Advances in next-generation sequencing platforms during the past decade have resulted in exponential increases in biological data generation. Besides applications in determining the sequences of genomes and other DNA elements, these platforms have allowed the characterization of cell-wide mRNA pools under different conditions and in different tissues. In 2009, Ingolia and colleagues developed an extension of high-throughput sequencing that provides a snapshot of all cellular mRNA fragments protected by translating ribosomes, dubbed ribosome profiling. This approach allows detection of differential translation activity, annotation of novel protein coding sequences and variants, identification of ribosome pause sites and estimates of de novo protein synthesis. As with other sequencing based methodologies, a major challenge of ribosome profiling has been sorting, filtering and interpreting the gigabytes of data produced during the course of a typical experiment. In this thesis, I developed and applied computational pipelines to interrogate ribosome profiling data in relation to gene expression in several viruses and eukaryotic species, as well as to identify sites of ribosomal pausing and sites of non-canonical translation activity. Specifically, I applied various control analyses for characterizing the quality of profiling data and developed scripts for visualizing genome-based (exon-by-exon) rather than transcript-based ribosome footprint alignments. I also examined the challenge of mapping footprints to repetitive sequences in the genome and propose ways to mitigate the associated problems. I performed differential expression analyses on data from coronavirus-infected murine cells, retrovirus-infected human cells and temperature-stressed Arabidopsis thaliana plants. Dissection of translational responses in Arabidopsis thaliana during heat shock or cold shock revealed several groups of genes that were highly upregulated within 10 minutes of temperature challenge. Analysis of the branches of the unfolded protein and integrated stress responses during coronavirus infection allowed for deconvolution of transcriptional and translational contributions. During the course of these analyses, I identified errors in a recently publicized algorithm for detection of differential translation, and wrote corrections that have now been pulled into the repository for this package. Comparison of the translational kinetics of the dengue virus infection in mosquito and human cell lines revealed host-specific sites of ribosome pausing and RNA accumulation. Analysis of HIV profiling data revealed footprint peaks which were in agreement with previously proposed models of peptide or RNA mediated ribosome stalling. I also developed a simulation to identify transcripts that are prone to generating RPFs with multiple alignments during the read mapping process. Together, the scripts and pipelines developed during the course of this work will serve to expedite future analyses of ribosome profiling data, and the results will inform future studies of several important pathogens and temperature stress in plants.

Les urgences écologiques actuelles, résultant des dérives de l'industrialisation et de la mondialisation, mènent la recherche fondamentale à se préoccuper hâtivement de la biologie de certains organismes comme les dinoflagellés. Ces organismes sont à la base de la chaîne alimentaire, et certaines espèces sont impliquées dans la formation des récifs coralliens. De plus, la prolifération incontrôlée de certaines espèces de dinoflagellés engendrée par l'eutrophisation industrielle est responsable des marées rouges qui menacent d'une part la flore et la faune aquatiques. Certaines des toxines produites par ces efflorescences algales ont un effet sur la santé publique, car elles peuvent rendre dangereuse la consommation alimentaire de poissons ou de fruits de mer contaminés. Nous avons examiné le comportement de l’espèce Lingulodinium polyedra à travers des cycles diurnes. Dans un premier projet, le protéome dans des extraits d'algues récoltés sur un cycle de 24 heures en conditions de 12 heures de jour suivies de 12 heures de nuit (un cycle LD) a été examiné. Nous avons identifié treize protéines clés qui ont montré des variations quantitatives synchronisées avec les temps de réalisation des rythmes biologiques les impliquant. Deux protéines déjà connues de varier faisait partie de ce groupe, tandis que les autres