La machinerie d'assemblage HSP90/R2TP est impliquée dans la biogenèse de complexes essentiels à l'expression génique et à la croissance cellulaire. Le complexe est constitué des protéines PIH1D1, RPAP3 et des AAA+ ATPases RVB1 et RVB2. Le R2TP, via son adaptateur NUFIP, permet l'assemblage de complexes ribonucléoprotéiques tels que les snoRNP à boîtes C/D, et le snRNP U4, tous deux impliqués respectivement dans la maturation des ARNr et ARNm. Le mode d'action du R2TP dans ces processus n'était pas bien compris. Pour étudier cela, une approche de protéomique, avec des tests d'interaction ARN/protéine et protéine/protéine, ainsi qu'une approche structurale, ont été utilisés. Un nouveau modèle a ainsi été établi. Le R2TP permettrait de former des pré-complexes d'assemblage contenant les protéines cœurs du complexe RNP avec des facteurs d'assemblage mais sans l'ARN. Les protéines RVB se détacheraient du R2TP pour rester associées à ce pré-complexe d'assemblage, par la suite, elles permettraient de le stabiliser tout en y incorporant de nouvelles protéines cœurs, et en relargant des facteurs d'assemblage ayant déjà accompli leur fonction dans le processus de biogenèse. Cette fonction de chaperon moléculaire des complexes en cours d'assemblage est très probablement régulée par hydrolyse de l'ATP par les ATPases RVB, et ceci sous le contrôle de co-facteurs, comme potentiellement la protéine BCD1. Dans le cas de l'assemblage des C/D snoRNP, il a été établi un modèle d'assemblage dans lequel le rôle des différents facteurs d'assemblage peut être prédit. ZNHIT3 aurait un rôle dans l'incorporation du snoARN naissant dans le pré-complexe, et NUFIP permettrait de garder la particule immature dans une conformation inactive afin de faciliter l'obtention de la structure active du snoRNP grâce aux RVB. Avec les travaux sur la biogenèse de la particule U4, il a été mis en évidence l'existence d'un pré-complexe cytoplasmique contenant PRP31/NUFIP/R2TP/complexe SMN qui serait important pour l'assemblage de la snRNP U4 avec non seulement les protéines Sm, mais aussi la protéine PRP31. / The HSP90/R2TP machinery is involved in the biogenesis of essential complex for gene expression and cell growth. The complex consists of proteins PIH1D1, RPAP3 and the AAA+ ATPases RVB1 and RVB2. The R2TP, via its NUFIP adaptator, allows assembly of ribonucleoprotein complexes like box C/D snoRNP, and the U4 snRNP, both involved in the maturation of mRNA and rRNA respectively. The mode of action of R2TP in these processes is not well understood. In this study, a proteomic approach, with tests of interaction RNA/protein and protein/protein and a structural approach, were used. A new model has been established. The R2TP would form an assembly pre-complex containing RNP core proteins with assembly factors but not RNA. RVB proteins detach from R2TP to remain associated with the assembly pre-complex, and then, would stabilize it while incorporating new core proteins. They would also release assembly factors that already have accomplished their function in the biogenesis process. This function of molecular chaperone complex during assembly is most likely regulated by ATP hydrolysis by the RVB ATPases, and this under the control of co-factors as potentially BCD1 protein. In the case of the assembly of box C/D snoRNP, it was established an assembly model in which the roles of the various assembly factors can be predicted. ZNHIT3 has a role in the incorporation of the nascent snoRNA in the pre-complex and NUFIP would keep the immature particle into an inactive conformation to facilitate the formation of the active structure of the snoRNP through RVB. With the study of the biogenesis of the U4 particle, it was revealed the existence of a cytoplasmic pre-complex containing PRP31/NUFIP/R2TP/SMN complex that would be important for the assembly of the U4 snRNP with not only Sm protein but also the PRP31 protein.

