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Molina, Elsa. "An investigation into the relationships between novel Y chromosome-linked long non-coding RNAs and coronary artery disease." Thesis, Federation University Australia, 2016. http://researchonline.federation.edu.au/vital/access/HandleResolver/1959.17/102986.
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Coronary artery disease (CAD) is the most common type of cardiovascular disease and is one of the leading causes of morbidity and mortality globally. However, the pathogenesis of atherosclerosis which leads to CAD and results in heart attacks, heart failure and death is not well understood. In this context, studies have demonstrated a positive correlation between increased hepatic free fatty acids (FFAs) in atherosclerosis and CAD. Although CAD has welldefined environmental risk factors, genome-wide association studies (GWAS) have demonstrated a genetic influence on CAD. Previous studies have shown that genetic variation within the human Y chromosome is associated with an increased risk of developing CAD independent of traditional cardiovascular risk factors; possibly through a modulating effect of an adaptive immunity and inflammatory response by macrophages in men. However, no Y chromosome-linked gene has been investigated in this disease so far. Long non-coding RNAs (lncRNAs) have recently gained focused attention as a new class of regulatory RNA molecules involved in cardiovascular function and associated disease, particularly long intergenic noncoding RNAs (lincRNAs), the largest class within the lncRNA group so far. To date, Y chromosome-linked lincRNAs are poorly characterised and the potential link between these non-coding RNA molecules and CAD in men has not been investigated. In this context, I hypothesised that Y chromosome-linked lncRNAs may regulate pathways involved in lipid metabolism and trigger an over accumulation of FFAs in coronary arteries contributing to atherosclerosis, the underlying cause of CAD. The main objective of this thesis was to therefore further investigate the relationship between the Y chromosome, lncRNAs and CAD in light of the deficiencies within the literature to better understand the causative molecular mechanisms of CAD pathophysiology in men. In my first study (Chapter 2), I identified for the first time through gene expression analysis (real-time PCR) the expression of the following (unannotated in PubMed) Y chromosomelinked lincRNA transcripts: lnc-KDM5D-4:1, lnc-ZFY-1:1, lnc-ZFY-1:3, lnc-ZFY-2:1, lnc- RBMY1B-1:1, lnc-RBMY1B-1:4, lnc-RBMY1J-1:1, lnc-RBMY1J-1:2, and lnc-RBMY1J- 1:3, across 21 different normal, human tissues such as adipose, bladder, brain, cervix, colon, esophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, trachea, and white blood cells (WBCs) (leukocytes). I found that Y-linked lincRNAs were expressed at low levels (with the lowest CT number equal at 24.5) with a high tissue-specificity for some. Also, the Y-linked RNA gene lnc-KDM5D-4 was widely expressed across male tissues while the Y-linked RNA gene lnc-RBMY1J-1 was specific to the testes. Furthermore, this study presents the first evidence through gene expression analysis that the Y chromosome-linked lincRNA transcripts, lnc-KDM5D-4:1, lnc- ZFY-1:1, lnc-ZFY-1:3, lnc-ZFY-2:1, lnc-RBMY1B-1:1, lnc-RBMY1B-1:4, and lnc- RBMY1J-1:3 are expressed in male leukocytes. Hence, these lincRNAs could be potential non-protein coding gene candidates for CAD research. Knowing that the Y chromosome contributes to lipid levels in humans, to further explore the potential function of these Y-linked lincRNAs in CAD in men, I then studied their expression in a fatty liver context (steatosis-associated atherosclerosis) (Chapter 3). This was performed using the human hepatocellular liver carcinoma cell line, HepG2; the human model of liver cells in CAD research. This study showed for the first time that the Y-linked lincRNA transcripts lnc-KDM5D-4:1, lnc-ZFY-1:1, lnc-ZFY-2:1, lnc-RBMY1B-1:1, and lncxix RBMY1B-1:4 were expressed in HepG2 cells, hence in hepatocellular carcinoma (HCC). Furthermore, this study demonstrated that lnc-KDM5D-4 is a nuclear-retained lincRNA using RNA fluorescence in situ hybridisation (RNA FISH), and is upregulated in palmitate-induced steatosis within hepatocytes (Fold Change = 2.16; p-value = 0.00216). The human Atherosclerosis RT2 Profiler™ PCR Array determined that the silencing of lnc-KDM5D-4 in HepG2 cells was triggering the upregulation of the inhibitor of apoptosis (IAP) gene baculoviral IAP repeat containing 3 (BIRC3) (Fold Change = 12.45, p-value = 0.000025), a well-described protein-coding gene expressed by vascular smooth muscle cells and macrophage foam cells of the inflamed vascular wall of atherosclerotic arteries. Furthermore, perilipin 2 (PLIN2), a gene known to be implicated in lipid metabolism, was also found upregulated. Therefore, this study provides the first evidence for the involvement of a Ychromosome- linked lincRNA, lnc-KDM5D-4, in steatosis-associated atherosclerosis and its retained-nuclear cellular localisation in human hepatocytes, suggesting a function which takes place in the cell nucleus and may play a role in regulating metabolic processes in the liver that are implicated in atherosclerosis. Having shown that a Y chromosome-linked lincRNA could be involved in the determination of lipid level and hence atherosclerosis in men, and to further explore the role of lnc-KDM5D- 4, the expression of this Y-linked lincRNA was studied in human coronary artery smooth muscle cells, especially in atherosclerotic coronary artery cells (Chapter 4). The expression of other non-coding RNAs were also studied such as the protein kinase, Y-linked, pseudogene (PRKY) - previously considered as a new functional candidate for the development of CAD. By analysing the transcriptome of human atherosclerotic and non-atherosclerotic coronary artery smooth muscle cells, I established evidence for the implication of the human Y chromosome in atherosclerosis and CAD. This study exposed the general underexpression of the transcripts from the Y chromosome in atherosclerotic cells implicating a loss or a repression of this chromosome in relation to CAD. Furthermore, this research determined by RNA sequencing a significant downregulation of seven transcripts from Y chromosome genes, including RPS4Y1, USP9Y, DDX3Y, TXLNGY, NLGN4Y and PRKY. RNA FISH determined the subcellular localisation of PRKY in smooth muscle cells by showing a nuclear and a cytoplasmic expression. Furthermore, qPCR gene expression analysis demonstrated that lnc- KDM5D-4 is significantly downregulated in atherosclerotic cells in comparison to the nonatherosclerotic cells. Together, these results showed that lnc-KDM5D-4 is a potential regulator of PLIN2 and BIRC3 genes. Therefore, the downregulation of lnc-KDM5D-4 in atherosclerotic coronary artery smooth muscle cells suggests that this downregulation could be linked to the inflammation of the vascular smooth muscle cells in pathophysiology of CAD via the inhibition of apoptosis of the vascular smooth muscle cells triggered by the upregulation of BIRC3 in these cells. Overall, this study is the first to emphasise a potential involvement of a Y-specific lincRNA, called lnc-KDM5D-4, as a potential contributor to physiology in males. Lnc-KDM5D-4 knockdown resulted in an upregulation of anti-apoptosis and lipid metabolism-related genes. Collectively, our data suggest that the male–specific lnc-KDM5D-4 may regulate key processes in cellular inflammation that trigger atherosclerosis and CAD in men. Accordingly, this data suggests that lnc-KDM5D-4 may provide a novel molecular biomarker for atherosclerotic arteries, and could potentially lead to revolutionary treatment modalities on Y-linked lincRNA as therapeutic agents to manipulate CAD-causing genes in men.Doctor of Philosophy
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Merry, Callie R. "Long Non-coding RNAs in Cancer." Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1467828387.
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Cabili, Nataly Moran. "Integrative Characterization of Human Long Non-Coding RNAs." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11409.
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Since its early discovery as a messenger, RNA has been shown to play a diverse set of regulatory, structural and even catalytic roles. The more recent understanding that the genome is pervasively transcribed stimulated the discovery of a new prevalent class of long non coding RNAs (lncRNAs). While these are lower abundant and relatively less conserved than other class of functional RNAs, lncRNAs are emerging as key players in different cellular processes in development and disease.
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Vučićević, Dubravka [Verfasser]. "Diverse regulatory functions of long non-coding RNAs / Dubravka Vučićević." Berlin : Freie Universität Berlin, 2017. http://d-nb.info/1137509899/34.
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Bussotti, Giovanni 1983. "Detecting and comparing non-coding RNAs." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/128970.
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In recent years there has been a growing interest in the field of non-coding RNA. This surge is a direct consequence of the discovery of a huge number of new non-coding genes, and of the finding that many of these transcripts are involved in key cellular functions. In this context, accurately detecting and comparing RNA sequences becomes extremely important. Aligning nucleotide sequences is one of the main requisite when searching for homologous genes. Accurate alignments reveal evolutionary relationships, conserved regions and more generally, any biologically relevant pattern. Comparing RNA molecules is, however, a challenging task. The nucleotide alphabet is simpler and therefore less informative than that of proteins. Moreover for many non-coding RNAs, evolution is likely to be mostly constrained at the structure level and not on the sequence level. This results in a very poor sequence conservation impeding the comparison of these molecules. These difficulties define a context where new methods are urgently needed in order to exploit experimental results at their full potential.
These are the issues I have tried to address in my PhD. I have started by developing a novel algorithm able to reveal the homology relationship of distantly related ncRNA genes, and then I have applied the approach thus defined in combination with other sophisticated data mining tools to discover novel non-coding genes and generate genome-wide ncRNA predictions.En los últimos años el interés en el campo de los ARN no codificantes ha crecido mucho a causa del enorme aumento de la cantidad de secuencias no codificantes disponibles y a que muchos de estos transcriptos han dado muestra de ser importantes en varias funciones celulares. En este contexto, es fundamental el desarrollo de métodos para la correcta detección y comparativa de secuencias de ARN. Alinear nucleótidos es uno de los enfoques principales para buscar genes homólogos, identificar relaciones evolutivas, regiones conservadas y en general, patrones biológicos importantes. Sin embargo, comparar moléculas de ARN es una tarea difícil. Esto es debido a que el alfabeto de nucleótidos es más simple y por ello menos informativo que el de las proteínas. Además es probable que para muchos ARN la evolución haya mantenido la estructura en mayor grado que la secuencia, y esto hace que las secuencias sean poco conservadas y difícilmente comparables. Por lo tanto, hacen falta nuevos métodos capaces de utilizar otras fuentes de información para generar mejores alineamientos de ARN. En esta tesis doctoral se ha intentado dar respuesta exactamente a estas temáticas. Por un lado desarrollado un nuevo algoritmo para detectar relaciones de homología entre genes de ARN no codificantes evolutivamente lejanos. Por otro lado se ha hecho minería de datos mediante el uso de datos ya disponibles para descubrir nuevos genes y generar perfiles de ARN no codificantes en todo el genoma.
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Schneider, Hugo Wruck. "Distinguishing long non-coding RNAs from protein coding transcripts based on machine learning techniques." reponame:Repositório Institucional da UnB, 2017. http://repositorio.unb.br/handle/10482/31264.
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Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Exatas, Departamento de Ciência da Computação, 2017.Submitted by Raquel Almeida (raquel.df13@gmail.com) on 2017-12-18T16:34:15Z No. of bitstreams: 1 2017_HugoWruckSchneider.pdf: 2108041 bytes, checksum: 6be494b560adf8d5114b2439b4b025c0 (MD5)
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Dentre as análises que devem ser realizadas nos projetos de sequenciamento, um problema importante é a distinção entre transcritos codificadores de proteinas (PCTs) e RNAs nãocodificadores longos (lncRNAs). Esse trabalho investiga potenciais características dos lncRNAs e propõe dois métodos para distinção dessas duas classes de transcritos (PCTs e lncRNAs). O primeiro método foi proposto com base em máquinas de vetores de suporte (SVM), enquanto o segundo utilizou técnicas de aprendizado semi-supervisionado. O mé- todo utilizando SVM obteve excelentes resultados, quando comparados a outras propostas existentes na literatura. Esse método foi treinado e testado com dados de humanos, camundongos e peixe-zebra, tendo atingido uma acurácia de ≈ 98% com dados de humanos e camundongos, e de ≈ 96% para os dados do peixe-zebra. Ainda, foram criados modelos utilizando várias espécies, que mostraram classificações melhores para outras espécies diferentes daquelas do treinamento, ou seja, mostraram boa capacidade de generalização. Para validar esse método, foram utilizados dados de ratos, porcos e drosófilas, além de dados de RNA-seq de humanos, gorilas e macacos. Essa validação atingiu uma acurácia de mais de 85%, em todos os casos. Por fim, esse método foi capaz de identificar duas sequências dentro do Swiss-Prot que puderam ser reanotadas. O método baseado em aprendizado semi-supervisionado foi treinado e testado com dados de humanos, camundongos, ornitorrincos, galinhas, gambás, orangotangos e rãs, tendo sido utilizadas cinco técnicas de aprendizado semi-supervisionado. A contribuição desse método foi que ele permitiu a redução do tamanho do conjunto de dados classificados, utilizados no treinamento. No melhor caso, somente 2 sequências bem anotadas foram usadas no treinamento, o que, comparado com outras ferramentas disponíveis na literatura, indica um ganho expressivo. A acurácia obtida pelo método nos melhores casos foram de ≈ 95% para dados de humanos e camundongos, ≈ 90% para dados de galinhas, gambás e orangutangos, e ≈ 80% para dados de ornitorrincos e rãs. Dados de RNA-seq foram utilizados para teste, tendo sido obtida acurácia de mais de 95%. Esses dados foram utilizados para treinamento dos modelos de orangotango e de rã, que também apresentaram acurácias excelentes.
Among the analyses that have to be performed in sequencing projects, an important problem to be addressed is the distinction of protein coding transcripts (PCTs) and long non-coding RNAs (lncRNA). This work investigates potential characteristics of the lncRNAs and proposes two methods for distinguishing these two classes of transcripts (PCTs and lncRNAs). The first methods was based on Support Vector Machine (SVM), while the second one used semi-supervised learning techniques. The SVM based method obtained excellent results when compared to other methods in the literature. This method was trained and tested with data from human, mouse and zebrafish, and reached accuracy of ≈ 98% for human and mouse data, and ≈ 96% for zebrafish data. Besides, models with multiple species were created, which improved the classification for species different from those used in the training phase, i.e., these models could also be used in the classification of species different from those that were used in the training phase. To validate this method, data from rat, pig and drosophila, and RNA-seq data from humans, gorillas and macaque were used. This validation reached an accuracy of more than 85% for all the species. Finally, this method was able to identify two sequences within the Swiss-Prot database that were reannotated. The semi-supervised based method was trained and tested with data from human, mouse, platypus, chicken, opossum, orangutan and xenopus, in five semi-supervised learning techniques. The contribution of this method was the reduction of the size of the classified training data set. In the best scenario, only two annotated sequences were used in the training phase, which is an expressive gain when compared to other tools available in the literature. Accuracies obtained by the method in the best cases were ≈ 95% for human and mouse datasets, ≈ 90% for chicken, opossum and orangutan datasets, and ≈ 80% for data platypus and xenopus datasets. RNA-seq data were used for testing, having obtained more than 95% of accuracy. This data was used to train the orangutan and xenopus models, also leading to an excellent accuracy.
