RNA is a molecular messenger of the cell, essential to many cellular pathways and processes. In order to maintain functionality, RNA is processed and modified by protein complexes such as the exosome and associated proteins. The exosome-mediated RNA processing or degradation both require a Ski-2 like helicase to function. One such helicase is the Frequency-interacting RNA Helicase (FRH), an essential RNA helicase from Neurospora Crassa. FRH is homologous to the Saccharomyces cerevisiae Mtr4 from the Ski2-like family of RNA helicases. Sequence alignments between FRH and Ski2-like family helicases predicted FRH to share the helicase core domains and the inserted arch domain a characteristic of the Mtr4-like proteins in this protein family. FRH is also a main component of the circadian oscillation pathway in N. crassa. The participation of FRH in circadian oscillation is not a shared role across RNA helicases. FRH forms a link between two major cellular pathways providing a unique system to study RNA surveillance. Here we present the 3.51Å and 3.25Å crystal structures of FRH which supports structural prediction by maintaining the core architecture found in Ski2-like helicases. These similarities are accompanied by significant flexibility of the arch domain and revealed a unique homodimer. Other known Ski2-like helicases have not been observed to form dimers and function biologically as monomers. Furthermore, the initial characterization of helicase activity of FRH on a poly-adenylated RNA substrate is presented. Also explored is the evidence of a dimer through crosslinking and size exclusion chromatography assays.

A proteína nucleolar Nop8p de levedura foi identificada inicialmente através de sua interação com Nip7p e está envolvida na formação da subunidade ribossomal 60S. A depleção de Nop8p em células de levedura leva à degradação prematura dos rRNAs, porém o mecanismo bioquímico responsável por este fenótipo ainda não é conhecido. Neste trabalho, mostramos que a interação de Nop8p com o rRNA 5.8S se dá através de sua região amino-terminal, enquanto que a porção carboxi-terminal é responsável pela interação com Nip7p e complementa parcialmente o defeito no crescimento observado na cepa mutante condicional Δnop8/GAL::NOP8. Além disso, Nop8p media a associação de Nip7p com as partículas pré-ribossomais. Nop8p também interage com a subunidade Rrp6p do exossomo e inibe a atividade do complexo in vitro, sugerindo que a diminuição dos níveis da subunidade ribosomal 60S detectada após a depleção de Nop8p pode ser resultado da degradação dos pré-rRNAs pelo exossomo. Estes resultados indicam que Nop8p pode regular a atividade do exossomo durante o processamento do pré-rRNA. / The yeast nucleolar protein Nop8p has previously been shown to interact with Nip7p and to be required for 60S ribosomal subunit formation. Although depletion of Nop8p in yeast cells leads to premature degradation of rRNAs, the biochemical mechanism responsible for this phenotype is still not known. In this work, we show that the Nop8p amino-terminal region mediates interaction with the 5.8S rRNA, while its carboxylterminal portion interacts with Nip7p and can partially complement the growth defect of the conditional mutant strain Δnop8/GAL::NOP8. Interestingly, Nop8p mediates the association of Nip7p to pre-ribosomal particles. Nop8p also interacts with the exosome subunit Rrp6p and inhibits the complex activity in vitro, suggesting that the decrease in 60S ribosomal subunit levels detected upon depletion of Nop8p may result from degradation of pre-rRNAs by the exosome. These results strongly indicate that Nop8p may control exosome function during pre-rRNA processing.

A proteína nucleolar Nop8p de levedura foi identificada inicialmente através de sua interação com Nip7p e está envolvida na formação da subunidade ribossomal 60S. A depleção de Nop8p em células de levedura leva à degradação prematura dos rRNAs, porém o mecanismo bioquímico responsável por este fenótipo ainda não é conhecido. Neste trabalho, mostramos que a interação de Nop8p com o rRNA 5.8S se dá através de sua região amino-terminal, enquanto que a porção carboxi-terminal é responsável pela interação com Nip7p e complementa parcialmente o defeito no crescimento observado na cepa mutante condicional Δnop8/GAL::NOP8. Além disso, Nop8p media a associação de Nip7p com as partículas pré-ribossomais. Nop8p também interage com a subunidade Rrp6p do exossomo e inibe a atividade do complexo in vitro, sugerindo que a diminuição dos níveis da subunidade ribosomal 60S detectada após a depleção de Nop8p pode ser resultado da degradação dos pré-rRNAs pelo exossomo. Estes resultados indicam que Nop8p pode regular a atividade do exossomo durante o processamento do pré-rRNA. / The yeast nucleolar protein Nop8p has previously been shown to interact with Nip7p and to be required for 60S ribosomal subunit formation. Although depletion of Nop8p in yeast cells leads to premature degradation of rRNAs, the biochemical mechanism responsible for this phenotype is still not known. In this work, we show that the Nop8p amino-terminal region mediates interaction with the 5.8S rRNA, while its carboxylterminal portion interacts with Nip7p and can partially complement the growth defect of the conditional mutant strain Δnop8/GAL::NOP8. Interestingly, Nop8p mediates the association of Nip7p to pre-ribosomal particles. Nop8p also interacts with the exosome subunit Rrp6p and inhibits the complex activity in vitro, suggesting that the decrease in 60S ribosomal subunit levels detected upon depletion of Nop8p may result from degradation of pre-rRNAs by the exosome. These results strongly indicate that Nop8p may control exosome function during pre-rRNA processing.

