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Ribosome profiling (RP) is a novel technique that exploits RNA sequencing and ribosome immobilization to quantify transcription and translation at different cell growth stages. Therefore, RP provides invaluable information for expression dynamics studies. Quantitative –omics studies are of crucial importance for identification of potential biomarkers of infection. An ideal parasite detection system should definitely establish the presence or absence of infection; determine the species involved; be detectable even in low concentrations; be proportional to parasite density; and determine the presence of antibiotic resistance. Here, we propose a simple workflow that attempts to identify a set of biomarkers that fulfill some of the above criteria for the ideal detection system. RP expression profiles were ranked for abundance, crosschecked with PlasmoDB for homogeneity along infection cycles and probed for availability of structural stability. The latter is of fundamental importance for the development of molecular biosensors to be give birth to rapid diagnostic kits. In addition, a simple biochemistry workflow was developed for easy production of the selected biomarkers in E. coli. Altogether, the present work provides two complementary and novel workflows that shall aid researchers to rapidly produce molecular biomarkers and develop biosensors based on antibodies or aptamers. / Tesis

Mutations in TSC2 cause the disorder tuberous sclerosis (TSC), which has a high incidence of autism and intellectual disability. TSC2 regulates mRNA translation required for group 1 metabotropic glutamate receptor-dependent synaptic long-term depression (mGluR-LTD), but the identity of mRNAs responsive to mGluR-LTD signaling in the normal and TSC brain is largely unknown. We generated Tsc2+/- mice to model TSC autism and performed ribosome profiling to identify differentially expressed genes following mGluR-LTD in the normal and Tsc2+/- hippocampus. Ribosome profiling reveals that in Tsc2+/-mice, RNA-binding targets of Fragile X Mental Retardation Protein (FMRP) are increased. In wild-type hippocampus, induction of mGluR-LTD caused rapid changes in the steady state levels of hundreds of mRNAs, many of which are FMRP targets. Moreover, mGluR-LTD signaling failed to promote phosphorylation of eukaryotic elongation factor 2 (eEF2) in Tsc2+/- mice, and chemically mimicking phospho-eEF2 with low cycloheximide enhances mGluR-LTD in the Tsc2+/- brain. These results suggest a molecular basis for bidirectional regulation of synaptic plasticity by TSC2 and FMRP. Furthermore, deficient mGluR-regulated translation elongation contributes to impaired synaptic plasticity in Tsc2+/- mice.

Alors que la biogénèse des ribosomes a étéconsidérée comme un mécanisme ubiquiste, lesétapes de ce processus ont récemment étédémontrées comme étant tissu-spécifiques. Letoit optique (OT) du poisson-zèbre est un modèleapproprié pour étudier la prolifération cellulairepuisque les cellules à différents états dedifférenciation se trouvent dans des domainesséparés.Au cours de mon doctorat, j'ai examiné si lesgènes de la biogenèse des ribosomes peuventavoir des rôles spécifiques dans les cellulesprogénitrices neuroépithéliales (CPNe). Profitantd'une analyse transcriptomique antérieure, j'aid'abord examiné les nouveaux candidatsaccumulés dans les CPNe. J'ai décidé de meconcentrer sur proliferation-associated 2G4(pa2G4/ebp1) qui est exprimé de manièrepréférentielle dans les CPNe.Ce gène favorise ou réprime la proliférationcellulaire dans des organismes normaux oupendant la tumorigénèse. J'ai conçu une stratégiepour l'expression inductible et cellule-spécifiquede ce gène.Fibrillarin (Fbl), une méthyltranférasenucléolaire est également préférentiellementexprimée dans CPNe. Ce gène joue un rôleimportant dans le cancer. J'ai montré que lesmutants fbl présentaient des défauts OTspécifiques,en lien avec une apoptose massive etune absence de différenciation neurale. J'aiégalement démontré une diminution de l'activitéde traduction des ribosomes. En outre, lesmutants fbl montrent une progression de la phaseS altérée. Nos données suggèrent que fbl estessentiel à la prolifération des progéniteursneuronaux du poisson-zèbre. / While ribosome biogenesis has been consideredas an ubiquitous mechanism, steps of thisprocess have recently been shown to be tissuespecific. Zebrafish optic tectum (OT) is asuitable model to study cell proliferation sincecells at different differentiation states arespatially partitioned.During my PhD, I examined whether ribosomebiogenesis genes may have specific roles inneuroepithelial progenitor cells (NePCs).Taking advantage of a previous transcriptomicanalysis, I first screened for new candidatesaccumulated in NePCs. I decided to focus onproliferation-associated 2G4 (pa2g4/ebp1),which was expressed preferentially in NePCs.This gene promotes or represses cellproliferation in normal organisms or duringtumorigenesis. I designed a strategy for theinducible expression and cell specificexpression of this gene.Fibrillarin (Fbl), a small nucleolarmethyltransferase is also preferentiallyexpressed in NePCs. It plays an important rolein cancer. I showed that fbl mutants displayedspecific OT defects linked to a massiveapoptosis and an absence of neuraldifferentiation. I also demonstrated deficienciesin the ribosome translational activity.Additionally, fbl mutants showed impaired Sphaseprogression. Our data suggest that fbl isessential for the proliferation of zebrafishneuronal progenitors.

