Das Herz ist das zentrale Kreislauforgan in unserem Körper und jede Abweichung seiner Funktion wirkt sich negativ auf die Homöostase des gesamten Körpers aus. Die Herzfunktion beruht auf der Synergie der Zellen, die das Organ bilden. Die detaillierte zelluläre Zusammensetzung sowie die Funktionalität der einzelnen Zellen müssen noch ermittelt werden, und diese Arbeit ist eine wichtige Ergänzung dieser Bemühungen. Dank der jüngsten Entwicklungen in den Einzelzelltechnologien sind wir nun in der Lage, Transkriptome einzelner Zellen aus komplexem Gewebe in beispiellosem Umfang zu charakterisieren. Im ersten Schritt eines solchen Experiments müssen die Zellen und Zellkerne aus dem Gewebe befreit und vereinzelt werden. Herzgewebe wirft in dieser Hinsicht einzigartige Herausforderungen auf, darunter die Knappheit des gesunden menschlichen Herzgewebes für die Forschung, das Vorhandensein von Kardiomyozyten, die aufgrund ihrer Größe nicht durch Microfluid-basierte Standardinstrumente passen und deren Multinukleation, sowie mögliche Voreingenommenheit verschiedener Methoden zur Gewebedissoziation. Hier präsentiere ich den umfassenden Zellatlas des gesunden erwachsenen menschlichen Herzens. Ich beginne mit der Methodenentwicklung zur Isolierung von einzelnen Zellen und Zellkernen aus Mausherzen. Um den Zellatlas des menschlichen Herzens zu erstellen, analysiere ich einen Datensatz von fast einer halben Million Einzelzellen und Zellkerne aus sechs Herzregionen von vierzehn gesunden Menschen. In diesem Atlas definieren wir 11 Hauptzelltypen und 62 Zellzustände des menschlichen Herzens. Ein tieferer Fokus wird auf das Herzgefäßsystem gelegt und die Zellen der arterio-venösen Achse sowie deren Wechselwirkungen und potenzielle Funktionalität werden definiert. Insgesamt präsentiert diese Dissertation einen komplex Datensatz aus menschlichem Herzgewebe und liefert neue Einblicke in die Biologie des gesunden Herzens mit Implikationen für kardiovaskuläre Erkrankungen. / The heart is the central circulatory organ in our bodies and any discrepancies of its function relative to healthy homeostasis negatively impact the whole body. Cardiac function relies on the synergy of all the cells that constitute the organ. The detailed cellular composition as well as the heterogeneity and functionality of the individual cells is yet to be established and this work is a major advance in this effort. Thanks to the recent developments in single cell genomics technologies, we are now able to profile transcriptomes from individual cells of complex tissues at unprecedented scale. In the first step of such an experiment, the single cells and nuclei need to be liberated from the tissue. Heart tissue presents a unique set of challenges in this regard, including the scarcity of healthy human cardiac tissue for research, large cardiomyocytes that do not fit into the standard droplet-based instruments, multinucleation of cardiomyocytes that might skew the proportions of the recovered nuclei as well as potential bias of tissue dissociation methods. Here I present a cell atlas of the free walls, apex and septum of the healthy adult human heart. I start with methods development for the isolation of single cells and single nuclei from mouse heart. Next, I move to the building of the atlas of the human cells and nuclei, where I describe the dataset of close to half a million single cells and nuclei sampled from 14 organ donors, defining 11 major cell types and 62 cell states of the heart. A deeper focus on the cardiac vasculature defined the cells of the arterio-venous axis as well as their interactions and potential functionality. Overall, this thesis presents a joined dataset of single cells and single nuclei from human cardiac tissues and provides new insights into cardiac biology in heath with implications for cardiovascular disease.

