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SMALL RNA EXPRESSION DURING PROGRAMMED REARRAGEMENT OF A VERTEBRATE GENOMEHerdy, Joseph R, III 01 January 2014 (has links)
The sea lamprey (Petromyzon marinus) undergoes programmed genome rearrangements (PGRs) during embryogenesis that results in the deletion of ~0.5 Gb of germline DNA from the somatic lineage. The underlying mechanism of these rearrangements remains largely unknown. miRNAs (microRNAs) and piRNAs (PIWI interacting RNAs) are two classes of small noncoding RNAs that play important roles in early vertebrate development, including differentiation of cell lineages, modulation of signaling pathways, and clearing of maternal transcripts. Here, I utilized next generation sequencing to determine the temporal expression of miRNAs, piRNAs, and other small noncoding RNAs during the first five days of lamprey embryogenesis, a time series that spans the 24-32 cell stage to the formation of the neural crest. I obtained expression patterns for thousands of miRNA and piRNA species. These studies identified several thousand small RNAs that are expressed immediately before, during, and immediately after PGR. Significant sequence variation was observed at the 3’ end of miRNAs, representing template-independent covalent modifications. Patterns observed in lamprey are consistent with expectations that the addition of adenosine and uracil residues plays a role in regulation of miRNA stability during the maternal-zygotic transition. We also identified a conserved motif present in sequences without any known annotation that is expressed exclusively during PGR. This motif is similar to binding motifs of known DNA binding and nuclear export factors, and our data could represent a novel class of small noncoding RNAs operating in lamprey.
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Expanding the repertoire of bacterial (non-)coding RNAsFindeiß, Sven 02 May 2011 (has links) (PDF)
The detection of non-protein-coding RNA (ncRNA) genes in bacteria and their diverse regulatory mode of action moved the experimental and bio-computational analysis of ncRNAs into the focus of attention. Regulatory ncRNA transcripts are not translated to proteins but function directly on the RNA level. These typically small RNAs have been found to be involved in diverse processes such as (post-)transcriptional regulation and modification, translation, protein translocation, protein degradation and sequestration.
Bacterial ncRNAs either arise from independent primary transcripts or their mature sequence is generated via processing from a precursor. Besides these autonomous transcripts, RNA regulators (e.g. riboswitches and RNA thermometers) also form chimera with protein-coding sequences. These structured regulatory elements are encoded within the messenger RNA and directly regulate the expression of their “host” gene.
The quality and completeness of genome annotation is essential for all subsequent analyses. In contrast to protein-coding genes ncRNAs lack clear statistical signals on the sequence level. Thus, sophisticated tools have been developed to automatically identify ncRNA genes. Unfortunately, these tools are not part of generic genome annotation pipelines and therefore computational searches for known ncRNA genes are the starting point of each study. Moreover, prokaryotic genome annotation lacks essential features of protein-coding genes. Many known ncRNAs regulate translation via base-pairing to the 5’ UTR (untranslated region) of mRNA transcripts. Eukaryotic 5’ UTRs have been routinely annotated by sequencing of ESTs (expressed sequence tags) for more than a decade. Only recently, experimental setups have been developed to systematically identify these elements on a genome-wide scale in prokaryotes.
The first part of this thesis, describes three experimental surveys of exploratory field studies to analyze transcript organization in pathogenic bacteria. To identify ncRNAs in Pseudomonas aeruginosa we used a combination of an experimental RNomics approach and ncRNA prediction. Besides already known ncRNAs we identified and validated the expression of six novel RNA genes.
Global detection of transcripts by next generation RNA sequencing techniques unraveled an unexpectedly complex transcript organization in many bacteria. These ultra high-throughput methods give us the appealing opportunity to analyze the complete RNA output of any species at once. The development of the differential RNA sequencing (dRNA-seq) approach enabled us to analyze the primary transcriptome of Helicobacter pylori and Xanthomonas campestris. For the first time we generated a comprehensive and precise transcription start site (TSS) map for both species and provide a general framework for the analysis of dRNA-seq data. Focusing on computer-aided analysis we developed new tools to annotate TSS, detect small protein-coding genes and to infer homology of newly detected transcripts. We discovered hundreds of TSS in intergenic regions, upstream of protein-coding genes, within operons and antisense to annotated genes. Analysis of 5’ UTRs (spanning from the TSS to the start codon of the adjacent protein-coding gene) revealed an unexpected size diversity ranging from zero to several hundred nucleotides. We identified and validated the expression of about 60 and about 20 ncRNA candidates in Helicobacter and Xanthomonas, respectively. Among these ncRNA candidates we found several small protein-coding genes that have previously evaded annotation in both species. We showed that the combination of dRNA-seq and computational analysis is a powerful method to examine prokaryotic transcriptomes.
Experimental setups are time consuming and often combined with huge costs. Another limitation of experimental approaches is that genes which are expressed in specific developmental stages or stress conditions are likely to be missed. Bioinformatic tools build an alternative to overcome such restraints. General approaches usually depend on comparative genomic data and evolutionary signatures are used to analyze the (non-)coding potential of multiple sequence alignments. In the second part of my thesis we present our major update of the widely used ncRNA gene finder RNAz and introduce RNAcode, an efficient tool to asses local protein-coding potential of genomic regions.
RNAz has been successfully used to identify structured RNA elements in all domains of life. However, our own experience and the user feedback not only demonstrated the applicability of the RNAz approach, but also helped us to identify limitations of the current implementation. Using a much larger training set and a new classification model we significantly improved the prediction accuracy of RNAz.
During transcriptome analysis we repeatedly identified small protein-coding genes that have not been annotated so far. Only a few of those genes are known to date and standard proteincoding gene finding tools suffer from the lack of training data. To avoid an excess of false positive predictions, gene finding software is usually run with an arbitrary cutoff of 40-50 amino acids and therefore misses the small sized protein-coding genes. We have implemented RNAcode which is optimized for emerging applications not covered by standard protein-coding gene annotation software. In addition to complementing classical protein gene annotation, a major field of application of RNAcode is the functional classification of transcribed regions. RNA sequencing analyses are likely to falsely report transcript fragments (e.g. mRNA degradation products) as non-coding. Hence, an evaluation of the protein-coding potential of these fragments is an essential task. RNAcode reports local regions of high coding potential instead of complete protein-coding genes. A training on known protein-coding sequences is not necessary and RNAcode can therefore be applied to any species. We showed this with our analysis of the Escherichia coli genome where the current annotation could be accurately reproduced. We furthermore identified novel small protein-coding genes with RNAcode in this extensively studied genome. Using transcriptome and proteome data we found compelling evidence that several of the identified candidates are bona fide proteins.
In summary, this thesis clearly demonstrates that bioinformatic methods are mandatory to analyze the huge amount of transcriptome data and to identify novel (non-)coding RNA genes. With the major update of RNAz and the implementation of RNAcode we contributed to complete the repertoire of gene finding software which will help to unearth hidden treasures of the RNA World.
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Developing the P19 Protein as a Tool for Studying the RNA Silencing PathwayDana, Foss January 2017 (has links)
RNA silencing is a cellular mechanism of post-transcriptional gene regulation which is highly conserved among the plant and animal kingdoms of life, and plays a critical part of developmental biology, maintenance of homeostasis, and host-pathogen interactions. The pathway is engaged by small double-stranded (ds)RNA molecules (small RNAs), which effect sequence specific gene silencing by targeting complementary RNA sequences. There are several classes of small RNAs which engage the pathway. MicroRNAs (miRNAs) are expressed in the genome as endogenous regulators of gene expression. Short-interfering RNAs (siRNAs) are usually from exogenous sources such as viral-derived short-interfering RNAs, or synthetic siRNAs which are applied to cells or organisms to inhibit expression of specific genes.
The p19 protein is a viral suppressor of RNA silencing (VSRS) endogenous to tombusviruses, which binds small RNA duplexes of any sequence with extremely high affinity. Because of its unique binding properties, recombinant p19 proteins are an excellent platform for tool development surrounding the RNA silencing pathway and are used extensively in novel applications for modulating the activity of small RNAs in living systems and for detecting small RNAs in biological samples. Herein we present work that has increased the breadth of p19’s utility as a biotechnology tool in three distinct realms. First, we present a chemical biology approach which combines p19 and small molecules for potent inhibition of the RNA silencing pathway in human cells. Secondly, we present the development of a novel fusion protein between p19 and a cell penetrating peptide (CPP), which functions as an siRNA delivery agent to allow gene knockdown in human cells. Thirdly, we have improved the utility of p19 for detecting and sequestering human miRNAs through rationally designing the binding surface; we describe mutations which dramatically enhance p19's affinity for human miRNA-122. The work presented here adds to the growing repertoire of engineered RNA binding proteins (RBPs) as tools for studying small RNA molecules and modulating their activity for applications in human therapeutics.
