The TRIB1 locus (8q24.13) is a novel locus associated with plasma TGs and CAD risk. Trib1 is a regulator of MAPK activity, and has been shown to regulate hepatic lipogenesis and VLDL production in mice. However, the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits is unknown; TRIB1 has not been identified as an eQTL. This cluster of SNPs falls within an intergenic region 25kb to 50kb downstream of the TRIB1 coding region. By phylogenetic footprinting analysis and DNA genotyping, we identified an evolutionarily conserved region (CNS1) within the risk locus that harbours two common SNPs in tight LD with GWAS risk SNPs and significantly associated with CAD. We investigated the regulatory function of CNS1 by luciferase reporter assays in HepG2 cells and demonstrate that this region has promoter activity. In addition, the rs2001844 risk allele significantly reduces luciferase activity, suggesting that altered expression of the EST-based gene may be associated with plasma TGs. We identified an EST within the risk locus directly downstream of CNS1. We performed 5'/3' RACE using HepG2 RNA, identified multiple variants of this EST-based gene, and confirmed its transcription start site within CNS1. We hypothesize that this EST is a long noncoding RNA due to low abundance, poor conservation, and absence of significant ORF. Over-expression of a short variant implicates its function in the regulation of target gene transcription, although the mechanism of action remains unknown. We conclude that the risk locus at 8q24.13 harbours a novel EST-based gene that may explain the relationship between GWAS SNPs at this locus and plasma lipid traits.

The innate immune system is a multicomponent response governed by intricate mechanisms of induction, regulation and resolution to elicit antimicrobial defenses. In recent years, the complexity of eukaryotic transcriptomes has become the subject of intense scrutiny and curiosity. It has been established, that RNA polymerase II (RNAPII) transcribes hundreds to thousands of long noncoding RNAs (lncRNAs), often in a stimulus and cell-type specific manner. However, the functional significance of these transcripts has been particularly controversial. While the number of identified lncRNAs is growing, our understanding of how lncRNAs themselves regulate other genes is quite limited. In chapter 2, a novel lncRNA is identified, more specifically, a natural antisense transcript, that mediates the transcription of the pro-inflammatory cytokine IL-1α. Through loss-of-function studies, I report the necessity of this transcript in mediating IL-1α mRNA expression by affecting RNAPII binding to the IL-1α promoter after toll-like receptor signaling. For the first time, I show that IL-1α is regulated at the transcriptional level. As a second independent component of this thesis, we explore the role of the innate immune response after infection by the malaria-causing parasite, Plasmodium berghei ANKA (PbA), and how innate immune components are both beneficial and detrimental to the host depending on when and where inflammation is triggered during infection. We attempt to identify the “malarial toxin” responsible for aberrations in the immune response that is detrimental for disease outcomes and the innate signaling pathways that are involved. Many pathogens induce pathological inflammatory conditions that lead to irreparable homeostatic imbalances and become fatal to the host. Here, type I Interferon signaling is required to dampen parasite load during liver-stage infections, but leads to host mobidity if these pathways are activated in the erythrocytic phase of infection. Together, this thesis provides new insights on how components of the innate immune system are regulated, and how dysregulation of immunity can potentially lead to adverse effects during active infections.

XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.