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de, Bony Eric James. "Novel insights into the function and regulation of coding and long non-coding RNAs." Doctoral thesis, Universite Libre de Bruxelles, 2018. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/268600.
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Le dogme central de la biologie repose sur la production de protéines à partir de notre ADN. L’ADN est d’abord transcrit en ARN et celui-ci est ensuite traduit en protéine. C’est donc en cette dernière qu’est localisé le “pouvoir exécutif” de la cellule, ce qui explique le fait que les protéines soient devenues le centre d’attention de la recherche. L’ARN, quant à lui, est donc depuis longtemps considéré comme une molécule intermédiaire, dont l’unique raison d’être est le transfert d’information entre l’ADN et les protéines. Pourtant, ces dernières années, les avancées technologiques ont révélé qu’une majeure partie de notre génome, notre ADN, est transcrit en ARNs dits « noncodants » ne donnant pas lieu à une protéine. Ceux-ci sont impliqués dans de nombreux processus cellulaires et de ce fait participent aux pathologies. D’autre part, de nouvelles technologies ont aussi mené à l’observation que le métabolisme des ARNs, codants ou non, est la cible de nouveaux mécanismes de régulation: les modifications chimiques des ribonucléosides. Analysées de manière conjointe, ces découvertes poussent à la révision du rôle des ARNs au sein des processus cellulaires. Dès lors, dans le cadre de cette thèse nous avons voulu mieux comprendre la fonction et la régulation des molécules d’ARN afin d’en révéler le rôle plus central qu’ils jouent dans les processus cellulaire et en particulier, la cancérogenèse. Pour ce faire cette thèse comporte deux parties, la première décrit comment certains ARNs, dit “longs ARNs non-codants” participent au développement et à l’hétérogénéité du cancer colorectal. En effet ces ARNs exercent des fonctions “exécutives” sans être la source d’une protéine. Nous avons identifié 282 long ARNs non-codants dont les profils d’expression reflètent les différentes caractéristiques rencontrées au travers des différents sous-types de tumeurs colorectales. De plus, nos analyses informatiques ont indiqué que ces ARNs font partie intégrante des réseaux de signalisations les plus importants et les plus souvent dérégulés dans les différents sous-types que présente ce cancer. Enfin, et ce via des expériences in vitro nous soutenons la validité de nos analyses informatiques en confirmant le rôle de lncBLID-5, un long ARN non-codant, dans la régulation du cycle cellulaire et de la transition épithéliale vers mésenchymale un processus cellulaire très important dans les cancers colorectaux. Dans la deuxième partie nous avons étudié la méthylation des cytosines de l’ARN, une modification très récemment identifiée. Nous avons découvert que la protéine SRSF2, un facteur général de l’épissage des ARNs, est capable de se lier aux cytosines méthylées et ce plus fortement qu’aux cytosines non-méthylées. Enfin, nous montrons que la mutation P95H de SRSF2, très fréquente chez les patients atteints de leucémie, empêche SRSF2 de favoriser sa liaison aux cytosines méthylées laissant entrevoir de nouvelles explications à l’épissage défectueux conduisant à ce type de cancer. En conclusion nos travaux apportent de nouvelles informations quant à l’implication et la régulation des ARNs codants et non-codants dans le cadre du cancer. Ces résultats devraient nous mener à revoir le rôle qu’occupe l’ARN au sein des processus cellulaires sains ainsi que pathologiques, ouvrant la porte sur une nouvelle dimension de cibles diagnostiques et thérapeutiques.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished
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Alvarez, Juan (Juan Rene Alvarez Dominguez). "Modulation of lineage-specific cell differentiation by long non-coding RNAs." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/97280.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student submitted PDF version of thesis.
Includes bibliographical references.
Mammalian genomes comprise thousands of non-protein-coding genes. These can produce small non-coding RNAs (such as rRNAs and tRNAs), as well as long non-coding RNAs (lncRNAs), which are >200nt and resemble mRNAs in their biogenesis. Although the functions of the vast majority of lncRNAs remain unknown, many are tissue- and developmental stage-specific, suggesting roles in lineage-specific development. We generated deep transcriptome surveys from differentiating mouse red blood cells, and implemented a computational strategy for de novo lncRNA discovery to comprehensively catalog erythroid-expressed lncRNAs. We found >100 previously unannotated loci, many of which are erythroid-specific and are induced by key erythroid transcription factors during differentiation. We exploited these features to select 12 candidates for loss-of-function studies, and found that depleting 10 out of 12 impaired red cell maturation, inhibiting cell size reduction and subsequent enucleation. To study how lncRNAs regulate erythropoiesis, we focused on EC6, an unpolyadenylated lncRNA needed for silencing neighboring loci encoding NF-kB activators. De-repression of these genes upon EC6 knockdown leads to activation of NF-kB and other immune pathways that antagonize erythropoiesis, resulting in impaired proliferation and elevated apoptosis during differentiation. We showed that EC6 is retained in chromatin and binds the nuclear matrix factor hnRNP U, which may enable co-localization with its targets to mediate their repression. Extending our work to a different lineage, we reconstructed transcriptomes from distinct mouse adipose tissues and identified ~1500 lncRNAs. These included many brown fat-specific loci induced during differentiation which are targets of key adipogenic factors. Inhibiting one of them, lnc-BATE1, compromised brown adipocyte development, impairing activation of brown fat genes, mitochondrial biogenesis, and thermogenic function. We showed that lnc-BATE1 acts in trans and binds hnRNP U, which is also required for proper brown adipocyte maturation. This work demonstrates that lncRNAs modulate lineage-specific cell differentiation by promoting or suppressing competing gene expression programs controlling cell fate.
by Juan Alvarez.
Ph. D.
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Chen, Li. "Functional and evolutionary characterization of flowering-related long non-coding RNAs." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/22833.
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Genomweite Bemühungen haben eine große Anzahl langer nichtkodierender RNAs (lncRNAs) identifiziert, obwohl ihre möglichen Funktionen weitgehend rätselhaft bleiben. Hier verwendeten wir ein System zur synchronisierten Blüteninduktion in Arabidopsis, um 4106 blütenbezogene lange intergene RNAs (lincRNAs) zu identifizieren. Blütenbezogene lincRNAs sind typischerweise mit funktionellen Enhancern assoziiert, die bidirektional transkribiert werden und mit verschiedenen funktionellen Genmodulen assoziiert sind, die mit der Entwicklung von Blütenorganen zusammenhängen, die durch Koexpressionsnetzwerkanalyse aufgedeckt wurden. Die Master-regulatorischen Transkriptionsfaktoren (TFs) APETALA1 (AP1) und SEPALLATA3 (SEP3) binden an lincRNA-assoziierte Enhancer. Die Bindung dieser TFs korreliert mit der Zunahme der lincRNA-Transkription und fördert möglicherweise die Zugänglichkeit von Chromatin an Enhancern, gefolgt von der Aktivierung einer Untergruppe von Zielgenen. Darüber hinaus ist die Evolutionsdynamik von lincRNAs in Pflanzen, einschließlich nicht blühender Pflanzen, noch nicht bekannt, und das Expressionsmuster in verschiedenen Pflanzenarten war ziemlich unbekannt. Hier identifizierten wir Tausende von lincRNAs in 26 Pflanzenarten, einschließlich nicht blühender Pflanzen. Ein direkter Vergleich von lincRNAs zeigt, dass die meisten lincRNAs speziesspezifisch sind und das Expressionsmuster von lincRNAs einen hohen Transkriptionsumsatz nahe legt. Darüber hinaus zeigen konservierte lincRNAs eine aktive Regulation durch Transkriptionsfaktoren wie AP1 und SEP3. Konservierte lincRNAs zeigen eine konservierte blütenbezogene Funktionalität sowohl in der Brassicaceae- als auch in der Grasfamilie. Die Evolutionslandschaft von lincRNAs in Pflanzen liefert wichtige Einblicke in die Erhaltung und Funktionalität von lincRNAs.Genome-wide efforts have identified a large number of long non-coding RNAs (lncRNAs), although their potential functions remain largely enigmatic. Here, we used a system for synchronized floral induction in Arabidopsis to identify 4106 flower-related long intergenic RNAs (lincRNAs). Flower-related lincRNAs are typically associated with functional enhancers which are bi-directionally transcribed and are associated with diverse functional gene modules related to floral organ development revealed by co-expression network analysis. The master regulatory transcription factors (TFs) APETALA1 (AP1) and SEPALLATA3 (SEP3) bind to lincRNA-associated enhancers. The binding of these TFs is correlated with the increase in lincRNA transcription and potentially promotes chromatin accessibility at enhancers, followed by activation of a subset of target genes. Furthermore, the evolutionary dynamics of lincRNAs in plants including non-flowering plants still remain to be elusive and the expression pattern in different plant species was quite unknown. Here, we identified thousands of lincRNAs in 26 plant species including non-flowering plants, and allow us to infer sequence conserved and synteny based homolog lincRNAs, and explore conserved characteristics of lincRNAs during plants evolution. Direct comparison of lincRNAs reveals most lincRNAs are species-specific and the expression pattern of lincRNAs suggests their high evolutionary gain and loss. Moreover, conserved lincRNAs show active regulation by transcriptional factors such as AP1 and SEP3. Conserved lincRNAs demonstrate conserved flower related functionality in both the Brassicaceae and grass family. The evolutionary landscape of lincRNAs in plants provide important insights into the conservation and functionality of lincRNAs.
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Coyne, Victoria. "Characterization of long non-coding RNAs in the Hox complex of Drosophila." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/characterization-of-long-noncoding-rnas-in-the-hox-complex-of-drosophila(733e3dec-3f7b-4d6e-a1bc-674a8786246d).html.
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Long non-coding RNAs (lncRNAs) are often defined as transcripts >200nts that have no discernable protein-coding ability (Quinn and Chang, 2016). Although relatively little is understood about the molecular mechanisms of lncRNA function, they have established roles in regulation of gene expression during development, cell differentiation and pluripotency (Fatica and Bozzoni, 2014; Luo et al., 2016; Quinn and Chang, 2016; Rinn and Chang, 2012) across vastly diverse organisms ranging from plants to humans (Ulitsky and Bartel, 2013). LncRNAs have also been associated with numerous pathological conditions, such as cancers (Brunner et al., 2012), cardiovascular disease and neurodegeneration (Chen et al., 2013). Investigations into lncRNAs in wide ranging organisms, have revealed that many influence gene activity by forming ribonucleoprotein complexes that affect the conformational state of chromatin (Rinn and Chang, 2012). A genomic region that has revealed several functional lncRNAs in diverse organisms is the Hox complex (Pauli et al., 2011; Pettini, 2012; Rinn et al., 2007). The Hox complex encodes a set of transcription factors (TFs), physically clustered in the genome, which provide morphological identity along the anterior to posterior axis of developing embryos (Mallo and Alonso, 2013), throughout the majority of bilatarian animals (Moreno et al., 2011). Misexpression or mutation of Hox genes causes morphological and pathophysiological defects (Quinonez and Innis, 2014). We investigated clustering of lncRNAs throughout the D. melanogaster genome using available annotations and carried out RNA-seq in D. virilis to expand the repertoire of lncRNAs and identify clusters of lncRNAs. We found the Hox complex to be heavily enriched with lncRNAs in both organisms, and syntenic transcripts from D. melanogaster could be identified in D. pseudoobscura and D. virilis. Several lncRNAs aligned with polycomb response elements (PREs); transcription of PREs has previously been linked to a switch in their activity (Herzog et al., 2014). However, we found that transcribed PREs in D. melanogaster move positions relative to the protein-coding genes in other drosophilids, whilst the transcriptional units remain in the same syntenic region. Conservation of syntenic transcripts without evidence of remaining a PRE suggest that the transcription is not linked to PRE function, agreeing with recent findings that transcription of PREs does not affect their function (Kassis and Muller, 2015). We investigated functions of a novel lncRNA and adjacent PRE in the Hox complex by ectopic expression and utilization of other genetic manipulation tools. Overexpression of either the lncRNA or PRE and partial duplication of the lncRNA caused phenotypes such as missing halteres and/or T3 legs, misshaped T3 legs or malformed abdominal segments. The observations that ectopic expression of this lncRNA and an adjacent regulatory element from the Hox complex causes phenotypes that can be linked to adjacent Hox gene misregulation, Antp and Ubx, suggest that they are likely to have roles in the regulation of at least one of these Hox genes.
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Pettini, Tom. "The role of novel long non-coding RNAs in Hox gene regulation." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-novel-long-noncoding-rnas-in-hox-gene-regulation(c8e44900-3ac0-40be-8ec6-b50179381d17).html.
Full textAbstract:
Whole genome transcriptome analysis has revealed that a large proportion of the genome in higher metazoa is transcribed, yet only a small proportion of this transcription is protein-coding. One possible function of non-coding transcription is that it enables complex and diverse body plans to evolve through variation in deployment of a relatively common set of protein-coding genes. Functional studies suggest that long non-coding RNAs (lncRNAs) regulate gene expression via diverse mechanisms, operating in both cis and trans to activate or repress target genes. An emerging theme common to lncRNA function is interaction with proteins that modify chromatin and mediate epigenetic regulation. The Hox gene complexes are particularly rich in lncRNAs and require precise and fine-tuned expression to deploy Hox transcription factors throughout development. Here we identify and functionally characterize two novel lncRNAs within the D. melanogaster Hox complex, in the interval between Scr and Antp. We use nascent transcript fluorescent in-situ hybridization (ntFISH) to characterize the embryonic expression patterns of each lncRNA with respect to flanking Hox genes, and to analyze co-transcription within individual nuclei. We find that the transcription of one lncRNA, ncX, is an initial response to early transcription factors and may activate Scr expression, while transcription of the other lncRNA, ncPRE is consistent with activation and/or maintenance of Scr expression. ntFISH performed in D.virilis embryos revealed the presence of a lncRNA ortholog with highly similar expression to ncX, indicating functional conservation of lncRNA transcription across ~60 million years of evolution. We identify the ncPRE lncRNA locus as a binding site for multiple proteins associated with Polycomb/Trithorax response elements (PREs/TREs) and show that DNA encoding the ncPRE lncRNA functions as a bona fide PRE, mediating trans-interactions between chromosomes and silencing of nearby genes. We find that transcription through the ncPRE DNA relieves silencing, suggesting a role for endogenous transcription of the ncPRE lncRNA in relieving Polycomb-silencing and enabling Scr activation. We demonstrate that both lncRNA transcripts are required for proper Scr expression, and over-expression of either lncRNAs from ectopic genomic loci has no effect on Scr expression, but ectopic expression at the endogenous locus is associated with ectopic Scr activation, indicating that the lncRNA-mediated regulation functions locally at the site of transcription on the chromosome. ncX may mediate transvection effects previously observed at the Scr locus, independent of the protein Zeste. Together our results support a model of competing mechanisms in the regulation of Scr expression - a background of Polycomb repression acting from the ncPRE locus, which in the first thoracic segment is counteracted by lncRNA transcription and Trithorax binding to ncPRE, enabling activation and maintenance of Scr expression. This work provides a functional insight into the complex regulatory interactions between lncRNAs and epigenetic mechanisms, essential to establish and maintain the precise expression pattern of Hox genes through development.
