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The mammalian heart is a remarkable organ in that it must provide for the cardiovascular needs of the organism throughout life, without pausing. Yet, through developmental growth to adulthood and into ageing, the mammalian heart undergoes extensive physiological, morphological and biochemical remodelling. Pivotal to the age-associated alterations in cardiac phenotype is a decline in the proliferative capacity of cardiac myocytes (CMs), which is insufficient to compensate for the basal rate of CM death over time. The terminally differentiated nature of adult CMs also underlies the inability of the heart to repair itself after myocardial damage, such as infarction. As a consequence, existing CMs mount a compensatory hypertrophic response to sustain cardiac output. In parallel, the proliferation rate of resident cardiac fibroblasts, which comprise approximately 60% of total cardiac cells, increases, replacing healthy myocardium with fibrotic scar tissue. Together, CM hypertrophy and fibroblast hyperplasia progressively reduces cardiac function and the ability of the heart to adapt to environmental stressors or damage. Under continued stress or through natural ageing, the heart progresses to a failing state in which cardiac output can no longer meet the demands of the body. The societal impact of ageing-associated decline in cardiac function is great, with heart failure affecting around 8% of over 65s and consuming approximately 2% of the NHS budget. These statistics are set to rise with an ageing population. The substantial phenotypic alterations characteristic of ageing and disease-associated cardiac remodelling requires a wholesale reprogramming of the CM transcriptome. In many biological systems, although yet to be established in adult myocytes, epigenetic mechanisms underlie the transcriptome changes that arise. I hypothesised that alterations in the epigenetic landscape of CMs mediate the transcriptome remodelling that determines the phenotypic transformations that occur in cardiac ageing, hypertrophy and disease. To test this hypothesis, I examined CM-specific changes in DNA cytosine modifications, long non-coding RNA (lncRNA) expression and histone tail lysine methylation marks – epigenetic marks with central roles in transcriptional regulation in many biological systems. I examined how these changes correlate with alterations in the CM transcriptome during disease and ageing. Understanding how alterations in the transcriptome and epigenome contribute to phenotypic changes using whole tissue data is confounded by the heterogeneous nature of the heart, coupled with ageing and disease-associated changes in relative cellular composition. To overcome this, I validated a method to isolate CM nuclei specifically from post-mortem heart tissue. This method also has the advantage that it could be applied to frozen tissue, allowing access to archived material. LncRNAs are functional RNA transcripts longer than 200 bases are emerging as important regulators of gene expression. Common mechanisms of gene expression regulation by lncRNAs include by antisense suppression, as guide/co-factor molecules to direct chromatin modifying components or splicing factors to locations in the genome. Transcriptome profiling in healthy and failing human CMs identified an increase in expression of the lncRNA MALAT-1, which was consistently observed in rodent models of pathology and in ageing. Loss-of-function investigations revealed a potential anti-hypertrophic function for this lncRNA. Specifically, MALAT-1 knock down in vitro in CMs incited spontaneous hypertrophy with features reflecting pathological remodelling in the heart and hypertrophy induced by pro-hypertrophic mediators in vitro. ix In addition, novel uncharacterised transcripts were identified as differentially expressed in cardiovascular disease, including a lncRNA at 4q35.2, which was found significantly downregulated in CMs from human failing hearts. DNA methylation is a stable epigenetic modification and is generally associated with transcriptional repression. It is established by de novo DNA methyltransferases (DNMTs) in early development to determine and maintain differentiated cell states and is ‘copied’ to daughter strands in DNA synthesis by the maintenance DNMT1. Methylcytosine (MeC) can be subject to further processing to hydroxymethylcytosine (hMeC) through a TET protein-mediated oxidation reaction. This serves as a means to actively remove methylation marks as well as hMeC being a novel epigenetic modification in its own right. For the first time, I identified the cardiac myocyte genome as having a high global level of hMeC, comparable with that in neurones. I also discovered an age-associated increase in gene body hMeC that coincided with the loss of proliferative capacity and plasticity of CMs. In parallel, gene body DNA MeC levels decrease in CM ageing. Both these phenomena in gene bodies corresponded with a non-canonical upregulation in expression of genes particularly relevant to cardiac function. This relationship between gene body methylation and transcription rate is strengthened with age in CMs. Recent work in the laboratory had identified the pervasive loss of euchromatic lysine 9 dimethylation on histone 3 (H3K9me2) as a conserved feature of pathological hypertrophy and associated with re-expression of foetal genes. Concurrently, expression and activity of the enzymes responsible for depositing H3K9me2, euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/GLP and EHMT2/G9a) were reduced. Consistently, microRNA-217-induced genetic or pharmacological inactivation of Ehmts was sufficient to promote pathological hypertrophy and foetal gene re-expression, while suppression of this pathway protected from pathological hypertrophy both in vitro and in mice. In summary, I provide new insight into CM-specific epigenetic changes and suggest the epigenome as an important mediator in the loss of plasticity and cardiac health in ageing and disease. Epigenetic mediators and pathways identified as responsible for this remodelling of the CM epigenome suggests opportunities for novel therapy approaches.

Introdução: o transplante renal é o tratamento de escolha para uma significativa porção dos pacientes com perda crônica terminal da função renal. A rejeição aguda é uma importante complicação pós-transplante e entre outras disfunções agudas tem na biópsia do enxerto o padrão ouro para o seu diagnóstico. No entanto as biópsias apresentam uma série de limitações e riscos sendo necessário que se desenvolva biomarcadores não invasivos capazes de identificar disfunções do enxerto. Objetivos: analisar e quantificar a expressão dos microRNAs miR-142-3p, miR-155 e miR-210 em amostras de sangue periférico, urina e tecido renal coletadas de pacientes que submetidos à transplante renal que desenvolveram disfunção do enxerto. Métodos: estudo com delineamento transversal e executado no Laboratório de Biologia Molecular aplicado à Nefrologia (LABMAN), do Centro de Pesquisa Experimental do Hospital de Clínicas de Porto Alegre. As amostras são de pacientes submetidos a transplante renal que necessitaram de biópsia, por critério clínico. A expressão dos miRNAs miR-142-3p, miR-155 e miR-210 nos materiais biológicos (tecido renal, sangue periférico e células do sedimento urinário) foi avaliada através da técnica de reação em cadeia da polimerase quantitativo em tempo real. Resultados: foi encontrada, no sangue periférico uma diminuição estatisticamente significativa na expressão do miR-142-3p no grupo de pacientes com rejeição aguda (n=23) quando comparado ao grupo com outras causas de disfunção do enxerto (n=68) (P = 0,01). Não houve diferença entre os grupos na expressão do miR-155 e do miR-210, tampouco para o miR142-3p nos demais compartimentos. Conclusão: miR-142-3p mostra uma expressão diferenciada de rejeição aguda de enxertos renais, há um envolvimento deste marcador no grupo de biomarcadores moleculares em potencial para a disfunção do enxerto renal. / Background: kidney transplantation is the treatment of choice for a significant portion of patients with end-stage kidney disease. Acute rejection is a major post-transplant complication among other acute disorders and has on graft biopsy the gold standard for diagnosis. Biopsy, however it is an invasive and potentially harmful procedure so it is desirable to develop new noninvasive markers for diagnosing graft dysfunction. Objective: to analyze and quantify the expression of microRNAs miR-142-3p, miR-155 and miR-210 in the peripheral blood, urinary sediment and kidney tissue obtained from patients who developed graft dysfunction after kidney transplantation. Methods: crosssectional study performed at the Laboratory of Molecular Biology applied to Nephrology (Labman), Center of Experimental Research from Hospital de Clinicas de Porto Alegre. The samples are from kidney transplant patients who undertook indication biopsies as a part of investigation of graft dysfunction. Micro-RNAs expression was evaluated by quantitative real-time polymerase chain reaction. Results: it was found that in peripheral blood, a significant decrease in the expression of miR-142-3p occurred in patients with acute rejection (n = 23) as compared to the group of patients with other causes of graft dysfunction (n = 68), (P = 0.01). No other significant differences were found in gene expression of miR-155 and miR-210, neither for miR142-3p in the other urine or kidney tissue. Conclusion: miR-142-3p presents differential expression in the peripheral blood of patients with rejecting kidney grafts. The role of miRNAs as biomarkers for kidney graft dysfunction is worth be further explored.

Chez les eucaryotes, le génome est organisé en chromatine, une structure nucléoprotéique essentielle pour la régulation de l’expression génique ainsi que pour le maintien de la stabilité du génome. Les ciliés sont d’excellents organismes modèles pour étudier les mécanismes généraux qui maintiennent l’intégrité du génomes eucaryote. Chez Paramecium tetraurelia, la différentiation du génome somatique à partir du génome germinal est caractérisée par des événements massifs et reproductibles d’élimination d’ADN. D’une part, des éléments répétés (transposons,régions minisatellites), de plusieurs kilobases de long, sont imprécisément éliminés.D’autre part, 45000 séquences courtes et uniques, appelées IES, sont précisément éliminées au nucléotide près. Une classe de petits ARN, appelé scnRNAs, est impliquée dans la régulation epigénétique de l’élimination d’ADN, mais comment les scnRNA contrôlent l’élimination d’ADN reste mystérieux. Nous avons testé l’hypothèse selon laquelle une organisation particulière de la chromatine, en particulier des modifications post-traductionelles des histones associées à des formes répressives de la chromatine, est impliquée dans le processus d’élimination d’ADN. Nous avons montré que la triméthylation de l’histone H3 sur la lysine 9 et la lysine 27 (H3K9me3 et H3K27me3)apparaît transitoirement dans le noyau somatique en développement au moment où se produisent les événements d’élimination d’ADN. Nous avons identifié la protéine de type Polycomb, Ezl1, et montré qu’elle est une histone methyltransferase qui présente une dualité de substrat et catalyse à la fois la mise en place de K9me3 et K27me3 sur l’histone H3. Nous avons montré que la déposition de H3K9me3 et H3K27me3 dans le noyau en développement requiert les scnRNAs. Des analyses de séquençage haut débit ont montré que Ezl1 est requise pour l’élimination des longues séquences répétées germinales, suggérant que les scnRNA guident la déposition des marques d’histones au niveau de ces séquences. Au contraire des régions répétées du génome, les IES montrent une sensibilité différente aux scnRNAs et à Ezl1, suggérant que plusieurs voies partiellement chevauchantes sont impliquées dans leur élimination. Notre étude montre que des caractéristiques intrinsèques des séquences d’ADN, telles que leur taille, peut contribuer à la définition des séquences germinales à éliminer. De manière intéressante, nous avons aussi montré que Ezl1 est requise pour la répression transcriptionnelle des éléments transposables. Nous suggérons que les voies H3K9me3et H3K27me3 coopèrent et contribuent à préserver le génome somatique de Paramecium des parasites génomiques. / Eukaryotic genomes are organized into chromatin, a complex nucleoprotein structureessential for the regulation of gene expression and for maintaining genome stability.Ciliates provide excellent model organisms with which to gain better understandinginto the regulation of genome stability in eukaryotes. In the ciliate Parameciumtetraurelia, differentiation of the somatic genome from the germline genome ischaracterized by massive and reproducible programmed DNA elimination events. Longregions of several kilobases in length, containing repeated sequences and transposableelements are imprecisely eliminated, whereas 45,000 short, dispersed, single-copyInternal Eliminated Sequences (IESs) are precisely excised at the nucleotide level. Aspecific class of small RNAs, called scnRNAs, is involved in the epigenetic regulation ofDNA deletion. How scnRNAs may guide DNA elimination in Paramecium remains tobe discovered. Here, we investigated whether chromatin structure, in particular histonepost-translational modifications known to be associated with repressive chromatin,might control DNA elimination. We showed that trimethylated lysine 9 and 27 onhistone H3 (H3K9me3 and H3K27me3) appear in the developing somaticmacronucleus when DNA elimination occurs. We identified the Polycomb-groupprotein, Ezl1, and showed that it is a dual histone methyltransferase that catalyzes bothH3K9me3 and H3K27me3 in vitro and in vivo. Genome-wide analyses show thatscnRNA-mediated H3K9me3 and H3K27me3 deposition is necessary for theelimination of long, repeated germline DNA. Conversely, single copy IESs displaydifferential sensitivity to depletion of scnRNAs and Ezl1, unveiling the existence ofpartially overlapping pathways in programmed DNA elimination. Our study revealsthat cis-acting determinants, such as DNA length, also contribute to the definition ofgermline sequences to delete. We further showed that Ezl1 is required fortranscriptional repression of transposable elements. We suggest that H3K9me3 andH3K27me3 pathways cooperate and contribute to safeguard the Paramecium somaticgenome against intragenomic parasites.

Les ARNs long non codants (ARNlnc) jouent un rôle clé dans les processus cellulaires vitaux, notamment le remodelage de la chromatine, la réparation de l'ADN et la traduction. Cependant, la taille et la complexité des ARNlnc présentent des défis sans précédent pour les études moléculaires mécanistiques, de sorte qu'il s'est avéré difficile jusqu'à présent de relier l'information structurelle à la fonction biologique pour les ARNlnc.Le gène 3 humain exprimé maternellement (de l’anglais "maternally expressed gene 3", MEG3), est un ARNlnc abondant, soumis à empreinte parentale et épissé alternativement. Pendant l'embryogenèse, MEG3 contrôle les protéines Polycomb, régulant la différenciation cellulaire, et dans les cellules adultes, MEG3 contrôle p53, régulant la réponse cellulaire aux stress environnementaux. Dans les cellules cancéreuses, MEG3 est régulé négativement, mais la surexpression ectopique de MEG3 réduit la prolifération incontrôlée, ce qui prouve que MEG3 agit comme un suppresseur de tumeur. Les données suggèrent que les fonctions de MEG3 pourraient être régulées par la structure de MEG3. Par exemple, on pense que MEG3 se lie directement aux protéines p53 et Polycomb. De plus, les différents variants d'épissage de MEG3, qui comprennent différents exons et possèdent ainsi des structures potentiellement différentes, présentent des fonctions différentes. Enfin, la mutagenèse par délétion, basée sur une structure de MEG3 prédit in silico, a permis d’identifier un motif MEG3 supposé structuré impliqué dans l'activation de p53. Cependant, au début de mes travaux, la structure expérimentale de MEG3 était inconnue.Pour comprendre la structure et la fonction de MEG3, j'ai utilisé des sondes chimiques in vitro et in vivo pour déterminer la structure secondaire de deux variants humains de MEG3 qui diffèrent par leurs niveaux d'activation de p53. À l'aide d'essais fonctionnels dans les cellules et de mutagenèse, j'ai systématiquement analysé la structure de MEG3 et identifié le noyau activant p53 dans deux domaines (D2 et D3) qui sont conservés structuralement dans les variants humains et conservés dans l’évolution chez les mammifères. Dans D2-D3, les régions structurales les plus importantes sont les hélices H11 et H27, car dans ces régions, j’ai pu supprimer l'activation de p53 grâce à des mutations ponctuelles, un degré de précision jamais atteint pour les autres ARNlnc jusqu’ici. J'ai découvert de manière surprenante que H11 et H27 sont reliés par des boucles connectées l’une à l’autre (de l’anglais "kissing loops") et j'ai confirmé l'importance fonctionnelle de ces interactions de structure tertiaire à longue distance par mutagenèse compensatoire. Allant au-delà de l’état de l’art, j'ai donc essayé de visualiser la structure 3D d’une isoforme de MEG3 longue de 1595 nucléotides, par diffusion de rayons X à petit angle (SAXS), microscopie électronique (EM) et microscopie à force atomique (AFM). Alors que le SAXS et l’EM sont limités par des défis techniques actuellement insurmontables, l’imagerie par AFM m’a permis d’obtenir la première structure 3D à basse résolution de MEG3 et de révéler son échafaudage tertiaire compact et globulaire. Plus remarquable encore, les mêmes mutations qui perturbent la connexion entre les «boucles» H11-H27 et qui inhibent la fonction de MEG3, perturbent aussi la structure 3D de cet ARNlnc, fournissant ainsi le premier lien direct entre la structure 3D et la fonction biologique pour un ARNlnc.Sur la base de mes découvertes, je peux donc proposer un mécanisme de l’activation de p53 basé sur la structure de MEG3, avec des implications importantes pour la compréhension de la cancérogenèse. Plus généralement, mes travaux prouvent que les relations structure-fonction des ARNlnc peuvent être disséquées avec une grande précision et ouvrent la voie à des études analogues visant à obtenir des informations mécanistes pour de nombreux autres ARNlnc d’importance médicale. / Long non-coding RNAs (lncRNAs) are key players in vital cellular processes, including chromatin remodelling, DNA repair and translation. However, the size and complexity of lncRNAs present unprecedented challenges for mechanistic molecular studies, so that connecting structural information with biological function for lncRNAs has proven difficult so far.Human maternally expressed gene 3 (MEG3) is an abundant, imprinted, alternatively-spliced lncRNA. During embryogenesis MEG3 controls Polycomb proteins, regulating cell differentiation, and in adult cells MEG3 controls p53, regulating the cellular response to environmental stresses. In cancerous cells, MEG3 is downregulated, but ectopic overexpression of MEG3 reduces uncontrolled proliferation, proving that MEG3 acts as a tumour suppressor. Evidence suggests that MEG3 functions may be regulated by the MEG3 structure. For instance, MEG3 is thought to bind p53 and Polycomb proteins directly. Moreover, different MEG3 splice variants, which comprise different exons and thus possess potentially different structures, display different functions. Finally, deletion mutagenesis based on a MEG3 structure predicted in silico identified a putatively-structured MEG3 motif involved in p53 activation. However, at the beginning of my work, the experimental structure of MEG3 was unknown.To understand the MEG3 structure and function, I used chemical probing in vitro and in vivo to determine the secondary structure maps of two human MEG3 variants that differ in their p53 activation levels. Using functional assays in cells and mutagenesis, I systematically scanned the MEG3 structure and identified the p53-activating core in two domains (D2 and D3) that are structurally conserved across human variants and evolutionarily conserved across mammals. In D2-D3, the most important structural regions are helices H11 and H27, because in these regions I could tune p53 activation even by point mutations, a degree of precision never achieved for any other lncRNA to date. I surprisingly discovered that H11 and H27 are connected by “kissing loops”, and I confirmed the functional importance of these long-range tertiary structure interactions by compensatory mutagenesis. Going beyond state-of-the-art, I thus attempted to visualize the 3D structure of a 1595-nucleotide long MEG3 isoform by small angle X-ray scattering (SAXS), electron microscopy (EM), and atomic force microscopy (AFM). While SAXS and EM are limited by currently-insurmountable technical challenges, single particle imaging by AFM allowed me to obtain the first low resolution 3D structure of MEG3 and reveal its compact, globular tertiary scaffold. Most remarkably, functionally-disrupting mutations that break the H11-H27 “kissing loops” disrupt such MEG3 scaffold, providing the first direct connection between 3D structure and biological function for an lncRNA.Based on my discoveries, I can therefore propose a structure-based mechanism for p53 activation by human MEG3, with important implications in understanding carcinogenesis. More broadly, my work serves as proof-of-concept that lncRNA structure-function relationships can be dissected with high precision and opens the field to analogous studies aimed to gain mechanistic insights into many other medically-relevant lncRNAs.

Les éléments transposables (ET) ont un impact majeur sur le fonctionnement etla dynamique des génomes, à l’échelle de l’individu et de l’espèce. Le cilié Parameciumest un modèle original pour l’étude des ET. Chaque individu unicellulaire a un génomegerminal qui subit, lors des processus sexuels, des réarrangements massifs, comprenantl’élimination des ET et de leurs vestiges à copie unique, pour former un génome somatiqueoptimisé pour l’expression des gènes. La programmation épigénétique de cesréarrangements implique des petits ARN dans un processus complexe de soustractiongénomique.Au cours de ma thèse, j’ai effectué des analyses bioinformatiques et biostatistiques dedonnées hétérogènes à l’échelle du génome pour : (i) Identifier et analyser des propriétésintrinsèques, de dizaines de milliers de vestiges d’ET à copie unique, appelés "InternalEliminated Sequences" (IES). (ii) Comprendre le rôle de déterminants génétiques et dedifférents facteurs épigénétiques dans le ciblage et l’élimination des IES.L’ensemble de ces analyses met en lumière la co-Évolution des ET et des mécan-Ismes de défense de l’hôte. / Transposable elements (TE) have major impact on the function and dynamicsof genomes, both at the level of the individual and of the species. The ciliate Parameciumprovides an original model for studies of TE. Each individual unicell has a germlinegenome that undergoes massive rearrangements at each sexual generation including thephysical elimination of TE and their single copy remnants, yielding a somatic genomestreamlined for gene expression. The epigenetic programming of the rearrangementsinvolves small RNAs in a complex process of genomic subtraction.During my thesis, I carried out bioinformatic and biostatistical analyses of heteroge-Neous, genome-Scale datasets in order to : (i) Identifiy and study the intrinsic propertiesof tens of thousands of TE remnants know as "Internal Eliminated Sequences" (IES).(ii) Explore the roles of genetic determinants and epigenetic factors in the targeting andelimination of the IESs.Taken together, the studies illustrate the co-Evolution of TE and host defense mecha-Nisms.

Los microRNAs (miRNAs) son unas pequeñas moléculas de RNA (20-25nt) que no codifican para proteína, sin embargo actúan inhibiendo la traducción a proteína de RNAs mensajeros mediante la unión a su región UTR 3'. Se ha visto que estos miRNAs juegan un papel esencial en la regulación de la diferenciación celular y en el mantenimiento del estado pluripotente en las células madre. De la misma manera se los ha visto desregulados en múltiples tumores, llegando a ser considerados en algunos casos oncogenes o genes supresores de tumores.En la presente tesis se realizado un análisis de la expresión de miRNAs maduros en dos modelos tumorales diferentes: por un lado se ha analizado el patrón de expresión de miRNAs en tejido tumoral y normal de pacientes afectos de cáncer colorectal y en muestras de colon embrionario humano de embriones de 7-12 semanas de desarrollo. Por otro lado se ha analizado el patrón de expresión de miRNAs en ganglios de pacientes afectos de linfoma de Hodgkin clásico y en ganglios normales. Para ello se han analizado un total de 156 miRNAs maduros mediante stem-loop RT-PCR y PCR a tiempo real(TaqMan). La validación de los resultados se ha realizado mediante el uso de líneas celulares (DLD1 de cáncer colorectal, y L-428, HD-MY-Z y L-1236 de linfoma de Hodgkin) y mediante hibridación in situ de miRNAs con sondas de tipo LNA(Exiqon) en la plataforma automática Bond Max (Vision Biosystems).En el análisis del cáncer colorectal y la embriogénesis del colon se ha detectado una expresión solapada de miRNAs entre la mucosa colónica embrionaria y el cáncer colorectal. Se ha determinado una firma de miRNAs que caracteriza al cáncer colorectal de estadio I y II. Se ha definido también el patrón de expresión de miRNAs durante la embriogénesis del colon. Además hemos demostrado que el cluster de miRNAs miR-17-92 y su proteína diana E2F1 comparten un patrón de expresión común entre la embriogénesis del colon humano y la carcinogénesis colorectal, actuando en la regulación del proceso de proliferación celular. En este sentido, la hibridación in situ confirmó la sobreexpresión de miR-17-5p en la base de las criptas colónicas, en el compartimiento proliferativo. Por otro lado, en el estudio del patrón de expresión de miRNAs en el linfoma de Hodgkin clásico (LHc), se ha encontrado una firma de 25 miRNAs que caracterizan a este LHc. Entre estos miR-21, miR-134 y miR-138 mediante hibridación in situ se los ha detectado en el citoplasma de las células de Hodgkin/Reed Sternberg (H/RS). La validación de los 25 miRNAs en las líneas celulares ha demostrado que 20 son expresados por las líneas celulares y únicamente 5 son de expresión exclusiva del microambiente reactivo. Por otro lado se han definido 32 miRNAs que permiten discriminar entre el subtipo histológico esclerosis nodular, del subtipo celularidad mixta, de los cuales 11 mostraban diferencias que caracterizaban a las células tumorales de un tipo u otro. Además se analizó si la presencia del virus de Epstein Barr (EBV) estaba alterando el patrón de miRNAs en los pacientes de LHc EBV+ respecto a los EBV-, y se encontraron un conjunto de 10 miRNAs diferencialmente expresados entre los dos grupos de pacientes. Finalmente se vio que miR-138 estaba sobreexpresado en estadios iniciales (I-II) y se infraexpresaba en estadios avanzados (III-IV).Los miRNAs se deben considerar como moléculas claves tanto en la embriogénesis del colon, como en la transformación neoplásica del epitelio colónico, así como en el LHc, pudiendo ser utilizadas en un futuro como herramienta terapéutica. / MicroRNAs (miRNAs) are negative regulators of gene expression that play an important role in hematopoiesis and tumorigenesis and have been shown to be essential to regulate cell differentiation and maintenance of the pluripotent cell state.To date, no evidence has linked miRNA expression in embryonic and tumor tissue. We assessed the expression of mature miRNAs in human embryonic colon tissue and in colorectal cancer and paired normal colon tissue. Overlapping miRNA expression was detected between embryonic colonic mucosa and colorectal cancer. We demonstrated that miR-17-92 cluster and expression of its target E2F1 share a common pattern between human colon embryogenesis and colonic carcinogenesis, regulating the proliferation process. In this sense, in situ hybridization assay confirmed the overexpression of miR-17-5p in the crypt progenitor compartment. miRNAs pathways must be considered to be included as major players contributing to both embryonic development and neoplastic transformation of colonic epithelium. In other hand, we analyzed miRNA expression in classic Hodgkin lymphoma (cHL) and the influence of Epstein-Barr virus (EBV) infection on the miRNA expression profiles. The expression of 157 miRNAs in lymph nodes from 49 cHL patients and 10 reactive lymph nodes (RLNs) was analyzed by real-time polymerase chain reaction (PCR). Hierarchic clustering revealed 3 well-defined groups: nodular sclerosis cHL, mixed cellularity cHL, and RLNs. A distinctive signature of 25 miRNAs differentiated cHL from RLNs, and 36 miRNAs were differentially expressed in the nodular sclerosis and mixed cellularity subtypes. These results were validated in a set of 30 cHLs and 5 RLNs, and in 3 cHL cell lines. miR-96, miR-128a, and miR-128b were selectively down-regulated in cHL with EBV. Our findings suggest that miRNAs play an important role in the biology of cHL and may be useful in developing therapies targeting miRNAs.

Bacterial pathogens rely on precise regulation of gene expression to coordinate host infection processes and resist invasion by mobile genetic elements. An interconnected network of protein and RNA regulators dynamically controls the expression of virulence factors using a variety of mechanisms. In this thesis, the role of selected regulators, belonging to the class of small RNAs (sRNAs), is investigated. Streptococcus pyogenes is a pathogen responsible for a wide range of human diseases. Genome-wide screenings have indicated that S. pyogenes encodes numerous sRNAs, yet only a limited number have been characterized. A major goal of this study was to identify and characterize novel sRNAs and antisense RNAs (asRNAs) using RNA sequencing analysis. We validated 30 novel sRNAs and asRNAs, and identified 9 sRNAs directly cleaved by the ribonucleases RNase III and/or RNase Y. Previous work from the laboratory has highlighted the role of sRNAs from the type II Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) systems in S. pyogenes. CRISPR-Cas systems provide adaptive immunity to prokaryotes against infection by mobile genetic elements. Two sRNAs, forming a complementary duplex (dual-RNA), are effectors of this system: the mature CRISPR RNAs (crRNAs) and the trans-activating crRNA (tracrRNA). The dual-RNA guides the Cas9 endonuclease to cleave both strands of the invading DNA in a sequence-specific manner. This RNA-programmable CRISPR-Cas9 system is now utilized for genome editing and engineering in a wide range of cells and organisms. To expand the potentialities of this tool, we both, searched for Cas9 orthologs and predicted numerous tracrRNA orthologs. We defined tracrRNA as a new family of sRNAs sharing the ability to base-pair to cognate crRNAs, without conservation of structure, sequence or location. We show that Cas9 and the dual tracrRNA:crRNAs are only interchangeable between closely related type II CRISPR-Cas systems. In summary, this thesis presents new insights into RNA-mediated regulatory mechanisms in S. pyogenes. We identified and described the expression of novel sRNAs, highlighting potential antisense RNAs. Focusing on the dual-RNA programmable type II CRISPR-Cas system, we provided evidence for co-evolution of the Cas9 enzyme with tracrRNA:crRNA, a basis for Cas9 multiplexing in genome editing.

RNA interference (RNAi) potently and specifically induces gene knockdown, and its potential for reverse genetics in Caenorhabditis elegans is enormous. However, even in these nematodes, RNAi can be induced more effectively via enhanced RNAi (Eri) mutant backgrounds. With advances in small RNA sequencing, evidence has suggested that the eri pathway plays an endogenous gene regulatory role, which competes with experimentally introduced RNAi triggers for limiting resources. However, the nature, cellular location, and physiological consequences of this small RNA pathways competition remain unclear. To answer these questions, I first fully characterized the genetic phenotypes of all known Eri mutants. I discovered that different components of the eri pathway have subtle differences upon mutation, which affects more than exogenous RNAi. I then attempted to screen for novel enhanced RNAi mutants, guided by hypothetical mechanisms or tissues of expression not associated with known mutants. After these attempts, I fully characterized the genetic mechanisms that account for enhanced RNAi. Surprisingly, I discovered that the nuclear Argonaute nrde-3 and the peri-nuclear P-granule component pgl-1 are necessary and sufficient for an Eri response. Finally, I examined the impact of the competition among microRNA, endogenous siRNA, and exogenous RNAi pathways. I discovered that C. elegans develops slower upon perturbations to its normal flux of small RNA pathways. Insights from these phenotypes and genetic mechanisms shed light on the importance of small RNA biology and offer a novel suite of tools for sensitizing RNAi in broader contexts, especially given the deep evolutionary conservation of most eri-associated genes.

Group I introns are structured RNAs that have been used extensively as model systems for RNA folding because they are experimentally tractable, yet complex enough to have folding challenges associated with larger RNAs. The Tetrahymena group I intron consists of a set of conserved core helices and a set of peripheral elements. Peripheral elements surround the core helices and form long range tertiary contacts between each other and to the core. Interestingly, a long-lived misfolded state is populated that has the same long range tertiary contacts as the native state but differs locally within the core. Our lab showed that the intact periphery is necessary to specify the correct core structure, as mutating tertiary contacts or removing the P5abc peripheral element dramatically destabilized the native ribozyme relative to the misfolded form. However, we also showed that the thermodynamic benefit peripheral structure provided is accompanied by kinetic liability in folding, apparently because native tertiary contacts formed by peripheral elements around the misfolded core must come apart to allow refolding of the misfolded RNA to the native state. In addition to peripheral elements, proteins also play a role in stabilizing the native structures of many group I introns. The CYT-18 protein, which occupies the same binding site as P5abc, stabilizes the functional structures of certain group I introns by using a set of insertions that are absent in other related bacterial and mitochondrial aminoacyl tRNA synthetases. Using the P5abc deletion variant of the Tetrahymena ribozyme, I sought to further define CYT-18 roles in RNA folding by probing its thermodynamic and kinetic effects on the native state formation relative to the misfolded state. I demonstrated that CYT-18, like P5abc, provided thermodynamic stability to the native state. However, unlike P5abc, CYT-18 had no apparent effect on the refolding kinetics, suggesting that a protein co-factor can stabilize the functional structure without acquiring the associated costs in RNA folding kinetics. Furthermore, I found that the mechanism of CYT-18 action appears to be distinct from P5abc. Disruption of the long-range contact P14, which is formed between P5c and L2 and is part of the network of peripheral contacts, dramatically weakened P5abc binding to the native ribozyme core by ~10⁸ fold. Interestingly, CYT-18 maintained specific and tight binding to these mutants, which suggests that CYT-18 does not rely on a circular network of contacts to specifically stabilize the native state. Instead, the specificity may arise from a more direct and intimate contact of CYT-18 with the ribozyme core. This study gives insight into an evolutionary advantage of protein co-factors in RNA folding; proteins may offer thermodynamic assistance without inhibiting folding kinetics. / text