Au cours des cinq dernières années l’existence d’un contrôle de la transcription par les ARN non-codants longs (lncRNAs) a été décrite dans une large variété d’eucaryotes. Cependant, les mécanismes par lesquels les lncRNAs régulent la transcription restent en grande partie méconnus. Les premiers travaux effectués dans le cadre de cette thèse ont participé à la caractérisation du mécanisme mis en jeu par un lncRNA, nommé nam1, dans le contrôle de l’entrée en différenciation sexuelle chez la levure Schizosaccharomyces pombe. Il a ainsi été montré qu’au cours de sa synthèse le lncRNA nam1 est ciblé par la protéine de liaison à l’ARN Mmi1 et une machinerie de surveillance des ARN qui comprend l’exosome, un complexe de dégradation des ARN conservé au cours de l’évolution. La fixation de Mmi1 au lncRNA nam1 contrôle la terminaison de la transcription de nam1 et empêche ainsi la transcription de se poursuivre et d’interférer alors avec la transcription du gène situé en aval (codant pour une MAP kinase essentielle à l’entrée en différenciation). Les travaux suivant montrent l’implication dans ce mécanisme de la protéine Cti1, un des co-facteurs connus de l’exosome. Fait marquant, ces travaux rapportent aussi l’existence d’un mode de production inédit pour un lncRNA. En effet, ils révèlent que la transcription non-interrompue d’un gène codant conduirait à la production d’un ARN bi-cistronique. La maturation co-transcriptionnelle de cet ARN bi-cistronique produirait, d’un côté, un ARN messager et, de l’autre, le lncRNA nam1. Enfin, ils ont permit la caractérisation initiale d’un nouveau composant de la machinerie de surveillance des ARN recrutée sur nam1 par Mmi1. Ainsi, dans leur ensemble, ces travaux contribuent à une meilleure connaissance des mécanismes pouvant être mis en jeu par un lncRNA et agissant en cis pour réguler l’expression génique et, à travers elle, des processus cellulaires majeurs, tel que la différenciation cellulaire. De plus, ils décrivent un nouveau mécanisme de biogénèse d’un lncRNA. / Over the last five years, the control of transcription mediated by long non-coding RNAs (lncRNAs) has been reported to take place in a wide variety of eukaryotes. However, the mechanisms by which lncRNAs regulate transcription remain relatively poorly described. The first work conducted in the context of this PhD thesis has contributed to the characterization of the mechanism used by a lncRNA, named nam1, to control entry into sexual differentiation of the fission yeast Schizosaccharomyces pombe. It was shown that, while the lncRNA nam1 is being produced, it is targeted by the RNA binding protein Mmi1 and a RNA surveillance machinery that includes the exosome, a conserved complex throughout evolution. The binding of Mmi1 to nam1 lncRNA controls the termination of transcription of nam1, which prevents this non-coding transcription from interfering with the transcription of the downstream gene, coding for a MAP kinase essential to entry into differentiation. The following work shows the importance of the protein Cti1, one of the known co-factor of the exosome, in the nam1-dependent control of sexual differentiation. Remarkably, it also strongly suggests the existence of a new way of producing a lncRNA. Indeed, it reveals that read-through transcription of a protein-coding gene leads to the production of a bi-cistronic RNA, which is co-transcriptionally matured to produce on one side a messenger RNA and on the other side the lncRNA nam1. Finally, this work initiated the characterization of a new component of the RNA surveillance machinery targeting nam1. Collectively, this work brings several insights into the mechanisms used by cis-acting lncRNAs to regulate gene expression and, thereby, major cellular processes such as cell differentiation. Moreover, it also provides insights into the biogenesis of lncRNAs by reporting a new mode of production of lncRNAs.

Le processus de recombinaison V(D)J des gènes d’immunoglobulines (Ig) est caractérisé par une grande imprécision des jonctions entre les segments variables (V), de diversité (D) et de jonction (J). Deux fois sur trois, un décalage du cadre de lecture apparaît, aboutissant à une jonction non productive dite « hors phase ». Plusieurs études ont démontré que les deux allèles productifs et non-productifs sont activement transcrits. Les transcrits matures issus des allèles non-productifs sont pris en charge par un mécanisme de surveillance des ARNm appelé NMD « Nonsense-Mediated mRNA Decay ». En dégradant efficacement les ARNm d’Ig contenant des codons non-sens, ce mécanisme prévient l’apparition des Ig tronquées au cours de l’ontogénie B. Néanmoins, aucune étude n’a jusqu’ici analysé l’impact de l’épissage alternatif des transcrits d’Ig non-productifs. Ce phénomène appelé NAS « Nonsense-associated Altered Splicing » peut conduire à une production d’Ig tronquées présentant des délétions internes du domaine variable (V).Les projets développés lors de cette thèse ont montré que la présence d’un codon non-sens, au niveau de l’exon variable (VJ) des transcrits Igκ, favorise le saut d’exon et la production de chaînes légères dépourvues de domaine variable (ΔV-κLCs). De façon intéressante, ces Ig tronquées provoquent un stress cellulaire et conduisent à l’apoptose des plasmocytes (Article 1). Ces observations ont permis d’identifier un nouveau point de contrôle agissant tardivement lors de la différenciation plasmocytaire : le TIE « Truncated-Ig Exclusion » checkpoint. Ce processus de contrôle provoque l’élimination des plasmocytes qui produisent des chaînes d’Ig tronquées. Nous avons également étudié l’épissage alternatif des transcrits d’Ig non-productifs en l’absence de TIE-checkpoint (Article 2). Cette étude a révélé que l’hypertranscription des gènes d’Ig dans les plasmocytes favorise l’épissage alternatif des transcrits d’Ig non-productifs. En utilisant un modèle d’expression forcée d’Ig tronquées, nous avons mis en évidence une coopération entre les mécanismes assurant la surveillance des ARNm (NMD) et la surveillance au niveau protéique (UPR : « Unfolded Protein Response », autophagie) (Article 3). Sur la base de ces résultats, nous avons mis au point une nouvelle approche thérapeutique qui consiste à forcer la production d’Ig tronquées en utilisant des oligonucléotides anti-sens (AON) capables de provoquer l’élimination de l’exon variable lors de l’épissage. Cette invention pourrait ouvrir des perspectives thérapeutiques pertinentes dans le traitement du Myélome Multiple et d’autres pathologies touchant les plasmocytes. / The recombination process V(D)J of immunoglobulin (Ig) genes is characterized by random junctions between the variable (V), diversity (D) and joining (J) segments. A frameshift mutation appears in two-third of cases, generating a non-productive or « out of frame » junction. Several studies have shown that both productive and non-productive alleles are actively transcribed. The mature transcripts from nonproductive alleles are usually considered sterile and innocuous as a result of an mRNA surveillance mechanism called NMD « Nonsense-Mediated mRNA Decay ». By degrading aberrant mRNA, this mechanism prevents the appearance of truncated Ig during B cell ontogeny. However, less is known about the impact of alternative splicing on non-productive Ig transcripts. This mechanism, called NAS « Nonsense-associated Altered Splicing » can lead to the production of truncated Ig with internal deletions of variable domain (V). During my thesis, we have shown that the presence of a stop codon, within the variable exon (VJ) of Igκ transcripts, promotes exon skipping and synthesis of V domain-less κ light chains (ΔV-κLCs). Interestingly, such truncated Ig causes cellular stress and leads to plasma cells apoptosis (Article 1). These findings have identified a new checkpoint acting late during plasma cell differentiation: TIE « Truncated-Ig Exclusion » checkpoint. This process ensures counter-selection of plasma cells producing truncated-Ig. We also studied the alternative splicing of non-productive Ig transcripts in the absence of TIE-checkpoint (Article 2). We found that hypertranscription of Ig genes in plasma cells promote alternative splicing of non-productive Ig transcripts. Using a model forcing the expression of truncated Ig, we identified a cooperative action between mRNA surveillance mechanisms (NMD) and those of protein surveillance (UPR « Unfolded Protein Response », autophagy) (Article 3). Based on these results, we have developed a new therapeutic approach by increasing the production of truncated Ig using antisense oligonucleotides (AON) that leads to the elimination of the variable exon during splicing. This invention could open new avenues for the treatment of Multiple Myeloma patients and other pathologies affecting plasma cells.