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Background Promoters are key players in gene regulation. They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG http://corg.molgen.mpg.de, a framework for studying upstream regions including untranslated exons (5' UTR). Description The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences. As of now, we have included 5 species in our analysis pipeline (man, mouse, rat, fugu and zebrafish). We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server http://tomcat.molgen.mpg.de:8080/das. The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set. Conclusion The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites).

Translation control is crucial during virus-host interaction, in which the host relies on the translation machinery to mount an antiviral response or induce the inflammation response to reduce virus spread, while the virus aims to take control of this system to thwart the host defense while producing viral progeny. The field of oncolytic virus (OV) therapy relies on replicating, engineered viruses that preferentially infect tumor cells to induce direct oncolysis or promote an antitumor immune response. Despite the importance of translation control in virus-host interaction, not much has been described on the interaction at the translation level between OV and cancer cells. I propose that this knowledge gap could reveal significant improvements in OV efficacy in treating cancer. In my first study, I set out to characterize the translatome of an infection-resistant breast cancer cell line infected by three clinically advanced OVs to identify residual antiviral activity in cancer cells regulated by translation control. I found the inositol phosphatase Inpp5e to be a novel antiviral gene that is translationally induced during infection via a transcript variant shift. Mechanistically, I showed that the majority of Inpp5e transcripts in uninfected cells contain a long 5’ UTR that harbor four translationally inhibitory upstream reading frames (uORF). Yet, OV infection induced the expression of a shorter 5’ UTR with a spliced intron that removes three uORFs, derepressing the translation of Inpp5e mRNA. CRISPR-Cas9 knockout of Inpp5e also enhanced the infectivity of oncolytic HSV1 and VSV. My study suggests the existence of a class of translationally regulated antiviral genes in cancer cells. In my second study, I sought to adapt the translation of transgenes to the unique translation condition imposed by the infecting virus via the incorporation of a viral 5’UTR. I identified HSV1 5’UTRs by locating the transcription start site of most HSV1 genes using RNA-seq data, then determined the 5’UTR of US11 as a potent translation enhancer during HSV1 infection. Incorporation of this 5’UTR into the transgene expression cassette inserted into the HSV1 genome enhanced transgene expression significantly at the translation level. In my third study, I set out to explore the mechanism of miR-223 mediated inflammation inhibition. miR-223 is a protective miRNA in the context of atherogenesis via suppressing inflammatory signaling. Using transcriptome and translatome profiling (RNA-seq and Ribo-seq), I found that the inhibitory effect of miR-223 on inflammation occurs primarily at the translation level. Overall, my work highlights the importance of translation control in OV-cancer cells interaction, as well as in inflammation-related diseases.

