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1
Engreitz, Jesse M. (Jesse Michael). "Genome regulation by long noncoding RNAs." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104616.
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Thesis: Ph. D. in Bioinformatics and Integrative Genomics, Harvard-MIT Program in Health Sciences and Technology, 2016.Cataloged from PDF version of thesis.
Includes bibliographical references.
Our genomes encode the molecular information that gives rise to life, yet we are just beginning to unravel how this information is organized, interpreted, and regulated. While the human genome contains -20,000 protein-coding genes, mammalian genomes also produce thousands of long non-coding RNAs (lncRNAs), some of which are now known to play essential roles in diverse biological processes including cellular differentiation and human disease. Recent studies show that many lncRNAs localize to the nucleus and interact with chromatin regulatory complexes, suggesting that some lncRNAs may represent a crucial missing component in our understanding of genome regulation. To test whether lncRNAs localize to and regulate specific sites in the genome, we developed genome-wide approaches to map lncRNA interactions with chromatin. Through studies of three conserved lncRNAs, we demonstrate that lncRNAs can exploit the three-dimensional architecture of the genome to identify their regulatory targets and, in turn, actively manipulate genome architecture to form subcompartments containing co-regulated genes. Thus, lncRNAs have unique capabilities as dynamic regulators that can locally amplify epigenetic signals. We next explored whether this model might apply to other long noncoding RNAs, many of which are not conserved across species and thus whose functions remain unclear. Through genetic dissection of their local regulatory functions, we show that many of these genomic loci participate in the local regulation of gene expression, but that these functions do not involve the IncRNA transcripts themselves. Instead, multiple mechanisms associated with RNA production including their promoters, the process of transcription, and RNA splicing - act in local networks of regulatory connections between spatially proximal genes, both protein-coding and noncoding. These findings reveal novel mechanistic explanations for the functions and evolution of noncoding transcription in mammalian genomes. Together these studies suggest a model in which mammalian gene regulation is organized into local neighborhoods defined by the spatial architecture of the genome. Within these neighborhoods, lncRNAs and DNA regulatory elements may function cooperatively to coordinate local gene expression. Dissecting this fundamental model for genome regulation may enable manipulation of the processes that interpret our genome sequence and galvanize efforts to develop new treatments for human disease.
by Jesse M. Engreitz.
Ph. D. in Bioinformatics and Integrative Genomics
2
Lagarde, Julien. "Genomic Characterization of Human Long Noncoding RNAs." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/668687.
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The human genome contains an astonishingly large fraction of noncoding DNA, which is pervasively transcribed into thousands of long noncoding RNAs (lncRNAs) -- long transcripts with no discernible protein-coding potential. However, little is known about lncRNAs' biological functions, and their genome annotations show evident signs of inadequacy: existing gene models are sketchy, and many lncRNAs remain uncatalogued. This annotation incompleteness hampers lncRNA functional characterization, notably by failing to accurately describe gene boundaries. To address this issue, the present work aims to advance towards a complete and accurate annotation of lncRNA genes in the human genome. Using a high-throughput, targeted long-read transcriptome sequencing methodology, this study uncovers thousands of novel lncRNAs, approximately doubling the annotated transcript complexity within targeted loci. The method presented vastly outperforms competing techniques in accuracy, and precisely maps many previously unknown, strongly supported lncRNA transcript boundaries. This augmented catalog provides the most definitive view of the genomic properties of lncRNAs to date, while contributing a robust foundation for future lncRNA functional characterization.
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Mohammad, Faizaan. "Long Noncoding RNA Mediated Regulation of Imprinted Genes." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-128882.
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Genomic imprinting is an epigenetic phenomenon that causes a subset of mammalian genes to be expressed from only one allele in a parent-of-origin manner. The defects in the imprinting regulation result in disorders that affect development, growth and metabolism. We have used the Kcnq1 imprinted cluster as a model to understand the mechanism of imprinted gene regulation. The imprinting at the Kcnq1 locus is regulated by a long noncoding RNA, Kcnq1ot1, whose transcription on the paternal chromosome is associated with the silencing of at least eight neighboring genes. By destabilizing Kcnq1ot1 in an episomal system, we have conclusively shown that it is the RNA and not the process of transcription that is required for the gene silencing in cis. Kcnq1ot1 RNA interacts with the chromatin modifying enzymes such as G9a and Ezh2 and recruits them to imprinted genes to establish repressive chromatin compartment and gene silencing. Using the episomal system, we have identified an 890 bp silencing domain (SD) at the 5’ end of Kcnq1ot1 RNA, which is required for silencing of neighboring reporter genes. The deletion of the SD in the mouse resulted in the relaxation of imprinting of ubiquitously imprinted genes (Cdkn1c, Kcnq1, Slc22a18, and Phlda2) as well as reduced DNA methylation over the somatic DMRs associated with the ubiquitously imprinted genes. Moreover, Kcnq1ot1 RNA interacts with Dnmt1 and recruits to the somatic DMRs and this recruitment was significantly affected in the SD mutant mice. By using a transgenic mouse, we have conditionally deleted Kcnq1ot1 promoter at different developmental stages and demonstrated that Kcnq1ot1 maintains imprinting of the ubiquitously imprinted genes by regulating DNA methylation over the somatic DMRs. Kcnq1ot1 is dispensable for the maintenance of repressive histone marks and the imprinting of placental-specific imprinted genes (Tssc4 and Osbpl5). In conclusion, we have described the mechanisms by which Kcnq1ot1 RNA establishes and maintains expression of multiple imprinted genes in cis.
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Richards, Edward J. "Function of Long Noncoding RNAs in Breast Cancer." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/5767.
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Breast cancer is a disease that will be diagnosed in about 1 in 10 women throughout their lifetime. The majority of breast cancers are originated from the epithelial cells of the mammary ducts, and this occurrence can be due to several factors including hereditary and acquired mutation. There are several major breast cancer subtypes, including estrogen receptor-α (ERα)-positive, HER2-enriched and triple-negative (TNBC). Patients diagnosed with ER+ tumors are generally treated with estrogen blockers (e.g., tamoxifen, letrozole and fulvestrant). Patients with HER2+ tumors are commonly administered with drugs that block HER2 signaling (e.g., trastuzumab) or inhibit HER2’s tyrosine kinase activity (e.g., lapatinib). For patients with TNBC, chemotherapies such as taxanes and anthracyclines are standard of care therapies.
However, for each breast cancer subtype, a significant number of patients develop resistance to these therapies and eventually die from metastasis, a process which accounts for ~90% of breast cancer mortality. Currently, metastatic breast cancer is incurable, and the short median survival of 3 years for patients with metastatic breast cancer has not significantly changed in over 20 years. Therefore identification of new molecules that are involved in breast cancer metastasis and development of more precisely targeted therapeutic strategies are urgently needed to improve the clinical outcome for this disease.
The transforming growth factor pathway beta (TGFβ) pathway has been show to play a key role in metastasis through induction of epithelial-mesenchymal transition (EMT), cell migration and invasion. Over more than a decade, this pathway has been studied across several cancers and it is now better established that it has context-dependent tumor suppressive and oncogenic qualities. In the early stages of breast cancer, TGFβ pathway is a suppressor of benign and early stage tumor growth. However, as disease progresses and corresponding levels of TGFβ ligands become elevated, a “switch” will take place and promote oncogenic phenotypes like EMT and cancer cell stemness which drive metastasis.
Long noncoding RNAs (lncRNAs) are an emerging subclass of RNA molecules in cancer biology. LncRNAs are >200nt and can influence target gene expression locally in “cis”, or along a distant chromosome in “trans”, through various mechanisms and interactions with other biological molecules. The contribution of TGFβ-regulated lncRNAs to associated phenotypes like EMT and cancer cell stemness has not been very well studied. The aim of this doctoral dissertation is to address the functional and mechanistic roles of lncRNAs in these processes. Using a well-established TGFβ-induced EMT model (e.g., mouse mammary epithelial cell NMuMG treated with TGFβ, we have identified 3 conserved lncRNAs (lncRNA-HIT, WDFY3-AS2 and TIL) that are significantly upregulated upon TGFβ-induced EMT. They all mediate TGFβ-induced EMT, cell migration and invasion. Overexpression of these lncRNAs is frequently detected during the breast cancer progression and is associated with high grade and late stage of breast cancer as well as metastatic lesion. We have also demonstrated that lncRNA-HIT positively regulates HOXA13 through “cis” mechanism and that WDFY3-AS2 induces WDFY3 and STAT3 expression at mRNA level by direct interaction with hnRNP-R. Interestingly, TIL stimulates C-MYC protein but not mRNA expression by promoting Akt phosphorylation of NF90 leading to its translation from the nucleus to cytosol where NF90 binds to C-MYC mRNA and enhances C-MYC translation. Importantly, we have shown that knockdown of lncRNA-HIT and WDFY3-AS2 significantly reduces breast cancer growth and lung metastasis in orthotopic breast cancer model. These findings indicate that these TGF-induced lncRNAs play critical role in EMT, metastasis, and are relevant in human patient tumors. Therefore, it is important to consider utilizing these molecules for clinical applications like diagnosis, monitoring recurrence, predicting a response to therapy, and even as a direct target for therapeutic intervention.
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Geisler, Sarah J. "Decapping of Long Noncoding RNAs Regulates Inducible Genes." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1340141951.
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Young, Rob. "Evolution and function of long noncoding RNAs in Drosophila." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:0eee0960-fe31-41ca-a6f9-0b29e0b9fed9.
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Not all transcribed DNA encodes protein, and some of these noncoding RNAs (ncRNAs), such as roX1 and roX2, may play important roles in the cell. The functional roles of the majority of these, however, remain largely unknown. In this thesis, I first used EST and mRNA evidence to define 2,788 lincRNA loci within the Drosophila melanogaster genome. I suggest that up to 1,652 of these are functional, as 1,411 show evidence for significant evolutionary constraint while 241 fast-evolving loci are enriched in short RNA species. A distinct set of 1,119 lincRNA loci were defined by RNA-seq, the vast majority of which show clear primary sequence constraint. Their expression profiles and enrichment in particular chromatin domains indicate that these lincRNAs are likely involved in developmental regulation. I also identified 42 potential analogous lincRNAs with shared genomic locations between Drosophila and mouse. Constrained, non-embryonic lincRNAs defined by ESTs are transcribed preferentially in the vicinity of protein-coding genes encoding transcription factors and I demonstrated that one of these, which I name dEvf-2, positively regulates the expression of its genomically adjacent transcription factor, Dll, in cell culture. Finally, I used a reverse genetics approach to search for lincRNA promoter mutations and examined the effect of these on lincRNA expression. My findings suggest that many, previously unknown, functional lincRNAs exist within the Drosophila genome and are worthy of further in-depth experimental investigation.
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Liapis, Stephen Constantine. "Discovery and In Vivo Characterization of Long Noncoding RNAs." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493297.