protéines sont impliquées dans des processus rythmiques nouveaux. Nous avançons l’hypothèse qu’une augmentation quantitative des protéines clés permettrait à l'accomplissement des différents processus cellulaires à différents moments de la journée. Dans un second projet, nous avons examiné la vitesse de synthèse des protéines qui pourrait expliquer ces variations quantitatives protéiques. En appliquant la technique du profilage ribosomal ( « ribosome profiling ») sur des échantillons algaux collectés d’abord à trois temps, puis ensuite sur tout le cycle LD, nous avons confirmé des variations de vitesse de synthèse des protéines identifiées dans notre étude protéomique et des protéines dont la vitesse de synthèse a déjà été mesurée par l’incorporation de méthionine radioactive in vivo. De plus, nous avons identifié plusieurs milliers d’autres séquences dont la vitesse de synthèse varie. Une classification des séquences dans les catégories GO nous a permis d’identifier un rythme diurne dans la synthèse des acides aminés qui pourrait aider à comprendre le métabolisme du nitrate. Nous proposons que des variations de vitesse de synthèse entraînent des augmentations quantitatives des protéines aux niveaux inférieurs à ce que l’on a pu détecter par la spectroscopie de masse. La variation dans les niveaux de protéines clés pourrait aider l'accomplissement des rythmes diurnes chez le dinoflagellé Lingulodinium. Ces avancées dans la compréhension de la régulation des rythmes biologiques du dinoflagellé Lingulodinium d'une part permettront de mieux penser la recherche concernant la lutte écologique contre les marées rouges et d'autre part ouvriront de nouvelles perspectives dans l'entendement de la régulation des rythmes biologiques des autres organismes y compris ceux de l'Homme dont les troubles impliquent de nombreuses maladies. / Industrialization and globalization has led to industrial eutrophication in many regions of the oceans resulting in different types of ecological emergencies. One of these is the uncontrolled proliferation of dinoflagellates which are responsible for the harmful algal blooms called “red tides”. Certain species release toxins which can harm aquatic flora and fauna and, through consumption of contaminated fish or seafood, can affect public health. Dinoflagellates are also at the base of the food chain in the marine environment, and certain species form symbioses with anthozoans thus allowing growth of coral reefs. There is thus a strong need for fundamental research on dinoflagellate biology. Using the dinoflagellate Lingulodinium polyedra as a model, we have examined how cells adapt to the daily changes in light and dark. In particular we have examined changes in protein amounts as well as changes in protein synthesis rates to gain insight into how cells differ during the day and night. In a first project, the daily proteome was examined by mass spectrometry using algae extracts harvested over a 24 hour cycle in conditions of 12 hours of day followed by 12 hours of night. Key proteins involved in biological rhythms have shown quantitative variations synchronized with the times of the biological rhythms involving them, ie a quantitative increase in key proteins is necessary for the accomplishment of the various cellular processes. Thirteen proteins were identified, of which two were previously documented to change in amount over the daily cycle. In a second project we addressed the origin of these quantitative protein variations. By applying the technique of ribosome profiling on algal samples, first collected in triplicate at three times and then at two hour intervals throughout the 24 h cycle, we have identified iv variations in the synthesis rate of thousands of proteins. These include the proteins found in the proteomic analysis as well as four proteins whose synthesis rate variations had already been observed using in vivo metabolic labeling. Interestingly, classification of the regulated proteins into GO categories also revealed a late night increase in the synthesis of many amino acid biosynthetic enzymes, potentially linking amino acid synthesis associated with the daily metabolism of nitrate. These advances in our understanding of the regulation of the daily rhythms of the dinoflagellate Lingulodinium will provide new tools in the ecological fight against red tides. They may also open new perspectives in understanding the daily rhythms of other organisms.

Die asymmetrische Verteilung von mRNA und Proteinen innerhalb einer Zelle definiert die Polarität. Dies ermöglicht eine strikte Regulierung der Genexpression in Raum und Zeit. Ich habe in dieser Arbeit untersucht, wie das Soma und die Neuriten in induzierten Neuronen sich hinsichtlich ihres Transkriptoms und Translatoms unterscheiden. Eine räumliche ribosomale Profilanalyse ergab, dass die Hälfte des lokalen Proteoms durch die mRNA-Lokalisierung und der lokalen Translation definiert wird. Dies sind Prozesse, die durch die synergistische Aktivität von trans- und cis-agierenden Elementen durchgeführt werden. In dieser Arbeit konzentrierte ich mich auf MOV10 als trans-agierendes Element und die alternativen 3′UTRs als cis-agierende Elemente, um ihre Rolle in der Asymmetrie zu untersuchen. MOV10 ist eine RNA-Helikase, welche an vielen Aspekten des RNA-Metabolismus beteiligt ist. Mit den Methoden RIP und PAR-CLIP konnte ich zeigen, dass sowohl MOV10-Ziele als auch MOV10 selbst in den Neuriten lokalisiert sind. Aus ̈erdem ist MOV10 möglicherweise an der translationalen Repression mitinvolviert. In der Tat konnte ich unter den MOV10-Protein-Interaktoren mehrere Proteine identifizieren, welche an der translationalen Repression beteiligt sind, wie z.Bsp. AGO2, FMR1, und TRIM71. Für die Identifizierung der cis-agierenden Elemente führte ich das "Mapping" von alternativen 3′UTRs durch. Diese Analyse zeigte mehrere Gene, die differentiell lokalisierte 3′UTR-Isoformen exprimieren. Insbesondere habe ich mich auf Cdc42 konzentriert. Ich konnte beweisen, dass die beiden Isoformen von Cdc42 auf mRNA-Ebene unterschiedlich lokalisiert sind und dass die 3′UTR der entscheidende Faktor für die mRNA- und Proteinlokalisierung ist. Darüber hinaus habe ich mehrere RBPs identifiziert, die an der Cdc42-Lokalisierung beteiligt sind. Diese Analyse zeigt, dass für die differenzierte Lokalisierung von funktional unterschiedlichen alternativen Protein-Isoformen die Verwendung von alternativen 3′UTR Isoformen als neu-entdeckter Mechanismus eine entscheidende Rolle spielt. / Asymmetric distribution of mRNA and proteins inside a cell defines polarity, which allow tight regulation of gene expression in space and time. In this thesis I investigated how asymmetric distribution characterizes the somatic and neuritic compartments of in induced neurons, in terms of transcriptome and translatome. Spatial ribosome profiling analysis revealed that half of the local proteome is defined by mRNA localization and local translation. These, are processes accomplished by the synergistic activity of trans- and cis-acting elements. I focused on MOV10 as trans-acting element, and on alternative 3′UTRs as cis-elements, to investigate their role in asymmetry. MOV10 is an RNA helicase which participates to many aspects of RNA metabolism. With RIP and PAR-CLIP I showed that MOV10 targets are localized to the neurites, consistently with MOV10-neuritic localization, and that MOV10 might be involved in translational repression. Indeed, among MOV10 protein interactors, I identified several proteins involved in translational repression, i.e. AGO2, FMR1, and TRIM71. On the side of cis-elements, I performed mapping of alternative 3′UTRs. This analysis identified several genes expressing differentially localized 3′UTR isoforms. In particular, I focused on Cdc42. I showed that the two isoforms of Cdc42 are differentially localized at mRNA level, and that the 3′UTR is the driver of mRNA and protein localization. Moreover, I identified several RBPs that might be involved in Cdc42 localization. This analysis points to usage of alternative 3′UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.