Erythroid terminal differentiation couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. I used Epor−/− mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. I found that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. Specifically, I found that high levels of EpoR signaling increase the size and shorten the cycle of early erythroblasts, which are amongst the fastest cycling somatic cells. I confirmed the effect of erythropoietin (Epo) on red cell size in human volunteers, whose mean corpuscular volume (MCV) increases following Epo administration. Our work shows that EpoR signaling alters the expected inverse relationship between cell cycle length and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress. The ability of EpoR signaling to increase cell size in rapidly cycling early erythroblasts suggests that these cells have exceptionally efficient EpoR-driven mechanisms for growth. I found evidence for this in ongoing work, where Epor−/− and Stat5−/− single-cell transcriptomes show dysregulated expression of ribosomal proteins and rRNA transcription and processing genes. Global rates of ribosomal rRNA transcription and protein synthesis increase in an EpoR dependent manner during a narrow developmental window in early ETD, coincident with the time of cell cycle shortening. Our work therefore suggests EpoR-driven regulation of ribosome biogenesis and translation orchestrating rapid cycling and cell growth during early ETD.

Le ribosome est un macro complexe moléculaire en charge de la synthèse de toutes lesprotéines de la cellule. Depuis les premiers essais de reconstruction in vitro d’un ribosome procaryote,des générations de chercheurs se sont succédées durant plusieurs décennies pour tenter d’éluciderles voies par lesquelles les ribosomes sont assemblés par la cellule. Un regain d’intérêt pour l’étude dela biogenèse du ribosome est advenu récemment suite à la mise en évidence de maladies liées à desmutations dans des acteurs de la biogenèse du ribosome mais également, et surtout, suite à la miseen évidence du rôle fondamental du ribosome dans les processus de transformation oncogénique.Le ribosome est composé de deux sous-unités, une petite et une grande, formées, ellesmêmes,d’un assemblage intriqué d’ARN ribosomique et de protéines ribosomiques. La formation d’unribosome, appelée biogenèse du ribosome, est un processus complexe, intégré et extrêmementhiérarchisé impliquant, chez la levure, plus 200 facteurs accessoires. Bien que les principes sousjacentsà la biogenèse du ribosome eucaryote établis à partir d’études réalisées chez la levure semblentconservés chez l’Homme, de nombreux éléments suggèrent qu’elle y soit plus complexe. Ainsi, latransposition directe chez l’Homme des connaissances acquises chez la levure quant à la biogenèse duribosome serait, tout du moins partiellement, inexacte.Au cours de ma thèse, j’ai poursuivi différents objectifs s’intégrant tous dans le cadre de labiogenèse du ribosome eucaryote. D’une part, chez la levure, j’ai poursuivi la caractérisation du rôlede la protéine Las1 dans la biogenèse de la grande sous-unité ribosomique. D’autre part, chezl’Homme, mon objectif premier a été de participer à l’identification de nouveaux facteurs accessoiresimpliqués dans la biogenèse du ribosome et à la caractérisation fonctionnelle de certains d’entre eux.Dans un second temps, je me suis concentré sur l’implication de deux particules ribonucléoprotéiquesnucléolaires, appelées snoRNP, dans la biogenèse du ribosome mais également dans le développementtumoral. Finalement, le dernier objectif de ma thèse a été de participer, dans le cadre d’un projettransverse réalisé chez la levure et chez l’Homme, à l’étude d’une protéine en charge d’une desmodifications que portent les ARN ribosomiques.Pris ensemble, les différents objectifs que j’ai poursuivis au cours de ma thèse, permettent desavancées fondamentales dans la connaissance du processus de la biogenèse du ribosome chez leseucaryotes mais aussi dans la caractérisation de l’impact clinique de certains acteurs de cette voie. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished