No description available.

Acetylation is emerging as an important posttranslational modification, which is found in thousands of proteins in eukaryotes, as well as prokaryotes. Global proteomic studies implicated acetylation in regulation of various processes like metabolism, gene expression, cell cycle or aging to name a few. In this work I set out to investigate the role of acetylation of a splicing regulatory protein SRSF5 by creating mutations in its acetylation site. I tested the hypothesis that acetylation influences SRSF5 interaction with RNA. I expressed acetylation-mimicking (Q) or non-acetylable (R) mutant of SRSF5 in HeLa cells and measured their interaction with RNA by RNA immunoprecipitation or in vitro by fluorescence anisotropy. Both approaches agreed that mutants interact with RNA less than the wild type protein and Q mutant bound RNA weaker than R mutant. I did not detect further difference in localization or dynamics among the proteins in vivo, which suggests that difference caused by weakened interaction of mutants with RNA was outweighed by other factors influencing SRSF5 behaviour, probably protein-protein interactions. I also found out that mutant SRSF5 proteins do not have a dominant effect on splicing of fibronectin alternative EDB exon. The data obtained give an indirect evidence for the hypothesis that...

Pour préserver l’intégrité de leur génome, les virus multipartite à ARN nécessitent une forte multiplicité d’infection qui représente un coût biologique inapproprié en terme de réplication virale. Dans cette étude, un réseau d’interaction entre ARN génomiques (ARNg), constitué d’au moins un type de chaque ARNg est proposé. Un tel réseau permet de réduire les coûts biologiques liés à la réplication en assurant une reconnaissance intermoléculaire et une mobilisation d’un complexe RNP modulaire maintenant l’intégrité du génome. Un tel complexe est considéré comme l’unité infectieuse mobile assurant la dissémination du virus dans la plante entière. Le but de cette thèse a été de démontrer l’existence d’interactions entre les ARNg du beet necrotic yellow vein virus (BNYVV) et de déterminer l’incidence de ces interactions sur le cycle viral. Une formule génomique a été déterminée pour différentes plantes et tissus. Les ARNg ont tous été co-détectés dans des cellules isolées issues de tissus infectés. Un domaine d’interaction entre l’ARN1 et 2 a été identifié in vitro et in silico puis évaluée in vivo par des approches de mutagenèse et de complémentation. / Multipartite RNA virus condition to provide a complete set of genomic segments in each infected cell implies a high level of MOI that results in an unsustainable biological cost in terms of viral replication. In the proposed model, to minimize the cost of the genome integrity preservation, a network of RNA/RNA interactions determines the recognition and the mobilization of at least one of each genomic RNAs in a modular RNP complex. Such complex must be considered as the mobile infectious unit of the segmented genome during viral spread in the plant. The Aim of this thesis was to experimentally determine the existence of RNA/RNA interactions between BNYVV RNAs and their implication in the viral cycle. BNYVV genomic segments have been co-detected within isolated single cells from systemic tissues where they accumulate to reach set point genome formulas. In the model where vRNAs interact each other to form the minimal mobile infective unit, RNA1 and RNA2 interaction domain has been identified in silico and in vitro. The rationale of such an interaction has been provided in vivo using BNYVV and Beet soil-borne mosaic virus chimeras.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb. 14, 2008). Includes bibliographical references.

La grippe, infection respiratoire virale fréquente, est due aux virus Influenza. Leur génome est constitué par huit molécules d’ARN de polarité négative retrouvés sous la forme de complexe ribonucléique (RNPv). Au cours du cycle viral, il a été démontré que les régions terminales des segments de gène étaient cruciales pour l’incorporation sélective des huit RNPv à l’intérieur des particules virales. Par des techniques d’interaction in vitro et de tomographie électronique, nous avons montré que les segments de gène du virus H3N2 interagissaient entre eux par des interactions ARN/ARN impliquant leurs régions de packaging. Nos résultats suggèrent que la mise en place de ce réseau permettrait la formation d’un complexe supra macromoléculaire multi-segmenté permettant l’incorporation d’un jeu complet des huit RNPv dans les particules virales néosynthetisées. En raison de la nature segmentée du génome viral, des phénomènes de réassortiment génétique peuvent avoir lieu lors d’une co-infection. Afin de définir les mécanismes responsables de la restriction observée lors de ce phénomène, nous avons évalué le taux de réassortiment génétique in vitro entre le virus humain H3N2 et le virus aviaire H5N2. Nos résultats suggèrent que le mécanisme gouvernant l’incorporation sélective des segments de gènes, régulerait le réassortiment génétique. Nous avons montré que la modulation de l’interaction ARN/ARN entre les segments de gènes HA et M permet d’augmenter le taux d’incorporation du segment de gène HA H5 dans le fond génétique du virus humain, prérequis pour l’émergence de virus pandémique / The Flu is a frequent viral infectious disease caused by the Influenza viruses. Their genomes are composed by eight negative single-stranded RNA organised as vRNPs. During the viral cycle, the terminal non-coding and coding regions of viral genome have been shown to be crucial for the selective incorporation of a complete set of the eight vRNPs into influenza viral particles. Band shift assay and electron tomography allowed us to show that all gene segments interact together by RNA/RNA interactions involving their packaging region. Our results suggest that the eight genomic vRNAs are selected and packaged as an organized supramolecular complex held together between identified packaging regions into neosynthesized virions. Due to genome segmented nature, genetic reassortment can occur during co-infection. In order to identify molecular mechanisms responsible for the observed restriction during the genetic reassortment, we have developed a new competitive reverse genetic strategy allowing us to evaluate the genetic reassortment between H3N2 and H5N2 viruses. Our results suggest that mechanism controlling the packaging should regulate genetic reassortment. We have shown that the modulation of RNA/RNA interaction between HA and M gene segment have allowed us to increase HA H5 gene segment incorporation rate into a viral human genetic background, prerequisite for pandemic virus emergency

