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.