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The noncoding genome, or the portion of the genome that does not encode for proteins, encompasses >95% of the human genome. It has been found that the majority of disease-associated genetic variants identified by genome-wide association studies (GWAS) are located in this noncoding 95%, where they have the potential to affect regions that control transcription (promoters, enhancers) and noncoding RNAs that also can influence gene expression. The discovery of these alterations has already contributed to a better understanding of the etiology of human diseases and has begun to yield insight into the function of these noncoding loci I am interested in studying how the noncoding genome functions and contributes to human development and disease pathology, especially when it is considered that our understanding of human disease is almost entirely contained within the realm of the <5% of the genome that is protein coding. Toward this end, I have focused my studies on one part of the noncoding genome, long noncoding RNAs. In order to identify whether long noncoding RNAs are important for mammalian development and disease, our lab created a set of lincRNA knockout animal models in which a cassette expressing beta-galactosidase (lacZ) replaces the lincRNA DNA sequence. I have used these models for the in vivo characterization of several lincRNAs, including Fendrr in the lungs, Brn1b in the brain, Tug1 in the testes, and Cox2 in the innate immune system. Each of these studies reveals perturbations in development induced by loss of function of the respective lincRNA locus, and demonstrates promising potential for further examination of the role these molecules play in human disease.Biology, Molecular and Cellular
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Jacob, Mathieu. "Functional Remodelling of the Nucleolus by Long Noncoding RNA." Thesis, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/30288.
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The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Though it is primarily known as the site of ribosomal biogenesis, the nucleolus is also capable of orchestrating the immobilization of a broad range of proteins under specific environmental conditions. This process, known as nucleolar sequestration, contributes to cell viability under stress. Despite the importance of this post-translational regulatory pathway, very little is known about the mechanisms that govern it. Here, we show that heat shock and acidosis, two physiological stimuli associated with nucleolar sequestration, induce the expression of long noncoding RNA (lncRNA) from stimulus-specific loci of the ribosomal intergenic spacer (IGS). These lncRNAs, in turn, immobilize proteins encoding a nucleolar detention sequence (NoDS) within a compartment of the nucleolus termed the detention centre (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate (ANS)-positive hydrophobic signature. Its formation is accompanied by a redistribution of nucleolar factors and an arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA, by environmental signals, operates as a molecular switch that regulates the structure and function of the nucleolus.
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Chodroff, Rebecca A. "Characterization of long noncoding RNAs in vertebrate brain development and evolution." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558292.
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Background: Long considered to be the building block of life, it is now apparent that protein is only one of many functional products generated by the eukaryotic genome. Indeed, more of the mammalian genome is transcribed into noncoding than into protein-coding sequence. This extensive and interleaved network of noncoding transcripts partially emerges from evolutionarily conserved genomic sequence, emphasizing its potential biological relevance. Nevertheless, the biological function of the vast majority of eutherian long intergenic noncoding RNAs (lincRNAs) has not been experimentally validated and the relationship between evolutionary sequence constraint and the role of non coding transcribed sequence remains unclear. To clarify the implications of evolutionary sequence conservation for biological function of lincRNAs, this thesis aims to (l) determine whether evolutionary sequence conservation is more often directed towards particular regions involved in the transcription of lincRNA loci, such as promoter and/or intron-exon boundaries; (2) identify and characterise the tissue expression patterns of lincRNA orthologs across diverse amniotes, ranging from mouse to chicken; and (3) describe the biological functions (if any) of four highly conserved brain-expressed lincRNAs. Results: Here, we performed a multi-disciplinary study of four highly conserved and brain-expressed transcripts selected from a list of mouse long intergenic noncoding RNA (lincRNA) loci that generally show pronounced evolutionary constraint within their putative promoter regions and across exon-intron boundaries. We identified some of the first lincRNA orthologs present in birds (chicken), marsupial (opossum) and eutherian mammals (mouse), and investigated whether they exhibit conservation of brain expression. In contrast to conventional protein-coding genes, the sequences, transcriptional start sites, exon structures and lengths for these noncoding genes are all highly variable. In a series of preliminary experiments, we found that these lincRNAs do not significantly contribute to global transcriptional regulation within a mouse cell line. Furthermore, a transgenic mouse model with a targeted deletion of one of these lincRNAs did not present a noticeable phenotype, suggesting that this lincRNA is not critical for survival. Conclusions: We identified four lincRNAs with evolutionary conservation in ex on structure and transcription, and similarities in brain expression pattern during embryonic and early postnatal stages across diverse amniotes. While tissue-specific expression patterns and evolutionary sequence constraint are suggestive of function, preliminary experiments investigating each transcripts' role did not provide significant proof for biological function. Nevertheless, the high levels of evolutionary sequence conservation and specific brain expression patterns among these four lincRNAs warrant further experimental inquiry.
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Keniry, Andrew James. "H19 and miR-675 : a long noncoding RNA conceals a growth suppressing microRNA." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609990.
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Pandey, Radha Raman. "Molecular Insights into Kcnq1ot1 Noncoding Antisense RNA Mediated Long Range Transcriptional Gene Silencing." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9392.
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Häfner, Sophia Julia. "Study of X-inactivation independent functions of the conserved long noncoding RNA Ftx." Paris 7, 2014. http://www.theses.fr/2014PA077015.
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Mon travail de thèse se focalise sur l'étude du 1ARNnc Ftx, dont le gène est situé dans le centre d'inactivation du chromosome X , région riche en gènes codant pour des IARNncs et responsable du processus d'inactivation du chromosome X chez les mammifères femelles. Le laboratoire a montré que l'expression de Ftx favorise l'expression des gènes voisins, lui conférant ainsi un rôle activateur dans le processus d'inactivation. Ftx est également exprimé dans l'organisme murin adulte, plus spécifiquement dans le cerveau, suggérant ainsi des fonctions indépendantes du processus d'inactivation. Ainsi, je me suis focalisée sur l'étude de l'implication potentielle d( Ftx dans le développement et/ou le fonctionnement du cerveau. L'expression de Ftx dans le cerveau est relativement homogène entre différentes régions, en revanche elle s'instaure que durant la période postnatale, plus précisément entre P7 et P21, où elle augmente brusquement. Cette période correspond à une importante phase de structuration du cerveau murin, à la fois en termes de myélination et de remaniement synaptique. Il est envisageable que Ftx participe à un de ces processus. En utilisant un modèle cellulaire basé sur des cellules souches embryonnaires murines sauvages et portant une délétion constitutive de Ftx, j'ai développé une technique de différenciation neurale in vitro. Malgré le fait que la perte de Ftx n'a pas d'impact majeur sur le potentiel de différenciation neurale des cellules, une analyse par puce ADN a révélé qu'elle induit une surexpression d'une grande quantité de gènes Hox. L'ensemble de ces travaux fortifient l'hypothèse initiale et donnent naissance à des pistes excitantesMy PhD project focuses on the study of the long RNAnc Ftx, whose gene is located in the X chromosome inactivation center, a region rich in genes encoding long RNAncs and in charge of the inactivation process of one X chromosome in female mammals. The team has shown that the expression of Ftx favors the expression of the neighboring genes, conferring it the role of an activator of the inactivation process. Ftx is also expressed in the adult murine organism, more specifically in the brain, suggesting thus functions independent of the inactivation process. As a consequence, I focused on the potential implication of Ftx in de development and/or the functions of the brain. Ftx expression in the brain is relatively homogeneous among different regions, although it is established only during the postnatal period, between P7 and P21, when it increases suddenly. This period corresponds to an important phase of restructuring of the murine brain like myelination and synaptic reorganization. Thus it is conceivable that Ftx takes part in one of these processes. Using a cellular model based on wild-type and Ftx-deleted mouse embryonic stem cells? I developed a technique of in vitro neural differentiation. Although the lors of Ftx does not impact in a visible way on the neural differentiation potential of the cells, an analysis by microarray revealed that it causes the overexpression of several Hox genes. These combined results reinforce the initial hypothesis and lay numerous exciting tracks
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Ard, Ryan Anthony. "Functional long non-coding RNA transcription in Schizosaccharomyces pombe." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/20396.
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Eukaryotic genomes are pervasively transcribed and frequently generate long noncoding RNAs (lncRNAs). However, most lncRNAs remain uncharacterized. In this work, a set of positionally conserved intergenic lncRNAs in the fission yeast Schizosaccharomyces pombe genome are selected for further analysis. Deleting one of these lncRNA genes (ncRNA.1343) exhibited a clear phenotype: increased drug sensitivity. Further analyses revealed that deleting ncRNA.1343 also disrupted a previously unannotated lncRNA, termed nc-tgp1, transcribed in the opposite orientation of the predicted ncRNA.1343 gene and into the promoter of the phosphate-responsive permease gene tgp1+. Detailed analyses revealed that the act of transcribing nc-tgp1 into the tgp1+ promoter increases nucleosome density and prevents transcription factor access. Decreased nc-tgp1 transcription permits tgp1+ expression upon phosphate starvation, while nc-tgp1 loss induces tgp1+ in repressive phosphate-rich conditions. Notably, drug sensitivity results directly from tgp1+ expression in the absence of nc-tgp1 transcription. Similarly, lncRNA transcription upstream of pho1+, another phosphate-regulated gene, increases nucleosome density and prevents transcription factor binding to repress pho1+ in phosphate-replete cells. Importantly, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription occurs in the absence of RNAi and heterochromatin components. Instead, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription resembles examples of transcriptional interference reported in other organisms. Thus, tgp1+ and pho1+ are the first documented examples of genes regulated by transcriptional interference in S. pombe.
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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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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.
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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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Tan, Jennifer Yihong. "Intergenic long noncoding RNAs provide a novel layer of post-transcriptional regulation in development and disease." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:708df26b-6e5b-4f6f-a0d7-6e3c8b1466ee.
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Recent genome-wide sequencing projects revealed the pervasive transcription of intergenic long noncoding RNAs (lincRNAs) in eukaryotic genomes (reviewed in Ponting et al. 2009). For the vast majority of lincRNAs, their mechanisms of function remain largely unrecognized. However, the genome-wide signatures of functionality associated with many lincRNAs, including apparent evolutionary sequence conservation, spatial and temporal-restricted expression patterns, strong associations with epigenetic marks, and reported molecular and cellular functions, reinforce their biological relevance. My work investigates lincRNAs that post-transcriptionally regulate gene abundance by competing for the binding of common microRNAs (miRNAs) with protein-coding transcripts, termed competitive endogenous RNAs (ceRNAs) acting lincRNAs (lnceRNAs). First, I examine the biological relevance of this post-transcriptional regulation of gene abundance by ceRNAs. Next, I estimate the genome-wide prevalence of lnceRNAs in mouse embryonic stem cells (mESCs) and characterize their properties. Finally, using two specific examples of lnceRNAs, I show the contributions of lnceRNAs to human monogenic and complex trait diseases. Collectively, these results illustrate that lnceRNAs provide a novel layer of post-transcriptional regulation via a miRNA-mediated mechanism that contributes to organismal and cellular biology.
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Siena, Ádamo Davi Diógenes. "Análise da expressão de RNAs longos não-codificadores em linhagens celulares de melanoma em diferentes estágios de progressão tumoral." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-04012017-084739/.
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Evidências sugerem que somente cerca de 2% do genoma codifica proteínas, mas que a maior parte dos 80% restante possui atividade transcricional. Por não ser codificadora de proteínas, essa fração do genoma foi considerada como \'DNA lixo\'. Entretanto, estudos mais recentes e análises pós-ENCODE vem demonstrando que parte significativa destes RNAs não-codificantes desempenham papéis importantes em processos biológicos essenciais e também em doenças. Os RNAs longos não codificadores (lncRNAs) embora tradicionalmente conhecidos pelo imprintinggenômico, vem demonstrando diversos mecanismos de regulação da expressão gênica, principalmente emnível pós transcricional. Um destes lncRNAs que está envolvido principalmente com a metastase em câncer é o HOTAIR. O melanoma tem sido utilizado como modelo de progressao do câncer por suas etapas bem definidas e por isso já tem apresentado alguns lncRNAs envolvidos na melanomagenese e progressão do melanoma, tal como o HOTAIR. Assim, neste trabalho foi analisado a expressão de lncRNAs de amostras de melanócito e melanoma, sendo que as amostras malignas representam as principais fases de progressão deste tipo de câncer. Foram analisados os níveis de expressão relativa. Além disso, foi realizado a expressão diferencial dos grupos representativos do melanoma. Foram encontrados lncRNAs com valores de expressão e significância (p-ajustado <0,01 e fold change >1) que podem ser indicativos de expressão associada a progressão do melanoma. Os lncRNAs mais diferencialmente expressos foram avaliados quanto a sua capacidade de interação proteína-RNA e literatura científica disponível e então foram selecionados para posteriores ensaios funcionais.Evidence suggests that only about 2% of the genome encodes protein, but most remaining 80% has transcriptional activity. Since they do not coding for proteins, this fraction of the genome was considered \'junk DNA\', However, recent studies and post-ENCODE analisys has shown that significant part of these non-coding RNAs play important roles in essential biological processes and in disease. Long noncoding RNAs (lncRNAs) although traditionally known for genomic imprinting, has demonstrated several mechanisms of regulation of gene expression, especially at the post transcriptional level. One of these lncRNAs that is involved primarily with metastasis in câncer is HOTAIR. Melanoma has been used as a model of câncer progression by its well-defined steps, and so it has been presented some lncRNAs involved in melanoma progression and melanomagenese, as HOTAIR was demonstrated. In this work it was analyzed the expression of lncRNAs of melanocyte and melanoma samples, and malignant samples represent the main stages of progression of this type of câncer. Relative expression levels were analyzed. Furthermore, it was performed differential expression of representative melanoma groups. lncRNAs found with expression values and significance (p-adjusted <0.01 and fold change> 1) may be indicative of expression associated with melanoma progression. The lncRNAs more differentially expressed were evaluated for their ability to interact protein-RNA and available scientific literature and then were selected for further functional assays.
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Sohiya, Yotsukura. "Computational Framework for the Dissection of Cancer Genomic Architecture and its Association in Different Biomarkers." 京都大学 (Kyoto University), 2016. http://hdl.handle.net/2433/217149.
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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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Tuck, Alex Charles. "Genome-wide identification of non-canonical targets of messenger RNA synthesis and turnover factors in Saccharomyces cerevisiae." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/11719.
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Pervasive transcription is widespread amongst eukaryotic genomes, and produces long noncoding RNAs (lncRNAs) in addition to classically annotated transcripts such as messenger RNAs (mRNAs). LncRNAs are heterogeneous in length and map to intergenic regions or overlap with annotated genes. Analogous to mRNAs, lncRNAs are transcribed by RNA polymerase II, regulated by common transcription factors, and possess 5’ caps and perhaps 3’ poly(A) tails. However, lncRNAs perform distinct functions, acting as scaffolds for ribonucleoprotein complexes or directing proteins to nucleic acid targets. The act of transcribing a lncRNA can also affect the local chromatin environment. Furthermore, whereas mRNAs are predominantly turned over in the cytoplasm, both nuclear and cytoplasmic pathways reportedly participate in lncRNA degradation. In this study, I address the question of when and how lncRNAs and mRNAs are distinguished in the cell. Messenger RNAs interact with a defined series of protein factors governing their production, processing and decay, and I hypothesised that lncRNAs might be similarly regulated. I therefore sought to determine which mRNA-binding proteins, if any, also bind lncRNAs. I reasoned that this would reveal the point at which lncRNAs and mRNAs diverge, and how differences in their biogenesis and turnover equip them for different roles. I selected factors from key stages of mRNA metabolism in Saccharomyces cerevisiae, and identified their transcriptome-wide targets using CRAC (crosslinking and analysis of cDNAs). CRAC can detect interactions with low abundance transcripts under physiological conditions, and reveal where within each transcript a protein is bound. Analyses of binding sites in mature mRNAs and intron-containing pre-mRNAs revealed the order in which the tested factors interact with mRNAs, and which region they bind. The poly(A)-binding protein Nab2 bound throughout mRNAs, consistent with an architectural role, whereas the cytoplasmic decay factors Xrn1 and Ski2 bound to poly(A) tails, which might act as hubs to coordinate turnover. The RNA packaging factors Tho2 and Gbp2, and nuclear surveillance factors Mtr4 and Trf4 bound abundantly to intron-containing premRNAs, indicating that they act during or shortly after transcription. The tested factors bound lncRNAs to various extents. LncRNA binding was most abundant for Mtr4 and Trf4, moderate for Tho2, Gbp2, the cap binding complex component Sto1, and the 3’ end processing factors Nab2, Hrp1 and Pab1, and lowest for Xrn1, Ski2 and the export receptor Mex67. This suggests that early events in lncRNA and mRNA biogenesis are similar, but unlike mRNAs, most lncRNAs are retained and degraded in the nucleus. Analyses of two documented classes of lncRNA, cryptic unstable transcripts (CUTs) and stable unannotated transcripts (SUTs), revealed some differences. SUTs were most similar to mRNAs, with canonical cleavage and polyadenylation signals flanking their 3’ ends, and poly(A) tails bound by the poly(A)-binding protein Pab1. CUTs lacked these characteristics, and in comparison to SUTs bound more abundantly to Mtr4 and Trf4 and less so to Ski2, Xrn1 and Mex67. Furthermore, CUTs accumulated upon Hrp1 depletion, suggesting that Hrp1 functions non-canonically to promote CUT turnover. Mtr4, Trf4 and Nab2 also bound abundantly to promoter-proximal RNA fragments generated from ~1000 protein coding genes. These fragments possessed short oligo(A) tails (hallmarks of nuclear surveillance substrates), were not bound to cytoplasmic factors, and apparently correspond to a population of ~150-200 nt promoter-proximal lncRNAs. Notably, CRAC analyses of Mtr4 and Sto1 targets in yeast subjected to a media shift revealed widespread changes in the abundance and surveillance of mRNAs, promoter-proximal transcripts and CUTs, which at many loci were arranged in a complex transcriptional architecture. Overall, the transcriptome-wide binding analyses presented here reveal that lncRNAs diverge from mRNAs prior to export, and are predominantly retained in the nucleus. Transcript fate is apparently determined during 3’ end processing, with CUTs diverging from mRNAs early in transcription via a distinct termination pathway coupled to rapid turnover, and SUTs diverging during or shortly after cleavage and polyadenylation, making them more stable and perhaps prone to escape to the cytoplasm. Promoter-proximal transcripts might arise from termination associated with an early checkpoint in Pol II transcription. The diverse behaviours of lncRNAs arise from their association with distinct subsets of RNA binding proteins, some of which perform different roles when bound to different types of transcript. In conclusion, my results provide the foundation for a mechanistic understanding of how distinct classes of non-coding Pol II transcripts are produced, and how they can perform diverse functions throughout the nucleus.
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Hu, Haiyang [Verfasser]. "Computational and Statistical Analysis of Sequence and Expression Features of MicroRNA and Long Noncoding RNA in Primate Brains / Haiyang Hu." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1093404175/34.
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Dixon, Maria S. "ANTISENSE AFP TRANSCRIPTS IN MOUSE LIVER AND THEIR POTENTIAL ROLE IN AFP GENE REGULATION." UKnowledge, 2017. http://uknowledge.uky.edu/microbio_etds/14.
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Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, ranking the sixth most common cancer and third most common cause of cancer mortality worldwide. Alpha-fetoprotein (AFP) is a plasma protein that is highly expressed in the fetal liver and shut off after birth. AFP expression is elevated in regenerating adult liver and HCC and has been used extensively as a diagnostic marker of liver cancer. We have been studying mouse liver gene regulation to better understand mechanisms by which changes in gene expression contribute to liver development, homeostasis and disease. Zinc Fingers and Homeoboxes 2 (Zhx2) has been identified as a repressor of AFP, but the mechanism of this regulation remains unknown. Interestingly, all targets of Zhx2 that have been identified to date, including H19, Glypican 3, Elovl3 and Cytochrome P450 (CYP) genes, are also known to be misregulated in HCC. Thus, a better understanding of the mechanism by which these genes are regulated by Zhx2 will likely lead to new insights into gene regulation during HCC progression.
Antisense transcripts belong to a diverse class of long noncoding RNA molecules > 200 nucleotides in length that often structurally resemble mRNAs, but do not encode proteins. While studying AFP mRNA regulation by Zhx2 in the mouse, our lab identified novel antisense AFP (asAFP) RNA transcripts that partially overlap the 3’ half of the mouse AFP gene. ENCODE tracks of ChIP-seq data for histone modifications in mouse liver show that the genomic region around the 5’ end of asAFP RNA has peaks for marks associated with promoters and enhancers. To better understand asAFP regulation, I identified the asAFP RNA 5’ end and the promoter elements that drive transcription. asAFP RNAs are ~5kb alternatively spliced, mainly cytoplasmic transcripts containing 2-4 exons. These transcripts were also detected in adult mouse liver RNA-seq data. asAFP is likely a noncoding RNA because it contains several small open reading frames that are 98 aa or smaller with no known functional domains or homology to known proteins. There is no evidence for similar transcripts in human liver. The abundance of asAFP RNA inversely correlates with AFP mRNA levels during postnatal liver development. Normally, asAFP RNA levels are high and AFP mRNA levels are low in the adult mouse liver. However, in the absence of Zhx2, AFP mRNA levels are higher and asAFP RNA levels are reduced, suggesting asAFP may be involved in the developmental regulation of AFP.
Antisense transcripts function through a variety of mechanisms to positively or negatively regulate the expression of target genes. To explore the role of asAFP RNA in AFP gene regulation, I expressed segments of asAFP RNA in a mouse liver cell line and measured endogenous AFP mRNA levels. My data revealed that all segments of asAFP repressed endogenous AFP mRNA in trans. To determine the mechanism by which asAFP RNA regulates AFP, I expressed asAFP segments that overlapped only with exons or introns of AFP. The asAFP segments that overlap with the exons showed greater repression of endogenous AFP mRNA levels than those overlapping with intronic sequences. Additionally, I considered whether asAFP RNA repression of AFP mRNA may involve RNA editing by Adenosine deaminase acting on RNA (ADAR). ADARs convert adenosine to inosine in double-stranded RNAs that results in RNA degradation. My data indicate that AFP and asAFP dsRNA is not extensively edited, suggesting ADAR mediated decay is not involved in the regulation of AFP mRNA expression. However, further studies are required to determine the mechanism of cytoplasmic AFP mRNA degradation. Together, my data characterizes the transcriptional regulation of novel mouse asAFP transcripts and provides a model system to investigate how these transcripts regulate AFP mRNA through RNA-RNA interaction.
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Jarroux, Julien. "Caractérisation fonctionnelle des longs ARN non codants associés à la transition épithélio-mésenchymateuse." Electronic Thesis or Diss., Paris Sciences et Lettres (ComUE), 2019. https://theses.hal.science/tel-02882448.
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Depuis dix ans, les techniques de séquençage haut-débit ont montré l’incroyable complexité du transcriptome humain, révélant une nouvelle classe d’ARN : les longs ARN non codants (lncARN). Depuis, leur expression a été démontré comme spécifique au type cellulaire ou aux variations pathologiques, et c’est notamment le cas du cancer où ils sont fortement dérégulés. Cependant, les mécanismes par lesquelles ils agissent dans le développement tumoral restent peu caractérisés. Aussi, ce projet vise à faire une caractérisation fonctionnelle des lncARN dans la transition épithélio-mésenchymateuse (TEM), un processus biologique associé à la métastase.A partir d’un modèle unique de TEM (Castro-Vega et al, 2013), l’analyse transcriptomique de fractions subcellulaires (cytoplasme et chromatine) ont permis d’identifier une signature de lncARN différentiellement exprimés dans les cellules épithéliales ou mésenchymateuses. Leur expression et localisation subcellulaire a ensuite été confirmé. Aussi, l’annotation de ces nouveaux transcrits a été validée par l’analyse des marques d’histones associées à leur régulation.Dans le but de déterminer quels lncARN identifiés sont fonctionnels dans la TEM, un crible d’activation par CRISPR (CRISPRa à l’échelle du génome a été mis au point en collaboration avec Neville Sanjana (NYU) pour activer l’expression de lncARN et définir leur rôle au travers de deux phénotypes associés à la TEM : les capacités d’invasion des cellules et les variations d’expression d’un marqueur de surface associée à l’identité épithéliale (EpCAM).En plus du crible, un nouveau lncARN a été identifié dans les cellules mésenchymateuses et sa caractérisation fonctionnelle a montré que son expression conduit à une perte de certains marqueurs épithéliaux ainsi qu’une augmentation drastique des capacités migratoires, lorsqu’il est surexprimé dans les cellules épithéliales.Pour résumer, de multiples approches haut-débit ont été utilisées pour caractériser le transcriptome non-codant associé à la transition épithélio-mésenchymateuse et ont permis d’identifier de nouveaux longs ARN non codants impliqués dans sa régulationIn the last decade, new high-throughput sequencing techniques have revealed the complexity of the human transcriptome, allowing the characterization of long non-coding (lnc)RNAs. These transcripts have been reported as very specific to tissues, developmental stages and pathological variations such as cancer. However, mechanisms through which they may act in the promotion of cancer are still poorly characterized. In my PhD project, I investigated the role of lncRNAs and their association to the epithelial to mesenchymal transition (EMT), a biological process which has been linked to metastasis and cancer progression.Using transcriptomic approaches from total and subcellular-fractionated RNA extracts from a cell system that models EMT (Castro-Vega et al, 2013), I identified over a thousand differentially expressed yet unannotated lncRNAs. Then I validated their expression, subcellular localization and the chromatin marks associated with their regulation.In order to assess whether these new lncRNA are functional in the EMT, I designed a CRISPR-activating (CRISPRa) screen using a dead Cas9 fused to transcription activating proteins (in collaboration with the lab of Neville Sanjana, NYU). This screen is based on two main phenotypes associated with the EMT: invasion capacity of the cells, and expression of an epithelial surface marker (EpCAM).In addition to the CRISPRa screening, I identified a novel lncRNA in mesenchymal cells which leads to a loss of epithelial markers and an increase in migration capacities when overexpressed in epithelial cells.Altogether, I used a combination of high-throughput methods to characterize the non-coding transcriptome associated to the EMT and identify yet unannotated transcripts which are functional in its regulation
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Romero, barrios Natali. "Non-codings RNAs, regulators of gene expression in Arabidopsis thaliana root developmental plasticity Noncoding Transcription by Alternative RNA Polymerases Dynamically Regulates an Auxin-Driven Chromatin Loop Battles and hijacks: noncoding transcription in plants Long noncoding RNA modulates alternative splicing regulators in Arabidopsis Detection of generic differential RNA processing events from RNA-seq data." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS128.
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Les techniques de séquençage à haut-débit développées ces dernières années ont permis d'identifier des milliers d’ARN non-codants et des événements tels que l’épissage ou l’édition. Cette approche est à l’origine d’une meilleure compréhension des mécanismes régulant l'expression des gènes. Les longs ARN non-codants (lncARN) ont ainsi émergé comme des acteurs clés de la régulation de divers processus développementaux. Ils agissent soit directement sous leur forme longue par des interactions lncARN-protéine(s) soit après une étape de maturation qui génère des siARN ou des miARN régulateurs, menant à l’extinction génique par clivage des ARNm, la répression de la traduction ou en entrainant des modifications épigénétiques (ADN/chromatine) de leurs cibles. L’objectif de cette thèse était d’élucider les mécanismes d'action de lncARNs dans le développement de la plante. J'ai contribué à l'analyse de l'action du lncARN APOLO dans la régulation de la topologie de la chromatine chez Arabidopsis thaliana. Ensuite, j’ai concentré mes efforts sur le lncARN ASCO (Alternative Splicing COmpetitor) qui interagit avec les protéines NSRs (Nuclear Speckles RNA-binding Proteins) et participent au patron d’épissage de certains gènes cibles. Lors d’un traitement par l’auxine, NSRb est induit alors qu’ASCO est réprimé dans les racines. Le même type de traitement, chez le double mutant nsra/b et les lignées surexprimant ASCO, entraine déficience partielle dans la formation des racines latérales. En utilisant un nouvel outil bio-informatique appelé "RNAprof", nous avons détecté 1885 ARN différentiellement maturés entre le mutant nsra/b et la lignée sauvage traités à l’auxine. Parmi ces gènes, nous avons identifié ARF19, un régulateur clé de la voie de signalisation de l’auxine au cours de l'initiation et le développement de la racine. J’ai démontré qu'ARF19 interagit directement avec les NSRs et qu’il est différentiellement polyadénylé dans le double mutant nsra/b, conduisant à une isoforme plus courte du transcrit ARF19. D’autre part, parmi les gènes dérégulés de manière transcriptionnelle chez le mutant des gènes impliqués dans la signalisation par l’éthylène ont été identifiés. J’ai ensuite montré que plusieurs de ces gènes sont aussi dérégulés dans les plantes mutantes arf19-1 et arf19-2 en réponse à l’auxine, soutenant un rôle d'ARF19 dans la réponse croisée entre l’auxine et l’éthylène. Le gène NSRb est induit par l'éthylène et l'inhibition de la synthèse d'éthylène par l'AVG complémente le phénotype de racine latérale du mutant nsra/b en réponse à l’auxine. De plus, l'AVG et la surexpression d’ASCO augmentent l'accumulation de l’isoforme courte d’ARF19. Cette étude met en avant la capacité du lncARN ASCO à moduler l’épissage par le détournement des NSRs et la capacité des ARN non-codants à moduler l’épissageIn the last years, high-throughput sequencing techniques have made possible to identify thousands of noncoding RNAs and a plethora of different mRNA processing events occurring in higher organisms. This led to a better understanding of different regulatory mechanisms controlling gene expression. Long noncoding RNAs (lncRNAs) are emerging as key players in the regulation of varied developmental processes. They can act directly in a long form by lncRNA-protein interactions or be processed into shorter small si/miRNAs, leading to mRNA cleavage, translational repression or epigenetic DNA/chromatin modification of their targets. In this study, we aim to understand the mechanism of action of lncRNAs in plant development. Initially, I contributed to the analysis of the action of the APOLO lncRNA in chromatin topology regulation. Then, I focused my work on the lncRNA ASCO (Alternative Splicing COmpetitor) that interacts with NSRs (Nuclear Speckles RNA-binding Proteins) to modulate the splicing pattern of NSR-regulated mRNA targets. Auxin treatment induces NSRb and represses ASCO expression in roots. The nsra/b double mutant and ASCO overexpressing lines treated with auxin are partially impaired in lateral root formation. Using a new bioinformatic tool called “RNAprof”, we detected 1885 differential RNA processing events genome-wide in auxin-treated nsra/b mutants compared to WT. Among them, we identified ARF19, a key regulator of auxin signaling in lateral root initiation and development. I demonstrated that ARF19 is directly bound by both NSRs and that in the nsra/b double mutant ARF19 is alternatively polyadenylated leading to a short transcript isoform. Furthermore, among the transcriptionally deregulated genes in the nsra/b mutant plants, I identified an important group related to ethylene response. I further showed that several of these genes are also deregulated in the arf19-1 and arf19-2 mutants plants in response to auxin, supporting a role of ARF19 in the auxin-ethylene crosstalk. NSRb is also induced by ethylene and the inhibition of ethylene synthesis by AVG rescues the nsra/b double mutant lateral root phenotype in response to auxin. Moreover, AVG and ASCO overexpression lead to increased accumulation of the ARF19 short isoform. Altogether, this study shed new light on the role of the lncRNA ASCO in the regulation of RNA processing by hijacking NSRs and the capacity of non-coding RNAs to modulate splicing
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Hansmeier, Nils Rouven [Verfasser], Jan-Wilhelm [Gutachter] Kornfeld, and Aleksandra [Gutachter] Trifunovic. "Decoding the Impact of Obesity Long Noncoding RNAs on Murine Liver Energy Homeostasis / Nils Rouven Hansmeier ; Gutachter: Jan-Wilhelm Kornfeld, Aleksandra Trifunovic." Köln : Universitäts- und Stadtbibliothek Köln, 2018. http://d-nb.info/1181324769/34.
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Tsuji, Shuhei. "Lionheart LincRNA alleviates cardiac systolic dysfunction under pressure overload." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263521.
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Canesin, Lucas Eduardo Costa 1988. "Identificação e caracterização de lncRNAs e genes codificadores linhagem-específicos em Andropogoneae = padrões comuns de evolução de genes emergentes = Identification and characterization lncRNAs and lineage specific coding genes in Andropogoneae : common patterns of evolution of emerging genes." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317251.
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Orientador: Renato Vicentini dos SantosDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Recentemente, a análise de dados de genômica comparativa, buscando elucidar melhor a hipótese nula de modelos evolutivos, i.e. evolução neutra, originou uma nova teoria que eleva o tamanho populacional como principal fator evolutivo. Populações pequenas estão sujeitas a forte influência de deriva genética, o que causa o aumento da entropia do genoma. A complexidade genômica, leia-se conteúdo de sequencias informativas, como genes, é então um subproduto do aumento da entropia e a seleção teria então um papel secundário, sobretudo como moduladora do processo evolutivo. Assumindo este modelo, a emergência e degeneração de transcritos linhagem-específicos estão submetidas primariamente a evolução neutra. A transcrição pervasiva, sobretudo em linhagens germinais, é o agente causal do nascimento de genes e a fixação destes, frente ao reduzido tamanho populacional de eucariotos multicelulares, como as plantas Saccarum officinarum e Sorghum bicolor, ocorre por deriva genética. A inserção de novos genes, que são inicialmente neutros ou levemente deletérios, em redes funcionais ainda é pouco compreendida. A integração se torna gradativamente mais robusta com a evolução individual destes loci. Neste contexto, este estudo buscou identificar genes codificadores e não-codificadores de proteínas de recente emergência em cana-de-açúcar e sorgo a fim de se elucidar a hipótese de que sua arquitetura gênica e integração em redes biológicas apresentam padrões evolutivos comuns. Para isso, realizamos a identificação de lncRNAs de cana a partir de bancos de cDNA, o que permitiu a caracterização da expressão desses transcritos contrastando seis variedades distintas. Em decorrência da disponibilidade do genoma de sorgo, a identificação de genes linhagem-específicos codificadores e não codificadores pode ser resolvida com maior precisão. Pudemos determinar uma correlação entre a sua arquitetura gênica e integração nas redes biológicas e sua idade relativa. Apesar da correlação encontrada, o efeito mais forte observado em transcritos não codificadores revelam outros fatores que devem estar influenciando sua evolução. Levantamos a hipótese de que o evento de tradução possa elevar a eficiência da seleção negativa sobre o transcrito emergente, o que resultaria no turnover mais acentuado de lincRNAs e maior conservação de genes linhagem-específicos
Abstract: Recently, comparative genomics studies, aiming to better elucidate the null hypothesis of models of evolution, i. e. the neutral evolution, originate a new theory that elects the population size as the main factor acting in evolution. Small populations are subject to stronger influence of genetic drift, which raises genomic entropy. Genomic complexity, which means the information content in genome, such as genes, is a byproduct of the high entropy levels and selection would then display a secondary role, mainly as a modulator of the evolutionary process. Assuming this model, the emergence and degeneration of lineage-specific transcripts are primarily subject to neutral evolution. The pervasive transcription, especially in germinal cell lines, is the causal agent of birth of genes and their fixation, in face to the reduced population size of multicellular eukaryotes, as Saccarum officinarum and Sorghum bicolor plant species, is ruled by genetic drift. The integration of new genes, initially neutral or weakly deleterious, in functional networks is still poorly understood. The integration becomes more robust with the individual historical evolutionary path of these loci. In this context, this study aimed identify protein coding and noncoding genes of recent emergence in in sugarcane and sorghum to elucidate the hypothesis that the gene architecture and integration in biological networks display common patterns of evolution. We then identified sugarcane lncRNAs from public cDNA databases that allowed us to characterize the expression of these transcripts in six different contrasting varieties of sugarcane. As sorghum bicolor genome is available, the identification of lineage-specific coding and noncoding could be done to a higher resolution. We could then determine a correlation between gene architecture and network integration with its relative age. Despite the correlation observed, a stronger effect seen in noncoding transcripts reveals other factors that may be influencing their evolution. We propose the hypothesis that the translation event may increase negative selection efficiency over the emerging transcript, what would result in the stronger turnover of lincRNAs and higher conservation levels of coding lineage-specific genes
Mestrado
Bioinformatica
Mestre em Genética e Biologia Molecular
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Hofmann, Patrick [Verfasser], Reinier [Akademischer Betreuer] Boon, Stefanie [Gutachter] Dimmeler, and Amparo [Gutachter] Acker-Palmer. "The long noncoding RNA H19 controls endothelial cell functions by STAT3 repression / Patrick Hofmann ; Gutachter: Stefanie Dimmeler, Amparo Acker-Palmer ; Betreuer: Reinier Boon." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2018. http://d-nb.info/1153572389/34.
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Zaniboni, Gabriel Francisco. "Implementação de abordagens computacionais para identificação de RNAs longos não codificadores envolvidos na diferenciação neural." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-02022016-150323/.
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Cada vez mais, RNAs longos não codificadores (lncRNAs) surgem como importantes reguladores da biologia celular, principalmente em processos de diferenciação durante o desenvolvimento. O interesse no estudo das funções e mecanismos de atuação dessa classe de transcritos durante esses processos é crescente, e mostra-se bastante relevante no processo de diferenciação neural, pelo qual são gerados neurônios e células da glia. A linhagem celular P19, uma célula pluripotente advinda de um tipo de carcinoma embrionário murino, é bem consolidada como modelo in vitro de diferenciação neural. Após tratamento com ácido retinóico, ela é capaz de se diferenciar em neurônios e células da glia (astrócitos e oligodendrócitos). Em busca de evidências que indiquem a atuação de lncRNAs durante o processo de diferenciação neural, nosso grupo realizou experimentos utilizando microarranjos para averiguar os níveis de expressão gênica de lncRNAs e genes codificadores de proteínas (mRNAs) durante a diferenciação de células P19 em neurônios (predominância após 10 dias de diferenciação) e glia (predominância em 14 dias de diferenciação). Em um primeiro momento foi realizada a reanotação das sondas referentes a esses lncRNAs da plataforma de microarranjo, visto que as informações presentes nos arquivos de anotação da mesma eram muito escassas e desatualizadas. Registros de lncRNAs e mRNAs foram obtidos a partir de bancos de dados públicos para esse fim, e ao final dessa etapa aproximadamente 25,0% das sondas que não tinham uma anotação foram reanotadas com identificadores advindos desses bancos de dados. A partir dos dados de expressão, foram identificados todos os lncRNAs e mRNAs que apresentaram expressão diferencial entre as diferentes condições estudadas. As informações dos mRNAs diferencialmente expressos foram então utilizadas para a realização de análises de enriquecimento de categorias gênicas do Gene Ontology, nas ontologias de processo biológico e função molecular. A partir das sondas reanotadas, foram realizadas análises de coexpressão entre lncRNAs e mRNAs. A partir do cruzamento das informações obtidas, foram selecionados lncRNAs que através dos princípios de guilt by association se mostraram propensos a desempenharem um papel regulatório na diferenciação neural. Assim, as informações geradas nesse trabalho servirão como base para estudos futuros de validação funcional desses lncRNAs.Increasingly, long noncoding RNAs (lncRNAs) emerge as important regulators of cell biology, especially in differentiation processes during development. The interest in the study of functions and mechanisms of action of this class of transcripts during these processes is growing, and shows quite relevant in the neural differentiation process by which neurons and glia are generated. The P19 cell line, pluripotent cells arising from a type of murine embryonal carcinoma, is well established as an in vitro model of neural differentiation. After treatment with retinoic acid, it is capable of differentiating into neurons and glial cells (astrocytes and oligodendrocytes). In search of evidence that indicate the action of lncRNAs during the neural differentiation process, our group conducted experiments using microarrays to assess gene expression levels of lncRNAs and protein coding genes (mRNAs) during differentiation of P19 cells into neurons (mainly after 10 days of differentiation) and glial cells (mainly after 14 days of differentiation). At first was performed the reannotation of the probes relating to these microarrays lncRNAs, as the information provided in the annotation files were very scarce or outdated. LncRNAs and mRNAs records were obtained from public databases for this purpose, and at the end of this stage approximately 25.0% of the probes without annotation were reannotated with identifiers arising from these databases. From the expression data, we identified all lncRNAs and mRNAs that showed differential expression between the different studied conditions. The information of differentially expressed mRNAs were then used to perform Gene Ontology enrichment, in the ontologies biological process and molecular function. From the reannotated probes, coexpression analyses were performed for lncRNAs and mRNAs. From the crosscheck of information obtained, we selected those lncRNAs that by the principles of guilt by association proved likely to play a regulatory role in neural differentiation. Thus, the information generated in this study will serve as a basis for future studies of functional validation of these lncRNAs.
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Meena, Shiv Kumar [Verfasser], Steffen Gutachter] Abel, Sven-Erik [Gutachter] Behrens, and Julia [Gutachter] [Kehr. "Characterization of cis-natural antisense long noncoding RNAs overlapping the UGT73C6 gene in Arabidopsis thaliana / Shiv Kumar Meena ; Gutachter: Steffen Abel, Sven-Erik Behrens, Julia Kehr." Halle (Saale) : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2020. http://d-nb.info/1217657223/34.
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Schmidt, Elena [Verfasser], Jan-Wilhelm [Gutachter] Kornfeld, Thorsten [Gutachter] Hoppe, and Lei [Gutachter] Sun. "Long noncoding RNA H19 protects from dietary obesity by promoting brown adipose tissue commitment and function / Elena Schmidt ; Gutachter: Jan-Wilhelm Kornfeld, Thorsten Hoppe, Lei Sun." Köln : Universitäts- und Stadtbibliothek Köln, 2018. http://d-nb.info/1172414653/34.
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Joshi, Parth Devesh [Verfasser], Gregor [Akademischer Betreuer] Eichele, Reinhard [Gutachter] Lührmann, and Ernst A. [Gutachter] Wimmer. "Molecular characterization and functional analysis of a novel long noncoding RNA in the mouse / Parth Devesh Joshi ; Gutachter: Reinhard Lührmann, Ernst A. Wimmer ; Betreuer: Gregor Eichele." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://d-nb.info/1187520225/34.
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De, Clara Etienne. "Etude des longs ARNs non codants dans la leucémie aiguë myéloblastique à caryotype normal." Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30280/document.
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Les longs ARN non codants (lncRNAs) sont définis comme des transcrits de plus de 200nt et n'ayant pas de potentiel codant. Des études récentes ont démontré que les lncRNAs pouvaient être impliqués dans la régulation de la transcription, de la traduction, de la différenciation cellulaire, de l'expression génique, du cycle cellulaire et des modifications de la chromatine. De plus, il a été montré un impact fonctionnel de certains lncRNAs dans le processus de cancérogenèse mais nos connaissances actuelles sur ces molécules dans le cancer, et plus particulièrement dans la leucémie, restent extrêmement limitées. Au cours de cette étude, nous avons analysé l'expression des lncRNAs par RNA-sequencing sur 40 patients atteints de leucémie aiguë myéloblastique (LAM) à caryotype normal. Parmi les 11065 lncRNAs exprimés dans nos échantillons, nous avons identifié une signature de lncRNAs associée à la mutation de NPM1. Afin de mettre en évidence les fonctions putatives des lncRNAs sélectionnés, nous avons utilisé un algorithme de prédiction d'interaction protéine/ARN. De manière intéressante, plus de la moitié des lncRNAs présentent des sites d'interactions potentiels à SUZ12, une sous unité du complexe PRC2 (Polycomb repressive complex 2), connu pour être recruté par des lncRNAs pour la régulation épigénétique de gènes cibles. Par RNA immunoprécipitation (RIP) de SUZ12, nous avons pu démontrer que le lncRNA XLOC_087120 interagissait avec SUZ12. De plus, son expression est anti-corrélée avec celle des gènes voisins codants des histones, suggérant un rôle dans la régulation négative des histones par ce lncRNA. L'impact de la dérégulation de XLOC_087120 sur les histones a été confirmé par des expériences de surexpression et d'inhibition de ce lncRNA dans des lignées de LAM. De plus, même si la mutation NPM1 ne semble pas affecter directement l'expression de ce lncRNA, des expériences d'infection de la forme mutée de NPM1 dans une lignée LAM ont montré que NPM1 pourrait réguler la localisation nucléaire/cytoplasmique de XLOC_087120 et moduler sa fonction de répresseur. En conclusion, ces données suggèrent que les lncRNAs sont des facteurs clés dans la pathogenèse des LAMsLong noncoding RNAs (lncRNAs) are defined as RNA transcripts that are larger than 200 nt but do not appear to have protein- coding potential. Recent studies have demonstrated that lncRNAs regulate many processes such as transcription, translation, cellular differentiation, gene expression regulation, cell cycle regulation, and chromatin modification. Cumulative evidence points towards an important role of lncRNAs in cancer initiation, development, and progression. However, our overall knowledge of lncRNAs in cancer, including leukemia, remains extremely limited. In this study, we investigated lncRNA expression by RNA-sequencing in 40 acute myeloid leukemia (AML) patients with normal karyotype. Among 11065 lncRNA expressed in our samples, we identified specific lncRNA signature associated with the presence of NPM1 mutation. To go further into the putative function of these lncRNAs, we used catRAPID Omics algorithm to predict potential protein partners. Interestingly, the majority of the selected lncRNAs contains putative SUZ12 binding sites, a PRC2 (Polycomb Repressive Complex 2) component known to be linked to lncRNAs and to epigenetically regulates target genes. By using SUZ12 RNA Immunoprecipitation, we identify one lncRNA named XLOC_087120 linked to SUZ12. XLOC_087120 is located in a region enriched in histone genes. Pearson correlation showed a significative anti-correlation between XLOC_087120 and histone neighboring coding gene expression suggesting a role of this lncRNA in the regulation of histone genes. The impact on histone genes expression was confirmed by overexpression and inhibition of XLOC_087120 in AML cell lines. Overexpression of NPM1 mutant in an AML cell line showed that NPM1 modulates the nuclear/cytoplasmic localization of XLOC_087120 and consequently its repressive function. Altogether, these data suggest that lncRNAs should be considered as key players in the pathogenesis of acute myeloid leukemias
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Beckedorff, Felipe César Ferrarezi. "Recrutamento do complexo repressivo polycomb 2 pelo RNA não codificador longo antissenso ANRASSF1 modula a expressão do gene RASSF1A e a proliferação celular." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-23042013-083641/.
Full textAbstract:
O gene supressor tumoral RASSF1A tem sido associado com redução da proliferação celular em diversos tumores. Sua expressão é regulada por eventos epigenéticos que envolvem o complexo repressivo polycomb (PRC2), no entanto os mecanismos moleculares da modulação do recrutamento deste modificador epigenético para este locus ainda são desconhecidos. Neste trabalho identificamos e caracterizamos ANRASSF1, um RNA não codificador longo (lncRNA) intrônico unspliced, que é transcrito na fita oposta do gene RASSF1A, em várias linhagem celulares e tecidos, e se liga a PRC2. ANRASSF1 é transcrito pela RNAPII, possui cap-5´ e cauda poli-A, além de localizar-se no núcleo e possuir uma meia-vida em média quatro vezes menor comparada com outros lncRNAs ligados à PRC2. A super-expressão ectópica de ANRASSF1 reduziu os níveis de RASSF1A e aumentou a taxa de proliferação em células HeLa, enquanto seu silenciamento provocou efeito oposto. Essas mudanças nos níveis de ANRASSF1 não afetaram a abundância da isoforma RASSF1C em nenhuma das condições. A super-expressão de ANRASSF1 provocou um grande aumento tanto da ocupação de PRC2 como da marca de histona repressiva H3K27me3 especificamente na região promotora RASSF1A. Nenhum efeito da super-expressão de ANRASSF1 foi detectado na ocupação de PRC2 e na histona H3K27me3 nas regiões promotoras de RASSF1C e de outros quatro genes vizinhos, incluindo dois genes supressores tumorais bem caracterizados. Além disso, foi demonstrado que ANRASSF1 forma um híbrido de RNA/DNA e recruta SUZ12, um componente do PRC2, para o promotor de RASSF1A. Notavelmente, foi detectado pelo ensaio de RNase-ChIP que a degradação de ANRASSF1 diminui a ocupação de PRC2 neste promotor. Esses resultados demonstram um novo mecanismo de repressão epigenética do supressor tumoral RASSF1A, envolvendo um lncRNA unspliced antissenso, onde ANRASSF1 reprime seletivamente a expressão da isoforma de RASSF1 que sobrepõe o transcrito antissenso de modo local e específico. Considerando uma perspectiva mais ampla, nossos resultados sugerem que outros lncRNAs intrônicos unspliced não caracterizados no genoma humano podem contribuir para uma modulação epigenética local e específica de cada região em que os lncRNAs são transcritos.Tumor-suppressor RASSF1A gene down-regulation has been implicated in increasing cell proliferation in several tumors. Its expression is regulated by epigenetic events involving polycomb repressive complex 2 (PRC2), however the molecular mechanisms modulating recruitment of this epigenetic modifier to the locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand of RASSF1 gene in several cell lines and tissues, and binds to PRC2. ANRASSF1 is transcribed by RNA Polymerase II, 5\'-capped, polyadenylated, displays nuclear localization, and has on average a four-fold shorter half-life compared to other lncRNAs that bind PRC2. ANRASSF1 ectopic overexpression decreases RASSF1A abundance and increases the proliferation rate of HeLa cells, whereas its silencing causes opposite effects. These changes in NRASSF1 levels do not affect RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase both in PRC2 occupancy and in histone H3K27me3 repressive mark specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression is detected on PRC2 occupancy and on histone H3K27me3 at the promoter regions of RASSF1C and of four other neighbor genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrate that ANRASSF1 forms an RNA/DNA hybrid, and recruits SUZ12, a PRC2 component, to the RASSF1A promoter. Notably, depletion of ANRASSF1 disrupts SUZ12 occupancy on RASSF1A promoter as measured by RNAse-ChIP assay. Together, these results show a new mechanism of epigenetic repression of RASSF1A tumor suppressor gene involving an antisense unspliced lncRNA, in which ANRASSF1 selectively represses expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome may contribute to a location-specific epigenetic modulation of 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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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.
Full textAbstract:
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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Pereira, Carlos de Ocesano. "INXS, um longo RNA não codificador de proteínas mediador da apoptose." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-20072015-144251/.
Full textAbstract:
O splicing alternativo do pré-mRNA de BCL-X produz duas isoformas de mRNAs com funções antagônicas, a pró-apoptótica BCL-XS e a anti-apoptótica BCL-XL, cujo balanço regula a homeostasia celular. Entretanto, o mecanismo que regula esse processamento ainda é desconhecido. Nesse trabalho, nós identificamos e caracterizamos um longo RNA não codificador de proteínas (lncRNA) nomeado INXS, que é transcrito a partir da fita oposta do locus genômico de BCL-X, sendo menos abundante em linhagens celulares tumorais e tecidos tumorais de pacientes quando comparados com os respectivos pares não tumorais. INXS é um RNA unspliced de 1903 nts, é transcrito pela RNA Polimerase II, possui cap 5\', está enriquecido na fração nuclear das células e se liga à proteína Sam68 do complexo modulador de splicing. O tratamento de células tumorais 786-O com cada um de três agentes indutores de apoptose aumentou a expressão endógena do INXS, levando ao aumento expressivo da proporção entre os mRNAs de BCL-XS / BCL-XL, e ativação das caspases 3, 7 e 9. Estes efeitos foram anulados na presença do knockdown do INXS. Da mesma forma, a superexpressão ectópica do INXS causou uma mudança no splicing favorecendo a isoforma BCL-XS e ativação das caspases, aumentando os níveis da proteína BCL-XS e conduzindo as células à apoptose. Utilizando um modelo in vivo, cinco injeções intra-tumorais do INXS durante 15 dias causaram uma regressão acentuada no volume dos xenotumores. Portanto, INXS é um lncRNA que induz a apoptose, sugerindo que essa molécula seja um possível alvo a ser explorado na terapia contra o câncer.BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL, whose balance regulates cell homeostasis. However, the mechanism that regulates the splice shifting is incompletely understood. Here, we identified and characterized a long noncoding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was less abundant in tumor cell lines and patient tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA Polymerase II, 5\'-capped, nuclear enriched and binds Sam68 splicing-modulator. The treatment of tumor cell line 786-O with each of three apoptosis-inducing agents increased endogenous INXS lncRNA, increased BCL-XS / BCL-XL mRNA ratio, and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing towards BCL-XS and activation of caspases, increasing the levels of BCL-XS protein and then leading the cells to apoptosis. In a mouse xenograft model, five intra-tumor injections of INXS along 15 days caused a marked regression in tumor volume. INXS is an lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.
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Junetha, Syed Jabarulla. "Chemical Biology Approaches for Regulating Eukaryotic Gene Expression." 京都大学 (Kyoto University), 2015. http://hdl.handle.net/2433/202664.
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Bhatta, Ankit. "Role of a Mitochondrial Micropeptide in Regulating Innate Immune Responses." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1108.
