Caractérisation de la protéine Naf1 chez Saccharomyces cerevisiae et chez l'homme

Ma thèse a porté sur la biogenèse des RNP H/ACA. Elles sont composées d'un ARN non-codant (ARN H/ACA) et de 4 protéines : Cbf5p, Gar1p, Nhp2p et Nop10p et sont impliquées dans la synthèse des ribosomes et des télomères. Au début de mon travail de thèse, des résultats suggérant l'implication de la protéine Naf1p de levure dans l'assemblage des RNP H/ACA. Pendant ma thèse, j'ai caractérisé l'orthologue humain (hNaf1) de la protéine Naf1p de Levure. hNaf1 peut fonctionnellement remplacer Naf1p endogène chez la levure. Chez l'homme, elle est requise pour l'accumulation des RNP H/ACA et interagi avec les scaARN H/ACA, les snoARN H/ACA et l'ARN de télomérase. Via la purification de Naf1p de levure, j'ai démontré qu'elle interagi avec la machinerie de transcription associée à l'ARN polymérase II. Puis, en collaboration avec l'équipe de Guillaume Chanfreau, nous avons mis en évidence que Naf1p est associée aux gènes codant les snoARN H/ACA suggérant que Naf1p pourrait permettre le recrutement de Cbf5p, Nhp2p et Nop10p sur les ARN H/ACA en cours de synthèse. En collaboration avec l'équipe de H. van Tilbeurgh, nous avons déterminé la structure cristallographique du domaine central de Naf1p. Ce domaine présente une structure similaire à la protéine aGar1. J'ai également démontré que ce domaine est essentiel in vivo et qu'il permet l'interaction avec la protéine Cbf5p in vitro. Nous proposons que Naf1p promeut l'assemblage des RNP H/ACA en recrutant Cbf5p, Nhp2p et Nop10p au niveau des sites de transcription des snoARN H/ACA. Puis Naf1p préviendrait une activité prématurée des complexes en cours d'assemblage en inhibant l'interaction de Cbf5p avec Gar1p.My PhD research focused on the biogenesis of box H/ACA ribonucleoprotein particles (RNPs). These RNPs are involved in essential cellular processes including ribosome and telomere synthesis. H/ACA RNPs are composed of one non-coding RNA (H/ACA RNA) and 4 proteins: Cbf5p, Gar1p, Nhp2p and Nop10p. At the beginning of my thesis work, several results suggested that the Naf1p protein, in yeast, is implicated in the H/ACA RNP assembly process. During my PhD training I characterized the human orthologous (hNaf1) of the yeast Naf1p. I showed that hNaf1 can functionally replace endogenous Naf1p in yeast and that, in human cells, hNaf1 is required for the accumulation of box H/ACA components. In addition, I demonstrated that hNaf1 interacts with different types of H/ACA RNAs including H/ACA scaRNA, snoRNA, and the telomerase RNA. In order to determine the exact role of Naf1p, I performed a purification of Naf1p containing complexes in yeast. I showed that Naf1p interact with the polymerase II machinery. Then, in collaboration with the lab of Guillaume Chanfreau we reveal that Naf1p interacts with H/ACA genes probably to allow the early assembly of Cbf5p, Nhp2p and Nop10p onto nascent H/ACA RNAs. Then, in collaboration with the H. van Tilbeurgh's lab, we determined the crystal structure of the central domain of Naf1p. This domain folds as the Gar1p protein. I showed that this domain is essential in vivo and that it mediates to interaction with Cbf5p in vitro. We propose that Naf1p brings Cbf5p, Nhp2p and Nop10p to the H/ACA RNA transcription site to promote the initial RNP assembly. Then Naf1p prevents premature catalytic activation of the RNPs by inhibiting Cbf5p-Gar1p interactions

Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance

© 2014 The Authors. The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A 165 b. Whereas flTIA-1 selectively bound VEGF-A 165 mRNA and increased translation of VEGF-A 165 b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy

Caractérisation de la protéine NAf1 chez Saccharomyces cerevisiae et chez l'homme

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Crosstalk between microRNA and DNA Methylation Offers Potential Biomarkers and Targeted Therapies in ALK-Positive Lymphomas

The discovery of microRNA (miRNA) has provided new and powerful tools for studying the mechanism, diagnosis and treatment of human cancers. The down-regulation of tumor suppressive miRNA by hypermethylation of CpG island (CpG is shorthand for 5′-C-phosphate-G-3′, that is, cytosine and guanine separated by only one phosphate) is emerging as a common hallmark of cancer and appears to be involved in drug resistance. This review discusses the role of miRNA and DNA methylation in drug resistance mechanisms and highlights their potential as anti-cancer therapies in Anaplastic Lymphoma Kinase (ALK)-positive lymphomas. These are a sub-type of non-Hodgkin’s lymphomas that predominantly affect children and young adults and are characterized by the expression of the nucleophosmin (NPM)/ALK chimeric oncoprotein. Dysregulation of miRNA expression and regulation has been shown to affect several signaling pathways in ALK carcinogenesis and control tumor growth, both in cell lines and mouse models. These data suggest that the modulation of DNA methylation and/or the expression of these miRNA could serve as new biomarkers and have potential therapeutic applications for ALK-positive malignancies