Bakterien haben eine Vielzahl an Strategien entwickelt, um sich an ständig wechselnde Umweltbedingungen anzupassen, darunter auch post-transkriptionelle regulatorische Mechanismen. Die Genexpression kann hierbei durch gezielten Abbau oder Stabilisierung von RNA durch Ribonukleasen (RNasen) reguliert werden. RNasen weisen je nach Spezies allerdings unterschiedliche Effekte auf Genexpression und bakterielle Physiologie, sowie verschiedene Strategien der Substraterkennung auf. Dies zeigt, dass unser Verständnis des RNA-Abbaus bei weitem nicht vollständig ist. Ziel dieser Arbeit ist es, die Eigenschaften und Funktionen der endoRNase Y des humanpathogenen Bakteriums Streptococcus pyogenes zu studieren. Um Einblick in Funktion und Spezifität dieser RNase zu gewinnen, wurden deren genomweite Schnittpositionen (“targetome”) mit Hilfe von RNA-Sequenzierung identifiziert. Zur weiteren Analyse des RNase Y-abhängigen RNA-Abbaus wurde dieses Ergebnis mit dem “targetome” der drei 3′-5′-Exoribonukleasen (ExoRNasen) PNPase, YhaM und RNase R verglichen. Schließlich wurden die Anforderungen für die Prozessierung durch RNase Y und deren Rolle in der Regulation von Virulenzgenen in vivo anhand der speB mRNA, die einen wichtigen Virulenzfaktor codiert, untersucht. Wir konnten in dieser Arbeit zeigen, dass RNase Y Substrate bevorzugt nach einem Guanosin schneidet und dieses Nukleosid essenziell für die Prozessierung der speB mRNA in vivo ist. Obwohl RNase Y die speB mRNA schneidet, unterstützen die Daten ein Modell nach dem RNase Y die Expression von speB auf transkriptioneller Ebene reguliert. Mit Hilfe des “targetome”-Vergleichs konnten wir ferner zeigen, dass RNase Y den RNA-Abbau in S. pyogenes initiiert und die dabei generierten 3′-Enden der RNA hauptsächlich von den 3′-5′-exoRNasen PNPase und/oder YhaM prozessiert werden. Zusammenfassend erweitern diese Erkenntnisse unser Verständnis der Funktionalität von RNase Y und des RNA-Abbaus in Gram-positiven Bakterien. / Bacteria have developed a plethora of strategies to cope with constantly changing environmental conditions, including post-transcriptional regulatory mechanisms. With this regard, regulation of gene expression can be achieved by either the rapid removal or stabilization of RNA molecules by ribonucleases (RNases). RNases exhibit species-specific effects on gene expression, bacterial physiology and different strategies of target recognition, indicating that our understanding of the RNA degradation machinery is not yet complete. The aim of this thesis was to investigate the features and functions of endoRNase Y from the strict human pathogen Streptococcus pyogenes. To gain insight into the role and specificity of this RNase, we identified RNase Y cleavage positions (i.e. targetome) genome-wide by RNA sequencing. Next, to investigate the RNA degradation pathway depending on RNase Y, we compared the RNase Y targetome with the ones of the three 3′-to-5′ exoribonuclease (exoRNases), namely PNPase, YhaM and RNase R. Finally, to dissect the requirements for RNase Y processing and to decipher the role of RNase Y in virulence gene regulation, we studied the impact of RNase Y on speB mRNA, encoding a major virulence factor. This study reveals that RNase Y preferentially cleaves RNAs downstream of a guanosine and for the first time we were able to show that the presence of a guanosine residue is essential for the processing of speB mRNA, in vivo. Although RNase Y cleaves the speB mRNA, our data underpin a model in which RNase Y-mediated regulation of speB expression occurs at the transcriptional level. Using the targetome comparative approach, we demonstrated that RNase Y initiates RNA decay in S. pyogenes and that the RNase Y-generated RNA 3′ ends are usually further trimmed by PNPase and/or YhaM. Overall, these findings increase our understanding of RNase Y functionality and RNA degradation in Gram-positive bacteria.