Full textAbstract:
Short ORF-encoded peptides (SEPs) are increasingly being identified as functional elements in various cellular processes. The current computational methods and experimental molecular biochemistry allow us to discover putative SEPs or micropeptides from proteogenomic datasets and experimentally validate them. Here, we identified a micropeptide produced from a putative long noncoding RNA (lncRNA) 1810058I24Rik which is downregulated in both human and murine myeloid cells exposed to lipopolysaccharide (LPS), as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed this transcript is localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with 35S-labeled methionine resulted in translation of a 47 amino acid micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene ‘Mitochondrial micropeptide-47 (Mm47)’. Functional studies using siRNA and Cripsr-cas9-mediated deletion in primary cells, showed that the transcriptional response downstream of TLR4 was not affected by Mm47 loss of function. In contrast, both the Crispr-cas9- and siRNA-targeted BMDM cells were compromised for Nlrp3 inflammasome responses. However, the primary macrophages derived from the Mm47 knockout mice do not require Mm47 for Nlrp3 activation, likely due to basal downregulation of a negative regulator microRNA of Nlrp3 called Mir-223. Notably, the Mm47-deficient mice are susceptible to influenza virus infection and succumb despite comparable antiviral and inflammatory response to wildtype mice. We hypothesize that the Mm47 deficiency may affect the antiviral resilience of mice due to secondary mitochondria dependent immunometabolic defect or failure of recovery from immune pathology, which warrants further investigation. This study therefore identifies a novel mitochondrial micropeptide Mm47 that is required for activation of the Nlrp3 inflammasome in cells and resistance to influenza virus infection. Broadly, this work highlights the presence of translatable ORFs is annotated noncoding RNA transcripts and underscores their importance in innate immunity and virus infection.
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Polović, Mirjana [Verfasser], Stefan [Gutachter] Hüttelmaier, Gerald [Gutachter] Schwerdt, and Zdravko [Gutachter] Lorković. "WISP1-AS1, a long noncoding RNA, upregulated in human renal cells exposed to the mycotoxin ochratoxin A and in human renal cancer cells / Mirjana Polović ; Gutachter: Stefan Hüttelmaier, Gerald Schwerdt, Zdravko Lorković." Halle (Saale) : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2020. http://d-nb.info/1222029332/34.
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Kolpa, Heather J. "XIST and CoT-1 Repeat RNAs are Integral Components of a Complex Nuclear Scaffold Required to Maintain SAF-A and Modify Chromosome Architecture: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/825.
Full textAbstract:
XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.
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Kolpa, Heather J. "XIST and CoT-1 Repeat RNAs are Integral Components of a Complex Nuclear Scaffold Required to Maintain SAF-A and Modify Chromosome Architecture: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/825.
Full textAbstract:
XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.
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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.
Full textAbstract:
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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Chan, Jennie. "Dissecting the Role of a lncRNA and Involvement of Plasmodium Infections in the Innate Immune Response: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/777.
Full textAbstract:
The innate immune system is a multicomponent response governed by intricate mechanisms of induction, regulation and resolution to elicit antimicrobial defenses. In recent years, the complexity of eukaryotic transcriptomes has become the subject of intense scrutiny and curiosity. It has been established, that RNA polymerase II (RNAPII) transcribes hundreds to thousands of long noncoding RNAs (lncRNAs), often in a stimulus and cell-type specific manner. However, the functional significance of these transcripts has been particularly controversial. While the number of identified lncRNAs is growing, our understanding of how lncRNAs themselves regulate other genes is quite limited. In chapter 2, a novel lncRNA is identified, more specifically, a natural antisense transcript, that mediates the transcription of the pro-inflammatory cytokine IL-1α. Through loss-of-function studies, I report the necessity of this transcript in mediating IL-1α mRNA expression by affecting RNAPII binding to the IL-1α promoter after toll-like receptor signaling. For the first time, I show that IL-1α is regulated at the transcriptional level. As a second independent component of this thesis, we explore the role of the innate immune response after infection by the malaria-causing parasite, Plasmodium berghei ANKA (PbA), and how innate immune components are both beneficial and detrimental to the host depending on when and where inflammation is triggered during infection. We attempt to identify the “malarial toxin” responsible for aberrations in the immune response that is detrimental for disease outcomes and the innate signaling pathways that are involved. Many pathogens induce pathological inflammatory conditions that lead to irreparable homeostatic imbalances and become fatal to the host. Here, type I Interferon signaling is required to dampen parasite load during liver-stage infections, but leads to host mobidity if these pathways are activated in the erythrocytic phase of infection. Together, this thesis provides new insights on how components of the innate immune system are regulated, and how dysregulation of immunity can potentially lead to adverse effects during active infections.
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Chan, Jennie. "Dissecting the Role of a lncRNA and Involvement of Plasmodium Infections in the Innate Immune Response: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/777.
Full textAbstract:
The innate immune system is a multicomponent response governed by intricate mechanisms of induction, regulation and resolution to elicit antimicrobial defenses. In recent years, the complexity of eukaryotic transcriptomes has become the subject of intense scrutiny and curiosity. It has been established, that RNA polymerase II (RNAPII) transcribes hundreds to thousands of long noncoding RNAs (lncRNAs), often in a stimulus and cell-type specific manner. However, the functional significance of these transcripts has been particularly controversial. While the number of identified lncRNAs is growing, our understanding of how lncRNAs themselves regulate other genes is quite limited. In chapter 2, a novel lncRNA is identified, more specifically, a natural antisense transcript, that mediates the transcription of the pro-inflammatory cytokine IL-1α. Through loss-of-function studies, I report the necessity of this transcript in mediating IL-1α mRNA expression by affecting RNAPII binding to the IL-1α promoter after toll-like receptor signaling. For the first time, I show that IL-1α is regulated at the transcriptional level. As a second independent component of this thesis, we explore the role of the innate immune response after infection by the malaria-causing parasite, Plasmodium berghei ANKA (PbA), and how innate immune components are both beneficial and detrimental to the host depending on when and where inflammation is triggered during infection. We attempt to identify the “malarial toxin” responsible for aberrations in the immune response that is detrimental for disease outcomes and the innate signaling pathways that are involved. Many pathogens induce pathological inflammatory conditions that lead to irreparable homeostatic imbalances and become fatal to the host. Here, type I Interferon signaling is required to dampen parasite load during liver-stage infections, but leads to host mobidity if these pathways are activated in the erythrocytic phase of infection. Together, this thesis provides new insights on how components of the innate immune system are regulated, and how dysregulation of immunity can potentially lead to adverse effects during active infections.
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Tahira, Ana Carolina. "Análise da expressão de RNAs não codificadores longos em adenocarcinoma de pâncreas." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-10062013-145054/.
Full textAbstract:
RNAs não codificadores longos (lncRNAs) compõem uma fração significativa do transcriptoma. Alterações na expressão de lncRNAs já foram observadas em vários cânceres humanos, mas ainda não foram exploradas no adenocarcinoma pancreático ductal (PDAC), uma doença devastadora e agressiva para a qual faltam métodos para diagnóstico precoce e tratamentos efetivos. Utilizando uma plataforma de microarranjo de cDNA com sondas para 984 lncRNAs e 2371 mRNAs, o presente estudo identificou conjuntos de lncRNAs expressos em 38 amostras clínicas pancreáticas. O enriquecimento de (i) elementos regulatórios associados às regiões promotoras (H3K4me3); (ii) possíveis inícios de transcrição (CAGE-tags); (iii) presença de elementos conservados sugere que ao menos uma fração desses RNAs seja originada a partir de unidades transcricionais independentes, reguladas e possivelmente funcionais. Foram identificadas assinaturas de expressão gênica compostas por mRNA e lncRNAs associadas ao tumor primário e à metástase pancreática. A assinatura gIenica associada à metástase apresentou enriquecimento RNAs intrônicos de loci gênicos associados à via MAPK quinase. O aumento de expressão dos transcritos intrônicos dos loci PPP3CB, MAP3K14 e DAPK1 foi confirmado por qPCR em metástases. Em conjunto, este trabalho aponta para a importância de lncRNAs intrônicos no PDAC e para a necessidade de estudos mais aprofundados para uma melhor compreensão do papel dessa classe de transcritos na biologia da doença.Long noncoding RNAs (lncRNAs) compose a significant fraction of transcriptome. Altered expression of lncRNAs has been observed in diverse human cancers, but has not being investigated in pancreatic ductal adenocarcinoma (PDAC), a devastating and aggressive disease that lack early diagnosis methods and effective treatments. Using a cDNA microarray platform with probes interrogating 984 lncRNAs and 2371 mRNA, the present study identified subsets of lncRNAs expressed in 38 pancreatic clinical samples. Enrichment of (i) regulatory elements associated to promoter region (H3K4me3); (ii) putative transcription start site (CAGEtags) and (iii) conserved elements, suggest that at least a fraction of these RNAs could be independent transcriptional unit, regulated, an possibly functional. Gene expression signatures comprised of mRNAs and lncRNAs and associated to primary or metastatic tumors were found. A gene signature associated to metastasis was enriched in intronic ncRNAs mapping to gene loci associated to the MAPK pathway. Over expression of intronic RNAs from PPP3CB, MAP3K14 and DAPK1 was confirmed by qPCR in metastatic samples. Taken together, this study points to the importance of intronic lncRNAs in PDAC and for the need to study this class of ncRNAs in greater detail to better understand its role in the biology of PDAC.
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Rege, Mayuri. "RNA Exosome & Chromatin: The Yin & Yang of Transcription: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/812.
Full textAbstract:
Eukaryotic genomes can produce two types of transcripts: protein-coding and non-coding RNAs (ncRNAs). Cryptic ncRNA transcripts are bona fide RNA Pol II products that originate from bidirectional promoters, yet they are degraded by the RNA exosome. Such pervasive transcription is prevalent across eukaryotes, yet its regulation and function is poorly understood.
We hypothesized that chromatin architecture at cryptic promoters may regulate ncRNA transcription. Nucleosomes that flank promoters are highly enriched in two histone marks: H3-K56Ac and the variant H2A.Z, which make nucleosomes highly dynamic. These histone modifications are present at a majority of promoters and their stereotypic pattern is conserved from yeast to mammals, suggesting their evolutionary importance. Although required for inducing a handful of genes, their contribution to steady-state transcription has remained elusive. In this work, we set out to understand if dynamic nucleosomes regulate cryptic transcription and how this is coordinated with the RNA exosome.
Remarkably, we find that H3-K56Ac promotes RNA polymerase II occupancy at a large number of protein coding and noncoding loci, yet neither histone mark has a significant impact on steady state mRNA levels in budding yeast. Instead, broad effects of H3-K56Ac or H2A.Z on levels of both coding and ncRNAs are only revealed in the absence of the nuclear RNA exosome. We show that H2A.Z functions with H3-K56Ac in chromosome folding, facilitating formation of Chromosomal Interaction Domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA levels, perhaps in part by regulating higher order chromatin structures. Together, these chromatin factors achieve a balance of RNA exosome activity (yin; negative) and Pol II (yang; positive) to maintain transcriptional homeostasis.
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Gourvest, Morgane. "Etude des longs ARNs non codants dans les leucémies aiguës myéloïdes : relevance clinique et caractérisation fonctionnelle." Thesis, Toulouse 3, 2020. http://www.theses.fr/2020TOU30117.