Die Genexpression wurde bisher hauptsächlich auf Transkriptions- und Proteinebene untersucht, wobei der Einfluss der Translation, die die Proteinhäufigkeit direkt beeinflusst, weitgehend außer Acht gelassen wurde. Um diese Rolle besser zu verstehen, habe ich Ribosomen-Profiling-Daten (Ribo-seq) verwendet, um die Translationsregulation zu untersuchen und neue Translationsvorgänge in 65 linksventrikulären Proben von DCM-Patienten im Endstadium und 15 Nicht-DCM-Kontrollen zu identifizieren. Dieser Datensatz half dabei, die Transkriptions- und Translationsregulation zwischen erkrankten und nicht betroffenen menschlichen Herzen zu sezieren und enthüllte Gene und Prozesse, die rein unter Translationskontrolle stehen. Darüber hinaus habe ich neue kardiale Proteine vorhergesagt, die von langen nicht-kodierenden RNAs (lncRNAs) und zirkulären RNAs (circRNAs) translatiert werden. Computergestützte Analysen dieser evolutionär jungen Proteine legten eine Beteiligung an verschiedenen molekularen Prozessen nahe, mit einer besonderen Anreicherung für den mitochondrialen Energiestoffwechsel. Schließlich identifizierte ich RNA-bindende Proteine (RBPs), deren Expression die Menge der Ziel-mRNA oder die Frequenz der Translationseffizienz (TE) beeinflusst. Für eine Untergruppe von 21 RBPs habe ich die Regulation auf beiden quantitativen Merkmalen beobachtet, was zu einer unterschiedlichen mechanistischen Basis der Expressionskontrolle für unabhängige Gensätze führte. Obwohl die genaue Umschaltung der RBP-Funktion wahrscheinlich durch eine Kombination von mehreren Faktoren erreicht wird, haben wir für drei Kandidaten eine starke Abhängigkeit von der Zielgenlänge und der 5'-UTR-Struktur beobachtet. Diese Arbeit präsentiert einen Katalog von neu identifizierten Translationsereignissen und einen quantitativen Ansatz zur Untersuchung der Translationsregulation im gesunden und kranken menschlichen Herzen. / Gene expression has primarily been studied on transcriptional and protein levels, largely disregarding the extent of translational regulation that directly influences protein abundance. To elucidate its role, I used ribosome profiling (Ribo-seq) data, obtained through ribosome profiling, to study translational regulation and identify novel translation events in 65 left ventricular samples of end-stage DCM patients and 15 non-DCM controls. This dataset helped dissect transcriptional and translational regulation between diseased and unaffected human hearts, revealing genes and processes purely under translational control. These would have remained undetected by only looking at the transcriptional level. Furthermore, I predicted novel cardiac proteins translated from long non-coding RNAs (lncRNAs) and circRNAs. Computational analysis of these evolutionary young proteins suggested involvement in diverse molecular processes with a particular enrichment for mitochondrial processes. Finally, I identified RNA-binding proteins (RBPs) whose expression influences target mRNA abundance or translational efficiency (TE) rates. For a subset of 21 RBPs, I have observed regulation on both quantitative traits, which resulted in different mechanistic basis expression control for independent sets of genes. Though the precise switch in RBP function is likely achieved by a combination of multiple factors, for three candidates we have observed a strong dependency on target length and 5’ UTR structure. This work presents a catalogue of newly identified translation events and a quantitative approach to study translational regulation in the healthy and failing human heart.

La localisation subcellulaire de l’ARN permet un déploiement prompt et spatialement restreint autant des activités protéiques que des ARN noncodant. Le trafic d’ARN est dirigé par des éléments de séquences (sous-séquences primaires, structures secondaires), aussi appelés motifs de régulation, présents en cis à même la molécule d’ARN. Ces motifs sont reconnus par des protéines de liaisons aux ARN qui médient l’acheminement des transcrits vers des sites précis dans la cellule. Des études récentes, chez l’embryon de Drosophile, indiquent que la majorité des ARN ont une localisation subcellulaire asymétrique, suggérant l’existence d’un « code de localisation » complexe. Cependant, ceci peut représenter un exemple exceptionnel et la question demeurait, jusqu’ici, si une prévalence comparable de localisation d’ARN est observable chez des cellules standards développées en culture. De plus, des informations facilement disponibles à propos des caractéristiques de distribution topologique d’instances de motifs à travers des transcriptomes complets étaient jusqu’à présent manquantes. Afin d’avoir un aperçu de l’étendue et des propriétés impliquées dans la localisation des ARN, nous avons soumis des cellules de Drosophile (D17) et de l’humain (HepG2) à un fractionnement biochimique afin d’isoler les fractions nucléaire, cytosolique, membranaire et insoluble. Nous avons ensuite séquencé en profondeur l’ARN extrait et analysé par spectrométrie de masse les protéines extraites de ces fractions. Nous avons nommé cette méthode CeFra-Seq. Par des analyses bio-informatiques, j’ai ensuite cartographié l’enrichissement de divers biotypes d’ARN (p. ex. ARN messager, ARN long non codant, ARN circulaire) et protéines au sein des fractions subcellulaires. Ceci a révélé que la distribution d’un large éventail d’espèces d’ARN codants et non codants est asymétrique. Une analyse des gènes orthologues entre mouche et humain a aussi démontré de fortes similitudes, suggérant que le processus de localisation est évolutivement conservé. De plus, j’ai observé des attributs (p. ex. la taille des transcrits) distincts parmi les populations d’ARN messagers spécifiques à une fraction. Finalement, j’ai observé des corrélations et anti-corrélations spécifiques entre certains groupes d’ARN messagers et leurs protéines. Pour permettre l’étude de la topologie de motifs et de leurs conservations, j’ai créé oRNAment, une base de données d’instances présumée de sites de liaison de protéines chez des ARN codants et non codants. À partir de données de motifs de liaison protéique par RNAcompete et par RNA Bind-n-Seq, j’ai développé un algorithme permettant l’identification rapide d’instances potentielles de ces motifs dans un transcriptome complet. J’ai pu ainsi cataloguer les instances de 453 motifs provenant de 223 protéines liant l’ARN pour 525 718 transcrits chez cinq espèces. Les résultats obtenus ont été validés en les comparant à des données publiques de eCLIP. J’ai, par la suite, utilisé oRNAment pour analyser en détail les aspects topologiques des instances présumées de ces motifs et leurs conservations évolutives relatives. Ceci a permis de démontrer que la plupart des motifs sont distribués de façon similaire entre espèces. De plus, j’ai discerné des points communs entre les sous-groupes de protéines liant des biotypes distincts ou des régions d’ARN spécifiques. La présence de tels patrons, similaires ou non, entre espèces est susceptible de refléter l’importance de leurs fonctions. D’ailleurs, l’analyse plus détaillée du positionnement d’un motif entre régions transcriptomiques comparables chez les vertébrés suggère une conservation synténique de ceux-ci, à divers degrés, pour tous les biotypes d’ARN. La topologie régionale de certaines instances de motifs répétées apparaît aussi comme évolutivement conservée et peut être importante afin de permettre une liaison adéquate de la protéine. Finalement, les résultats compilés avec oRNAment ont permis de postuler sur un nouveau rôle potentiel pour l’ARN long non codant HELLPAR comme éponge de protéines liant l’ARN. La caractérisation systématique d’ARN localisés et de motifs de régulation en cis présentée dans cette thèse démontre comment l’intégration d’information à l’échelle transcriptomique permet d’évaluer la prévalence de l’asymétrie, les caractéristiques distinctes et la conservation évolutive de collections d’ARN. / The subcellular localization of RNA allows a rapid and spatially restricted deployment of protein and noncoding RNA activities. The trafficking of RNA is directed by sequence elements (primary subsequences, secondary structures), also called regulatory motifs, present in cis within the RNA molecule. These motifs are recognized by RNA-binding proteins that mediate the transport of transcripts to specific sites in the cell. Recent studies in the Drosophila embryo indicate that the majority of RNAs display an asymmetric subcellular localization, suggesting the existence of a complex "localization code". However, this may represent an exceptional example and the question remained, until now, whether a comparable prevalence of RNA localization is observable in standard cells grown in culture. In addition, readily available information about the topological distribution of pattern instances across full transcriptomes has been hitherto lacking. In order to have a broad overview of the extent and properties involved in RNA localization, we subjected Drosophila (D17) and human (HepG2) cells to biochemical fractionation to isolate the nuclear, cytosolic, membrane and insoluble fractions. We then performed deep sequencing on the extracted RNA and analyzed through mass spectrometry the proteins extracted from these fractions. We named this method CeFra-Seq. Through bioinformatics analyses, I then profiled the enrichment of various RNA biotypes (e.g. messenger RNA, long noncoding RNA, circular RNA) and proteins within the subcellular fractions. This revealed the high prevalence of asymmetric distribution of both coding and noncoding RNA species. An analysis of orthologous genes between fly and human has also shown strong similarities, suggesting that the localization process is evolutionarily conserved. In addition, I have observed distinct attributes (e.g. transcript size) among fraction-specific messenger RNA populations. Finally, I observed specific correlations and anti-correlations between defined groups of messenger RNAs and the proteins they encode. To study motifs topology and their conservation, I created oRNAment, a database of putative RNA-binding protein binding sites instances in coding and noncoding RNAs. Using data from protein binding motifs assessed by RNAcompete and by RNA Bind-n-Seq experiments, I have developed an algorithm allowing their rapid identification in a complete transcriptome. I was able to catalog the instances of 453 motifs from 223 RNA-binding proteins for 525,718 transcripts in five species. The results obtained were validated by comparing them with public data from eCLIP. I then used oRNAment to further analyze the topological aspects of these motifs’ instances and their relative evolutionary conservation. This showed that most motifs are distributed in a similar fashion between species. In addition, I have detected commonalities between the subgroups of proteins linking preferentially distinct biotypes or specific RNA regions. The presence or absence of such pattern between species is likely a reflection of the importance of their functions. Moreover, a more precise analysis of the position of a motif among comparable transcriptomic regions in vertebrates suggests a syntenic conservation, to varying degrees, in all RNA biotypes. The regional topology of certain motifs as repeated instances also appears to be evolutionarily conserved and may be important in order to allow adequate binding of the protein. Finally, the results compiled with oRNAment allowed to postulate on a potential new role for the long noncoding RNA HELLPAR as an RNA-binding protein sponge. The systematic characterization of RNA localization and cis regulatory motifs presented in this thesis demonstrates how the integration of information at a transcriptomic scale enables the assessment of the prevalence of asymmetry, the distinct characteristics and the evolutionary conservation of RNA clusters.

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