<p dir="ltr">Skeletal muscle accounts for 40% of total body weight and the homeostasis of muscle tissue is critical in maintaining proper body function. Skeletal muscle develops during the embryonic stages from the muscle progenitor cells derived from the dermomyotome structure. The myogenic progenitor cells contribute to the primary myogenesis by forming the primary myotubes which are the founding structures that the secondary myogenesis continues to build on. A portion of the myogenic progenitor cells makes up the adult muscle stem cells residing in homeostatic muscle tissue. The adult muscle stem cells contribute substantially for the adult muscle regeneration. Due to the significance of the muscle tissue and the importance of muscle stem cells, dysregulation of the muscle homeostasis or the muscle stem cell homeostasis will result in severe pathological conditions such as myopathy.</p><p dir="ltr">Mitochondria are cellular organelles that are responsible for generating energy needed for cellular processes, especially for muscle tissue where muscle contraction requires the presence of ATP. On the other hand, mitochondria also serve as signaling molecules and provide macromolecules for the biosynthesis. FAM210A (Family With Sequence Similarity 210 Member A) protein was shown to impact the lean mass of human subjects yet a detailed study on the effect of FAM210A in skeletal muscle was not performed, nor has the molecular mechanisms through which FAM210A function been elucidated. Therefore, I take on the task to unveil the function of FAM210A in muscle development, muscle homeostasis and muscle stem cell behavior by using a combination of mouse models with different myogenic promoters to target <i>Fam210a</i> at different developmental stages.</p><p dir="ltr">In the first part of the thesis, I investigated the role of FAM210A in post differentiation myofibers. Using the <i>Myl1</i>^<em>Cre</em> driven deletion of <i>Fam210a</i>, I found that <i>Fam210a</i>^<em>MKO</em> had normal development before 3 weeks of age, but the growth was stagnant from 4 weeks on, and the mice did not survive past 8 weeks of age. I found that the assembly of the ribosomes in the <i>Fam210a</i>^<em>MKO</em> was defective, leading to impaired translation which attenuated the muscle atrophy phenotype. I identified through proteomics that the mitochondrial autophagy and proteostatic control pathways were significantly induced yet mitochondrial organization and energetic proteins were downregulated. Metabolomics analysis showed that the signaling metabolite acetyl-CoA was increased in the <i>Fam210a</i>^<em>MKO</em> which led to increased protein acetylation, specifically, we showed that the ribosomal proteins were hyperacetylated, and that the acetylation increase was elicited by the <i>Fam210a</i>-null mitochondria.</p><p dir="ltr">In the second part of the thesis, I investigated the function of FAM210A in muscle progenitor cells. In the <i>FamMKO</i> mice, I found that deletion of <i>Fam210a</i> from embryonic myogenic progenitor cells led to developmental arrest and postnatal death at day 6. In the <i>FamPKO</i> mice, I found that <i>Fam210a</i> is needed for adult muscle stem cell to contribute to regeneration. Loss of <i>Fam210a</i> leads to the regenerative defects when the muscle was exposed to injury cues. We further showed that <i>Fam210a</i> deletion in muscle stem cells resulted in disruption of the proteostatic control over muscle stem cell activation, thereby forbidding the translational increase necessary to facilitate activation and proliferation. Furthermore, I showed that <i>Fam210a</i> deletion leads to excessive OPA1 cleavage, which contributes to the regenerative failure of muscle stem cells as fusion is required for the mitochondrial network remodeling during regeneration. Therefore, <i>Fam210a</i> safeguards the mitochondrial network and proteostasis during regeneration.</p><p dir="ltr">In summary, my studies characterized the functional contribution of FAM210A during embryonic muscle development, muscle mass maintenance and adult muscle stem cell homeostasis. The regulation of FAM210A in these three processes impinge on the translational regulation. My studies further demonstrated the importance of mitochondrial regulated protein translation in skeletal muscle and muscle stem cells.</p>

Increasing evidence of non-canonical protein translation has sparked interest in their identification and characterization for use in immunotherapy. In addition, recent studies on the repertoire of major histocompatibility complex class I (MHC-I) associated peptides (MAPs or immunopeptidome), have suggested that MAPs derived from these translations are potential targets for cancer immunotherapy. Therefore, the aim of this study was to assess the impact of these MAPs in cancer by developing methods to facilitate their identification and their validation as potential targets for immunotherapy. To facilitate the identification of non-canonical proteins, we developed Ribo-db, a proteogenomic approach that combines RNA sequencing, ribosome profiling and mass spectrometry. This approach enables the generation of specific databases aimed at including protein diversity. The use of Ribo-db to analyze diffuse large B-cell lymphoma (DLBCL) samples revealed that approximately 10% of MAPs were derived from non-canonical proteins. These proteins had distinct properties compared to those derived from canonical proteins. They had shorter lengths and lower stability, but greater efficiency in generating MAPs. Importantly, we found limited overlap between the non-canonical proteins detected in the immunopeptidome and those detected in the whole proteome suggesting the existence of two distinct non-canonical protein repertoires. Knowing that non-canonical MAPs can be effective targets for cancer immunotherapy, we developed BamQuery, a tool to assess their expression in tissues to determine whether they can be used in a vaccine. BamQuery aims to predict the probability of MHC-I presentation of each peptide in different tissues based on its RNA expression. Using BamQuery, we found that previously identified tumor antigens (TA) would be highly expressed in healthy tissues, making them poor candidates for immunotherapy. In addition, we also identified highly potential immunotherapeutic targets in DLBCL that were derived from non-canonical translations. These targets showed promising as they were poorly expressed in normal tissues but highly expressed and shared in tumor samples. Thus, BamQuery proved to be a useful tool for identifying and prioritizing potential immunotherapeutic targets. Overall, our research indicated that non-canonical regions of the genome increase the diversity of MAPs that can be recognized by T cells. Furthermore, the expression of MAPs in tissues can be used as a predictor of their presentation to MHC I to identify reliable targets for immunotherapy, for which BamQuery is an effective tool. / Les preuves de plus en plus nombreuses de la traduction des protéines non canonique ont suscité l'intérêt pour leur identification et leur caractérisation en vue de leur utilisation dans les immunothérapies. En outre, des études récentes sur le répertoire des peptides associés au complexe majeur d'histocompatibilité de classe I (CMH-I, connus sous le nom de MAPs ou immunopeptidome), ont suggéré que les MAPs dérivés de ces traductions sont des cibles potentielles pour l'immunothérapie du cancer. L'objectif de cette étude était donc d'évaluer l'impact de ces MAP dans le cancer en développant des méthodes pour faciliter leur identification et leur validation en tant que cibles potentielles pour l'immunothérapie. Afin de faciliter l'identification des protéines non canoniques, nous avons développé Ribodb, une approche protéogénomique qui combine le séquençage de l'ARN, le profilage ribosomal et la spectrométrie de masse. Cette approche permet de générer des bases de données spécifiques visant à inclure la diversité des protéines. Notre analyse avec Ribo-db d'échantillons de lymphome diffus à grandes cellules B (DLBCL) a révélé qu'environ 10% des MAP étaient dérivés de protéines non canoniques. Ces protéines avaient des propriétés distinctes par rapport à celles dérivées de protéines canoniques. Elles étaient plus courtes et avaient une stabilité plus faible, mais une plus grande efficacité dans la génération de MAPs. Fait important, nous avons constaté un chevauchement limité entre les protéines non canoniques détectées dans l'immunopeptidome et celles détectées dans le proteome entier, ce qui suggère l'existence de deux répertoires distincts de protéines non canoniques. Sachant que les MAP non canoniques peuvent être des cibles efficaces pour l'immunothérapie du cancer, nous avons développé BamQuery, un outil permettant d'évaluer leur expression dans les tissus afin de déterminer s'ils peuvent être utilisés dans un vaccin. BamQuery vise à prédire la probabilité de présentation au CMH-I de chaque MAP dans différents tissus sur la base de son expression ARN. En utilisant BamQuery, nous avons découvert que des antigènes tumoraux (TA) précédemment identifiés seraient fortement exprimés dans les tissus sains, ce qui en fait de mauvais candidats pour l'immunothérapie. En outre, nous avons également ii identifié des cibles immunothérapeutiques très potentielles dans DLBCL qui étaient dérivées de traductions non canoniques. Ces cibles se sont révélées prometteuses car elles étaient peu exprimées dans les tissus normaux mais fortement exprimées et partagées dans les échantillons tumoraux. Ainsi, BamQuery s'est avéré être un outil utile pour identifier et hiérarchiser les cibles immunothérapeutiques potentielles. Dans l'ensemble, nos recherches ont indiqué que les régions non canonique du génome augmentent la diversité des MAPs qui peuvent être reconnues par les cellules T. De plus, l'expression des MAPs dans les tissus peut être utilisée comme un prédicteur de leur présentation au CMH I afin d'identifier des cibles fiables pour l'immunothérapie, ce pour quoi BamQuery est un outil efficace.