La dyskératose congénitale

La dyskératose congénitale (DC), ou syndrome de Zinsser-Cole-Engman, est une maladie héréditaire, trop souvent létale, décrite pour la première fois sur le plan dermatologique par Zinsser en 1906. Il s’agit d’une maladie très polymorphe sur le plan clinique, variable dans son mode de transmission et dont certains symptômes cliniques apparaissent tardivement, rendant son diagnostic clinique difficile. À cette hétérogénéité clinique est associée une hétérogénéité génétique : on sait que plusieurs gènes, quatre étant identifiés à ce jour, sont impliqués ; néanmoins, pour la majorité des patients, le gène en cause n’est pas connu. Le point remarquable est que les quatre gènes connus (DKC1, hTERC, hTERT et NOP10) codent des composants de la télomérase tous impliqués dans le maintien de la longueur des télomères, faisant de la DC un modèle clinique unique pour la compréhension du rôle et de l’importance de la télomérase et des télomères. En outre, les protéines codées par les gènes DKC1 et NOP10 sont également des composants des particules dites H/ACA impliquées dans la synthèse des ribosomes et des ARN messagers matures. Des altérations de ces deux processus pourraient contribuer aux symptômes des patients atteints de DC due à des mutations de DKC1 ou NOP10

hNaf1 is required for accumulation of human box H/ACA snoRNPs, scaRNPs, and telomerase

The human telomerase ribonucleoprotein particle (RNP) shares with box H/ACA small Cajal body (sca)RNPs and small nucleolar (sno)RNPs the proteins dyskerin, hGar1, hNhp2, and hNop10. How dyskerin, hGar1, hNhp2, and hNop10 assemble with box H/ACA scaRNAs, snoRNAs, and the RNA component of telomerase (hTR) in vivo remains unknown. In yeast, Naf1p interacts with H/ACA snoRNP proteins and may promote assembly of Cbf5p (the yeast ortholog of dyskerin) with nascent pre-snoRNAs. Here we show that the human HsQ96HR8 protein, thereafter termed hNaf1, can functionally replace endogenous Naf1p in yeast. HeLa hNaf1 associates with dyskerin and hNop10 as well as box H/ACA scaRNAs, snoRNAs, and hTR. Reduction of hNaf1 steady-state levels by RNAi significantly lowers accumulation of these components of box H/ACA scaRNP, snoRNP, and telomerase. hNaf1 is found predominantly in numerous discrete foci in the nucleoplasm and fails to accumulate within Cajal bodies or nucleoli. Altogether, these results suggest that hNaf1 intervenes in early assembly steps of human box H/ACA RNPs, including telomerase

The anti-angiogenic isoforms of VEGF in health and disease

First Proposal: The artist requested that a number of earth-movers be placed at his disposal during the day alloted for his work. Second Proposal: The artist planned to ride a number of quarter horses along a white line across Sir Winston Churchill Square. Each horse was to be ridden by the artist until tired out, and the piece considered finished when the artist himself became too tired to ride and a path had been worn in the grass along the white line. Actual Work: Executed at the G Bar E Ranch about 25 miles east of Edmonton. The artist rode several quarter horses, starting at 11 A.M., along a line between two posts about 200 yards apart. At about 2 P.M. the work was interrupted when the owner of the ranch became aware that a path was being worn into the grass. Statement by the Artist: "For each horse ridden, one set of 9 sequential photographs should be taken. These may be enlarged and shown later as a record of the event, tigether with two photographs looking down the white line: one before I begin, one after I finish."full view, executed at the G Bar E Ranch about 25 miles east of Edmonto

MiR-497 suppresses cycle progression through an axis involving CDK6 in ALK-positive cells.

International audienceAnaplastic large-cell lymphoma, a T cell neoplasm, is primarily a pediatric disease. 75% of pediatric anaplastic large-cell lymphoma cases harbor the chromosomal translocation t(2;5)(p23;q35) leading to the ectopic expression of NPM-ALK, a chimeric tyrosine kinase. NPM-ALK consists of an N-terminal nucleophosmin (NPM) domain fused to an anaplastic lymphoma kinase (ALK) cytoplasmic domain. Pediatric NPM-ALK(+) anaplastic large-cell lymphoma is often a disseminated disease and young patients are prone to chemoresistance or relapse shortly after chemotherapeutic treatment. Furthermore, there is no gold standard protocol for the treatment of relapses. To the best of our knowledge, this is the first study on the potential role of the microRNA, miR-497, in NPM-ALK(+) anaplastic large-cell lymphoma tumorigenesis. Our results show that miR-497 expression is repressed in NPM-ALK(+) cell lines and patient samples through the hypermethylation of its promoter and the activity of NPM-ALK is responsible for this epigenetic repression. We demonstrate that overexpression of miR-497 in human NPM-ALK(+) anaplastic large-cell lymphoma cells inhibits cellular growth and causes cell cycle arrest by targeting CDK6, E2F3 and CCNE1 - the three regulators of the G1 phase of the cell cycle. Interestingly, we show that a scoring system based on CDK6, E2F3 and CCNE1 expression could help identify relapsing pediatric patients. In addition, we demonstrate the sensitivity of NPM-ALK(+) cells to CDK4/6 inhibition using for the first time a selective inhibitor, Palbociclib. Together, our findings suggest that CDK6 could be a therapeutic target for the development of future treatments for NPM-ALK(+) anaplastic large-cell lymphoma