Non-coding RNAs (ncRNAs) have emerged as highly diverse and powerful key players in the cell, the range of capabilities spanning from catalyzing essential processes in all living organisms, e.g. protein synthesis, to being highly specific regulators of gene expression. To fully understand the functional significance of ncRNAs, it is of critical importance to identify and characterize the repertoire of ncRNAs in the cell. Practically every genome-wide screen to identify ncRNAs has revealed large numbers of expressed ncRNAs and often identified species-specific ncRNA families of unknown function. Recent years' advancement in high-throughput sequencing techniques necessitates efficient and reliable methods for computational identification and annotation of genes. A major aim in the work underlying this thesis has been to develop and use computational tools for the identification and characterization of ncRNA genes. We used computational approaches in combination with experimental methods to study the ncRNA repertoire of the model organism Dictyostelium discoideum. We report ncRNA genes belonging to well-characterized gene families as well as previously unknown and potentially species-specific ncRNA families. The complicated task of de novo ncRNA gene prediction was successfully addressed by developing a method for nucleotide composition-based gene prediction using maximal-scoring partial sums and considering overlapping dinucleotides. We also report a substantial heterogeneity among human spliceosomal snRNAs. Northern blot analysis and cDNA cloning, as well as bioinformatical analysis of publicly available microarray data, revealed a large number of expressed snRNAs. In particular, U1 snRNA variants with several nucleotide substitutions that could potentially have dramatic effects on splice site recognition were identified. In conclusion, we have by using computational approaches combined with experimental analysis identified a rich and diverse ncRNA repertoire in the eukaryotes D. discoideum and Homo sapiens. The surprising diversity among the snRNAs in H. sapiens suggests a functional involvement in recognition of non-canonical introns and regulation of messenger RNA splicing.

Après 30 années de recherche, de nombreux obstacles s'opposent encore à la conception d'un vaccin contre le SIDA. En effet, il n'existe pas de consensus sur les corrélats immunitaires de protection qu'il devra induire ni sur les mécanismes à l'origine de la progression vers le SIDA chez les individus infectés. Dans un premier temps, nous avons cherché à concevoir un virus hybride structuralement semblable au VIH-1 et capable de se répliquer exclusivement dans le cytoplasme des cellules infectées. Dans cet objectif, nous avons développé des nouveaux vecteurs bi et tri-cistroniques dérivés du poliovirus et contenant les séquences des gènes gag et/ou env du VIH-1. Nous avons montré que ces réplicons permettaient l'expression des protéines structurales du VIH-1 sous leur forme mature. Dans un second temps, nous avons mis en évidence une corrélation indiquant que, plus la composition nucléotidique (% A/T/G/C) d'un lentivirus diverge de celle de son hôte, plus la probabilité qu'il soit pathogène est élevée. Nous avons montré que l'optimisation artificielle de la composition nucléotidique de séquences d'ARN lentivirales diminuait leur capacité d'induction d'interféron (IFN-I) après transfection. Nous avons ensuite synthétisé un virus de l'immunodéficience simienne (VIS) dont la séquence a été artificiellement optimisée à la composition nucléotidique moyenne du macaque. Ce virus présente une capacité d'induction d'IFN-I in vitro réduite par rapport au VIS sauvage. Ces données indiquent pour la première fois un lien entre la composition nucléotidique du génome des lentivirus et la progression vers le SIDA. Elles suggèrent de nouvelles stratégies vaccinales d'atténuation du VIH-1. / After over thirty years of AIDS epidemic, we still need to identify immunological correlates of protection against AIDS and we do not properly understand how HIV causes AIDS in infected individuals. In order to reproduce the protective capacity of live attenuated viruses, we first aimed at generating a hybrid virus structurally similar to HIV-1 and able to replicate exclusively in the cytoplasm of infected cells. We developed new polioviral pluricistronic vectors that contain HIV-1 packaging sequences, gag gene and/or env gene. We then showed that the use of these replicons was compatible with the production of processed and mature HIV structural proteins. Secondly, we investigated the consequences of the lentivirus nucleotide composition (% A/T/G/C) bias on their pathogenicity. We found a correlation, indicating that AIDS results from infection by primate lentiviruses having the most divergent nucleotide composition compared to their hosts, whereas less divergent lentiviruses cause non-pathogenic infections. A strong type I interferon (IFN-I) response during the chronic phase of infection is a typical feature of lentiviral pathogenic infection. We showed that nucleotide optimization of lentiviral RNA sequences dramatically reduce their in vitro capacity to induce IFN-I. We synthesized a simian immunodeficiency virus (SIV), whose genome sequence was artificially optimized to the macaque average nucleotide composition. This virus showed a reduced capacity to stimulate IFN-I in vitro than wt SIV. These data indicate for the first time a link between the nucleotide composition of lentiviruses and their pathogenicity. They suggest new vaccine attenuation strategies against AIDS.

No description available.