Der eukaryotische Zellzyklus ist ein streng regulierter Prozess, für dessen zeitlichen Ablauf unter anderem oszillierende Genexpression notwendig ist. Die Regulation und die zeitliche Koordination des Zellzyklus sind nach wie vor fundamentale Fragen der Zellbiologie. Spezifische Ereignisse, wie DNA Replikation und Zellkernteilung, können vier Zellzyklusphasen zugeordnet werden, welche durch Cyclin-abhängige Kinasen, Cycline und deren Inhibitoren reguliert werden. Während in Saccharomyces cerevisiae Cyclin-abhängige Kinasen (Cdc28, Pho85) über den gesamten Zellzyklus zu Verfügung stehen, werden Cycline und ihre Inhibitoren nur in spezifischen Phasen exprimiert. In S. cerevisiae sind drei wichtige G1-Cycline (Cln1-Cln3) in die oszillierende Genexpression involviert. In dieser Arbeit wurde die zeitaufgelöste, transkriptomweite Genexpression im Wildtyp und in Cyclindeletionsmutanten gemessen. Um die Rolle der G1-Cycline für die Feinabstimmung des Zellzykluses zu verstehen, wurden Gene nach charakteristischen Expressionsprofilen geclustert, Expressionsmaxima detektiert, ein Transkriptionsfaktornetzwerk integriert und Zellzyklusphasendauern bestimmt. Um Unterschiede zwischen der Rolle der Cycline zu verstehen, wurden die Zellen zusätzlich Osmostress ausgesetzt. Des Weiteren wurde mit Hilfe von RNA-Fluorescence In Situ Hybridization (FISH) die Expression zweier Cycline (PCL1 und PCL9), die an Pho85 binden, auf Einzelzellniveau gemessen. Um die Expression in spezifischen Zellzyklusphasen zu quantifizieren, wurden einzelne Zellen mithilfe von Zellzyklusmarkern spezifischen Zellzyklusphasen zugeordnet. Nachdem die Expression unter normalen Wachstumsbedingungen gemessen wurde, wurde zusätzlich Osmostress angewandt. Durch die Kombination einer Einzelzellquantifizierung und einer transkriptomweiten Methode konnten spezifische Aufgaben der Cycline, Cln1, Cln2 und Cln3, erforscht werden. Zusätzlich konnten backup Mechanismen für die Zellzyklusregulation entschlüsselt werden. / The eukaryotic cell cycle is a highly ordered process. For its timing and progression, oscillating gene expression is crucial. The stability of cell cycle regulation and the exact timing is still a fundamental question in cell biology. Specific events, like DNA replication and nuclear division can be assigned to four distinct phases. These events are regulated by cyclin-dependent kinases, cyclins and their inhibitors. In Saccharomyces cerevisiae cyclin-dependent kinases (Cdc28, Pho85) are present throughout the cell cycle, while cyclins and their inhibitors are only expressed and active during specific phases. The G1 cyclins Cln1-3 are essential players to induce oscillating gene expression and are thereby involved in the fine-tuning of the cell cycle. To understand the role of the G1 cyclins for exact cell cycle timing and oscillating gene expression, time-resolved, transcriptome-wide gene expression in wild type and cyclin deletion mutants were measured. Characteristic expression profiles were clustered, precise peak times for each gene were estimated, a transcription factor network was integrated and cell cycle phase durations were defined. To further understand the role and differences of each cyclin osmostress was applied. Furthermore the expression of two cyclins (PCL1 and PCL9) corresponding to the cyclin-dependent kinase Pho85 was measured in single cells. Using RNA-Fluorescence In Situ Hybridization (FISH) and cell cycle progression markers, high and low expression phases and absolute numbers of mRNAs were obtained. Gene expression was quantified under normal and osmostressed growth conditions to understand the necessity of the cyclins for osmostress adaptation in different cell cycle phases. By the combination of a single cell and a transcriptome-wide approach distinct roles of G1 cyclins Cln1, Cln2 and Cln3 were deciphered and an insight in the backup mechanisms during cell cycle progression for normal and osmostressed growth conditions were proposed.

Artenübergreifende Interaktionen helfen uns, Krankheitsmechanismen zu verstehen und Targets für Therapien zu finden. Die Koexpression von Genen, gemessen an der mRNA-Häufigkeit, kann Interaktionen zwischen Wirt und Pathogen aufzeigen. Die RNA-Sequenzierung von Wirt und Pathogen wird als "duale RNA-Sequenzierung" bezeichnet. Malaria ist eine der am besten untersuchten parasitären Krankheiten, so dass eine Fülle von RNA-seq-Datensätzen öffentlich zugänglich ist. Die Autoren führen entweder duale RNA-seq durch, um den Wirt und den Parasiten gleichzeitig zu untersuchen, oder sie erhalten kontaminierende Sequenzierungs-Reads aus dem Nicht-Zielorganismus. Ich habe eine Meta-Analyse durchgeführt, bei diese beiden Arten von RNA-seq-Studien verwendet wurden, um über korrelierte Genexpression auf Wirt-Parasit-Interaktionen zu schließen. Ich habe Studien mit Homo sapiens, Mus musculus und Macaca mulatta als Wirte und ihre Plasmodium-Parasiten einbezogen. Ich benutzte orthologe Einzelkopien von Genen, um ein Repertoire von Interaktionen bei Malaria und in diesen Modellsystemen zu erstellen. Ich verknüpfte die Daten von 63 Plasmodium-Phasen-spezifischen Studien und reduzierte die Zahl der Interaktionen von potenziell 56 Millionen auf eine kleinere, relevantere Menge. Die Zentralität in den Netzwerken der Blutphasen konnte die Essentialität der Plasmodium-Gene erklären. Das aus den verketteten Daten sagte die Genessenzialität besser vor als die einzelnen Studien - ein Vorteil der Meta-Analyse. Neutrophile und Monozyten Immunmarkergene waren überrepräsentiert, was auf eine Fülle von phagozytären und respiratorischen Reaktionen hindeutet. Die Analyse der Leberphase ergab Wirts- und Parasitenprozesse in frühen und späten Entwicklungsphasen. Ich fand bekannte Wirt-Parasit-Interaktionen, die für beide Phasen gleich sind, sowie bisher unbekannte Interaktionen. Dieses Prinzip lässt sich auch auf andere Krankheiten anwenden, um Mechanismen und therapeutische Ziele zu verstehen. / Cross-species interactions help us understand disease mechanisms and find targets for therapy. Gene co-expression, measured by mRNA abundance, can identify host-pathogen interactions. The RNA-sequencing of host and pathogen is termed “dual RNA-sequencing”. Malaria is one of the most studied eukayotic parasitic diseases, making an abundance of RNA-seq data sets publicly available. Authors either perform dual RNA-seq to study the host and parasite simultaneously or acquire contaminant sequencing reads from the non-target organism. I performed a meta-analysis using these two kinds of RNA-seq studies to infer host-parasite interactions using correlated gene expression. I included studies of Homo sapiens, Mus musculus and Macaca mulatta as hosts and their corresponding Plasmodium parasites. I used single-copy orthologous genes to generate a repertoire of interactions in human malaria and in these model systems. I found 63 malaria RNA-seq studies. I concatenated sequencing runs from Plasmodium stage-specific studies and reduced the number of interactions from a potential 56 million to a smaller, more relevant set. Centrality in the blood stage networks was able to explain Plasmodium gene essentiality. The network from the concatenated data predicted gene essentiality better than the individual studies, indicating a benefit of the meta-analysis. Immune marker genes for neutrophils and monocytes were over-represented, suggesting an abundance of phagocytic and respiratory burst-related responses. The liver stage analysis revealed linked host and parasite processes at early stages until late developmental stages. I found linked host and parasite processes that are common to the two stages, e.g. parasite cell gliding and invasion and host response to hypoxia and immune response. I showed that existing data can be explored for new information. This principle can be applied to other diseases to understand mechanisms and therapeutic targets.

Macular neovascularization type 3, formerly known as retinal angiomatous proliferation (RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However, the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly, MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG significantly altered their transcriptional profile during RAP formation, activating immune-associated processes and exhibiting enhanced proliferation, however, without showing substantial upregulation of angiomodulatory factors.

Immunosenescence is considered a possible factor in the development of age-related macular degeneration and choroidal neovascularization (CNV). However, age-related changes of myeloid cells (MCs), such as microglia and macrophages, in the healthy retina or during CNV formation are illdefined. In this study, Cx3cr1-positive MCs were isolated by fluorescence-activated cell sorting from six-week (young) and two-year-old (old) Cx3cr1GFP/+ mice, both during physiological aging and laser-induced CNV development. High-throughput RNA-sequencing was performed to define the age-dependent transcriptional differences in MCs during physiological aging and CNV development, complemented by immunohistochemical characterization and the quantification of MCs, as well as CNV size measurements. These analyses revealed that myeloid cells change their transcriptional profile during both aging and CNV development. In the steady state, senescent MCs demonstrated an upregulation of factors contributing to cell proliferation and chemotaxis, such as Cxcl13 and Cxcl14, as well as the downregulation of microglial signature genes. During CNV formation, aged myeloid cells revealed a significant upregulation of angiogenic factors such as Arg1 and Lrg1 concomitant with significantly enlarged CNV and an increased accumulation of MCs in aged mice in comparison to young mice. Future studies need to clarify whether this observation is an epiphenomenon or a causal relationship to determine the role of immunosenescence in CNV formation.