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Karlsson, Joakim. "Differential and co-expression of long non-coding RNAs in abdominal aortic aneurysm." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-236141.
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This project concerns an exploration of the presence and interactions of long non-coding RNA transcripts in an experimental atherosclerosis mouse model with relevance for human abdominal aortic aneurysm development. 187 long noncoding RNAs, two of them entirely novel, were found to be differentially expressed between angiotensin II treated (developing abdominal aortic aneurysms) and non-treated apolipoprotein E deficient mice (not developing aneurysms) harvested after the same period of time. These transcripts were also studied with regards to co-expression network connections. Eleven previously annotated and two novel long non-coding RNAs were present in two significantly disease correlated co-expression groups that were further profiled with respect to network properties, Gene Ontology terms and MetaCore© connections.
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Javad, F. "Long non-coding RNAs as regulators of human microtubule-associated protein tau (MAPT)." Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1531024/.
Full textAbstract:
Aggregation of the neuronal microtubule-binding protein, tau, is associated with cognitive decline and neuronal degeneration in tauopathies, including Alzheimer’s disease and frontotemporal dementia. In particular, changes in tau gene (MAPT) expression and splicing are directly linked with pathogenesis. Most genes, including MAPT, are now known to be associated with one or more sense/antisense non-coding RNA genes (ncRNA). We identified alternatively spliced variants of MAPT-AS1 that overlap in antisense with the MAPT 5’ promoter region, producing three transcripts, tau-NAT1, tau-NAT2s and tau-NAT2l (tNAT1, tNAT2s and tNAT2l, respectively) and investigated the role of these alternate isoforms in functional regulation of MAPT under physiological and stress conditions. The tau-NATs, with MAPT, are predominantly expressed in the brain and with increasing levels during neuronal differentiation of human fibroblast-derived iPSCs. Compared to tNAT1 and tNAT2l, the tNAT2s variant is most abundant and correlates with MAPT transcript levels in different brain regions. Using RNA-FISH we show that the tau-NATs are mostly present in the cytoplasm with some in nuclear spots that could be the sites of active transcription. To study the role of the tau-NATs in MAPT regulation, we created SH-SY5Y neuroblastoma clones stably expressing the tau-NAT variants and targeted deletions and mutants. Although full-length tNAT1 and tNAT2l do not effect MAPT gene transcription, they significantly repress tau expression at the translational level. By targeted deletions, we demonstrated that this translational repression requires the regions of 5’-5’ antisense overlap with MAPT as well as an MIR-repeat element in the 3’-domain of tNAT1 and tNAT2l. On the other hand, tNAT2s enhances tau translation due to additional regulatory elements. In this work, we further demonstrate that tNAT2s may have a selective role in heat-shock response whereby endogenous tNAT2s levels in SH-SY5Y cells are significantly elevated during heat shock with an accompanying increase in tau protein levels. Our data support a possible physiological role of MAPT-AS1 in post-transcriptional regulation of tau expression with a specific role of tNAT2s in cellular stress response.
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Chen, Li [Verfasser]. "Functional and evolutionary characterization of flowering-related long non-coding RNAs / Li Chen." Berlin : Humboldt-Universität zu Berlin, 2021. http://d-nb.info/1233678590/34.
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Bogu, Gireesh K. 1984. "Understanding the transcriptional landscape of non-coding genome in mammals." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/572043.
Full textAbstract:
Widespread transcription in mammals revealed unexpected discovery of non-coding
elements like long noncoding RNAs (lncRNAs) and repetitive elements. First,
lncRNAs were previously identified in limited number of tissues or cell lines in mouse
and the discovery of lncRNAs was still pending in many other tissues in mouse. To
address this, we applied a computational pipeline that discovered 2,803 highconfidence
novel lncRNAs by mapping and de novo assembling billions of RNA-Seq
reads in eight tissues and a primary cell line in mouse. Further, we integrated this
catalog of lncRNAs with chromatin state maps and found many regulatory lncRNAs
(promoter-associated and enhancer-associated lncRNAs). Second, more than half of
the human genome contains repetitive elements. However, it is not clear how they
are expressed across all mammalian tissues. To address this, as a part of Genotype-
Tissue Expression (GTEx) project, we profiled repetitive elements using 8,551 poly-A
RNA-Seq datasets from 53 tissues across 550 individuals and found various repeat
families transcribed across multiple human tissues in a tissue-specific manner. In
summary, to understand the transcriptional landscape of non-coding genome, we
mainly analyzed RNA-Seq datasets across many tissues in mammals and show that
the non-coding elements like lncRNA and repetitive elements are not only
transcribed but also tissue-specific. Together, this thesis work defines a unique
collection of non-coding elements that are transcribed and tissue-specific in
mammalian tissues.Una gran parte del genoma de mamiefores se expresa en forma de ARNs y se conoce hoy en dia que una gran parte de estos transcritos son no codificantes llamados lncRNAs y que contienen elementos repetitivos. En ratones, estos han sido identificados recientemente en un número limitado de tejidos y líneas celulares. Esta tesis presenta un trabajo exhaustivo de estudio de lnRNAs en ratón en ocho tejidos y una línea celular. En este trabajo se descubrieron 2803 nuevos lncRNAs a los cuáles se les asignó una función reguladora (asociados a promotores o activadores “enhancers”) en el genoma usando datos del estado de la cromatina. Asimismo, más de la mitad del genoma humano contiene elementos repetitivos. Desafortunadamente no se conoce el patrón de expresión de estos elementos repetitivos en los tejidos mamíferos. Como miembros del proyecto GTEx (GenotypeviTissue Expression), analizamos la expresión de estos elementos repetitivos en 8,551 muestras de polyA RNA-Seq en 53 tejidos de 550 individuos. Encontramos que muchas familias de elementos repetitivos son expresadas en tejidos específicos en varios individuos, y representan una característica peculiar de la identidad de cada tejido en humanos.
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Hawkes, Emily. "Conservation and function of COOLAIR long non-coding RNAs in Brassica flowering time control." Thesis, University of East Anglia, 2017. https://ueaeprints.uea.ac.uk/67797/.
Full textAbstract:
Since their discovery long non-coding RNAs (lncRNAs) have in turn been described as essential genomic regulators or as transcriptional noise. Examples of lncRNAs with experimentally-validated function are limited, with poor nucleotide sequence conservation calling apparent functionality into question. COOLAIR lncRNAs are transcribed in the antisense direction at the Arabidopsis thaliana floral repressor gene, Flowering Locus C (FLC). Previous work has revealed a role for COOLAIR antisense RNAs in regulation of the FLC protein-coding sense transcript and, consequently, flowering time. FLC homologues are wide-spread in flowering plants, but nucleotide sequence conservation of COOLAIR-specific regions is low. COOLAIR has a complex secondary structure, and covariant base-pair mutations predict strong conservation of this secondary structure across flowering plants. Syntenic transcription of COOLAIR was confirmed in vivo for several species within the family Brassicaceae, including three commercially important Brassica crops: B. rapa, B. oleracea and B. napus. COOLAIR transcription was detected from at least three of four ancient FLC clades within the latter three polyploid and paleopolyploid species. Each FLC homologue has distinct nucleotide sequence, expression patterns, and COOLAIR isoforms. Further variation in COOLAIR was identified between winter and spring cultivars. It was hypothesised that this could affect cis-regulation of FLC. Correlation between COOLAIR modifications and flowering time was tested by introducing antisense splice site mutations into Brassica FLC transgenes in A. thaliana. These experiments suggested that a shorter COOLAIR isoform with a disrupted structural motif was a weaker negative regulator of FLC. This work supports conservation of COOLAIR expression, structure and function in Brassica crop plants, and a role for RNA structure in COOLAIR function. We propose an evolutionarily conserved lncRNA that is neither essential regulator nor transcriptional noise, but rather adapts with the environment to fine-tune the transition to flowering.
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Gadekar, Veerendra Parsappa. "Functional exploration of antisense long non-coding RNAs containing transposable elements : a bioinformatics approach." Thesis, Open University, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701364.
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Long non-coding RNA (lncRNAs) show a wide range of regulatory functions at the transcriptional and post-transcripltional levels both in the nucleus and cytoplasm. Recently, antisense lncRNAs (ASlncRNAs) were reported to up-regulate protein synthesis post-transcriptionally through a mechanism depending on an embedded inverted SINE B2 and 5' overlap to the target mRNAs. Such ASlncRNAs are also referred as SINEUPs. Synthetic SINEUPs with identical modular organization were also demonstrated to exert the same activity suggesting a functional relationship between SINE repetitive elements and ASlncRNAs. In order to gain a broader insight on the contribution of transposable elements (TEs) in the sequence composition of ASlncRNAs, I have developed a bioinformatic pipeline that can identify and characterize. transcripts containing TEs and analyze TEs coverage for different classes of coding/non-coding sense/antisense (S/AS) pairs. I aimed at identifying if the functional activity of SINEUPs could be a widespread phenomenon across multiple similar natural ASlnRNAs in the transcriptomes of the extensively studied model organisms that have a well annotated catalog of lncRNAs. From my initial analysis I identified human and mouse are the two species that showed a significant coverage enrichment of SINE repeats among ASlncRNAs. I further performed several functional enrichment analysis for the sense coding genes overlapping to ASlncRNAs taking into consideration of different characteristics of the 5' binding domain and the 3' embedded SINE repetitive elements. This permitted me to identify the effect of these modular features over the functional associations of sense coding genes. The results of the analysis showed that the products of coding genes associated to ASlncRNAs containing SINEs are significantly enriched for rnitochondriallocalization. Further, to determine if these ASlncRNAs could exert SINEUP-like activity during stress, I analyzed the data from a published custom rnicroarray experiment study, that were associated to the polysome fractions of MRCS cell lysates in control and oxidative stress condition. The results revealed that the ASlncRNA carrying inverted or direct SINE repeats and their corresponding sense coding genes do not show any significant differential polysome loading in stress with respect to normal conditions, which is not a desired characteristic of a potential SINEUP. However, ASlncRNAs with inverted and direct SINE repeats corresponding to high translating polysome fractions showed a significantly higher ratio of means for RNA levels in stress over control, in contrast to noASlncRNA. This suggests that the ASlncRNA containing SINE elements are the key RNA molecules that are active during stress, although to determine if they are also involved in the increased polysome loading of their respective sense coding mRNAs, there is a need of further experimentation and exploration. Altogether, the work presented in this thesis provides a novel bioinformatics approach to study
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Hewitt, Rachael Nicole. "Investigation of the role of long non-coding RNAs in oncogene induced cellular senescence." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/7143/.
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Cellular senescence is a stable arrest of cell proliferation induced by several factors such as activated oncogenes, oxidative stress and shortening of telomeres. Senescence acts as a tumour suppression mechanism to halt the progression of cancer. However, senescence may also impact negatively upon tissue regeneration, thus contributing to the effects of ageing. The eukaryotic genome is controlled by various modes of transcriptional and translational regulation. Focus has therefore centred on the role of long non- coding RNAs (lncRNAs) in regulating the genome. Accordingly, understanding how lncRNAs function to regulate the senescent genome is integral to improving our knowledge and understanding of tumour suppression and ageing. Within this study, I set out to investigate the expression of lncRNAs’ expression within models of senescence. Through a custom expression array, I have shown that expression of multiple different lncRNAs is up-regulated and down regulated in IMR90 replicative senescent fibroblasts and oncogene-induced senescent melanocytes. LncRNA expression was determined to be specific to stable senescence-associated cell arrest and predominantly within the nucleus of senescent cells. In order to examine the function of lncRNA expression in senescence, I selected lncRNA transcript ENST0000430998 (lncRNA_98) to focus my investigations upon. LncRNA_98 was robustly upregulated within multiple models of senescence and efficiently depleted using anti-sense oligonucleotide technology. Characterisation and unbiased RNA-sequencing of lncRNA_98 deficient senescent cells highlighted a list of genes that are regulated by lncRNA_98 expression in senescent cells and may regulate aspects of the senescence program. Specifically, the formation of SAHF was impeded upon depletion of lncRNA_98 expression and levels of total pRB protein expression severely decreased. Validation and recapitulation of consequences of pRB depletion was confirmed through lncRNA_98 knock-out cells generated using CRISPR technology. Surprisingly, inhibition of ATM kinase functions permitted the restoration of pRB protein levels within lncRNA_98 deficient cells. I propose that lncRNA_98 antagonizes the ability of ATM kinase to downregulate pRB expression at a post-transcriptional level, thereby potentiating senescence. Furthermore, lncRNA expression was detected within fibroblasts of old individuals and visualised within senescent melanocytes in human benign nevi, a barrier to melanoma progression. Conversely, mining of 337 TCGA primary melanoma data sets highlighted that the lncRNA_98 gene and its expression was lost from a significant proportion of melanoma samples, consistent with lncRNA_98 having a tumour suppressor functions. The data presented in this study illustrates that lncRNA_98 expression has a regulatory role over pRB expression in senescence and may regulate aspects of tumourigenesis and ageing.
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Nowicki-Osuch, Karol Piotr. "Identification and characterisation of long non-coding RNAs expressed downstream of EGF-induced signalling programme." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/identification-and-characterisation-of-long-noncoding-rnas-expressed-downstream-of-egfinduced-signalling-programme(fd52d235-1a50-4347-bdb1-fdba4fdb912d).html.
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It has recently become apparent that cells encode a large number of novel non-protein-coding genes called long non-coding RNAs (lncRNAs). Whilst the biological function of many lncRNAs remains unknown, recent evidence has suggested that lncRNAs may be important regulators of cellular growth, differentiation and may play a significant role in cancer. Epidermal growth factor (EGF) – an activator of the ERK1/2 signalling cascade – is an important spatio-temporal regulator of transcription and, ultimately, of cellular growth and movement. EGF stimulation triggers a wave-like expression of immediate-early genes (IE genes), followed by delayed-early genes (DE genes) and secondary-response genes (SR genes). Over the years, considerable effort has been made to unravel the regulatory loops downstream of EGF signalling. This study investigated whether lncRNAs are sensitive to EGF signalling and whether they play a role in the transcriptional programme associated with EGF signalling. In order to identify lncRNAs regulated by EGF signalling, I sequenced nuclear RNA in the presence or absence of EGF stimulation. RNA-seq data showed that 173 lncRNAs are upregulated by EGF, of which 89 were intergenic lncRNAs (lincRNAs). The time-dependent expression profile of EGF-upregulated lincRNAs followed the well-established expression pattern of IE genes. Finally, investigation of the expression of lincRNAs in primary breast and lung cancer cells showed that EGF-upregulated lincRNAs were differentially expressed in cancer. The EGF-dependent induction profile and cancer enrichment were particularly strong for one of the transcripts – EGF-induced lncRNA 1 (EIN1) – and I selected it for further studies. Firstly, using bioinformatics and biochemical approaches, I confirmed the non-coding status of the EIN1 transcript. Secondly, I confirmed that EIN1 transcription is ERK1/2-dependent and is independent of protein synthesis. Investigation of EIN1 expression in normal tissues showed its high enrichment in the human cardiovascular system. At the cellular level, the EIN1 transcript was predominantly found in the nucleus. Functionally, the depletion of endogenous EIN1 transcripts (using the newly developed CRISPRi approach) led to changes in the EGF-dependent transcription programme. EIN1 downregulation resulted in the addition of normally EGF-independent genes into the EGF-dependent expression programme. Collectively, these results show that EGF (via the ERK1/2 pathway) can regulate transcription of lincRNAs. The EIN1 example suggests that lincRNAs may play a crucial role in the modulation of the EGF-dependent expression programme by limiting of the scope of the programme.
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Bertrand, Claire. "Long non-coding RNAs in cancer : the role of HOTAIR in Epithelial-to-Mesenchymal Transition." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066632/document.
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Le génome humain est largement transcrit en milliers d’ARN non traduits en protéines. Les longs ARN non-codants (ARNlnc) ont un rôle majeur dans la régulation du génome, au cours du développement et lors de la progression de nombreuses maladies, dont les cancers. La transition épithélio-mésenchymateuse (TEM), donnant à une cellule la capacité de former des métastases, semble être un processus crucial transformant une tumeur bénigne en maladie mortelle. Certains ARNlnc ont été associés à ce phénomène, mais leur fonction reste à définir.Un modèle in vitro de TEM et des approches de séquençage d’ARN à très haut débit, nous ont permis de définir un catalogue d’ARNlnc dérégulés entre cellules épithéliales et mésenchymateuses. Parmi eux, nous avons identifié HOTAIR, étudié pour son expression aberrante dans les tumeurs métastasées et son interaction avec les complexes PRC2 et LSD1/CoREST/REST. Par des approches de perte et de gain de fonction, nous avons montré que HOTAIR n’est pas impliqué dans l’initiation de la TEM mais est un régulateur majeur de la prolifération cellulaire ainsi que des capacités de migration et d’invasion des cellules. Nous avons généré des lignées cellulaires sur-exprimant HOTAIR privé de son domaine d’interaction avec PRC2 ou LSD1. L’étude de leur phénotype et l’établissement de leur transcriptome ont permis de montrer que le domaine d’interaction avec le complexe LSD1/CoREST/REST est crucial pour la régulation de nombreux gènes par HOTAIR. Ces résultats permettent une meilleure compréhension du rôle des ARNlnc dans la TEM, et de la fonction cruciale de HOTAIR dans l’acquisition d’un phénotype métastatique par des cellules cancéreuses épithélialesThe human genome is pervasively transcribed into thousands of non-coding transcripts. Numerous studies underline the diversity and importance of long non-coding RNAs (lncRNAs) in genome regulation and their impact on development and diseases. Processes of cancer progression are extensively studied, in particular the Epithelial-to-Mesenchymal Transition (EMT) that enables epithelial cancer cells to invade other tissues to form metastases. If several lncRNAs have been associated with EMT, their molecular function is not clearly defined. Using a well-established in vitro cell model of EMT and high-throughput RNA sequencing approaches, we defined a catalogue of annotated and novel lncRNAs significantly deregulated between epithelial and mesenchymal states of HEK cells. Among them, we identified HOTAIR, linked to cancer metastasis and described as a scaffold RNA guiding chromatin-modifying complexes PRC2 and LSD1/CoREST/REST. Using loss- and gain-of-function approaches, we showed that HOTAIR is not an inducer of the EMT per se but a major regulator of cell proliferation rate, migratory and invasive capacities. We generated stable cell-lines over expressing HOTAIR transcripts lacking PRC2- or LSD1-interacting domains. Transcriptome analysis and phenotypic studies showed that LSD1-binding domain is crucial for HOTAIR-mediated gene regulation. Altogether, our results give new insights into lncRNAs role in EMT, with a better understanding of HOTAIR-mediated gene regulation mechanism and its role in the acquisition of a metastatic phenotype by cancer cells. Further studies will be performed to deeper investigate lncRNAs role in EMT, particularly for previously unannotated lncRNAs
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Nelson, Andrew D. L., Upendra K. Devisetty, Kyle Palos, Asher K. Haug-Baltzell, Eric Lyons, and Mark A. Beilstein. "Evolinc: A Tool for the Identification and Evolutionary Comparison of Long Intergenic Non-coding RNAs." FRONTIERS MEDIA SA, 2017. http://hdl.handle.net/10150/624658.
Full textAbstract:
Long intergenic non-coding RNAs (lincRNAs) are an abundant and functionally diverse class of eukaryotic transcripts. Reported lincRNA repertoires in mammals vary, but are commonly in the thousands to tens of thousands of transcripts, covering similar to 90% of the genome. In addition to elucidating function, there is particular interest in understanding the origin and evolution of lincRNAs. Aside from mammals, lincRNA populations have been sparsely sampled, precluding evolutionary analyses focused on their emergence and persistence. Here we present Evolinc, a two-module pipeline designed to facilitate lincRNA discovery and characterize aspects of lincRNA evolution. The first module (Evolinc-I) is a lincRNA identification workflow that also facilitates downstream differential expression analysis and genome browser visualization of identified lincRNAs. The second module (Evolinc-II) is a genomic and transcriptomic comparative analysis workflow that determines the phylogenetic depth to which a lincRNA locus is conserved within a user-defined group of related species. Here we validate lincRNA catalogs generated with Evolinc-I against previously annotated Arabidopsis and human lincRNA data. Evolinc-I recapitulated earlier findings and uncovered an additional 70 Arabidopsis and 43 human lincRNAs. We demonstrate the usefulness of Evolinc-II by examining the evolutionary histories of a public dataset of 5,361 Arabidopsis lincRNAs. We used Evolinc-II to winnow this dataset to 40 lincRNAs conserved across species in Brassicaceae. Finally, we show how Evolinc-II can be used to recover the evolutionary history of a known lincRNA, the human telomerase RNA (TERC). These latter analyses revealed unexpected duplication events as well as the loss and subsequent acquisition of a novel TERC locus in the lineage leading to mice and rats. The Evolinc pipeline is currently integrated in CyVerse's Discovery Environment and is free for use by researchers.
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Marques, Lucas Farinazzo. "Busca e análise de lncRNA (long non-coding RNAs) importantes para a tolerância ao etanol em Saccharomyces cerevisiae." Botucatu, 2019. http://hdl.handle.net/11449/191041.
Full textAbstract:
Orientador: Guilherme Targino ValenteResumo: A levedura Saccharomyces cerevisiae é o microrganismo mais utilizado para a produção de etanol devido a sua alta capacidade fermentativa e resistência aos estresses oriundos desse processo. Entretanto, a própria concentração de etanol é um dos fatores mais limitantes no processo de produção desse combustível. Os aspectos da genômica funcional relacionada à tolerância ao etanol são ainda pouco esclarecidos, e nem mesmo se sabe se os lncRNAs tem papel nesse processo. Poucos lncRNAs foram identificados em S. cerevisiae, e nem mesmo se conhece as redes lncRNAs-proteínas nessa espécie e nem se podem codificar micropeptídeos. Nesse contexto, este trabalho visa identificar lncRNAs em linhagens de S. cerevisiae com diferentes níveis de tolerância ao etanol. Para isso, foi realizado a montagem dos lncRNAs, predição de ligações lncRNA-proteínas, buscas de micropepetídeos, análises de conservação genômica, estrutural e funcional dos lncRNAs, avaliação da influência do lncRNAs em regular as expressões de seus vizinhos e comparação dos resultados entre linhagens mais e menos tolerantes ao etanol. As análises de enriquecimento ontológico apontam para uma relação próxima entre os lncRNAs e a tolerância ao etanol e uma conservação funcional, embora os dados não reportem nenhuma conservação nem genômica nem estrutural. Além disso, variados tipos de prováveis regulações foram sugeridas, sendo a regulação em trans majoritariamente inversa entre os lncRNAs e seus genes-alvo, diferentemente da ma... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The yeast Saccharomyces cerevisiae is the most used microorganism for ethanol production due to its high fermentative capacity and resistance to different stressors along this process. However, the ethanol concentration is one of the most limiting factors of fuel production. The functional genomics aspects related to the ethanol tolerance are still unclear, and it is not clear if the lncRNAs really have a role in this process. Few lncRNAs were identified in S. cerevisiae, lncRNA-protein networks of this species are still unknown and also if they can code micropeptides. In this context, this thesis aims to identify lncRNAs and evaluate their roles in S. cerevisiae ethanol tolerance. Then, it was performed the assembling of lncRNAs, predictions of lncRNA-protein interactions, searches for potential micropeptides coding-lncRNAs, analysis of genomic, structural and functional conservation of lncRNAs, evaluation of the lncRNAs influence in regulating the expressions of their neighbors, and comparison between strains that are more and less tolerant to the ethanol. Moreover, many putative regulatory pathways were here suggested, being that most trans regulations act on an inversely manner between the expression of the lncRNAs and their target-genes, unlike observed in most of cis regulations. The current literature confirms the lncRNAs functional conservation here observed, and the role of these non-coding molecules as regulators. Finally, here we suggest that lncRNAs are acting to ... (Complete abstract click electronic access below)
Mestre
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Chakraborty, Debojyoti. "Systematic dissection of long non coding RNAs involved in the regulation of embryonic stem cell pluripotency." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-134658.
Full textAbstract:
Living organisms portray diverse patterns of growth and developmental regulation. The entire process, beginning from a single cell to the formation of tissues and organ systems and the final culmination in a form that characterizes a fully grown organism is closely guarded by numerous molecular pathways. Like a master conductor, the genetic material of the cell which is stored in its DNA (deoxyribonucleic acid), determines the fate of each individual cell and demarcates its developmental direction. Although in an organism, this genetic material is normally identical in every cell, there are differences in the ways cells respond to them. For example, skin cells behave differently from muscle cells and their developmental processes are highly variable in space and time.
It is now known that based on intrinsic or extrinsic environmental cues, the information passed on from DNA is converted into a functional protein product through an RNA (ribonucleic acid) intermediate. Thus, different genes fire at different times leading to diverse patterns of developmental regulation among cells. However, there exists a stark contrast between the size of the genome (DNA), the transcriptome (transcribed RNAs) and the proteome (functional protein products) inside a cell. While there are abundant RNA molecules that are transcribed from the DNA, only few give rise to proteins.
The search for function of RNAs that do not code for proteins is a relatively new topic in molecular biology. With advancements in sequencing methodologies, there is a rapid surge in the discovery of such molecules but due to the nonavailability of systematic tools to study them, the functional characterization of these RNAs has been relatively slow. Even among the non coding RNAs, there exists small and long varieties of which the long non coding RNAs (lncRNAs) have more heterogenous functional attributes. The roles that these lncRNAs play in development is only recently emerging, especially in the field of embryonic stem (ES) cell biology.
ES cells are of particular interest to researchers due to their properties of replicating indefinitely in culture and giving rise to all the germ layers that eventually constitute an organism. These unique abilities make them perfect models to study essential cellular developmental processes and also contribute to the understanding of the molecular pathways that ultimately lead to diseases like like other processes, is orchestrated by a host of different factors in which lncRNAs are slowly emerging as important players. Although there are thousands of lncRNAs identified, only a few have been implicated in pluripotency. I reasoned that there should be more such candidates and to study them one needs to develop a strategy to functionally investigate several lncRNAs simultaneously.
Loss-of-function screens have been extremely successful for dissecting the functions of protein coding genes. Among the triggers for conducting such screens, endoribonuclease-prepared small interfering RNAs (esiRNAs) have been demonstrated as effective mRNA depletion agents with minimum silencing of non-intended targets. Since these RNA interference (RNAi) agents had not been comprehensively tested on lncRNAs, I used them for conducting a screen to discover lncRNAs involved in pluripotency. Using a combination of RNAi and localization strategies, I here report the discovery of a novel lncRNA called Panct1 which through interaction with other factors takes part in the ES cell pluripotency programme. In the process of characterization of Panct1, I have also identified and partially characterized a potential DNA binding protein called CXORF23 which might emerge as an important player in the determination of stem cell fate. These discoveries hint towards the presence of more such lncRNA protein interactions and further widen our understanding of stem cell biology.
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Al-Mnaseer, Zainab Ahmed Mustafa. "Investigation into the role of the long non-coding RNAs NEAT1 and MIAT in breast cancer." Thesis, Keele University, 2018. http://eprints.keele.ac.uk/4541/.
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Several long non-coding RNAs (lncRNAs) have been identified to play key, rate-limiting roles in malignancies, and the mechanisms involved are now being elucidated. This study addressed the roles of NEAT1 and MIAT lncRNAs, in breast cancer. The short isoform NEAT1 _1 was found to be significantly up-regulated in advanced stages of breast cancer samples and in the ER/PR +ve and HER –ve molecular subtype, where its expression correlated positively with that of its neighbouring gene, MALAT1. NEAT1 transcripts in breast cancer cell lines were detected in both nuclear and cytoplasmic compartments. Silencing of cytoplasmic NEAT1 led to an increase in the expression of nuclear NEAT1, where such overexpression inhibited apoptosis and increased cell survival. Consistent with this, siRNA and ASO mediated knockdown of NEAT1 transcript levels decreased cell survival and migration and promoted cell death induced by different apoptotic stimuli including chemotherapeutic agents and UV-C irradiation. Reduced NEAT1 expression levels were also associated with changes in the expression of genes involved in the regulation of cell proliferation and survival. More importantly, it was found that NEAT1_1 regulates gene expression in cis and trans. MIAT expression levels were significantly increased in triple negative breast cancer samples and its expression correlated with NEAT1 expression. In breast cancer cell lines, MIAT expression was found to correlate with the expression of the transcription factor Oct4. MIAT down-regulation in breast cancer cells enhanced the basal apoptosis level and inhibited short and long-term survival. Induction of cell death by UV-C irradiation and chemotherapeutic drugs was also augmented in cells transfected with MIAT specific siRNA. Taken together, the outcome of this study reveals important roles for NEAT1 and MIAT lncRNAs in breast cancer. Future work should explore the potential of these lncRNAs in the development of therapeutic drugs and as diagnostic and prognostic markers.
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Surappa-Narayanappa, Ananth Prakash. "The evolution, modifications and interactions of proteins and RNAs." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/269851.
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Proteins and RNAs are two of the most versatile macromolecules that carry out almost all functions within living organisms. In this thesis I have explored evolutionary and regulatory aspects of proteins and RNAs by studying their structures, modifications and interactions. In the first chapter of my thesis I investigate domain atrophy, a term I coined to describe large-scale deletions of core structural elements within protein domains. By looking into truncated domain boundaries across several domain families using Pfam, I was able to identify rare cases of domains that showed atrophy. Given that even point mutations can be deleterious, it is surprising that proteins can tolerate such large-scale deletions. Some of the structures of atrophied domains show novel protein-protein interaction interfaces that appear to compensate and stabilise their folds. Protein-protein interactions are largely influenced by the surface and charge complementarity, while RNA-RNA interactions are governed by base-pair complementarity; both interaction types are inherently different and these differences might be observed in their interaction networks. Based on this hypothesis I have explored the protein-protein, RNA-protein and the RNA-RNA interaction networks of yeast in the second chapter. By analysing the three networks I found no major differences in their network properties, which indicates an underlying uniformity in their interactomes despite their individual differences. In the third chapter I focus on RNA-protein interactions by investigating post-translational modifications (PTMs) in RNA-binding proteins (RBPs). By comparing occurrences of PTMs, I observe that RBPs significantly undergo more PTMs than non-RBPs. I also found that within RBPs, PTMs are more frequently targeted at regions that directly interact with RNA compared to regions that do not. Moreover disorderedness and amino acid composition were not observed to significantly influence the differential PTMs observed between RBPs and nonRBPs. The results point to a direct regulatory role of PTMs in RNA-protein interactions of RBPs. In the last chapter, I explore regulatory RNA-RNA interactions. Using differential expression data of mRNAs and lncRNAs from mouse models of hereditary hemochromatosis, I investigated competing regulatory interactions between mRNA, lncRNA and miRNA. A mutual interaction network was created from the predicted miRNA interaction sites on mRNAs and lncRNAs to identify regulatory RNAs in the disease. I also observed interesting relations between the sense-antisense mRNA-lncRNA pairs that indicate mutual regulation of expression levels through a yet unknown mechanism.
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Aznaourova, Marina [Verfasser], and Leon [Akademischer Betreuer] Schulte. "Long non-coding RNAs involved in myeloid cell differentiation and macrophage activation / Marina Aznaourova ; Betreuer: Leon Schulte." Marburg : Philipps-Universität Marburg, 2021. http://d-nb.info/1232405868/34.
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Tachtsidi, Alexandra. "Nuclear organization and regulation of gene expression in mouse Embryonic Stem Cells by long non-coding RNAs." Electronic Thesis or Diss., Sorbonne université, 2018. http://www.theses.fr/2018SORUS444.
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Le noyau est extrêmement structuré et il a été démontré que les longs ARN non-codants (lncRNAs) sont impliqués dans l'organisation nucléaire en établissant la compartimentation nucléaire, par les domaines sous-nucléaires ou des interactions à longue portée dans l'espace nucléaire. Une approche robuste pour l'identification de telles molécules fait toutefois défaut. Nous avons mis en place une approche expérimentale permettant d'identifier les lncRNAs «structurels» à l'échelle du génome. Basé sur la propriété biochimique de certains lncRNAs à résister l’extraction de la matrice nucléaire, quand l’ADN et les molécules solubles sont enlevés, nous avons effectué des préparations de matrice nucléaire en utilisant des cellules souches embryonnaires murines, purifié la fraction d’ARN et exploré ses constituants par RNA-seq. Nous avons identifié des transcrits (non-extracted RNAs, nextRNAs) potentiellement impliqués dans l'organisation fonctionnelle du noyau. Nous avons notamment détecté des transcrits précédemment pas annotées et axé notre travail sur deux gènes: NextC1 (Next Candidate 1) et 2. Nous les avons caractérisées au niveau fonctionnel et phénotypique en explorant leur profil d’expression dans différents contextes de culture et lignées cellulaires dérivées de l'embryon. Leur localisation subcellulaire a été évaluée par RNA-FISH. Des analyses de perte et de gain de fonction ont été effectuées en ciblant leurs régions promotrices avec le système CRISPR/Cas9 et les systèmes dérivés de CRISPR pour l'inhibition ou l'activation de la transcription. Nombreuses de ces analyses fonctionnelles ont ensuite été soumis à RNA-seq et une analyse de données intégrative est en coursThe nucleus is a highly structured organelle and long non-coding RNAs (lncRNAs) have been shown to be involved in nuclear organization by establishing and maintaining nuclear compartmentalization, by the formation of subnuclear domains or the establishment of long range interactions in the nuclear space. A robust approach for the identification of “nuclear organizers” molecules is currently lacking though. We established an experimental approach that would allow us to identify such “structural” lncRNAs on a genome-scale level. Based on the biochemical property of some nuclear organizing lncRNAs to resist the so called nuclear matrix preparation, where DNA and soluble molecules are largely removed, we performed nuclear matrix preps on mouse Embryonic Stem Cells (mESCs), purified the RNA fraction and explored its constituents by RNA-seq. We identified a subset of transcripts (non-extracted RNAs, nextRNAs) potentially involved in the functional organization of the nucleus. Notably, we detected previously non-annotated transcripts with our original RNA-seq datasets and focused our work on two of them: NextC1 (Next Candidate 1) and 2. We characterized them on a functional and phenotypical level by monitoring their expression profile in different culturing conditions and embryo-derived cell types. Their subcellular localization was assessed by RNA-FISH. Loss- and gain-of-function assays were performed targeting their promoter regions with the CRISPR/Cas9 system for genome editing and CRISPR-derived systems for transcription inhibition or activation. Many of these functional assays were subsequently RNA-sequenced and an integrative data analysis is currently ongoing
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Basu, Swaraj. "Conservation and synteny of long non-coding RNAs in vertebrate genomes and their identification in novel transcriptomes." Thesis, Open University, 2013. http://oro.open.ac.uk/54717/.
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Long non-coding RNAs (IncRNAs) are a biological entity defined by what they are not, rather than by what they are. This indicates that our knowledge about them is sensibly limited. The aim of my PhD is to gain insights into the evolution and the functions of IncRNAs through computational approaches and the usage of large scale functional genomics dataset. I developed an annotation pipeline, which can effectively identify IncRNAs in entire transcriptomes. The pipeline is able to accurately annotate the coding genes while predicting a conservative estimate of the IncRNA population. It allowed me to show, for the first time, the presence of lncRNA transcription in a diverse range of organisms. Further, I analysed sequence and positional conservation of lncRNAs, demonstrating the presence of short segments of conserved sequence in IncRNAs and the existence of several syntenically conserved non-coding transcripts over large evolutionary distances. However, I also demonstrate that positional conservation of lncRNAs with a flanking coding gene is generally independent from the conservation of the lncRNA expression with respect to the coding gene. Finally, I have characterised the diversity of lncRNA transcription in specific cells and developmental stages of two teleost fishes. In summary, the work presented in the thesis provides novel findings and contributions in the field of lncRNAomics.
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Furió, Tarí Pedro. "Development of bioinformatic tools for massive sequencing analysis." Doctoral thesis, Universitat Politècnica de València, 2020. http://hdl.handle.net/10251/152485.
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[EN] Transcriptomics is one of the most important and relevant areas of bioinformatics. It allows detecting the genes that are expressed at a particular moment in time to explore the relation between genotype and phenotype. Transcriptomic analysis has been historically performed using microarrays until 2008 when high-throughput RNA sequencing (RNA-Seq) was launched on the market, replacing the old technique. However, despite the clear advantages over microarrays, it was necessary to understand factors such as the quality of the data, reproducibility and replicability of the analyses and potential biases.
The first section of the thesis covers these studies. First, an R package called NOISeq was developed and published in the public repository "Bioconductor", which includes a set of tools to better understand the quality of RNA-Seq data, minimise the impact of noise in any posterior analyses and implements two new methodologies (NOISeq and NOISeqBio) to overcome the difficulties of comparing two different groups of samples (differential expression). Second, I show our contribution to the Sequencing Quality Control (SEQC) project, a continuation of the Microarray Quality Control (MAQC) project led by the US Food and Drug Administration (FDA, United States) that aims to assess the reproducibility and replicability of any RNA-Seq analysis.
One of the most effective approaches to understand the different factors that influence the regulation of gene expression, such as the synergic effect of transcription factors, methylation events and chromatin accessibility, is the integration of transcriptomic with other omics data. To this aim, a file that contains the chromosomal position where the events take place is required. For this reason, in the second chapter, we present a new and easy to customise tool (RGmatch) to associate chromosomal positions to the exons, transcripts or genes that could regulate the events.
Another aspect of great interest is the study of non-coding genes, especially long non-coding RNAs (lncRNAs). Not long ago, these regions were thought not to play a relevant role and were only considered as transcriptional noise. However, they represent a high percentage of the human genes and it was recently shown that they actually play an important role in gene regulation. Due to these motivations, in the last chapter we focus, first, in trying to find a methodology to find out the generic functions of every lncRNA using publicly available data and, second, we develop a new tool (spongeScan) to predict the lncRNAs that could be involved in the sequestration of micro-RNAs (miRNAs) and therefore altering their regulation task.[ES] La transcriptómica es una de las áreas más importantes y destacadas en bioinformática, ya que permite ver qué genes están expresados en un momento dado para poder explorar la relación existente entre genotipo y fenotipo. El análisis transcriptómico se ha realizado históricamente mediante el uso de microarrays hasta que, en el año 2008, la secuenciación masiva de ARN (RNA-Seq) fue lanzada al mercado y comenzó a desplazar poco a poco su uso. Sin embargo, a pesar de las ventajas evidentes frente a los microarrays, resultaba necesario entender factores como la calidad de los datos, reproducibilidad y replicabilidad de los análisis así como los potenciales sesgos. La primera parte de la tesis aborda precisamente estos estudios. En primer lugar, se desarrolla un paquete de R llamado NOISeq, publicado en el repositorio público "Bioconductor", el cual incluye un conjunto de herramientas para entender la calidad de datos de RNA-Seq, herramientas de procesado para minimizar el impacto del ruido en posteriores análisis y dos nuevas metodologías (NOISeq y NOISeqBio) para abordar la problemática de la comparación entre dos grupos (expresión diferencial). Por otro lado, presento nuestra contribución al proyecto Sequencing Quality Control (SEQC), una continuación del proyecto Microarray Quality Control (MAQC) liderado por la US Food and Drug Administration (FDA) que pretende evaluar precisamente la reproducibilidad y replicabilidad de los análisis realizados sobre datos de RNA-Seq. Una de las estrategias más efectivas para entender los diferentes factores que influyen en la regulación de la expresión génica, como puede ser el efecto sinérgico de los factores de transcripción, eventos de metilación y accesibilidad de la cromatina, es la integración de la transcriptómica con otros datos ómicos. Para ello se necesita generar un fichero que indique las posiciones cromosómicas donde se producen estos eventos. Por este motivo, en el segundo capítulo de la tesis presentamos una nueva herramienta (RGmatch) altamente customizable que permite asociar estas posiciones cromosómicas a los posibles genes, transcritos o exones a los que podría estar regulando cada uno de estos eventos. Otro de los aspectos de gran interés en este campo es el estudio de los genes no codificantes, especialmente los ARN largos no codificantes (lncRNAs). Hasta no hace mucho, se pensaba que estos genes no jugaban ningún papel fundamental y se consideraban como simple ruido transcripcional. Sin embargo, suponen un alto porcentaje de los genes del ser humano y se ha demostrado que juegan un papel crucial en la regulación de otros genes. Por este motivo, en el último capítulo nos centramos, en un primer lugar, en intentar obtener una metodología que permita averiguar las funciones generales de cada lncRNA haciendo uso de datos ya publicados y, en segundo lugar, generamos una nueva herramienta (spongeScan) que permite predecir qué lncRNAs podrían estar secuestrando determinados micro-RNAs (miRNAs), alterando así la regulación llevada a cabo por estos últimos.
[CA] La transcriptòmica és una de les àrees més importants i destacades en bioinformàtica, ja que permet veure quins gens s'expressen en un moment donat per a poder explorar la relació existent entre genotip i fenotip. L'anàlisi transcriptòmic s'ha fet històricament per mitjà de l'ús de microarrays fins l'any 2008 quan la tècnica de seqüenciació massiva d'ARN (RNA-Seq) es va fer pública i va començar a desplaçar a poc a poc el seu ús. No obstant això, a pesar dels avantatges evidents enfront dels microarrays, resultava necessari entendre factors com la qualitat de les dades, reproducibilitat i replicabilitat dels anàlisis, així com els possibles caires introduïts. La primera part de la tesi aborda precisament estos estudis. En primer lloc, es va programar un paquet de R anomenat NOISeq publicat al repositori públic "Bioconductor", el qual inclou un conjunt d'eines per a entendre la qualitat de les dades de RNA-Seq, eines de processat per a minimitzar l'impact del soroll en anàlisis posteriors i dos noves metodologies (NOISeq i NOISeqBio) per a abordar la problemàtica de la comparació entre dos grups (expressió diferencial). D'altra banda, presente la nostra contribució al projecte Sequencing Quality Control (SEQC), una continuació del projecte Microarray Quality Control (MAQC) liderat per la US Food and Drug Administration (FDA) que pretén avaluar precisament la reproducibilitat i replicabilitat dels anàlisis realitzats sobre dades de RNA-Seq. Una de les estratègies més efectives per a entendre els diferents factors que influïxen a la regulació de l'expressió gènica, com pot ser l'efecte sinèrgic dels factors de transcripció, esdeveniments de metilació i accessibilitat de la cromatina, és la integració de la transcriptómica amb altres dades ómiques. Per això es necessita generar un fitxer que indique les posicions cromosòmiques on es produïxen aquests esdeveniments. Per aquest motiu, en el segon capítol de la tesi presentem una nova eina (RGmatch) altament customizable que permet associar aquestes posicions cromosòmiques als possibles gens, transcrits o exons als que podria estar regulant cada un d'aquests esdeveniments regulatoris. Altre dels aspectes de gran interés en aquest camp és l'estudi dels genes no codificants, especialment dels ARN llargs no codificants (lncRNAs). Fins no fa molt, encara es pensava que aquests gens no jugaven cap paper fonamental i es consideraven com a simple soroll transcripcional. No obstant això, suposen un alt percentatge dels gens de l'ésser humà i s'ha demostrat que juguen un paper crucial en la regulació d'altres gens. Per aquest motiu, en l'últim capítol ens centrem, en un primer lloc, en intentar obtenir una metodologia que permeta esbrinar les funcions generals de cada lncRNA fent ús de dades ja publicades i, en segon lloc, presentem una nova eina (spongeScan) que permet predeir quins lncRNAs podríen estar segrestant determinats micro-RNAs (miRNAs), alterant així la regulació duta a terme per aquests últims.
Furió Tarí, P. (2020). Development of bioinformatic tools for massive sequencing analysis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/152485
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Wagner, Simone [Verfasser], and Bettina [Akademischer Betreuer] Kempkes. "Identification of cellular long non-coding RNAs regulated by the EBV nuclear antigen EBNA2 / Simone Wagner ; Betreuer: Bettina Kempkes." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1199265721/34.
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Hadjicharalambous, Marina. "Characterisation of the role of long non-coding RNAs in lung fibroblasts from control and idiopathic pulmonary fibrosis patients." Thesis, University of Bath, 2018. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.767572.
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Ray, Mridula Kumari. "Long non-coding RNAs interact with PRC1 to impact Polycomb group protein recruitment and expression of Polycomb regulated genes." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11246.
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Long non-coding RNAs (lncRNAs) are increasingly recognized as important regulators of genomic processes and cellular specification. Many lncRNAs regulate chromatin by functionally impacting the epigenetic state through direct interactions with chromatin-modifying proteins. We developed a protocol to enrich for chromatin-lncRNA interactions and used this technique to identify several candidate lncRNAs that interact with the Polycomb group (PcG) proteins. Our immunoprecipitation protocol uses a crosslinked chromatin fraction as the input and employs stringent washes and cross-validation techniques to dramatically decrease mRNA signal (as a metric of transient interactions or false positives), and increase the dynamic range of conventional RNA immunoprecipitation protocols. Applying this protocol to the PRC1 component Bmi1, we have identified 11 PcG-interacting lncRNA candidates whose expression impacts the transcription of many other chromatin factors and PcG targets. We focus on knockdown of one lncRNA candidate, CAT7, which increases expression of several homeobox-containing transcription factors as well as chromatin interacting proteins, including Trithorax group proteins, Jumanji-domain containing proteins, and PcG-like proteins in HeLa cells. Consistent with the observed increase in gene expression, knockdown of CAT7 decreases PcG binding (Suz12, H3K27me3 and Bmi1) at the promoter of the homeodomain protein Mnx1, located at the boundary of an adjacent gene desert. During early motor neuron differentiation from embryonic stem cells, knockdown of CAT7 is accompanied by changes in expression of master regulators of neuronal specification: increased upregulation Mnx1, upregulation of Isl1, and downregulation of Irx3, as well as changes in expression to several other PcG-regulated targets. Overall, this protocol is the first of its kind to efficiently identify de novo interactions between the PcG proteins and lncRNAs which impact PcG binding or PcG target gene expression.
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Uroda, Tina. "Caractérisation structurale et fonctionnelle de l’ARN long non codant MEG3." Thesis, Université Grenoble Alpes (ComUE), 2019. http://www.theses.fr/2019GREAV014.
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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 médicaleLong 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
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Amaral, Murilo Sena. "Identificação de RNAs longos não-codificadores de proteínas regulados por micro-RNAs." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22102014-102412/.
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Estudos recentes têm revelado que a maior parte dos transcritos gerados em células humanas é composta por RNAs não-codificadores de proteínas (ncRNAs). Uma parte desses ncRNAs compreende a classe de RNAs curtos, que possuem menos que 200 nucleotídeos. Os micro-RNAs (miRNAs) fazem parte dessa classe e têm sido alvo de grande interesse, pois são preditos como possíveis reguladores de mais de 60% dos RNAs mensageiros (mRNAs) humanos. Outra classe dos ncRNAs é composta por ncRNAs longos (lncRNAs, com mais de 200 nucleotídeos), que são transcritos a partir de regiões intergênicas e intrônicas do genoma humano e possuem várias funções, muitas delas relacionadas ao controle da expressão de mRNAs. Recentemente, os lncRNAs têm sido caracterizados quanto à sua estrutura e função. No entanto, muito pouco se sabe sobre os mecanismos pelos quais os lncRNAs são regulados. Este trabalho teve como objetivo avaliar se lncRNAs são regulados por miRNAs em células humanas. Para tanto, identificamos lncRNAs ligados ao complexo de silenciamento induzido por RNA (RISC) em células da linhagem HeLa, utilizando um método aqui desenvolvido de geração de bibliotecas de cDNA direcionadas para sequenciamento em larga escala na plataforma 454/Roche. Em paralelo, sequenciamos os miRNAs ligados ao RISC nestas mesmas células. Os resultados obtidos mostram que centenas de lncRNAs de diversas classes se ligam ao RISC em células HeLa, juntamente com milhares de mRNAs e várias centenas de miRNAs. Entre os miRNAs, encontramos 37 que são preditos como alvejando os lncRNAs detectados. Estes miRNAs constituem possíveis reguladores dos lncRNAs e, portanto, nosso trabalho estabelece um mapa experimental de interações diretas entre lncRNAs e miRNAs. Dentre os lncRNAs identificados ligados ao RISC neste trabalho, destaca-se o TUG1, lincRNA sabidamente envolvido na regulação de genes relacionados à apoptose e ao ciclo celular. Mostramos por ensaio de super-expressão de miRNAs e qPCR que TUG1 é regulado pelo miRNA-148b, um dos miRNAs por nós detectados que possui um sítio alvo altamente conservado em mamíferos localizado na extremidade 3\' de TUG1. Em conjunto, este trabalho contribui para o entendimento da regulação dos níveis de expressão de lncRNAs em células humanas e abre perspectivas para a modulação de miRNAs como estratégia de regulação dos níveis e das funções de lncRNAs.Recent studies have revealed that the largest fraction of the transcripts generated in human cells is composed of non-protein coding RNAs (ncRNAs). A portion of these RNAs encompasses the class of short RNAs, which are less than 200 nucleotides in length. Micro-RNAs (miRNAs) are part of this class and are of great interest, as they are predicted to target over 60% of the human messenger RNAs (mRNAs). Another class of ncRNAs is composed of long ncRNAs (lncRNAs, longer than 200 nucleotides), which are transcribed from intergenic and intronic regions of the human genome and have several functions, many of them related to the control of the mRNA expression. Recently, the structure and function of lncRNAs have been characterized. However, little is known about the mechanisms involved in lncRNA regulation. This work aimed to evaluate whether lncRNAs are regulated by miRNAs in human cells. For this purpose, we identified lncRNAs bound to the RNA-induced silencing complex (RISC) in HeLa cells using a method developed here for the generation of strand-specific cDNA libraries for large scale RNA-sequencing in the 454/Roche plataform. In parallel, we sequenced the miRNAs bound to RISC in these cells. Our results show that hundreds of lncRNAs from diverse classes are bound to RISC in HeLa cells, along with thousands of mRNAs and several hundred miRNAs. Among the miRNAs we identified 37 that are predicted to target the detected lncRNAs. These miRNAs are possible regulators of the lncRNAs, and therefore our work establishes an experimental map of direct interactions between lncRNAs and miRNAs. The lncRNA TUG1, a lincRNA involved in the regulation of genes related to apoptosis and cell cycle, was identified among the lncRNAs bound to RISC. We showed by miRNA over-expression and qPCR that TUG-1 is regulated by the miRNA-148b, which is one of the miRNAs detected in our sequencings and has a binding site highly conserved in mammals located at the TUG1 3` end. Taken together, our results contribute to the understanding of the regulation of the lncRNA expression levels in human cells and open perspectives for the modulation of miRNAs as a strategy to regulate the levels and functions of lncRNAs.
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Hösler, Nadine. "Bedeutung nicht-kodierender RNAs im Immunsystem." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-174927.
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Immer mehr Berichte deuten darauf hin, dass nicht-kodierende RNAs an der Regulation des Immunsystems beteiligt sind. In dieser Arbeit wurden nicht-kodierende RNAs identifiziert, die durch zwei unterschiedliche immunologische Prozesse in zwei verschiedenen Zelltypen reguliert wurden. Zum einen wurde das Transkriptom von Multiplen Myelom-Zellen in Abhängigkeit von der Interleukin 6-Stimulation untersucht. Dabei wurden einige sehr lange, IL 6-regulierte macroRNAs identifiziert, die STAIRs (STAT3-induced RNAs). Bei den STAIRs handelt es sich wahrscheinlich um funktionelle, kontinuierliche, nicht-kodierende macroRNAs, die im Zellkern angereichert sind. Einige STAIRs dienen eventuell zusätzlich oder ausschließlich als Primärtranskript für gespleißte, lange ncRNAs (lncRNAs), die weitere Funktionen in der Zelle ausüben können. Die STAIRs weisen eine große Bandbreite an Gewebsspezifität auf und bei den Untersuchungen in dieser Arbeit zeigten sich Hinweise, dass sie sich für verschiedene Krebserkrankungen als Biomarker eignen könnten. Die zweite Transkriptomanalyse wurde bei der Aktivierung naiver T Zellen durchgeführt. Dabei offenbarte sich, dass die Zellen bei diesem Prozess einen dramatischen Wechsel ihres Transkriptionsprogrammes vollziehen und eine Vielzahl nicht Protein-kodierender Gene reguliert werden. Es wurde die Regulation von ncRNAs, die bisher noch nicht im Zusammenhang mit T Zellen beschrieben wurden, beobachtet und erneut unbekannte, differentiell exprimierte Bereiche identifiziert. Im Anschluss wurde STAIR18, eine nicht-kodierende RNA, die durch die beiden untersuchten Signalwege reguliert wird, eingehender untersucht. Es zeigte sich, dass STAIR18 im menschlichen Genom dupliziert ist und beide Loci die gespleißte, lange ncRNA152 in diversen Varianten transkribieren. ncRNA152 ist hauptsächlich im Zytoplasma lokalisiert und befindet sich dort anscheinend in perinukleären Aggregaten. Die verschiedenen ncRNA152-Isoformen scheinen unter-schiedliche Funktionen auszuführen. Einerseits ist eine Wirkung als competing endogenous RNA wahrscheinlich. Eine weitere Aufgabe der ncRNA152 scheint darin zu bestehen, das STAT3-Primärtranskript zu stabilisieren oder dessen Prozessierung zu fördern.
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Kuo, Chao-Chung [Verfasser], Filho Ivan Gesteira [Akademischer Betreuer] Costa, Martin [Akademischer Betreuer] Zenke, and Björn [Akademischer Betreuer] Usadel. "Computational detection of triple helix binding domains in long non-coding RNAs / Chao-Chung Kuo ; Ivan Gesteira Costa Filho, Martin Zenke, Björn Usadel." Aachen : Universitätsbibliothek der RWTH Aachen, 2019. http://d-nb.info/1211487601/34.
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Kuo, Chao-Chung Verfasser], Filho Ivan Gesteira [Akademischer Betreuer] [Costa, Martin [Akademischer Betreuer] Zenke, and Björn [Akademischer Betreuer] Usadel. "Computational detection of triple helix binding domains in long non-coding RNAs / Chao-Chung Kuo ; Ivan Gesteira Costa Filho, Martin Zenke, Björn Usadel." Aachen : Universitätsbibliothek der RWTH Aachen, 2019. http://d-nb.info/1211487601/34.
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Neumann, Philipp [Verfasser], Michaela [Akademischer Betreuer] Müller-McNicoll, Stefanie [Gutachter] Dimmeler, and Michaela [Gutachter] Müller-McNicoll. "Identification and characterization of hypoxia-regulated long non-coding RNAs in endothelial cells / Philipp Neumann ; Gutachter: Stefanie Dimmeler, Michaela Müller-McNicoll ; Betreuer: Michaela Müller-McNicoll." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2018. http://d-nb.info/1208248731/34.
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Bürger, Matheus Carvalho. "Análise transcricional de RNAs não codificadores longos em pacientes com dengue." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-24012018-214001/.
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A dengue é uma infecção viral sistêmica que pode se manifestar clinicamente de diversas formas, desde febres leves a hemorragia e síndrome do choque, condições potencialmente fatais. Diversos estudos já foram publicados investigando as mudanças globais de expressão que ocorrem durante a evolução da doença nesses diferentes quadros clínicos. Porém, nenhum desses estudos analisou o papel dos RNAs não codificadores longos (lncRNAs) na progressão da doença. Neste projeto, foi realizada uma metanálise dos dados de expressão provenientes desses estudos de dengue focando na expressão de lncRNAs e seus possíveis mecanismos de regulação gênica. Foram identificados dezenas de lncRNAs cuja expressão aumenta ou diminui em pacientes infectados com dengue comparado com pessoas saudáveis. Através de análise de \"guilty-by-association\", procurou-se identificar genes codificadores de proteína possivelmente regulados por esses lncRNAs ou genes que os regulem. Nossos resultados fornecem evidência de novos mecanismos de regulação entre lncRNAs e mRNAs.Dengue fever is a systemic viral infection that can manifest clinically in a variety of ways, from mild fever to potentially fatal conditions such as hemorrhage and shock syndrome. Several studies have already been published investigating the global changes in expression that occur during the evolution of the disease in these different clinical settings. However, none of these studies analyzed the role of long non-coding RNAs (lncRNAs) in disease progression. In this project, we performed a meta-analysis of transcriptome data obtained from these dengue studies and focused on the expression of lncRNAs and their possible mechanisms of gene regulation. Dozens of lncRNAs have been identified whose expression increases or decreases in patients infected with dengue compared to healthy individuals. Through guilty-by-association analysis, we identified several lncRNAs that possibly regulate protein coding genes. Our results provide evidence of novel regulatory mechanisms between lncRNAs and mRNAs.
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Yu, Ai-Ming, Magnus Ingelman-Sundberg, Nathan J. Cherrington, Lauren M. Aleksunes, Ulrich M. Zanger, Wen Xie, Hyunyoung Jeong, et al. "Regulation of drug metabolism and toxicity by multiple factors of genetics, epigenetics, lncRNAs, gut microbiota, and diseases: a meeting report of the 21 st International Symposium on Microsomes and Drug Oxidations (MDO)." INST MATERIA MEDICA, CHINESE ACAD MEDICAL SCIENCES, 2017. http://hdl.handle.net/10150/623278.
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Variations in drug metabolism may alter drug efficacy and cause toxicity; better understanding of the mechanisms and risks shall help to practice precision medicine. At the 21st International Symposium on Microsomes and Drug Oxidations held in Davis, California, USA, in October 2-6, 2016, a number of speakers reported some new findings and ongoing studies on the regulation mechanisms behind variable drug metabolism and toxicity, and discussed potential implications to personalized medications. A considerably insightful overview was provided on genetic and epigenetic regulation of gene expression involved in drug absorption, distribution, metabolism, and excretion (ADME) and drug response. Altered drug metabolism and disposition as well as molecular mechanisms among diseased and special populations were presented. In addition, the roles of gut microbiota in drug metabolism and toxicology as well as long non-coding RNAs in liver functions and diseases were discussed. These findings may offer new insights into improved understanding of ADME regulatory mechanisms and advance drug metabolism research. (C) 2017 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.
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Chakraborty, Debojyoti [Verfasser], Frank [Akademischer Betreuer] Buchholz, and Francis [Akademischer Betreuer] Stewart. "Systematic dissection of long non coding RNAs involved in the regulation of embryonic stem cell pluripotency / Debojyoti Chakraborty. Gutachter: Francis Stewart ; Frank Buchholz. Betreuer: Frank Buchholz." Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://d-nb.info/1068445300/34.
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Yunusov, Dinar. "Characterization of HIPSTR highlights the heterogeneous expression pattern of lncRNAs in human embryos and stable cell lines." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22082016-083421/.
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There is a growing appreciation that eukaryotic genomes are transcribed into numerous, previously undetected - and thus uncharacterized regulatory long non-coding RNAs (lncRNAs). Recent studies are primarily focused on lncRNAs transcribed from intergenic regions and enhancers, leaving antisense lncRNAs the least studied group of lncRNAs. At the same time, antisense transcription occurs in up to 74 % of human gene loci, frequently - from the opposite strand of genes encoding proteins involved in regulation of transcription. Here, we identified HIPSTAR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel conserved lncRNA that is transcribed antisense to the TFAP2A gene. Unlike previously reported antisense lncRNAs, HIPSTR expression does not correlate with the expression of its antisense counterpart. Although HIPSTAR and TFAP2A are co-expressed in in vitro derived neural crest and trophoblast cells, only HIPSTAR and not TFAP2A is specifically expressed in a subset of cells within 8-cell- and morula-stage human embryos. We show that, similar to HIPSTAR, in the individual cells of developing human embryos or of stable cell lines the expression of lncRNAs is more highly heterogeneous than the expression of mRNAs. Finally, we demonstrate that HIPSTAR depletion in HEK293 and H1BP, a human embryonic stem cell line, predominantly affects the expression levels of genes involved in early organismal development and cell differentiation. Together, we show that expression of HIPSTAR and hundreds other lncRNAs is highly heterogeneous in human embryos and cell lines. We use HIPSTAR to exemplify the functional relevance of lncRNAs with heterogeneous and developmental stage-specific expression patterns.Tem sido cada vez mais reconhecido que a transcrição dos genomas eucarióticos produz múltiplos transcritos novos, anteriormente não detectados e ainda não caracterizados, sendo que a maioria é constituida de RNAs não-codificantes longos (lncRNAs) regulatórios. Estudos recentes estão focados principalmente nos lncRNAs transcritos de regiões intergênicas e enhancers; assim, o grupo dos lncRNAs antisenso permanece o menos estudado de todos. Ao mesmo tempo, a transcrição antisenso ocorre em até 74% dos loci de genes humanos, frequentemente - a partir da fita oposta de genes que codificam proteínas envolvidas na regulação da transcrição. No presente trabalho, nós identificamos HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), um lncRNA novo conservado que é transcrito a partir da fita antisenso do gene TFAP2A. Ao contrário do anteriormente relatado para os lncRNAs antisenso, a expressão de HIPSTR não está correlacionada com a expressão do gene da fita oposta. HIPSTR e TFAP2A são co-expressos em células da crista neural e em trofoblastos derivadas in vitro, mas somente HIPSTR e não TFAP2A está especificamente expresso num subconjunto de células de embriões humanos nos estágios de 8-células e mórula. Mostramos que, semelhante a HIPSTR, a expressão de lncRNAs é mais altamente heterogênea que a expressão de mRNAs em células individuais de embriões humanos em desenvolvimento ou em linhagens estáveis de células. Finalmente, nós demonstramos que a depleção de HIPSTAR em células HEK293 e H1BP, uma linhagem de células tronco embrionárias humanas, afeta predominantemente os níveis de genes envolvidos no início do desenvolvimento do organismo e na diferenciação de células. No conjunto, nós mostramos que a expressão de HIPSTR e de centenas de outros lncRNAs é altamente heterogênea em embriões humanos e linhagens celulares. Usamos HIPSTR para exemplificar a relevância funcional de lncRNAs com padrões de expressão heterogêneos e estágio-de-desenvolvimento específicos.
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Robinson, Emma. "Control of cardiac remodelling during ageing and disease by epigenetic modifications and modifiers." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275733.
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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.
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Militti, Cristina 1982. "Drosophila UNR regulates dosage compensation through modulation of RNA-protein interactions." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/283476.
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En Drosophila, el desequilibrio en cuanto al contenido de genes ligados al cromosoma X entre hembras (XX) y machos (XY) es corregido mediante la duplicación de la transcripción del único cromosoma X del macho. Este proceso, llamado compensación de dosis, es mediado por un ensamblaje molecular compuesto por al menos cinco proteínas (MSL1, MSL2, MSL3, MLE y MOF) y dos RNAs largos no codificantes (roX1 y roX2), llamado complejo de compensación de dosis (DCC). La compensación de dosis requiere dos condiciones fundamentales: el reconocimiento específico del cromosoma X por el DCC, y la restricción del proceso a moscas macho.
La proteína de unión a RNA Upstream-of-N-Ras (UNR) está implicada en la consecución de ambas condiciones, y aquí hemos estudiado los mecanismos moleculares por los que UNR actúa. Hemos encontrado que, en machos, UNR promueve la compensación de dosis facilitando la asociación de roX2 a MLE, necesaria para una correcta formación del DCC y para su unión al cromosoma X. En hembras, UNR inhibe la compensación de dosis, al menos en parte, promoviendo la unión de SXL al extremo 3’ UTR del mRNA que codifica para msl2, lo que resulta en represión de la traducción de msl2 e inhibición de la formación del DCC.In Drosophila, the imbalance in X-linked gene content between females (XX) and males (XY) is restored through the 2-fold hypertranscription of the single male X-chromosome. This process, which is called dosage compensation, is mediated by the action of the dosage compensation complex (DCC), a ribonucleoprotein assembly composed of at least five proteins (MSL1, MSL2, MSL3, MLE and MOF) and two long non-coding RNAs (roX1 and roX2). Two features are essential for correct dosage compensation: the specific recognition of the X-chromosome by the DCC and the confinement of the DCC function to the male organism. The RNA binding protein Upstream of N-ras (UNR) is involved in the regulation of these two processes and we have dissected the molecular mechanisms by which this regulation occurs. We have found that, in male flies, UNR promotes dosage compensation by facilitating the association of roX2 with MLE, which is required for correct DCC formation and X-chromosome targeting. In female flies, UNR represses dosage compensation in part by enhancing the binding of SXL to the 3’UTR of msl2 mRNA, thus ensuring tight msl2 translational repression and subsequent inhibition of DCC formation.
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Riquier, Sébastien. "Dans les abysses du transcriptome : découverte de nouveaux biomarqueurs de cellules souches mésenchymateuses par analyse approfondie du RNAseq." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTT004.
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Le développement du séquençage ARN, ou RNAseq, a permis l'essor de la recherche intensive de biomarqueurs dans de nombreux domaines de la biologie. L’information complète du transcriptome contenue dans les données de sorties, permet à un bioinformaticien assidu de dépasser les connaissances actuelles et d’accéder, grâce à des pipelines informatiques avancés, à d’innombrables signatures d’intérêts inédites. Dans cette thèse nous mettons en avant que ces marqueurs potentiels, essentiellement explorés pour répondre à des problématiques clinique en conditions pathologiques, peuvent être utilisés pour affiner la caractérisation de types de cellules sans marqueurs strictement spécifiques. Nous nous sommes intéressés aux cellules souches mésenchymateuses (MSCs), un type de cellules souches adultes multipotentes, fortement utilisées en clinique mais ne possédant pas de marqueurs positifs strictement spécifiques.Notre étude se concentre sur la recherche des ARN longs non-codants non annotés. Ces ARNs, aussi nommés "lncRNA", constituent une classe émergente de transcrits encore peu explorée à ce jour. De plus, cette catégorie démontre une spécificité conditionnelle et tissulaire élevée. Nous avons élaboré un pipeline d’analyse RNAseq optimisé pour la reconstruction et la quantification de lncRNAs non annotés.En utilisant les données publiques de RNAseq, venant de différentes sources de MSCs et d'autres types de cellules, nous avons identifié de nouveaux lncRNA non annotés exprimés spécifiquement dans les MSCs.Nous avons développé pour ce projet Kmerator.jl, un outil qui permet de décomposer un transcrit en sous séquences spécifiques (k-mers) afin de chercher et quantifier plus rapidement la signature de nos candidats dans un grand nombre de données RNAseq. Kmerator a également été utilisé dans d'autres applications pour tester la qualité des données RNA-seq disponibles en accés public.Après validation de ces nouveaux biomarqueurs de MSCs par qPCR, nous avons eu recours à plusieurs outils informatiques pour prédire leurs fonctions potentielles. Enfin, nous avons analysé des données RNAseq « single-cell » pour aborder l’hétérogénéité d’expression au sein des populations MSCsThe development of RNA sequencing, or RNAseq, have opened the path of intensive biomarkers research in many areas of biology. The complete information of the transcriptome contained in the output data, allows a bioinformatician to surpass the current knowledge and to access, thanks to advanced computer pipelines, to signatures of new interest. In this thesis, we are showing that these potential markers, classically used in clinical and pathological conditions, can be used to characterize cell types without extensive markers profile. We have studied mesenchymal stem cells, a type of adult multipotent stem cells, strongly used in clinics but without strickly specific positive markers. Our study mainly focuses on the search for non-annotated, long non-coding RNAs. These RNAs, also called "lncRNA", constitute an emerging class of transcripts and are still lightly explored.In addition, this category presents a highly tissue-related specificity. We have developed an optimized RNAseq pipeline for the reconstruction and quantification of non-annotated lncRNAs.Using public data from RNAseq, coming from different sources of MSC and other cell types, we have identified new non-annotated lncRNAs clearly and specifically expressed in MSCs. to complete this project, we developed Kmerator.jl, a bioinformatical tool that allows to decompose a transcript in k-mer, and select specific sub-sequences, in order to search and quantify at a faster rate the signature of our candidates in a large number of RNAseq dataset. After validation of these new biomarkers of MSCs by qPCR, we used several computer tools to predict their potential functions. Finally, we analyzed single-cell RNAseq data to address the heterogeneity of expression within MSC populations
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Sutaria, Dhruvitkumar S. "INVESTIGATION OF DIFFERENTIALLY EXPRESSED NONCODING RNAS IN PANCREATIC DUCTAL ADENOCARCINOMA." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480550158159039.
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Tran, Van Giang. "Régulation de l'expression du gène Igf2 : nouveaux promoteurs et implication de longs ARN non-codants." Thesis, Montpellier 2, 2014. http://www.theses.fr/2014MON20022/document.
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L'expression du gène Igf2, qui est soumis à l'empreinte génomique parentale chez les mammifères, est hautement régulée au cours du développement embryonnaire et de la période périnatale grâce à divers mécanismes transcriptionnels et post-transcriptionnels. Ces mécanismes mettent à contribution de longs ARN non codants produits au sein même du locus, dont le plus connu est l'ARN H19. En utilisant une approche de complémentation génétique par un transgène H19 dans myoblastes H19 KO de souris, nous démontrons l'existence de plusieurs nouveaux promoteurs d'Igf2. L'un de ces promoteurs, qui est conservé chez l'homme, peut être activé par un ARN ectopique antisens d'H19 (lncARN 91H) en dépit d'une méthylation complète de la région de contrôle empreinte située en cis sur le même allèle. Nous montrons également que les lncARN 91H présentent une certaine spécificité tissulaire et que leur transcription peut être initiée à partir des séquences conservées CS4, CS5 et CS9 situées en aval du gène H19. Quant à l'ARN H19, qui est l'ARN non codant majeur du locus, il semble pouvoir réguler ses transcrits antisens dans les myoblastes H19 KO complémentés par le transgène H19, mais surtout il participe activement à la régulation post-transcriptionnelle du gène Igf2 chez la souris. Nous observons en effet qu'il favorise la coupure endoribonucléolytique de l'ARN Igf2 par un mécanisme qui reste à découvrir. Enfin, nous mettons en évidence l'existence d'un l'arrêt de l'élongation de la transcription du gène d'Igf2, pour lequel nous proposons un modèle de régulation faisant intervenir un autre long ARN non codant du locus: le lncARN PIHit. Au-delà des mécanismes qui restent à explorer, nos résultats renforcent l'idée que la structure tridimensionnelle de la chromatine participe à la régulation de l'expression des gènes chez les mammifèresIn mammals, the expression of the Igf2 gene, which is subject to parental genomic imprinting, is tightly regulated during embryonic development and the perinatal period through several transcriptional and post-transcriptional mechanisms. These mechanisms are involving long non-coding RNAs (lncRNAs) produced within the locus; among them the best known is probably the H19 RNA. Using a genetic complementation assay consisting in transfections of an H19 transgene into H19 KO myoblasts, we discovered several novel Igf2 promoters in the mouse. One of these promoters, that is conserved in the human, can be activated by ectopic H19 antisens RNAs (91H lncRNAs) despite a complete methylation of the Imprinting-Control Region located in cis on the same allele. We also show that the 91H lncRNAs possess some tissue-specific features and that their transcription can be initiated from the CS4, CS5 and CS9 conserved sequences located downstream of the H19 gene. On the other hand, the H19 RNA, that is the major lncRNA of the locus, appears to regulate its antisense transcripts in H19 KO myoblasts complemented with the H19 transgene, but its major function seems to be in regulating post-transcriptionally the Igf2 gene expression. Indeed, we have observed that it favours the endoribonucleolytic cleavage of the Igf2 messenger RNAs through a mechanism that remains to be elucidated. Finally, we reveal the existence of a premature transcriptional elongation stop of the Igf2 gene, for which we propose a regulation model involving another lncRNA of the locus: the PIHit lncRNA. Beyond the mechanisms that remain to be explored, our results strengthen the idea that, in mammals, the three-dimensional organization of the chromatin is involved in regulating gene expression
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Chadourne, Manon. "La suppression de Topaz1 perturbe la méiose et l'expression des ARN non-codant longs testiculaires au cours de la spermatogenèse murine. Topaz1, an Essential Gene for Murine Spermatogenesis, Down-Regulates the Expression of Many Testis-Specific Long Non-Coding RNAs." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL021.
