Un nouveau rôle de la tyrosine kinase oncogénique NPM-ALK dans le contrôle de l'expression génique au niveau post-trancriptionnel

La protéine hybride à activité tyrosine kinase, NPM-ALK est exprimée dans 75% des Lymphomes Anaplasiques à Grandes Cellules (LAGC). Bien que le phénotype tumoral de ces lymphomes soit en partie associé à l'activation constitutive de nombreuses voies de signalisation (MAPK, PI3K/AKT, Jak/STAT et PLC-gamma), l'identification de nouveaux partenaires de NPM-ALK a permis d'envisager l'existence de nouveaux mécanismes moléculaires participants à son pouvoir oncogénique. Ainsi, la découverte d'interactions entre NPM-ALK et des protéines de liaisons aux ARNm (RNA-Binding protein, RBPs), nous a poussé à émettre l'hypothèse, qu'outre son effet sur la transcription, la tyrosine kinase oncogénique NPM-ALK pouvait également moduler l'expression génique au niveau post-transcriptionnel. Dans la première partie de mon travail (article n°1), j'ai montré que HuR, une AUBP (AU-binding protein qui contrôle le devenir d'ARNm dont l'extrémité 3' non traduite présente des motifs riches en Adénines et Uridines (AU-rich element, ARE), augmente la stabilité et le niveau de traduction de l'ARNm-ARE C/EBPb-beta dans les LAGC ALK+. J'ai également démontré que la tyrosine kinase NPM-ALK augmente l'activité de HuR en modulant ses propriétés biologiques telles que son affinité pour ses ARNm cibles et son recrutement au niveau des polysomes. J'ai enfin déterminé que NPM-ALK et HuR co-localisent dans des granules cytoplasmiques et que HuR est phosphorylée sur résidus tyrosine dans les LAGC ALK+. Dans la deuxième partie de mon travail (manuscrit en préparation), et grâce à la génération d'une série de mutants de HuR, j'ai : 1/ identifié les résidus tyrosine, phosphorylés dans les LAGC ALK+; 2/ démontré l'implication directe de la tyrosine kinase NPM-ALK dans cette phosphorylation; 3/ recherché l'impact de ces phosphorylations sur les propriétés biologiques de HuR (affinité envers ses cibles et localisation subcellulaire). Plus particulièrement, j'ai montré que la phosphorylation du résidu tyr26, localisé dans le motif de reconnaissance aux ARN (RRM1), joue un rôle essentiel dans l'affinité de HuR pour ses ARNm cibles. Il reste aujourd'hui à préciser le rôle de ces phosphorylations dans l'adressage de HuR et de ses cibles aux polysomes. Enfin, il faut démontrer la relevance fonctionnelle de cette phosphorylation sur l'émergence et le maintien des LAGC ALK+. Parallèlement à ce projet qui a été au centre de ma thèse, j'ai également participé à un travail de l'équipe qui a permis de mettre en évidence l'implication de la tyrosine kinase NPM-ALK dans le contrôle de l'expression d'un miRNA, par méthylation de son promoteur. Ce travail s'est basé sur l'exemple du contrôle de l'expression de la protéine anti-apoptotique Mcl1 par le miR-29a. Ces différents travaux sont à l'origine d'une part, de la découverte de deux nouveaux acteurs de la lymphomagénèse dépendante de ALK, l'AUBP HuR et les miRNAs. Ils valident d'autre part le rôle original de NPM-ALK dans la régulation de l'expression génique au niveau post-transcriptionnel.The NPM-ALK chimeric protein is expressed in 75% of Anaplastic Large Cell Lymphomas. Although the oncogenic features of these lymphomas are in part due to the constitutive activation of many signalling pathways such as MAPK, PI3K/AKT, Jak/STAT et PLC-gamma, the identification of new partners of NPM-ALK would allow to consider new molecular mechanisms that could also participate to this phenotype. Thereby, interactions between NPM-ALK and RNA-Binding Proteins (RBPs) led us to postulate that, in addition to its recognized role in transcriptional activation, the oncogenic tyrosine kinase NPM-ALK could also modulate gene expression at the post-transcriptional level. In the first part of my work (1st article), I have shown that HuR, an AU-rich Binding Protein (AUBP), that bind to Adenine and Uridine rich elements (ARE) in the 3' untranslated region of some mRNAs, controls the stability and the level of translation of C/EBP-beta mRNA in ALK+ ALCL. I have also demonstrated that the tyrosine kinase NPM-ALK increases HuR activity by modulating its biological properties such as its binding affinity on its mRNA targets or its recruitment into actively translating polysomes. Lastly, we have determinated that NPM-ALK and HuR colocalize into cytoplasmic granules and that HuR is phosphorylated on tyrosine residus in ALK+ ALCL. In the second part of my work (publication in prep.), by testing different point mutated versions of HuR, I have: 1/ identified the tyrosine residues that are phosphorylated in ALK+ ALCL; 2/ demonstrated the direct involvement of the tyrosine kinase NPM-ALK in this phosphorylation event; 3/ measured the impact of these phosphorylations on HuR biological properties (affinity toward its targets mRNAs and subcellular localization). More particularly, I have shown that the phosphorylation on tyrosine residue 26 within the RNA recognition motif (RRM) 1 is essential for NPM-ALK-mediated HuR binding to ARE-mRNAs. It remains now to clarify the role of these phosphorylations in the recruitment of HuR into polysomes and to demonstrate the functional relevance of these phosphorylations on the emergence and the maintenance of ALK+ ALCL. In the same time, I have taken part in another work in the team dealing with the role of the tyrosine kinase NPM-ALK in the control of miRNA expression, by methylation events. This project focused on the example of the control of the expression of the anti-apoptotic MCL-1 by miR-29a