In oxygenic phototrophs, the photosynthetic machinery is located in thylakoid membrane (TM), a specialized endogenous membrane system. How TM are synthesized remains however mostly unknown. The aim of this thesis was to clarify a link between the synthesis of chlorophyll (Chl)-binding proteins, the main protein component of TM, and the formation of TM system in the model cyanobacterium Synechocystis PCC 6803. During the project, the analysis of TM under various growth conditions and in Chl-deficient mutants has demonstrated that a sufficient amount of de novo produced Chl molecules is crucial for the TM biogenesis. Particularly, the synthesis of the photosystem II subunit CP47 and trimeric photosystem I appeared to be sensitive to a shortage in de novo made Chl molecules. Interestingly, a specialized ribosome-binding protein (Pam68) has been identified to facilitate the insertion of Chl molecules into CP47. The synthesis of Chl-proteins and the biogenesis of TM have been further explored in cells recovering from long-term nitrogen depletion. Using this approach, it was possible to identify a large structure in the cell cytosol, which is very likely the site of TM biogenesis, and to correlate the appearance of this structure with the restored biogenesis of Chl-binding proteins.

No description available.

<p>Ribosome assembly is a complex process, facilitated by more than 20 protein factors in bacteria. GTPases and ATPases represent the energy driving force of these factors. In my research as a PhD student, I studied the function of two GTPases, YjeQ and RbgA, involved in the assembly of the small and the large ribosomal subunits, respectively.</p> <p>We isolated and characterized <em>in-vivo</em> assembled immature small (30S) and large (50S) subunits using a perturbation in the genes coding for these proteins. We observed that both subunits contained an incomplete ribosomal protein content, mainly lacking late-binding r-proteins. Additionally, we observed distortions in the functional core of the immature ribosomal subunit, particularly in the mRNA decoding center of the 30S subunit, the peptidyltransferase center of the 50S subunit, and tRNA binding sites.</p> <p>Additionally, we have determined that the YjeQ protein interacts with the 30S subunit through its N-terminal OB-fold domain, and C-terminal Zn-finger motif. The binding site of YjeQ on the 30S subunit prevents the interaction with tRNAs, translation factors, and the 50S subunit.</p> <p>Finally, we uncovered a novel functional interplay between RbgA and the ribosomal protein L16 during late stages of ribosomal assembly. We proposed that recruitment of L16 to the assembling 50S subunit would induce a conformational rearrangement that would ultimately promote the GTP-dependent release of RbgA.</p> <p>The function of the assembly factors associated with the process of <em>in-vivo</em> ribosome assembly is not known, and thus a framework on how ribosomes are built is still elusive. I believe the research presented in this thesis provides novel insights into the role of YjeQ and RbgA in the assembly of ribosomes</p> / Doctor of Philosophy (PhD)

No description available.

Plusieurs études ont permis la caractérisation de la structure et de la fonction du ribosome. En ce qui attrait à la biogénèse du ribosome, nombreux aspects restent à être découverts et compris de façon plus dynamique. En effet, cette biogénèse englobe une variété de voies de modifications et d’assemblages requises pour la maturation des ARNr et pour leurs liaisons avec les protéines ribosomales. De ce fait, les protéines Noc ont été caractérisées comme des facteurs d’assemblages et ont permis la découverte d’une des premières indications sur l’ordre spatio-temporel de la maturation du ribosome. Ainsi, en utilisant la levure comme modèle, notre objectif est d’étudier d’avantage l’échange des complexes composés des protéines Noc ainsi que leur localisation intranucléaire. Ainsi, la nature des interactions de Noc2p avec Noc1p et Noc3p et l’influence de l’arrêt du transport intranucléaire ont été étudiés en utilisant des promoteurs inductibles, la microscopie à fluorescence, des immunobuvardages, qRT-PCR et des purifications par affinité. / Several studies have been performed to characterize the ribosome as far as to understand its structure and its function. However, major aspects of ribosome biogenesis remain elusive or gave only a static picture of the process. In fact, ribosome biogenesis involves dynamic processing and assembly pathways that are required for rRNA modification and folding, in addition to rRNA binding with some ribosomal proteins. One set of assembly factors, the Noc proteins, allowed one of the first indications about the spatio-temporal ordering of ribosome maturation. By using yeast as model, our objective is to provide a dynamic picture of the Noc proteins complexes exchange and nuclear localization by determining the nature of Noc2p interactions with Noc1p and Noc3p and by studying the influence of reversibly arrested intranuclear transport on these proteins and on Rix7p, an AAA-ATPase. In order to achieve these aims, inducible promoter, fluorescent microscopy, western blot, qRT-PCR and affinity purification analyses were used.