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Modified oligonucleotides and mimics have been developed with improved physicochemical and biological properties and are utilised in a wide range of applications including antisense and RNAi. Notably, new conjugation strategies have been developed to aid delivery and cellular localisation. Modified oligonucleotides are also now widely used for gene functional analysis, as probes for bioanalytical applications and as building blocks for assembly of higher-order nanostructures. The Micklefield group fIrst introduced pyrrolidine-amide oligonucleotide mimics (pOM) in 2000; the modifications involved the replacement of the ribose sugar of native RNA with a pyrrolidine ring and the phospho diester backbone by a rigid amide linker. The cationic nature of POM should also aid water solubility, cellular uptake and binding affinities due to electrostatic attraction between the cationic POMs and the anionic native DNAIRNA. , . Short POM homo-oligomers and longer mixed sequence POM oligonucleotides have shown' promising binding aff'mity towards both DNA and RNA. Homo-pyrimidine sequences have shown anti-parallel and parallel binding towards native'DNA and RNA. However, longer mixed sequence POM have also showed rates of association/dissociation that are noticeably slower than those typically observed with short oligonucleotides or PNA. Backbone extended pyrrolidine-amide oligonucleotide mimic (bePOM) monomers were prepared with the Boc protected backbone nitrogen and exocyclic amines of the nucleobases protected with the Cbz group. The bePOM oligomers were assembled using adapted PNA Boc/Cbz solid-phase peptide synthesis protocols. The bePOM oligomers were purified by reverse-phase HPLC and characterised by MALDI-ToF mass spectrometry. The DNAIRNA binding properties of the bePOMs have been studied by UV thermal denaturation, circular dichroism and isothermal titration calorimetry. Further to this a non-enzymatic DNA template directed synthesis approach has been developed that allows for the morpholino nucleoside extension of a PNA primer. Equilibration of the ribonucleosides with the template-primer complex, in a noncovalent fashion, was shown to be essential for high selectivity. The morpholino nucleotide extension products consisted of a seven membered amide backbone and the subsequent Fmoc protected thyminyl monomer was prepared for the assembly of homo-thymine oligomer via solid phase peptide synthesis. The morpholino oligomers were purifIed by reverse-phase HPLC and characterised by MALDI-ToF mass spectrometry. The DNAIRNA. binding properties of the amide linked morpholino oligomers were studied by UV thermal denaturation and circular dichroism.

Ετέρωση, γνωστή επίσης ως υβριδική ευφορία, αναφέρεται στην φαινοτυπική ανωτερότητα ενός ετερόζυγου υβριδίου σε σύγκριση με τους ομόζυγους γονείς του. Πρόσφατες πρόοδοι στην χαρτογράφηση γενετικών τόπων ποσοτικών χαρακτήρων σε Saccharomyces cerevisiae έχουν αποκαλύψει αρκετούς στόχους που συνδέονται με την ετέρωση, όπως το γονίδιο που κωδικοποιεί την ριβοσωματική πρωτεΐνη L39. Για να διερευνήσουμε περαιτέρω τη δυνητική επίδραση της ετέρωσης στην ευκαρυωτική πρωτεϊνική σύνθεση, χρησιμοποιήσαμε τα ομοζυγωτικά γονικά στελέχη 6x6 και BYxBY, σε σύγκριση με τα υβρίδια 6xBY και 6xBY6, και το ημιζυγωτικό στέλεχος Δ6xBY στο οποίο λείπει ένα από τα δύο αλληλόμορφα του γονιδίου. Μετά από πειράματα in vitro, ελέγξαμε την poly(U) εξαρτώμενη δραστηριότητα πολυμερισμού φαινυλαλανίνης, τη μεταφραστική πιστότητα, τη δραστικότητα της πεπτιδυλοτρανσφεράσης και επιπλέον την ευαισθησία έναντι του αντιβιοτικού κυκλοεξιμίδιο. Σύμφωνα με τα στοιχεία μας, τόσο τα υβριδικά στελέχη όσο και το ημιζυγωτικό στέλεχος έδειξαν αυξημένη συχνότητα λάθους, ενώ η δράση της ΡΤάσης παρουσίασε σημαντική αύξηση μόνο στην περίπτωση του υβριδικού στελέχους 6xBY. Επιπλέον, η ανθεκτικότητα έναντι του κυκλοεξιμιδίου ήταν αυξημένη για όλα σε σύγκριση με τα γονικά στελέχη, υποδεικνύοντας ότι η ετέρωση μπορεί να συνδέεται απ 'ευθείας με τον μηχανισμό της πρωτεϊνοσύνθεσης, ανοίγοντας έτσι ένα άλλο νέο παράθυρο για να διερευνηθεί περαιτέρω αυτό το σημαντικό φαινόμενο. / Heterosis, known also as hybrid vigor, refers to the