Data for the generation of RNA spatiotemporal distributions and interpretation of Chk1 and SLBP protein depletion phenotypes during Drosophila embryogenesis

The data presented in this article is related to the research article entitled “Biochemical Fractionation of Time-Resolved Drosophila Embryos Reveals Similar Transcriptomic Alterations in Replication Checkpoint and Histone mRNA Processing Mutants” (Lefebvre et al., 2017) [1]. This article provides a spatiotemporal transcriptomic analysis of early embryogenesis and shows that mutations in the checkpoint factor grapes/Chk1 and the histone mRNA processing factor SLBP selectively impair zygotic gene expression. Here, lists of transcripts enriched in early syncytial embryos, late blastoderm embryos, cytoplasmic and nuclear extracts of blastoderm embryos are made public, along with transcription factor motif occurrence for genes enriched in each context. In addition, extensive lists of genes down-regulated upon Chk1 and SLBP protein depletion in embryos are released to enable further analyses

Loss of function of the Drosophila Ninein-related centrosomal protein Bsg25D causes mitotic defects and impairs embryonic development

The centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) play significant roles in microtubule stability, nucleation and anchoring at the centrosome in mammalian cells. Here, we investigate Blastoderm specific gene 25D (Bsg25D), which encodes the only Drosophila protein that is closely related to Nin and Nlp. In early embryos, we find that Bsg25D mRNA and Bsg25D protein are closely associated with centrosomes and astral microtubules. We show that sequences within the coding region and 3′UTR of Bsg25D mRNAs are important for proper localization of this transcript in oogenesis and embryogenesis. Ectopic expression of eGFP-Bsg25D from an unlocalized mRNA disrupts microtubule polarity in mid-oogenesis and compromises the distribution of the axis polarity determinant Gurken. Using total internal reflection fluorescence microscopy, we show that an N-terminal fragment of Bsg25D can bind microtubules in vitro and can move along them, predominantly toward minus-ends. While flies homozygous for a Bsg25D null mutation are viable and fertile, 70% of embryos lacking maternal and zygotic Bsg25D do not hatch and exhibit chromosome segregation defects, as well as detachment of centrosomes from mitotic spindles. We conclude that Bsg25D is a centrosomal protein that, while dispensable for viability, nevertheless helps ensure the integrity of mitotic divisions in Drosophila

Loss of function of the Drosophila

The centrosome-associated proteins Ninein (Nin) and Ninein-like protein (Nlp) play significant roles in microtubule stability, nucleation and anchoring at the centrosome in mammalian cells. Here, we investigate Blastoderm specific gene 25D (Bsg25D), which encodes the only Drosophila protein that is closely related to Nin and Nlp. In early embryos, we find that Bsg25D mRNA and Bsg25D protein are closely associated with centrosomes and astral microtubules. We show that sequences within the coding region and 3′UTR of Bsg25D mRNAs are important for proper localization of this transcript in oogenesis and embryogenesis. Ectopic expression of eGFP-Bsg25D from an unlocalized mRNA disrupts microtubule polarity in mid-oogenesis and compromises the distribution of the axis polarity determinant Gurken. Using total internal reflection fluorescence microscopy, we show that an N-terminal fragment of Bsg25D can bind microtubules in vitro and can move along them, predominantly toward minus-ends. While flies homozygous for a Bsg25D null mutation are viable and fertile, 70% of embryos lacking maternal and zygotic Bsg25D do not hatch and exhibit chromosome segregation defects, as well as detachment of centrosomes from mitotic spindles. We conclude that Bsg25D is a centrosomal protein that, while dispensable for viability, nevertheless helps ensure the integrity of mitotic divisions in Drosophila

Developmentally Regulated Elimination of Damaged Nuclei Involves a Chk2-Dependent Mechanism of mRNA Nuclear Retention

SummaryThe faithful execution of embryogenesis relies on the ability of organisms to respond to genotoxic stress and to eliminate defective cells that could otherwise compromise viability. In syncytial-stage Drosophila embryos, nuclei with excessive DNA damage undergo programmed elimination through an as-yet poorly understood process of nuclear fallout at the midblastula transition. We show that this involves a Chk2-dependent mechanism of mRNA nuclear retention that is induced by DNA damage and prevents the translation of specific zygotic mRNAs encoding key mitotic, cytoskeletal, and nuclear proteins required to maintain nuclear viability. For histone messages, we show that nuclear retention involves Chk2-mediated inactivation of the Drosophila stem loop binding protein (SLBP), the levels of which are specifically depleted in damaged nuclei following Chk2 phosphorylation, an event that contributes to nuclear fallout. These results reveal a layer of regulation within the DNA damage surveillance systems that safeguard genome integrity in eukaryotes

Oncologic trogocytosis of an original stromal cells induces chemoresistance of ovarian tumors

Background: The microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours. Methodology/Principal Findings: We isolated an original type of stromal cells, referred to as ‘‘Hospicells’ ’ from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance

A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs