Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.

Non-protein coding eukaryotic genome sequences often referred to as junk DNA are estimated to encode several non-coding RNAs (ncRNAs) which may account for nearly 98% of all genomic output in humans. The output of such a wide spread transcription in eukaryotes consists of intronic, antisense and small RNAs. In addition to the classical ncRNAs such as rRNA, tRNA and small nucleolar RNAs, the eukaryotic genome encodes two distinct categories of ncRNAs, referred to as small ncRNAs and long mRNA–like ncRNAs (mlncRNAs). The long ncRNAs, which are transcribed by RNA Polymerase II, spliced and polyadenylated, are implicated in a number of regulatory processes such as imprinting, X-chromosome inactivation, DNA demethylation, transcription, RNA interference, chromatin structure dynamics and antisense mediated regulation. Expression of noncoding RNAs is altered during stress conditions and a large number of such transcripts have been identified of late. This study has identified a novel ncRNA whose expression is upregulated during viral infection of mouse brain. While we have named this RNA as VINC or virus inducible ncRNA, others have named it as NEAT1 (Hutchinson et al., 2007) and Men (Sunwoo et al., 2008). VINC/NEAT1/Men is associated with a distinct nuclear domain called paraspeckles Using a cell line as well as an animal model system we have investigated VINC in great detail and based on these studies we report that VINC is a nuclear ncRNA that localizes to paraspeckles and it interacts with the paraspeckle protein, P54nrb in both cell line model system as well as in animal tissues by a combination of in vitro and in vivo methods. We have also mapped the domains within VINC that are involved in P54nrb interactions. Till date, the only other RNA known to localise to paraspeckles is CTN-RNA. While CTN-RNA is a protein coding RNA, VINC does not code for a protein and thus VINC is the first ncRNA to be localized to paraspeckles. Further, the mechanism of nuclear retention of these two paraspeckle RNAs appears to be distinct. In case of CTN-RNA, it has been clearly shown that it is A-I edited and such hyperedited RNAs are retained by the p54/nrb mediated complex in nucleus (Zhang and Carmichael, 2001). However the mechanism by which VINC is retained in nucleus is not clear. There is apparently no A-I editing in VINC and hence VINC retention in the nucleus by binding to nuclear proteins such as p54/nrb might involve a different mechanism. It is well established of late that nuclear matrix retains RNAs and that there is a population of poly (A) RNA that is retained in nucleus (Huang et al.,1994 ; Carter et al.,1991). However the significance of such retention is not clear but it is believed that it might be important for some constitutive functions in nucleus (Nickerson et al., 1989). More investigations are needed to understand the exact functions of nuclear RNAs such as VINC in supporting the nuclear architecture. P54nrb is a multi functional nuclear protein that mediates most of its functions in association with PSF (Shav-Tal and Zipori, 2002). Phosphorylation status of P54nrb is a key determinant for its localisation to various nuclear regions. P54nrb is a multiphosphorylated protein during mitosis and its phosphorylation is mediated by PIN-1 at its C-terminus (Proteau et al., 2005). Tyrosine phosphorylation of P54nrb is essential for it to be retained in nuclear matrix (Otto et al., 2001). The N-terminal phosphorylation is speculated but not much has been investigated. The protein has two distinct RNA recognition motifs (RRMs) in its N-terminus that are responsible for its RNA binding activity. The significance of the p54/nrb-PSF heterodimer cannot be undermined as they have been shown to be important during HIV replication. The dimer is recruited by viral machinery and P54nrb has been shown to be exported to cytosol for binding to replicative complexes (Zolotukhin et al., 2003). During adenoviral replication in nucleus many SR proteins are recruited to viral replication foci and rearrangement of speckle components happen. It has been shown with respect to speckles that nuclear domains are highly dynamic and exchange of proteins depends upon the transcriptional status of cell (Lamond and Spector, 2003). Flaviviral replication complexes are hosted in nucleus and ~20% of this complex docks in nucleus and serves as an alternate site for viral replication. The presence of viral replicative complexes alters the nuclear organisation and hence modulation of gene expression is expected (Uchil et al., 2006). The up regulation of nuclear ncRNA such as VINC is definitively one of those events associated with viral replication and definitively one needs to study the various changes carefully to understand the role of VINC in virus life cycle and/or viral pathogenesis. VINC interaction with the multi-functional nuclear protein P54nrb raises interesting aspects related to function of P54nrb in JEV infection. Knockdown of P54nrb in human myeloid cell line results in abnormal size of paraspeckles and impairs chondrogenesis (Hata et al., 2008). PSF-P54nrb complex can divert many of HIV gag RNA complexes to paraspeckles thus trying to restrict viral replication. However the exact relationship between paraspeckles and its constituent proteins is not clear. The presence of ncRNA adds another new dimension to paraspeckles. It is unclear whether the ncRNA VINC is essential for paraspeckle structure but a recent study indicates that Men (VINC/NEATI) RNA may be essential for paraspeckle formation (Sunwoo et al., 2008). The exact function VINC in neuronal as well as non-neuronal cell nuclei remains elusive and more investigations are need to understand these aspects.

This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci. / QC 20100723

Non-coding RNAs are highly abundant regulators of gene expression in all kingdoms of life that often play important roles in vital cellular functions. In bacteria, small regulatory RNAs (sRNAs) usually act post-transcriptionally by regulating mRNAs through base pairing within ribosome binding sites (RBS), thereby inhibiting translation initiation. tisB encodes a toxin, TisB, whose synthesis is controlled by the sRNA IstR-1. Intriguingly, IstR-1 base pairs far upstream of the RBS but nevertheless inhibits translation initiation. The tisB mRNA is unusual in that ribosomes cannot access the RBS directly, but instead need an unstructured upstream region. This is precisely where IstR-1 exerts its inhibitory effect. We propose this region to serve as a ribosome loading site (standby site) which permits ribosomes to overcome the obstacle of inhibitory RBS-containing structures. Sequence-independent ribosome binding to the standby site allows for efficient relocation to the RBS structure when it is transiently open. Thus, standby sites are translation enhancer elements. I also characterized TisB-mediated toxicity. The hydrophobic protein TisB is targeted to the inner membrane and causes damage. This decreases the intracellular ATP concentration and entails decreased  replication, transcription and translation rates. It is likely that this toxin is involved in multidrug tolerance under certain conditions. We identified the sRNA MicF as a negative regulator of lrp expression. Lrp is a global transcription factor that controls genes involved in amino acid metabolism and transport of small molecules. Interestingly, Lrp also downregulates MicF. Thus, this study established that the mutual downregulation of MicF/Lrp creates a positive feedback loop which gives a switch-like behavior important for fast adaptations.

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

Le virus de la maladie de Marek (MDV) est un virus oncogène responsable des lymphomes T chez les poulets. L´infection par ce virus est divisée en une phase lytique dépendante de l´expression du gène très précoce ICP4 et une phase latente, caractérisée par l´expression de l’ARN long non codant LAT localisé en antisens. Nous avons montré que l’expression différentielle des miARN du cluster mdv1-miR-M8-M10 était directement corrélée à l’épissage alternatif de l’intron 1 du LAT et plus particulièrement à la biogenèse par le splicéosome du premier mirtron viral. La présence du mirtron mdv1-miR-M6 au milieu du cluster est associée à une cinétique d’expression des miARN. En parallèle, nous avons identifié deux promoteurs alternatifs de type Sp1, quatre signaux poly-A et trois exons associés à la régulation de la transcription du transcrit ICP4. Nous avons prédits cinq isoformes potentielles pour la protéine ICP4 et avons pu observer par immunodétection que la protéine était exprimée principalement dans le cytoplasme des cellules infectées en phase lytique ou de réactivation. / The Marek disease virus (MDV) is an oncogenic herpesvirus responsible of T-cell lymphoma in chicken. MDV infections are divided into a lytic phase, depending on the expression of immediate early gene like ICP4, and a latent phase characterized by the expression of the long non-coding RNA LAT localized in antisense. In this study, we have shown the differential expression of the cluster of miRNA mdv1-miR-M8-M10 was directly correlated with the alternative splicing of LAT’s intron 1 and more specifically with the first viral mirtron biogenesis by the spliceosome. The location of the mirtron mdv1-miR-M6 inside of the cluster is associated with a two-step biogenesis of the miARN of the cluster. On the other hand, we have identified a dual promoter that responded to Sp1, four poly-A signals and three exons that are responsible of transcriptional regulation of ICP4 transcript. We also have predicted five potential isoproteines for ICP4 and were able to observe by immunodetection that ICP4 was mainly expressed in the cytoplasm of infected cells during the lytic phase or the reactivation one.

Condensine est un complexe protéique organisateur du génome qui conduit l’assemblage des chromosomes et promeut leur transmission fidèle en anaphase. De nombreuses études ont rapporté des changements dans les niveaux des ARNs cellulaires quand Condensine est défaillante, suggérant un rôle pour Condensine dans la régulation de l’expression génique. Cependant, les mécanismes sous-jacents sont demeurés énigmatiques, et l’on ignore dans quelle mesure le rôle joué par Condensine dans l’expression génique est lié ou non à sa fonction dans l’organisation des chromosomes. Lors de ma thèse, j’ai étudié l’activité de Condensine dans la régulation de l’expression génique en utilisant la levure S. pombe comme organisme modèle. Contrairement à l’idée communément admise, mes résultats montrent que Condensine ne joue aucun rôle direct dans le maintien du transcriptome, ni en interphase, ni en mitose chez cette levure. En accord avec les études précédentes, j’observe des changements de niveau et de qualité des ARNs dans les cellules mutantes pour Condensine au sortir de la mitose ; des ARNs non codants et des ARNs aberrants, étendus en 3’, s’accumulent. En revanche, je démontre que ces changements sont la conséquence de défauts de transmission des chromosomes en anaphase. L’inactivation de Condensine cause la non-disjonction de l’ADN ribosomique et du nucléole, entrainant une déplétion de l’ARN-exosome des cellules filles, lesquelles accumulent alors des ARNs normalement dégradés par l’ARN-exosome. De façon cruciale, je montre qu’empêcher les anomalies de migration des chromosomes restaure une expression normale des gènes malgré l’inactivation de Condensine, démontrant que c’est l’instabilité chromosomique qui est source des changements d’expression génique observés quand Condensine est défaillante, et non le complexe Condensine en tant que tel. Ce travail remet en question le concept de régulation de l’expression génique par les complexes Condensine et appelle à la prudence lorsque l’on cherche à étudier les fonctions de ces complexes en dehors de la condensation de la chromatine en mitose. / Condensin is a genome organiser that shape chromosomes and promote their accurate transmission in anaphase. Several studies have related changes in RNA level when Condensin is defective, suggesting that the complex has also a role in gene expression. However, the mechanisms have remained enigmatic and we still don’t know to what extent it is related to its role in chromosome organization. During my thesis, I studied the role played by Condensin in the regulation of gene expression using S. pombe as a model system. In contrast to previous studies, my results provide compelling evidence that Condensin plays no direct role in the maintenance of the transcriptome, neither during interphase nor during mitosis in this yeast. Accordingly to previous studies, I observed changes in RNA level in cells mutated for Condensin; non coding and 3’ extended RNA accumulate. However, I showed that the changes in gene expression in post-mitotic fission yeast cells that result from Condensin inactivation are largely a consequence of chromosome missegregation during anaphase, which notably depletes the RNA-exosome from daughter cells. Crucially, preventing karyotype abnormalities in daughter cells restores a normal transcriptome despite Condensin inactivation. Thus, chromosome instability, rather than a direct role of Condensin in the transcription process, changes gene expression. This work challenges the concept of gene regulation by canonical Condensin complexes and ask for caution when studying Condensin role outside chromosome condensation in mitosis.

No final do século passado, avanços ocorridos no campo da Biologia Molecular levantaram questionamentos sobre como organismos complexos, com poucos genes a mais que organismos relativamente mais simples, regulariam seu desenvolvimento e funções celulares tão mais intrincadas. A descoberta dos RNAs não codificadores de proteínas e suas funções lançou nova luz ao entendimento da regulação da expressão gênica em organismos superiores. Apesar do conhecimento adquirido nos últimos anos, pouco ainda é sabido sobre a regulação destes RNAs. MicroRNAs, por outro lado, são uma espécie bem estudada de pequenos RNAs preditos como possíveis reguladores de mais de 60 % dos genes codificadores de proteínas no genoma humano, considerados importantes reguladores da expressão gênica em nível pós-transcricional. O presente projeto estudou a possível regulação do gene não codificador de proteínas TUG1, envolvido em proliferação celular e apoptose, por miRNAs e o papel desta regulação em processos tumorigênicos. Para isto foram utilizadas técnicas que superexpressaram e silenciaram miRNAs e técnicas de PCR quantitativo em tempo real para medir o nível de TUG1 nas amostras tratadas. Verificou-se a possibilidade de regulação do TUG1 por microRNAs em diferentes linhagens celulares sendo que, no entanto, esta regulação não parece ser importante em nível fisiológico / At the end of the last century, advances occurred in the Molecular Biology field raised questions about how complexes organisms, with few more genes than relatively simpler organisms, regulate it so intricate development and cellular functions. The discovery of long non-protein coding RNAs and it functions gave light to the understanding of gene expression regulation in superior organisms. Despite the knowledge acquired in the last years, few is yet known about the regulation of these RNAs. MicroRNAs, other way, are a well-studied tiny RNAs specie. They are predicted as possible regulators of more than 60 % of protein-coding genes in the human genome and considered important regulators of gene expression regulation at post-transcriptional level. This project studied the possible regulation of the non protein-coding gene TUG1, involved in cell proliferation and apoptosis, by microRNAs and the role of this regulation in tumorigenic processes. In order to do this we used techniques that superexpressed and silenced miRNAs and techniques of real time quantitative PCR to measure the TUG1 levels in the treated samples. We find the possibility of regulation of TUG1 by microRNAs in different cell lineages but this regulation does not seems to be important in a physiologic context.

Récemment, des analyses dans divers organismes eucaryotes ont révélé que l'ensemble du génome est transcrit et produit en plus des ARNs messagers, une grande variété d’ARNs non codants de différentes longueurs. Les ARNs non codants de plus de 200 nucleotides, classés comme longs ARNs non codants (LARNnc), représentent la classe la plus abondante de transcripts non codants. Les études des fonctions des LARNnc suggèrent que beaucoup d'entre eux seraient impliqués dans la régulation de la transcription. L'objectif de ma thèse de doctorat était d'élucider les mécanismes de la régulation transcriptionnelle médiée par des LARNnc dans différents systèmes eucaryotes. Dans mon premier projet, j'ai étudié le rôle d'un long ARN non codant antisens dans la régulation transcriptionnelle du gène PHO84, codant un transporteur de phosphate à haute affinité, chez S. cerevisiae. Des études antérieures ont montré que la suppression d’une proteine de l’exosome Rrp6 entraîne une augmentation de l'expression antisens et la répression de PHO84. Il a été suggéré que la perte de Rrp6 entraîne une stabilisation antisens au locus PHO84, entraînant le recrutement de l'histone de-acétylase Hda1 et la répression de PHO84. Cependant, le mécanisme par lequel Rrp6p régule la transcription de PHO84 n’était pas connu. En combinant des méthodes à l’échelle de cellule unique, des approches biochimiques et génétiques, nous avons montré que les niveaux d'ARN antisens sont régulés principalement lors de l'élongation par le complexe Nrd1-Nab3-Sen1, qui nécessite Rrp6 pour un recrutement efficace à l`extrémité 3`de PHO84. De plus, nous révélons l'expression anticorrelé du sens et de l'antisens, En résumé, nos données suggèrent que la transcription antisens régule le seuil d'activation du promoteur PHO84. Dans mon second projet, j'ai étudié les rôles des ARNs dérivés des amplificateurs (ARNa) dans la regulation de la transcription. En utilisant les cellules de cancer du sein MCF7 comme système modèle, nous avons cherché à déterminer comment les ARNa induits par l'oestrogène (E2) participent à la régulation de la transcription médiée par le recepteur d’oestrogène (ERα) au niveau de l'allèle unique. À l'aide de l’hybridation fluorescente à l’échelle de molécule unique (smFISH), nous avons révélé qu`après induction d'E2, les ARNa sont induits avec une cinétique similaire à celle des ARNm cibles, sont localisés exclusivement dans le noyau, principalement associés à la chromatine, et sont moins abondants que les ARNm. De manière surprenante, nous avons constaté que les ARNa sont rarement co-transcrits avec leurs loci cibles, indiquant que la transcription active des gènes ne nécessite pas la synthèse continue ou l'accumulation d'ARNa sur l'amplificateur. En outre, en utilisant des mesures de la distance à sous-diffraction, nous avons démontré que la cotranscription des ARNa et des ARNm se produit rarement dans une boucle amplificateurpromoteur. De plus, nous avons révélé que la transcription basale d'ARNa n'exige pas ERα ou l'histone méthyltransférase MLL1 qui active l'amplificateur par la mono-méthylation H3K4. Dans l'ensemble, nos résultats ont montré que les ARNa peuvent jouer un rôle lors de l'activation du promoteur, mais ne sont pas nécessaires pour maintenir la transcription de l'ARNm ou pour stabiliser les interactions amplificateur-promoteur. / Transcription is the initial step in gene expression and is subject to extensive regulation. Recently, analyses in diverse eukaryotes have revealed that in addition to protein coding genes, transcription occurs throughout the noncoding genome, producing non-coding RNAs of various lengths. Non-coding RNAs longer than 200 nucleotides, classified as long non-coding RNAs (lncRNAs), represent the most abundant class of non-coding transcripts, whose functions however are poorly understood. Recent studies suggest that many lncRNAs might have roles in transcription regulation. The goal of my PhD thesis was to elucidate the mechanisms of lncRNA mediated transcription regulation in different eukaryotic systems. For my first project, I investigated the role of an antisense long noncoding RNA in transcription regulation of the high-affinity phosphate transporter gene PHO84 in the unicellular eukaryote S. cerevisiae. Previous studies showed that deletion of the nuclear exosome component Rrp6 results in increased antisense expression and repression of PHO84. It was suggested that the loss of Rrp6 results in antisense stabilization at the PHO84 locus, leading to recruitment of the histone de-acetylase Hda1 and repression of PHO84. However, most of the mechanistic details of how Rrp6p functions in regulating PHO84 transcription were not understood. Combining single cell methods with biochemical and genetic approaches, we showed that antisense RNA levels are regulated primarily during transcriptional elongation by the Nrd1-Nab3-Sen1 complex, which requires Rrp6 for efficient recruitment to the 3’end of PHO84. Furthermore, we reveal anti-correlated expression of sense and antisense, which have distinct modes of transcription. In summary, our data suggest a model whereby antisense transcriptional read-through into the PHO84 promoter regulates the activation threshold of the gene. For my second project, I investigated the roles of enhancer derived RNAs (eRNAs). eRNAs are lncRNAs transcribed from enhancers that have been suggested to regulate transcription through different mechanisms, including enhancer-promoter looping, RNA polymerase elongation, and chromatin remodeling. However, no coherent model of eRNA function has yet emerged. Using MCF7 breast cancer cells as a model system, we sought to determine how estrogen (E2) induced eRNAs participate in estrogen receptor alpha (ERα) mediated transcription regulation at the single allele level. Using single molecule fluorescent in situ hybridization (smFISH), we revealed that upon E2 induction eRNAs are induced with similar kinetics as target mRNAs, but are localized exclusively in the nucleus, mostly chromatin associated, and are less abundant than mRNAs. Surprisingly, we found that eRNAs are rarely co-transcribed with their target loci, indicating that active gene transcription does not require the continuous synthesis or accumulation of eRNAs at the enhancer. Furthermore, using sub-diffraction-limit distance measurements, we demonstrated that co-transcription of eRNAs and mRNAs rarely occurs within a closed enhancer-promoter loop. Moreover, we revealed that basal eRNA transcription does not require ERα or the histone methyltransferase MLL1, which activates the enhancer through H3K4 mono-methylation. Altogether, our findings showed that eRNAs may play a role during promoter activation, but are not required to sustain mRNA transcription or stabilize enhancer-promoter looping interactions.

The Central Dogma of molecular biology describes a framework for how genetic information is transferred in cells, placing RNA as a messenger between DNA and translated proteins. During the last years, interest in RNA research has grown tremendously due to the increasing understanding and recognition of the importance of RNA in regulation of gene expression, biochemical catalysis, and genome integrity surveillance. Most importantly, RNA content, unlike DNA, changes constantly, fine-tuning the cellular response to match the environmental conditions. There is a clear potential for RNA biomarkers, reflecting both the natural and pathological conditions in vivo. Various methods have been developed to study RNA, of which the most common tools and techniques are described in this thesis. Since many of these gold standard methods are based on detecting RNA derivative (cDNA), there is a wide scope for efficient alternative tools directly targeting RNA. In Paper I, the spatiotemporal expression of human adenovirus-5 mRNA in epithelial and blood cells infected with the virus has been studied. For this, padlock probes and rolling circle amplification (RCA) were used to visualize, quantify and analyse both viral and host cell cDNAs in different infection scenarios, at single cell level. In Paper II, direct RNA detection fidelity has been evaluated using padlock probes. A novel type of probe (iLock) that is activated on RNA via invasive cleavage mechanism, prior to RCA was developed in this approach. Using iLocks, a substantial improvement of direct RNA sensing fidelity has been observed. In Paper III, RNA modifications were introduced in otherwise DNA iLock probes to enhance the probes’ efficiency on miRNAs. Using chimeric iLock probes, multiplexed differentiation of conserved miRNA family members were performed with next- generation sequencing-by-ligation readout. Efficient replication of chimeric probes used in Paper III implies that the Phi29 DNA polymerase readily accepts RNA-containing circles as amplification substrates. In Paper IV, real-time RCA monitoring for measurement of amplification rates and analysis of amplification patterns of various RNA-containing circles was achieved. Moreover, the RCA products were sequenced as a proof for the reverse-transcriptase activity of the Phi29 DNA polymerase. This thesis effectively contributes to a better understanding of mechanisms influencing RNA detection with, but not limited to, padlock probes. It expands the available RNA analyses toolkit with novel strategies and solutions, which can be potentially adapted for RNA-focused research, in general and molecular diagnostics, in particular. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>