoprecipitated RNA, a specific association to the unspliced tRNA glycine (UCC) was shown in vivo. The analysis of ppr5 maize mutants demonstrated a loss of stability of the tRNA precursor in mutants. It was concluded that the interaction with PPR5 protects the unspliced tRNA from endonucleolytic decay. In addition, close relatives of PPR5 were identified in maize (PPR2, PPR50, and PPR51) by phylogenetic means and maize mutants were isolated. A future characterization of four paralogous PPR proteins might answer whether closely related PPR proteins have similar functions or RNA targets. The analysis of PPR54 in maize demonstrated that PPR5 is needed for the splicing of the ndhA intron in maize as it is in Arabidopsis (Tillich, not published). Three important conclusions concerning the function of PPR proteins in general were drawn from the studies of chosen PPR proteins presented here. First, plastid PPR proteins that are essential in embryo development in eudicots like Arabidopsis should be necessary for plastid translation in most cases. Second, the characterization of PPR5 revealed a possibly ancient functional mechanism of PPR proteins which does not invoke the recruitment of additional catalytic factors but relies on the passive binding of RNA elements. Last, the conservation of function of orthologous PPR proteins in monocots and eudicots, which was shown in the case of PPR54, was demonstrated experimentally for the first time.

Mechanisms of post-transcriptional control are critical to deploy RNAs and proteins asymmetrically to a discrete region of cytoplasm at the posterior of the Drosophila oocyte and embryo, called the pole plasm and thus allow differentiation of the germline. Research presented in this thesis investigates the post-transcriptional control of Drosophila pole plasm component germ cell-less (gcl ). Maternal gcl activity is required for germ cell specification and gcl RNA and protein accumulate asymmetrically in the pole plasm. gcl RNA, but not Gcl protein, is also detected in somatic regions of the embryo, and ectopic expression of Gcl in the soma causes repression of somatic patterning genes suggesting that gcl RNA is subject to translational control. I find that Gcl is expressed during oogenesis, where its expression is regulated by translational repressor Bruno (Bru). Increased levels of Gcl are observed in the oocyte when Bru is reduced (i.e., in an arrest heterozygote) and Bru overexpression reduces the amount of Gcl. Consistent with this, reduction of the maternal dosage of Bru leads to ectopic Gcl expression in the embryo, which, in turn, causes repression of anterior huckebein RNA expression. Bruno binds directly to the gcl3'UTR in vitro, but surprisingly, this binding is largely independent of a Bruno Response Element (BRE) in the gcl 3'UTR and depends upon a novel site. Furthermore, the gcl BRE-like region is not required to repress Gcl expression during oogenesis or embryogenesis. I concluded that Bru regulates gcl translation in a BRE-independent manner. In addition, I established the role of the gcl 3'UTR in gcl RNA localization and translation using transgenes that replace the endogenous 3'UTR with the alpha-tubulin 3'UTR or place it in tandem to the bicoid 3'UTR. I find that accumulation of gcl RNA in the embryonic pole plasm requires the gcl 3'UTR. Moreover, Gel is restricted to the pole plasm by translational repression mediated by the gcl 3'UTR and a limiting pool of trans-acting translational repressors. The phenotypic consequences of loss of this translational control are relatively mild, suggesting that gcl translation does not require stringent repression in the soma.

Chromosomal alterations involving the RUNXI or CBFB genes are specifically and recurrently associated with human acute myeloid leukemia (AML). One such chromosomal alteration, a pericentric inversion of chromosome 16, is present in the majority of cases of the AML subtype M4Eo. This inversion joins CBFB with the smooth muscle myosin gene MYH11 creating the fusion CBFB-MYH11. Knock-in studies in the mouse have demonstrated that expression of the protein product of the Cbfb-MYH11fusion, Cbfβ-SMMHC, predisposes mice to AML and that chemical mutagenesis both accelerates and increases the penetrance of the disease (Castilla et al., 1999). However, the mechanism of transformation and the associated collaborating genetic events remain to be resolved. As detailed in Chapter 2, we used retroviral insertional mutagenesis (RIM) to identify mutations in Cbfb-MYH11 chimeric mice that contribute to AML. The genetic screen identified 54 independent candidate cooperating genes including 6 common insertion sites: Plag1, Plagl2, Runx2, H2T23, Pstpip2, and Dok1. Focusing on the 2 members of the Plag family of transcription factors, Chapter 3 presents experiments demonstrating that Plag1 and Plagl2 independently cooperate with Cbfβ-SMMHC in vivo to efficiently trigger leukemia with short latency in the mouse. In addition, Plag1 and PLAGL2 increased proliferation and in vitro cell renewal in Cbfβ-SMMHC hematopoietic progenitors. Furthemore, PLAG1 and PLAGL2 expression was increased in 20% of human AML samples suggesting that PLAG1 and PLAGL2 may also contribute to human AML. Interestingly, PLAGL2was preferentially increased in samples with chromosome 16 inversion, t(8;21), and t(15;17). To define the mechanism by which PLAGL2 contributes to leukemogenesis, Chapter 4 presents studies assessing the role of the Mp1 signaling cascade as a Plagl2 downstream pathway in leukemia development. Using microarray analysis we discovered that PLAGL2 induces the expression of Mp1 transcript in primary bone marrow cells that express Cbfβ-SMMHC and that this induction is maintained in leukemogenesis. We have also performed luciferase assays to confirm that the Mp1 proximal promoter can be directly bound and activated by PLAGL2. Furthermore, we demonstrate increased Mp1 expression leads to hypersensitivity to the Mp1 ligand thrombopoietin (TPO) in PLAGL2/Cbfβ-SMMHC leukemic cells. To test the functional relevance in leukemia formation, we performed a bone-marrow transplantation assay and demonstrate that overexpression of Mp1 is indeed sufficient to cooperate with Cbfβ-SMMHC in leukemia induction. This data reveals that PLAGL2 cooperates with Cbfβ-SMMHC at least in part by inducing the expression of the cytokine receptor Mp1. Thus, we have identified the Mp1 signal transduction pathway as a novel target for therapeutic intervention in AML.