Full textAbstract:
Topaz1 (pour Testis and Ovary specific PAZ domain gene 1) est un gène très conservé chez les vertébrés qui présente une expression spécifique dans les cellules germinales. La caractérisation du modèle murin dépourvu du gène Topaz1 a mis en évidence son rôle indispensable pour la fertilité mâle. Les souris mutantes présentent un arrêt de la spermatogenèse lors de la première division méiotique. Les spermatocytes Topaz1-/- bloqués en fin de prophase I de méiose présentent un défaut d’alignement des chromosomes le long de la plaque métaphasique. D'un point de vue histologique, les différences observées entre les testicules de souris Topaz1-/- et Topaz1+/+ apparaissent entre 15 (P15) et 20 (P20) jours après la naissance. Une première analyse transcriptomique par puce à ADN a montré que 10% des gènes dérégulés (DEGs) à P20 sont des ARN non codants longs (lncRNAs). Au cours de cette thèse, des analyses transcriptomiques à haut débit (RNAseq) ont été réalisées à P16 et P18 afin de mieux caractériser le phénotype testiculaire des souris dépourvues du gène Topaz1. Dès P16, le transcriptome testiculaire est perturbé et le nombre de DEGs est multiplié par 10 à P18. Des gènes associés au centrosome, centriole, à la dynamique des microtubules et à la spermatogenèse appartiennent aux voies moléculaires les plus perturbées. De plus, un quart des DEGs sont des lncRNAs. Trois d'entre eux, dérégulés à P16 et à P18, ont été étudiés par hybridation in situ et biologie moléculaire et sont spécifiques des cellules germinales. Puis une lignée de souris exempt d'un de ces lncRNA a été générée grâce à la technologie CripsR/Cas9. Ces animaux mutants se développent normalement et sont fertiles pour les deux sexes.Néanmoins, les souris mâles mutantes présentent une diminution de plus de 50% de la concentration de spermatozoïdes épipidymaires ainsi qu’une modification de paramètres de motilité par rapport à des souris sauvages. Des nouvelles analyses RNAseq ont été réalisées afin d'étudier le transcriptome testiculaire de ces souris. Elles mettent en évidence que ce lncRNA régule un nombre important de gènes codants pour les protéines (environ 80% des DEGs à P18). Là encore, certains d'entre eux régulent la dynamique des microtubules, la spermatogenèse et la génération des gamètes haploïdes.En conclusion, le gène Topaz1 murin est donc essentiel pour la mise en place d’un fuseau bipolaire en fin de prophase I de méiose et son absence empêche la première division méiotique. La dérégulation d’un nombre important de gènes codants pour des protéines du centrosome, des microtubules et de la spermatogenèse ainsi que la forte répression de l'expression de lncRNAs au sein du testicule murin laisse à penser que des complexes ARNs-protéines se forment au cours de la méiose.Dans cette étude, la suppression d'un de ces lncRNA ne perturbe pas la fertilité des souris bien que la concentration spermatique soit réduire de moitié. Chez l'homme, une telle baisse pourrait conduire à des infertilités masculines. Une mutation du gène Topaz1 chez l'homme pourrait aussi induire une azoospermie non obstructive. L'étude des complexes ARN-protéines pourrait représenter un nouveau champ d’investigation dans l’étude des infertilités et notamment de la régulation de la méioseTopaz1 (Testis and Ovary specific PAZ domain gene 1), a germ cell specific factor, is a highly conserved gene in vertebrates. The study of the Topaz1-inactivation mouse model demonstrated its essential role for male fertility. The absence of Topaz1 in mutant mice caused spermatogenesis arrest during the first meiotic division. Topaz1-/- spermatocytes, blocked at the end of meiotic prophase I, showed chromosome misalignment along the metaphase I plate. Histological experiments specified that the differences observed between Topaz1-/- and Topaz1+/+ mouse testes appeared between 15 (P15) and 20 (P20) days post-partum. Previously, transcriptomic analyses using a whole-genome expression array indicated that 10% of P20-deregulated genes (DEGs) were long non-coding RNAs (lncRNAs). During this thesis, high throughput transcriptomic analyses (RNAseq) were performed at P16 and P18 in order to better characterise the testicular phenotype of mice lacking the Topaz1 gene. From P16, the testicular transcriptome was disturbed and the DEGs number was multiplied by 10 at P18. Genes associated with centrosome, centriole, microtubule dynamics and spermatogenesis belonged to the most disturbed molecular pathways. Moreover, a quarter of DEGs were lncRNAs. Three of them, deregulated at P16 and P18, were studied by in situ hybridization and molecular biology techniques. They were germ cell specific. Thus, a new mouse model deleted for one of these lncRNAs was generated using CRISPR/Cas9 technology. These mutant mice developed normally and were fertile in both sexes. However, mutant male mice presented a more than 50% decrease in the epididymal sperm concentration as well as a change in motility parameters compared to wild-type mice. New RNAseq analyses were realised to study testicular transcriptome of these mice. These showed that this lncRNA regulates a large number of protein-coding genes (approximately 80% of the DEGs at P18). There again, some of them regulated microtubule dynamics, spermatogenesis and haploid gamete generation.In conclusion, this work shows that the murine Topaz1 gene is therefore essential for the establishment of the bipolar spindle during the transition from late prophase I to metaphase I and its absence prevents the first meiotic division. The deregulation of a significant number of protein-coding genes of the centrosome, microtubule movements and spermatogenesis, as well as the strong repression of lncRNAs expression within mouse testis, suggests that RNAs-proteins complexes are formed during meiosis.In this study, deletion of one of these lncRNA did not affect fertility in mice even though sperm concentration was halved. In men, such a decrease could lead to male infertility. A mutation of the Topaz1 gene in men could also induce non-obstructive azoospermia. The study of RNAs-proteins complexes could represent a new field of investigation in the understanding of infertility, particularly in meiotic regulation
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Andric, Vedrana. "Study of the mechanisms of sexual differentiation in the fission yeast Schizosaccharomyces pombe Formation of S. pombe Erh1 homodimer mediates gametogenic gene silencing and meiosis progression A scaffold lncRNA shapes the mitosis to meiosis switch." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL056.
Full textAbstract:
Chez la levure fissipare Schizosaccharomyces pombe, un sous-ensemble de gènes méiotiques est transcrit de manière constitutive au cours de la mitose. Afin d’éviter l'expression prématurée du programme méiotique et l'initiation de la différenciation sexuelle, les cellules ont développé un système de dégradation de l'ARN qui élimine sélectivement les transcrits méiotiques correspondants. Ce processus nécessite la protéine de liaison à l'ARN Mmi1 (à domaine YTH), qui reconnaît en cis les molécules d'ARN (motifs UNAAAC) et les cible pour la dégradation par l'exosome nucléaire. Au début de la méiose, Mmi1 est séquestrée au sein d’une particule ribonucléoprotéique composée de la protéine de liaison à l'ARN Mei2 et du long ARN non-codant (lncRNA) meiRNA, permettant ainsi l'expression des gènes méiotiques et le déroulement de la méiose. Mon travail de thèse a consisté à étudier les mécanismes par lesquels Mmi1 assure la dégradation des transcrits méiotiques et qui régulent son activité au cours des cycles mitotiques et méiotiques. Pendant la croissance végétative, Mmi1 s'associe étroitement à la protéine conservée Erh1 pour former le complexe hétérotétramérique Erh1-Mmi1 (EMC) qui est essentiel pour la dégradation des transcrits méiotiques. Par des approches de biologie structurale et de biochimie, nous avons montré qu'Erh1 s'assemble en homodimère in vitro et in vivo, en accord avec des analyses récentes. Des mutations qui empêchent l'homodimérisation d'Erh1 mais préservent son interaction avec Mmi1 entraînent l'accumulation de transcrits méiotiques en raison d'un défaut de liaison de Mmi1 à ses cibles ARN. L'homodimérisation d’Erh1 est également nécessaire pour séquestrer Mmi1 dans le complexe Mei2-meiRNA et assurer la progression de la méiose. Ainsi, l'assemblage d’EMC est essentiel pour la reconnaissance et la dégradation des transcrits méiotiques par Mmi1 dans les cellules mitotiques et contribue à l'inactivation de cette dernière au début de la méiose. Des travaux antérieurs ont montré que, pendant la croissance végétative, Mmi1 recrute le complexe Ccr4-Not pour ubiquitinyler et limiter l’accumulation de son propre inhibiteur Mei2, maintenant ainsi son activité dans la dégradation des ARNs méiotiques. Nous avons identifié un lncRNA, différent de meiRNA et appelé mamRNA (Mmi1- and Mei2-associated RNA), qui sert de plateforme à Mmi1 pour cibler Mei2 vers le complexe Ccr4-Not. Réciproquement, lorsque cette régulation négative de Mei2 est défectueuse, mamRNA est nécessaire pour l'inactivation de Mmi1 par les niveaux élevés de Mei2. Des expériences d’hybridation in situ par fluorescence en molécules uniques (smFISH) ont également montré que mamRNA est localisé dans un corps nucléaire contenant Mmi1, suggérant que le contrôle mutuel de Mmi1 et Mei2 est confiné dans l’espace. mamRNA peut également relayer meiRNA pour inhiber Mmi1 et favoriser la progression de la méiose. mamRNA apparait donc comme un régulateur critique des activités de Mmi1 et Mei2 pour ajuster la dégradation des ARNs méiotiques et modeler la transition de la mitose vers la méioseIn the fission yeast S. pombe, a subset of meiosis-specific genes is constitutively transcribed during the mitotic cell cycle. To prevent untimely expression of the meiotic program and premature initiation of sexual differentiation, cells have evolved an RNA degradation system that selectively eliminates the corresponding meiotic transcripts. This process requires the YTH-family RNA-binding protein Mmi1, which recognizes cis-elements within RNA molecules (UNAAAC motifs) and targets them for degradation by the nuclear exosome. At the onset of meiosis, Mmi1 is sequestered in a ribonucleoparticle composed of the RNA-binding protein Mei2 and the long non-coding RNA (lncRNA) meiRNA, thereby allowing expression of meiotic genes and meiosis progression. My PhD work consisted in studying the mechanisms by which Mmi1 promotes the degradation of meiotic transcripts and how its activity is regulated during both the mitotic and meiotic cell cycles. During vegetative growth, Mmi1 tightly associates with the evolutionarily conserved Erh1 protein to form the heterotetrameric Erh1-Mmi1 complex (EMC) that is essential for the degradation of meiotic transcripts. Using biochemical and structural approaches, we have shown that Erh1 assembles as a homodimer in vitro and in vivo, consistent with recent analyses. Mutations that disrupt Erh1 homodimerization but preserve interaction with Mmi1 result in the accumulation of meiotic transcripts due to inefficient binding of Mmi1 to its RNA targets. Erh1 homodimerization is also required for Mmi1 luring by the Mei2-meiRNA complex and meiosis progression. Thus, EMC assembly is essential for the recognition and degradation of meiotic transcripts by Mmi1 in mitotic cells and contributes to Mmi1 inactivation at meiosis onset. Previous work showed that, during vegetative growth, Mmi1 recruits the conserved Ccr4-Not complex to ubiquitinylate and downregulate a pool of its own inhibitor Mei2, thereby maintaining its activity in meiotic RNA degradation. We have identified a lncRNA, different from meiRNA and termed mamRNA (Mmi1- and Mei2-associated RNA), to which Mmi1 associates to target Mei2 to the Ccr4-Not complex. Conversely, when Mei2 downregulation is impaired, mamRNA is necessary for Mmi1 inactivation by increased Mei2 levels. Single molecule RNA FISH experiments also indicated that mamRNA localizes to a nuclear body enriched in Mmi1, suggesting that the mutual control of Mmi1 and Mei2 is spatially confined. mamRNA can also take over meiRNA to inhibit Mmi1 and promote meiosis progression. Therefore, mamRNA emerges as a critical regulator of Mmi1 and Mei2 activities to fine tune meiotic RNA degradation and shape the mitosis to meiosis transition
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Laugier, Laurie. "Identification de marqueurs de susceptibilité dans les formes chroniques de la maladie de Chagas." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0226.
Full textAbstract:
La maladie de Chagas est une maladie parasitaire causée par le protozoaire Trypanosoma cruzi et transmise par des insectes hématophages . Elle est composée de 2 phases : la phase aiguë et la phase chronique. Parmi les individus infectés, 30 % développent la forme chronique de la maladie. Les patients présentent des atteintes cardiaques, digestives (œsophage, côlon) et cardiodigestives. Notre étude a été focalisée sur les patients atteints de cardiomyopathie chagasique (CCC). Notre objectif est d’identifier des gènes de susceptibilité pouvant être impliqués dans le développement des formes chroniques. Notre étude a permis de mettre en évidence une variation d’expression de certains gènes entre les CCC et les contrôles. Nous nous sommes également intéressés aux processus épigénétiques pouvant réguler l’expression des gènes. Une étude de la méthylation de l’ADN croisée avec l’étude du transcriptome nous ont permis d’identifier des gènes présentant à la fois des variations d’expression et de méthylation. Pour certains de ces gènes, nous avons démontré que la méthylation est responsable de la variation d’expression observée. Enfin, nous avons étudié un ARN long non-codant, MIAT. Nous avons démontré qu’il est surexprimé chez les CCC par rapport aux contrôles et dans un modèle murin infecté par T. cruzi. De plus, l’analyse de l’expression de micro-ARNs couplée à une analyse de transcriptome nous a permis d'identifier plusieurs micro-ARNs indispensables à la régulation de l’expression des gènes. Enfin, une étude protéomique nous a permis de mettre en évidence une augmentation de la production de protéine pour certains gènes, en lien avec l’augmentation de l’expression observéeChagas disease is a parasitic disease caused by the protozoan Trypanosoma cruzi and transmitted by the hematophagous insects. The disease is composed by acute and chronic phases. Among the infected individuals, 30 % develop chronic form. They suffer from heart, digestive (esophagus, colon) and cardiodigestives injury. Our study was focused on patients with dilated chagasic cardiomyopathy (CCC). Our goal is to identify susceptibility genes that may be involved in the development of chronic forms. Our study revealed a variation in the expression of certain genes between CCC group and controls. We are also interested in epigenetic processes that can regulate the expression of genes. A study of the DNA methylation crossed with the transcriptome allowed us to identify genes presenting both variations in expression and methylation. For some of these genes we demonstrated that methylation is responsible for the expression variation observed. Finally, we studied a long non-coding RNA called MIAT. Our study demonstrated that it is overexpressed in CCC compared to controls and in a murine model infected by T. cruzi. Furthermore, the analysis of the expression of micro-RNAs crossed with transcriptome analysis allowed us to identify several micro-RNAs whose functions are essential in the regulation of gene expression. Finally, a proteomic study allowed us to demonstrate an increase in the production of protein for certain genes, correlated with the increase in expression levels observed