phenotypic superiority of an heterozygous hybrid compared to its homozygous parents. Recent advances in quantitative trait loci (QTL) mapping in Saccharomyces cerevisiae have uncovered several targets associated with heterosis, such as the gene encoding ribosomal protein L39 (RPL39 or spb2)(?). To further explore the potential effect of heterosis on eukaryotic protein synthesis, we used the homogygous parental strains 6x6 and BYxBY, in comparison to hybrids 6xBY and 6xBY6, and the hemizygous strain Δ6xBY lacking one of the two alleles of RPL39 (spb2). Following in vitro experiments, we tested the poly(U) dependent phenylalanine polymerization activity, the translational fidelity, the peptidyl-transferase activity and additionally the susceptibility versus the antibiotic cycloeximide. According to our data, both hybrid and hemizygous strains showed increased error frequency while the PTase activity exhibited significant increase only in the case of the hybrid strain 6xBY. Moreover, the immunity versus the antibiotic cycloeximide was increased for all compared to the parental strains, indicating that heterosis may be directly associated with protein synthesis machinery, thus opening another new window to explore further this important phenomenon.

2-Oxoglutarate (2OG) and Fe(II) dependent oxygenases have a broad range of substrates, extending from histones to fatty acids. Several 2OG oxygenases have nucleic acid substrates, with members of the AlkB subfamily being responsible for nucleic acid modification and repair. The AlkB protein itself is part of the Escherichia coli adaptive response, protecting the DNA from methylation damage. Methyl lesions are repaired by a direct removal mechanism via a hydroxylated intermediate, with release of formaldehyde. Homologues of AlkB have been identified throughout the vertebrates, with nine known human homologues: AlkB homologue 1-8 (ABH1-8) and Fat, mass and obesity associated protein (FTO). ABH2, ABH3 and FTO catalyse similar reactions to AlkB, whereas ABH8 methylates then hydroxylates modified wobble-position uridines in tRNA. The remaining homologues are of unknown function. The FTO gene is associated with obesity in humans, a link confirmed by mouse models; mice lacking FTO are thinner than wildtype individuals, whereas overexpression of FTO leads to increased mass. Investigation of recombinant FTO identified a novel C terminal helical domain which appears to mediate protein dimerisation in vitro. A loss of function mutation in this C terminal domain produces a lean phenotype in mice, emphasising the importance of this domain for the protein’s function in vivo. The FTO protein was further studied in cells, and localisation of several protein variant constructs were studied by immunofluorescence. Cell lysis and immunoprecipitation techniques were developed that enable proteomic analyses of proteins with which FTO may interact in cells. No protein interactors were confidently identified, suggesting that FTO may not interact with specific proteins in cells, and instead may preferentially interact with nucleic acids. Studies were initiated on two further members of the ABH family, ABH1 and ABH7. Recombinant proteins were prepared and characterised as 2OG oxygenases, however initial attempts to identify potential histone or nucleic acid substrates were not successful. Both proteins were found to be localised in the mitochondria, however proteomic analysis was unable to identify proteins interacting with either protein in cells. Selective inhibitors are required for in vivo inhibition of the ABH proteins. AlkB and ABH2 proteins were purified and characterised, and a formaldehyde dehydrogenase-coupled assay was developed to follow activity of these DNA demethylases. A dynamic combinatorial mass spectrometry method was employed to identify novel inhibitor scaffolds for AlkB, leading to the successful discovery of the first series of potent and selective inhibitors for this class of enzymes. Crystal structures of AlkB in complex with the most potent compounds were obtained, rationalising the inhibition observed. This work therefore suggests that therapeutic inhibition of this family of 2OG oxygenases is likely to be tractable.