Full textAbstract:
Les longs ARNs non codants (lncRNAs) sont définis comme des transcrits ayant une taille supérieure à 200 nucléotides et dépourvus de potentiel codant. Longtemps considérés comme inutiles, leur étude récente a démontré qu’ils jouent un rôle important dans l’expression de nos gènes. On compte d’ailleurs de plus en plus d’exemples de ces lncRNAs dérégulés dans les cancers. Notre étude visait à évaluer l’existence de profils d’expression particuliers de lncRNAs au sein des leucémies aiguës myéloïdes à caryotype normal (LAM-CN), dont l’implication dans cette pathologie n’est que peu décrite. Le séquençage des ARNs que l’on a effectué sur une cohorte de 40 patients atteints de LAM-CN nous a permis de faire ressortir une signature minimale de 12 lncRNAs différentiellement exprimés chez les patients porteurs de la mutation dans le gène de la nucléophosmine (NPM1). Ces résultats ont été validés par RT-qPCR (Fluidigm) sur une cohorte indépendante composée de 134 nouveaux patients atteints de LAM-CN. Parmi cette signature, nous avons identifié un biomarqueur potentiel, le XLOC_109948, dont la faible expression est associée à un bon pronostic, particulièrement chez les patients NPM1 mutés. De plus, l’inhibition de ce lncRNA par transfection transitoire de gapmeRs dans une lignée cellulaire de LAM NPM1 muté augmente l’apoptose de ces cellules traitées à l’aracytine, suggérant un rôle du XLOC_109948 dans la sensibilité au traitement. Nous avons également caractérisé un autre lncRNA de la signature NPM1, baptisé LONA (LncRNA Overexpressed in NPM1-Mutated AML patients). Nous avons remarqué d’une part, que la mutation NPM1 induit une délocalisation nucléaire du lncRNA LONA, ce qui impacte ses fonctions cellulaires. Des stratégies perte et gain de fonctions ont montré que LONA aurait un rôle oncogénique en contexte de LAM NPM1 muté où il est impliqué in vitro et in vivo dans les processus de différentiation myéloïde et de croissance cellulaire en régulant l’expression de gènes cruciaux tels que THBS1, ASB2 ou MAFB. A l’inverse, la dérégulation de LONA en contexte de LAM NPM1 sauvage induit des effets opposés et suppresseurs de tumeurs, suggérant des régulations différentes en fonction du statut mutationnel de NPM1. D’autre part, le locus du lncRNA LONA est situé sur le chromosome 6 humain, au sein du cluster HIST1 codant des gènes des histones canoniques. Chez les patients NPM1 muté, l’expression du lncRNA LONA est inversement corrélée à celle de gènes voisins codant des histones. De manière cohérente, la diminution du lncRNA LONA dans notre lignée de LAM NPM1 muté est associée à une augmentation de l’expression de certaines des histones proximales du cluster. Par immunoprécipitation d’ARN, nous avons montré que LONA interagit avec le complexe de répression Polycomb (PRC2), suggérant sa contribution dans les régulations épigénétiques de la transcription des histones. De manière plus préliminaire, le lncRNA LONA pourrait également réguler l’étape de maturation des messagers des histones canoniques, en séquestrant telle une éponge moléculaire le snRNA U7, un petit ARN régulateur impliqué dans la maturation des extrémités 3’ des ARNs messagers des histones. L’ensemble de ces données suggère que les lncRNAs pourraient être considérés comme des biomarqueurs potentiels robustes, et apparaissent comme des acteurs clés dans le développement des leucémies aiguës myéloïdesLong noncoding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides without protein-coding potential. Long considered as useless, their recent study has demonstrated that lncRNAs have important roles in gene expression regulation. Cumulative evidence points toward the implication for lncRNAs deregulation in tumorigenesis. In this study, we sought to evaluate specific lncRNAs expression profiles among cytogenetically normal AML patients (CN-AML), their involvement in this pathology being barely referenced. The RNA sequencing that we performed on forty CN-AML patients allowed us to highlight a minimal set of 12 differentially expressed lncRNAs in AML patients bearing the mutation in the Nucleophosmin gene (NPM1). These results were confirmed by RT-qPCR (Fluidigm) on a validation set of 134 CN-AML patients. Among these, we identified one putative biomarker, the lncRNA XLOC_109948, whose low expression indicates a good prognosis, especially for NPM1-mutated patients. Consistently, the downregulation of XLOC_109948 using GapmeRs in a NPM1-mutated AML cell line enhances apoptosis of these cells treated with aracytine, suggesting the role of XLOC_109948 in drug sensitivity. We also functionally characterized another lncRNA of the NPM1 signature, that we named LONA (lncRNA overexpressed in NPM1-mutated AML patients). On one hand, we observed that the mutation of NPM1 leads to a nuclear delocalization of LONA lncRNA, which consequently modulates its cellular functions. Loss and gain of functions strategies allowed us to show that LONA seems to have oncogenic effects in a NPM1 mutated AML context, where it is implicated in vitro in myeloid differentiation and in vivo cellular growth processes by regulating the expression of master genes such as THSB1, ASB2, and MAFB. At the contrary, the deregulation of LONA lncRNA in a NPM1 wild type AML context leads to opposite and tumor suppressor effects, suggesting a different regulation depending on the mutational status of NPM1. On the other hand, the LONA’s genomic locus is located on chromosome 6, within a cluster of histone coding genes. In NPM1 mutated AML patients, we observed that the expression of LONA inversely correlates with the expression of some neighboring histones genes. Consistently, the downregulation of LONA lncRNA by using GapmeRs in a NPM1 mutated AML cell line leads to the upregulation of some proximal histones genes of the cluster. By RNA immunoprecipitation, we showed that LONA interacts with the Polycomb Repressive Complex 2 (PRC2), suggesting its contribution to epigenetic regulation of histone genes transcription and chromatin remodeling. More preliminary, we also think that LONA could regulate the maturation step of histone messengers by sequestrating, as a molecular sponge, the snRNA U7, a small regulatory RNA implicated in the maturation of histone messengers 3’ ends. Altogether, these data suggest that lncRNAs could be considered as strong prognostic biomarkers and emerged as key players in the pathogenesis of Acute Myeloid Leukemia
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Sahu, Divya, and 笛芙亞. "Mining MYCN Regulated Long Noncoding RNAs in Neuroblastoma." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/fud3mr.
Full textAbstract:
博士國立交通大學
生物資訊及系統生物研究所
106
Despite the discovery of protein therapeutic targets and advancement in multimodal therapies, the survival chance of patients with high-risk neuroblastoma is still less than 50%. MYCN amplification is one of the established drivers of neuroblastoma and, when combined with high-risk displays, worsens outcomes. Growing high-throughput transcriptomics suggest long noncoding RNAs (lncRNAs) dysregulation in cancers, including neuroblastoma. However, lncRNAs that are altered by MYCN amplification and associated with outcome in high-risk neuroblastoma patients are largely unknown. In this thesis, I present two related studies of the systematic discovery of lncRNAs. In the first study, we examined the expression profiles of lncRNAs and protein-coding genes (PCGs) between MYCN amplified and MYCN nonamplified neuroblastoma from microarray (n = 47) and RNA-seq data sets (n = 493). We identified 6 lncRNAs in common that were differentially expressed (fold change ≥ 2 and adjusted P ≤ 0.05) and subsequently validated by RT-qPCR. The co-expression analysis revealed lncRNA SNHG1 and PCG TAF1D highly co-expressed in neuroblastoma. Kaplan-Meier survival curves showed higher expression of SNHG1 significantly associated with poor patient survival. Importantly, multivariable Cox regression analysis confirmed high expression of SNHG1 as an independent prognostic marker for event-free survival [EFS] (HR = 1.58, P = 2.36E-02). Thus, this study unveils SNHG1 is up-regulated by MYCN amplification and could be a potential prognostic biomarker for high-risk neuroblastoma intervention. Current clinical risk factors stratify patients with neuroblastoma for appropriate treatments, yet patients with similar clinical behavior evoke variable responses. In the second study, we investigated RNA-seq based expression profiles of lncRNAs in MYCN status and risk status in discovery cohort (n = 493) and validated in three independent cohorts. In the discovery cohort, 16 lncRNAs that were differentially expressed (fold change ≥ 2 and adjusted P ≤ 0.01) integrated into a prognostic signature using the risk score method. A novel risk score threshold selection criteria developed to stratify patients into risk groups. Kaplan-Meier survival curves showed high-risk group of lncRNA signature had poor EFS (P < 1E-16). ROC curve revealed the lncRNA signature had higher accuracy for EFS prediction (AUC = 0.788). Notably, multivariable Cox regression analysis identified lncRNA signature as an independent predictor for EFS (HR = 3.21, P = 5.95E-07). The findings were confirmed in independent cohorts (P = 2.86E-02, P = 6.18E-03, P = 9.39E-03, respectively). Conclusively, in this study we report the first (to our knowledge) RNA-seq 16-lncRNAs prognostic signature for neuroblastoma that may contribute to precise clinical stratification and EFS prediction.
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Dill, Tiffany Loren. "Long noncoding RNA Meg3 regulates myoblast plasticity and skeletal muscle regeneration." Thesis, 2021. https://hdl.handle.net/2144/42644.
Full textAbstract:
Skeletal muscle formation is among the most striking examples of cellular plasticity in animal tissue development, where mononucleated, lineage-restricted progenitor cells are epigenetically reprogrammed to produce multinucleated myofibers. While some mediators of epithelial-mesenchymal transition (EMT) have been shown to function in myogenesis, regulation of this process at the interface of multipotency and myogenic differentiation remains poorly understood. The long noncoding RNA (lncRNA) Meg3 is processed from the >200 kb Dlk1-Dio3 polycistron, and while many encoded miRNAs have been shown to regulate skeletal muscle differentiation, regeneration, and aging, the functional relevance of encoded lncRNAs in skeletal muscle remains elusive. Here, I demonstrate that Meg3 is enriched in proliferating post-natal myoblasts, where it epigenetically modulates aspects of cellular plasticity to facilitate myogenic differentiation in vitro, skeletal muscle regeneration in vivo, and safeguard myogenic identity. Chronic inhibition of Meg3 in C2C12 myoblasts compromised cytoarchitectural and transcriptomic cell-state transitions required for myogenic fusion and differentiation. These differentiation defects were primarily driven by TGFβ-dependent Snai2 activation, which correlated with irregular Ezh2 activity and abnormal epigenetic marks in differentiating C2C12 cells. Similarly, adenoviral Meg3 knockdown compromised muscle regeneration in vivo, which manifested as abnormal mesenchymal gene expression, fibrosis, and interstitial cell proliferation in the regenerating milieu. Comparison of Meg3-depleted C2C12 myoblasts and injured skeletal muscle to literature-derived gene sets suggest that Meg3-deficient samples deviate from controls towards abnormal transcriptional states, including immature satellite cell activation, muscle aging, and adoption of an osteoblast-like cell ontology. Thus, Meg3 regulates myoblast identity to maintain proper cell state transitions in postnatal myogenesis.