Le génome des virus influenza de type A est composé de huit segments de gènes (ARNv) de polarité négative retrouvés sous la forme de complexes ribonucléiques (RNPv). L'incorporation sélective des huit RNPv dans les particules virales néosynthétisées se fait par un mécanisme moléculaire qui fait intervenir des signaux d'encapsidation dites « région de packaging ». Nous avons montré que les segments de gènes interagissaient entre eux via des interactions de type ARN/ARN permettant la formation d'un réseau d'interactions. Nous avons de plus montré que les régions de packaging décrites dans la littérature semblent héberger les régions impliquées dans la mise en place du réseau d'interactions. Cette étude a été réalisée pour le virus humain H3N2 et le virus aviaire H5N2. Le mécanisme d'incorporation sélective des segments de gènes semble également réguler le réassortiment génétique, processus génétique responsable de l'émergence de virus réassortants. Nous avons montré qu'une restriction génomique impliquant les régions de packaging semble être responsable du taux de réassortiment génétique faible observé in-vitro et in-vivo. La modulation du réseau d'interactions ARN/ARN semble être nécessaire pour l'incorporation de segments aviaire dans le fond génétique du virus humain. Pour finir, nous avons montré que la composition génomique des virus réassortants vaccinaux joue un rôle central dans la réplication virale et dans la production des antigènes vaccinaux. Par une stratégie de cryo-microscopie, nous avons montré que la protéine PB1 joue un rôle central dans l'optimisation de la production des antigènes de surface / The genome of the influenza A virus (IAV) comprises eight single-stranded negativesense RNA segments (vRNAs). All eight vRNAs are selectively packaged into each progeny virion via packaging signal sequences that are located at both ends of the vRNAs. How these signals ensure packaging of all eight vRNAs remains unclear. It was hypothesized that selective packaging might be driven by direct interactions between vRNAs. Combination of biochemical and reverse genetic approaches allowed us to identify short nucleotide regions on vRNAs interacting with each other in vitro. Here, we demonstrated the importance of these interactions in the packaging process of the human H3N2 and avian H5N2 viral genomes. Furthermore, our results suggest that the packaging process could regulate genetic reassortment. Indeed, we observed that the genetic reassortment between H3N2 and H5N2 viruses is restricted as the avian vRNA HA cannot be incorporated into the human genetic background. Our investigations indicated that (i) the packaging signals are crucial for genetic reassortment and (ii) the modulation of the vRNAs interaction network may be required for the incorporation of the avian HA gene into the human genetic background. Characterization of seed viruses showed that the genetic composition is important for both high growth ability and antigen production. Indeed, cryo-electronic microscopy observations of reassortant virus indicated that the PB1 gene can strongly influence the antigen glycoprotein spike density

Since the time RNA has been discovered by the nature scientist Miescher the structure and function of it has been forgotten for a long time. The prime role in science had always DNA. An increase of interest in RNA came with the discovery of the tRNA structure and its catalytic and enzymatic properties. These discoveries led to a great development wave of bioinformatics and structure and function analysis of RNA.

Les huit ARN viraux (ARNv) génomiques des influenzavirus de type A sont sélectivement empaquetés dans les virions, vraisemblablement sous la forme d’un complexe supramoléculaire maintenu par des appariements ARN/ARN entre signaux d’empaquetage. Afin d’améliorer la compréhension des règles qui gouvernent ce mécanisme, nous avons déterminé le réseau d’interactions formé entre les ARNv de la souche modèle de laboratoire A/Puerto Rico/8/34 (H1N1). Nous avons ensuite défini, au nucléotide près et par deux approches in vitro, les séquences des ARNv impliquées dans la formation de certaines interactions. Le rôle fonctionnel de deux d’entre elles a été testé en contexte infectieux. Nous avons également poursuivi l’étude d’une interaction identifiée au laboratoire entre deux ARNv de la souche A/Moscou/10/99 (H3N2) circulante. Enfin, nous avons collaboré avec le Dr L. Brown (Australie) sur l’étude du rôle d’une interaction entre deux ARNv de la souche A/Udorn/307/72 (H3N2). / The genome of influenza A viruses comprises eight viral RNAs (vRNAs) likely to be selectively packaged into progeny virions as organized supramolecular complexes where vRNAs are held together by base pairing within the packaging signals. To better understand the rules governing this mechanism, we investigated the vRNA/vRNA interaction network of the A/Puerto Rico/8/34 (H1N1) strain. We then identified, at the nucleotide level, using two in vitro approaches, the vRNA sequences involved in several of the interactions. Two of them were functionally tested in an infectious context. We also studied an interaction previously identified in the laboratory between two vRNAs belonging to the circulating A/Moscow/10/99 (H3N2) strain. Finally, we collaborated with Dr L. Brown (Australia) in order to assess the role of an interaction between two vRNAs of the A/Udorn/307/72 (H3N2) strain.

MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...

RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...