De nombreuses fonctions cellulaires essentielles telles que la traduction, l'épissage, la biogenèse des ribosomes et la réplication des télomères font appels aux particules RNP non codantes. La biogenèse de ces dernières chez les eucaryotes est un processus très complexes qui fait intervenir de nombreux facteurs cellulaires. La biogenèse du ribosome nécessite au moins 150 facteurs. Ceux-ci sont importants pour faciliter mais également contrôler la biogenèse de cette machinerie cellulaire essentielle qu'est le ribosome. Parmi ces facteurs, nous avons les snoRNP à boîtes C/D. Ces RNP sont impliqués dans la maturation des pré-ARNr (méthylation post-transcriptionnelle des riboses et clivages endonucléolytiques). Récemment notre laboratoire a participé à la découverte de facteurs d'assemblage de ces RNP. Il s'agit entre autre des protéines Rsa1p, du complexe R2TP (Rvb1p, Rvb2p, Tah1p et Pih1p) et de Hit1p chez la levure Saccharomyces cerevisiae. En utilisant une approche de co-expression à haut débit, nos travaux ont révélé un réseau complexe d'interactions entre les protéines constitutives des snoRNP et leurs facteurs d'assemblage. Couplé à une stratégie de protéolyse ménagée, la co-expression nous a permis d'obtenir des sous-complexes protéiques Snu13p/Rsa1p et Rsa1p/Hit1p qui font actuellement l'objet d'une étude structurale par RMN. En collaboration avec l'équipe de F. Allain (ETH Zurich), nous avons également déterminé la structure tridimensionnelle de la protéine Tah1p, ainsi que de son complexe avec le peptide C-terminale de la protéine chaperonne Hsp90 à haute résolution. Ces travaux ont révélé un mode d'association particulier entre le domaine TPR de la protéine et le peptide / A lot of essential cellular functions like translation, splicing, ribosome biogenesis and telomere replication need the activity of non coding RNPs. The biogenesis of non coding RNPs in eukaryotes is a complex pathway involving numerous cellular factors. For instance, ribosome biogenesis requires more than 150 factors. They are important to facilitate and to control the biogenesis of this essential cellular machinery. These factors include the C/D box snoRNPs. These RNPs are involved in pre-rRNA maturation (post-transcriptional ribose methylation and endo-nucleolytic cleavages). Recently, our laboratory participated to the discovery of snoRNP assembly factors: the Rsa1p protein, R2TP complex (Rvb1p, Rvb2p, Tah1p and Pih1p) and Hit1p in the yeast Saccharomyces cerevisiae. Using a high throughput co-expression approach, we deciphered a network of interactions between RNP core proteins and the assembly factors. Coupled with a limited proteolysis strategy, the co-expression method allowed us to obtain proteins sub-complexes Snu13p/Rsa1p and Rsa1p/Hit1p which are currently studied by NMR. In collaboration with the F. Allain team (ETH Zurich), we also determined the tridimensional structure of protein Tah1p and its complex with the chaperon Hsp90 C-terminal peptide at high resolution. The data obtained reveal a particular mode of association of the Tah1p TPR domain with the Hsp90 peptide

Les particules ribonucléoprotéiques (RNP) sont impliquées dans divers mécanismes cellulaires : les UsnRNP, la SRP, les RNP à boîtes C/D et H/ACA dans la modification des ARN et la maturation des ARN ribosomiques, et la télomérase dans le maintien des extrémités chromosomiques. L'assemblage de ces RNP est un processus complexe faisant intervenir de nombreux facteurs, dont le complexe SMN. Un déficit de l’une des protéines de ce complexe conduit à l’amyotrophie spinale. Il est essentiel à la survie cellulaire et est nécessaire à l’assemblage des UsnRNP et de la SRP. Il est suggèré que le complexe SMN joue un rôle dans la biogenèse des RNP à boîtes C/D et H/ACA. Nous avons montré des interactions in vitro et des associations in cellulo entre le complexe SMN et la protéine NUFIP (un facteur d’assemblage de ces RNP). Ces résultats suggèrent l’existence d’un lien fonctionnel entre le complexe SMN et NUFIP dans l'assemblage des RNP à boîtes C/D et H/ACA et de la snRNP U4. Des interactions in vitro entre le complexe SMN et la protéine NAF1 (un facteur d’assemblage des RNP à boîtes H/ACA) ont révélés, que le complexe SMN est capable de s’associer avec la RNP à boîtes H/ACA en formation. Si le complexe SMN intervient dans l’assemblage des RNP, on peut supposer que cet assemblage soit défectueux dans la SMA. Nous avons montré que certains ARN sont accumulés dans la moelle épinière et le cerveau de souris SMA. La télomérase est réactivée dans les cellules cancéreuses. En collaboration avec l’équipe de J-M Vignaud (CHU central, Nancy), nous avons montré une augmentation du taux des protéines cœur des RNP à boîtes H/ACA et de NUFIP dans les cellules tumorales / Ribonucleoprotein particles (RNPs) are involved in various cellular mechanisms in eukaryotic cells: UsnRNP, SRP, C/D and H/ACA box RNPs in RNA modifications and rRNA maturation and telomerase in the synthesis of the chromosome extremities. RNP assembly is a very complex process, which involves numerous factors. One of these factors is the SMN complex. Decreased level of one of its components leads to spinal muscular atrophy. It is essential for cell survival and necessary for UsnRNP and SRP assembly. It is suggested that the SMN complex plays a role in C/D and H/ACA RNP biogenesis. We showed in vitro interactions and in cellulo associations between the SMN complex and the protein NUFIP (an assembly factor of these RNP). These results suggest the existence of a functional link between the SMN complex and NUFIP in the assembly of the C/D and H/ACA box RNPs and the U4 snRNP. In vitro interactions between the SMN complex and the protein NAF1 (an assembly factor of the H/ACA boxes RNPs) revealed, that the SMN complex is capable of joining with the H/ACA boxes RNPs in formation. If the SMN complex intervenes in the RNPs assembly, we can suppose that this assembly is defective in the SMA. We showed that any ARN is accumulated in the spinal cord and the brain of SMA mouse. The telomerase is reactivated in cancer cells. In association with the team of J-M Vignaud (CHU central, Nancy), we showed an increase of H/ACA box RNP proteins and NUFIP in these cancer cells

Chez les eucaryotes, l’information génétique est transcrite en ARN messager qui subit plusieurs étapes de maturation et évènements d’assemblage avant d’être exporté hors du noyau. Ces modifications du transcrit sont effectuées par de nombreux facteurs protéiques recrutés au transcrit naissant, formant ainsi une particule ribonucléoprotéique (mRNP). La biogenèse du mRNP est étroitement liée avec la transcription et le contrôle qualité afin d’assurer l’efficacité et l’exactitude de la production de mRNPs matures. Des études récentes suggèrent que les membres du complexe THO-Sub2 pourraient être des facteurs cruciaux dans le couplage de la transcription, de la biogénèse du mRNP et de l’export. Dans notre groupe, nous avons mis en oeuvre un essai novateur pour étudier la biogénèse du mRNP et le contrôle qualité, basé sur l’expression du facteur Rho bactérien dans Saccharomyces cerevisiae. Rho interfère avec l’assemblage adéquat du mRNP et génère des transcrits aberrants qui sont dégradés par la machinerie de dégradation nucléaire. Dans cette étude, nous avons utilisé le système expérimental Rho pour mieux comprendre Rrp6 et l’implication de l’exosome dans la dégradation des transcrits liée au contrôle qualité, ainsi que pour mieux caractériser le rôle et la fonction du complexe THO-Sub2 dans le processus de biogénèse du mRNP. Les résultats obtenus révèlent une différence intéressante dans le comportement des membres du complexe THO sous l’action de Rho et dévoilent leur dépendance à la liaison à l’ARN, ce qui n’aurait pas pu être observé avec d’autres techniques expérimentales. Cela confirme le potentiel attendu du système expérimental basé sur Rho dans l’étude des facteurs protéiques impliqués dans la biogénèse et le contrôle qualité du mRNP. / In eukaryotes, the genetic information is transcribed into messenger RNA which undergoes various processing and assembly events prior to its export from the nucleus. These transcript modifications are performed by numerous protein factors recruited to the nascent transcript, thus making a messenger ribonucleoprotein particle (mRNP). mRNP biogenesis is tightly interconnected with both transcription and quality control to ensure efficiency and accuracy in production of mature mRNPs. Recent findings suggest that members of THO-Sub2 complex might be crucial factors in coupling transcription, mRNP biogenesis and export. In our group, we have implemented an innovative assay to study mRNP biogenesis and quality control, based on the expression of the bacterial factor Rho in Saccharomyces cerevisiae. Rho interferes with proper mRNP assembly and generates aberrant transcripts degraded by the nuclear degradation machinery. In this study, we use Rho experimental system to expand our findings on Rrp6 and exosome involvement in quality control degradation of transcripts, as well as to better characterize the role and function of THO-Sub2 complex in the process of mRNP biogenesis. Obtained results reveal an interesting difference in behavior of THO complex members upon Rho action and disclose their dependence on binding to the RNA, which could not be observed by other experimental techniques. This substantiates the expected potential of Rho-based experimental system in the study of protein factors involved in mRNP biogenesis and quality control.