Single-cell RNA-sequencing makes possible to study the gene expression at the level of individual cells. However, one of the main challenges of the single-cell RNA-sequencing analysis today, is the identification and annotation of cell types. The current method consists in manually checking the expression of genes using top differentially expressed genes and comparing them with related cell-type markers available in scientific publications. It is therefore time-consuming and labour intensive. Nevertheless, in the last two years,numerous automatic cell-type identification and annotation tools which use different strategies have been created. But, the lack of specific comparisons of those tools in the literature and especially for immuno-oncologic and oncologic purposes makes difficult for laboratories and companies to know objectively what are the best tools for annotating cell types. In this project, a review of the current tools and an evaluation of R tools were carried out.The annotation performance, the computation time and the ease of use were assessed. After this preliminary results, the best selected R tools seem to be ClustifyR (fast and rather precise) and SingleR (precise) for the correlation-based tools, and SingleCellNet (precise and rather fast) and scPred (precise but a lot of cell types remains unassigned) for the supervised classificationtools. Finally, for the marker-based tools, MAESTRO and SCINA are rather robust if they are provided with high quality markers.

Transkription är stokastisk, där utbrottsmässiga episoder av RNA-transkription genererar RNA-molekyler. Trots att detta är en kärndel av eukaryotiskt liv, är lite känt om hur DNA-bindande transkriptionsfaktorer och transkriptionella kofaktorer formar gen-specifik transkriptionell utbrottskinetik. Syftet med detta examensarbete var att tyda rollerna hos kofaktorerna Med14 och P300/CBP inom transkriptionell utbrottskinetik. För detta ändamål användes Auxin inducible degron systemet för snabb nedbrytning av Med14 eller P300/CBP-proteiner i HCT116-celler, följt av Smart-seq3xpress single cell-RNA-sekvensering. Ett särskild fokus i denna avhandling var även att utvärdera förmågan att härleda direkta  genuttrycksförändringar genom analys av introniska reads – detta då introner ko-transkriptionellt splitsas och dess nyttjande skulle fånga effekter av mycket närliggande transkription. Resultaten visar en tidsberoende minskning av introniskt innehåll och en nedreglering av genuttryck för majoriteten av generna i de behandlade cellinjerna, medan opåverkade kontroller inte visar sådana trender. Utbrottskinetikresultaten indikerar att det inte finns någon korrelation mellan P300/CBP-pertuberade cellers geners ursprungliga utbrottsstorlek och några trender i genuttryckets relativa förändring, medan detsamma kan sägas för Med14-pertuberade cellers geners utbrottsfrekvens. Svaga trender från P300/CBP-påverkade cellers utbrottskinetik och uttrycksändring kan antyda att deras utbrottsfrekvens och inte utbrottsstorlek har påverkats. Resultaten antyder att perturbationen var framgångsrik och att P300/CBP inte påverkar utbrottsstorlek samt att Med14 kan reglera utbrottsfrekvensen för alla påverkade gener i lika hög grad. Vidare forskning behövs inom utbrottskinetikdata för att utöka vår förståelse av denna studies implikationer gällande Med14:s och P300/CBP:s reglerande roller på transkriptionella utbrott. / Transcription is stochastic with episodes of RNA transcription generating bursts of RNA molecules. Despite being a core part of eukaryotic life, little is known about how DNA-binding transcription factors and transcriptional co-factors shape gene-specific transcriptional bursting kinetics. The aim of this thesis was to decipher the roles of the co-factors Med14 and P300/CBP in transcriptional burst kinetics. To this end, the Auxin inducible degron system was used for rapid Med14 or P300/CBP protein degradation in HCT116 cells, followed by Smart-seq3xpress single-cell RNA-sequencing. A particular focus of this thesis was to evaluate the abilities to infer direct gene expression changes by analysis of intronic reads – since introns are co-transcriptionally spliced and would capture very recent transcription. Results show a time dependent decrease of intronic contents and a downregulation in gene expression for a majority of genes in the perturbed cell lines, while unperturbed controls show no such trends. Bursting kinetics results indicate that there is no correlation between P300/CBP perturbed cells’ gene’s original bursting size and any trends in gene expression fold change while the same can be said for Med14 perturbed cell’s gene’s burst frequency. Weak trends from P300/CBP perturbed cells’ bursting kinetics and expression fold change could imply that their bursting frequency and not bursting size has been affected. The results imply that the perturbation was successful and that P300/CBP does not affect bursting size as well as that Med14 could regulate bursting frequency for all affected genes to an equal degree. Further research is needed into the bursting kinetics data to expand our understanding of this study’s implications regarding regulatory roles of Med14 and P300/CBP on transcriptional bursting.

Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.

La dystrophie myotonique de type 1 (DM1) est une maladie dégénérative impliquant des symptômes d’atrophie musculaire et de myotonie. Au niveau moléculaire, elle est caractérisée par une expansion aberrante de CUG dans la région 3’UTR de l’ARNm de DMPK (Dystrophia Myotonica protein kinase). Ces répétitions CUG forment des agrégats toxiques (appelés foci) majoritairement nucléaires dans les cellules de patients DM1 et causent la séquestration anormale de ribonucléoprotéines (RBP), tel que le facteur «Muscleblind-like 1» (MBNL1), qui lieraient normalement les motifs CUG d’autres ARN. Les fonctions normales de ces RBPs seraient alors perturbées. En plus de leur rôle dans l’épissage alternatif, MBNL a récemment été caractérisé pour son implication dans la localisation intracellulaire de ses ARN cibles. Ceci suggèrerait que la pathogénèse de la DM1 pourrait résulter de l’effet perturbateur des répétitions CUG sur la localisation d’ARN précis et de protéines RBPs. À cet effet, un criblage basé sur de la microscopie fluorescente de 322 RBPs dans des myoblastes de patients DM1 a permis d’identifier des nouveaux facteurs qui colocaliseraient avec les expansions pathogéniques CUG. De plus, ces myoblastes DM1 ont été fractionnés et un séquençage-ARN a par la suite permis l’identification de transcrits délocalisés. Les deux banques de données ainsi générées, tant par le criblage que par le fractionnement/séquençage-ARN, pourraient ouvrir des nouvelles avenues de recherches dans la compréhension des anomalies moléculaires associées à la DM1, et potentiellement d’autres maladies à expansions microsatellites. / Myotonic dystrophy of type 1 (DM1) is a degenerative disorder implicating symptoms of muscular atrophy and myotony. In a molecular level, it is caused by the aberrant expansion of CUG repeats in the 3’-UTR region of the DMPK mRNA (Dystrophia Myotonica protein kinase). Excessive CUG repeats then form toxic aggregates (foci) enriched within the nucleus of DM1 patient cells. These RNA foci cause the abnormal sequestration of RNA Binding Proteins (RBP), in particular members of the Muscleblind-like protein 1 (MBNL), that normally bind the CUG motif of other target RNAs, and will hence alter their normal functions. In addition to their role in alternative splicing, MBNL1 has recently been implicated in the intracellular localisation of its RNA targets. It remains elusive whether the pathogenesis of DM1 could result from the deregulating effect of CUG repeats on the localisation of specific RNAs and RBP proteins. In this thesis, a fluorescent imaging-based screening of 322 RBPs in DM1 patient’s myoblasts has been conducted and this had led to the identification of new factors that may colocalize with pathogenic CUG expansions. Moreover, these DM1 myoblasts have been fractionated and subsequent RNA-sequencing has permitted the identification of transcripts that are delocalised between subcellular compartments. From the two large datasets generated from the RBP imaging-based screening and fractionation/RNA-sequencing, new avenues of research can be initiated to further understand not only DM1, but perhaps also other disorders that implicate microsatellite expansions.