Kürzlich wurde mithilfe von Ribo-seq Experimenten die Translation hunderter Mikroproteine in menschlichen Herzen entdeckt. Diese blieben zuvor aufgrund ihrer geringen Größe (< 100 Aminosäuren) unentdeckt, und ihre physiologische Rolle ist noch weitgehend unbekannt. Ziel dieser Promotionsarbeit ist es, potentielle Funktionen dieser neuartigen Mikroproteine zu entschlüsseln. Dabei sollen insbesondere die Aufklärung ihrer evolutionären Konservierungssignatur, subzellulären Lokalisierung und ihres Proteininteraktoms helfen. Die Konservierungsanalyse ergab, dass fast 90% der Mikroproteine nur in Primaten konserviert ist. Weiterhin konnte ich die Produktion von Mikroproteine in vitro und in vivo nachweisen, die subzelluläre Lokalisierung von 92 Mikroproteinen definieren, und Interaktionspartner für 60 Mikroproteine identifizieren. Dutzende dieser Mikroproteine lokalisieren in Mitochondrien. Dazu gehörte ein im Herzen angereichertes Mikroprotein, das aufgrund der Interaktions- und Lokalisationsdaten einen neuartigen Modulator der mitochondrialen Proteintranslation darstellen könnte. Der Interaktom-Screen zeigte außerdem, dass evolutionär junge Mikroproteine ähnliche Interaktionsfähigkeiten wie konservierte Kandidaten haben. Schließlich wurden kurze Sequenzmotive identifiziert, die Mikroprotein-Protein-Wechselwirkungen vermitteln, wodurch junge Mikroproteine mit zellulären Prozessen – wie z.B. Endozytose und Spleißen – in Verbindung gebracht werden konnten. Zusammenfassend wurde die Produktion vieler kleiner Proteine im menschlichen Herzen bestätigt, von denen die meisten lediglich in Primaten konserviert sind. Zusätzlich verknüpften umfangreiche Lokalisierungs- und Interaktionsdaten mehrere Mikroproteine mit Prozessen wie Spleißen, Endozytose und mitochondrialer Translation. Weitere Untersuchungen dieses zuvor verborgenen Teils des Herzproteoms werden zu einem besseren Verständnis von evolutionär jungen Proteinen und kardiologischen Prozessen beitragen. / Recently, the active translation of hundreds of previously unknown microproteins was detected using ribosome profiling on tissues of human hearts. They had remained undetected due to their small size (< 100 amino acids), and their physiological roles are still largely unknown. This dissertation aims to investigate these novel microproteins and validate their translation by independent methods. Particularly, elucidating their conservation signature, subcellular localization, and protein interactome shall aid in deciphering their potential biological role. Conservation analysis revealed that sequence conservation of almost 90% of microproteins was restricted to primates. I next confirmed microprotein production in vitro and in vivo by in vitro translation assays and mass spectrometry-based approaches, defined the subcellular localization of 92 microproteins, and identified significant interaction partners for 60 candidates. Dozens of these microproteins localized to the mitochondrion. These included a novel cardiac-enriched microprotein that may present a novel modulator of mitochondrial protein translation based on its interaction profile and subcellular localization. The interactome screen further revealed that evolutionarily young microproteins have similar interaction capacities to conserved candidates. Finally, it allowed identifying short linear motifs that may mediate microprotein-protein interactions and implicated several young microproteins in distinct cellular processes such as endocytosis and splicing. I conclude that many novel small proteins are produced in the human heart, most of which exhibit poor sequence conservation. I provide a substantial resource of microprotein localization and interaction data that links several to cellular processes such as splicing, endocytosis, and mitochondrial translation. Further investigation into this hidden part of the cardiac proteome will contribute to our understanding of recently evolved proteins and heart biology.