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Étude physiopathologique des petits ARN dérivés du clivage des YRNA dans les macrophages dans le contexte de l’athérosclérose / Physiopathologic study of the YRNA-derived small RNA in atherosclerotic macrophagesHizir, Zoheir 16 December 2016 (has links)
La récente découverte de nouvelles classes de petits ARN a ouvert la voie permettant l’exploration de nouvelles régulations des évènements physiopathologiques. Nous avons récemment démontré que les petits ARN dérivés des RNY (s-RNY) forment une classe indépendante de biomarqueurs permettant la détection de lésions coronariennes et sont associés au fardeau athérosclérotique. Ici, nous étudions le rôle des s-RNY dans les monocytes/macrophages humains et murins et avons démontré que dans les monocytes/macrophages chargées en graisse l’expression des s-RNY est corrélée dans le temps avec l’activation de la mort cellulaire dirigée par les caspases et de l’inflammation via la voie NF-κB. Des expériences de gain ou de perte de fonction ont démontré que l’expression même des s-RNY active la caspase 3 et l’activation de NF-κB. Etant donné que, dans les maladies cardiovasculaires, les s-RNY sont associés à Ro60 et circulent dans le sang de patients, nous avons étudié la fonction extracellulaire du complexe Ro60/s-RNY. Nos données démontrent que ce complexe induit la mort cellulaire ainsi que l’inflammation, lorsqu’il est ajouté au milieu de culture de monocytes/macrophages. Enfin, nous démontrons aussi que la fonction des s-RNY se fait par l’intermédiaire du récepteur de type Toll, TLR7. En utilisant un inhibiteur spécifique des TLR7, nous avons bloqué les effets intracellulaires aussi bien que les effets extracellulaires des s-RNY. Ces résultats positionnent les s-RNY en tant que molécules significatives qui vont impacter la physiopathologie des macrophages, indiquant leur rôle potentiel en tant que médiateur des maladies inflammatoires comme l’athérosclérose / The recent discovery of new classes of small RNAs has opened unknown territories to explore new regulations of physiopathological events. We have recently demonstrated that RNY-derived small RNAs (referred to as s-RNYs) are an independent class of clinical biomarkers to detect coronary artery lesions and are associated to atherosclerosis burden. Here, we have studied the role of s-RNYs in human and mouse monocytes/macrophages and have shown that in lipid-laden monocytes/macrophages s-RNY expression is timely correlated to the activation of both NF-κB and caspase 3-dependent cell death pathways. Loss- or gain-of- function experiments demonstrated that s-RNYs activate caspase 3 and NF-κB signaling pathways ultimately promoting cell death and inflammatory responses. Since, in atherosclerosis, Ro60-associated s-RNYs generated by apoptotic macrophages are released in the blood of patients, we have investigated the extracellular function of the s-RNY/Ro60 complex. Our data demonstrated that s-RNY/Ro60 complex induces caspase 3-dependent cell death and NF-κB-dependent inflammation, when added to the medium of cultured monocytes/macrophages. Finally, we have shown that s-RNY function is mediated by Toll-like receptor 7 (TLR7). Indeed using chloroquine, which disrupts signaling of endosome-localized Toll-like receptors (TLRs) or a more specific TLR7 antagonist, we blocked the effect of either intracellular or extracellular s-RNYs. These results position s-RNYs as relevant novel functional molecules that impacts on macrophage physiopathology, indicating their potential role as mediators of inflammatory diseases, such as atherosclerosis
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Évolution de la tolérance aux Hydrocarbures Aromatiques Polycycliques (HAPs) chez les spartines polyploïdes : analyses physiologiques et régulations transcriptomiques par les micro-ARNs / Evolution of tolerance to Polycyclic Aromatic Hydrocarbons (PAHs) in polyploid spartinas : physiological analyses and transcriptomic regulations by micro-RNAsCavé-Radet, Armand 19 December 2018 (has links)
Cette étude vise à explorer les mécanismes de tolérance des plantes aux xénobiotiques organiques de la famille des HAPs (phénanthrène), à travers l’analyse de l’impact des évènements de spéciation par hybridation et duplication génomique (allopolyploïdie). Nous avons pour cela mené une approche comparative sur un modèle de spéciation allopolyploïde récente, constitué des espèces parentales hexaploïdes S. alterniflora et S. maritima, et de l’allopolyploïde S. anglica qui résulte de la duplication du génome de leur hybride F1 S. x townsendii. Une approche intégrative basée sur des analyses physiologiques et moléculaires nous a permis de montrer que chez Spartina l’hybridation et le doublement du génome augmentent la tolérance aux xénobiotiques. Le parent paternel S. maritima se montre particulièrement sensible au phénanthrène par rapport au parent maternel S. alterniflora. Différentes analyses transcriptomiques ont permis l’identification de novo de transcrits spécifiquement exprimés en condition de stress, et l’annotation des petits ARNs (miARNs, leurs gènes cibles, et siARNs) agissant en tant que régulateurs de l’expression des gènes et la régulation des éléments transposables. Les analyses d’expression différentielle en réponse au stress ont permis de générer un modèle de régulation (miARN/gènes cibles) en réponse aux HAPs, testé par validation fonctionnelle en système hétérologue chez Arabidopsis. Un travail exploratoire de profilage du microbiome de la rhizosphère des spartines exposées au phénanthrène a été réalisé pour préciser les mécanismes de dégradation des xénobiotiques dans l’environnement en vue d’une application dans les stratégies de remédiation verte. / We explored mechanisms involved in tolerance to organic xenobiotics belonging to PAHs (phenanthrene), in the context of allopolyploid speciation (hybrid genome duplication). We developed a comparative approach, using a recent allopolyploidization model including the hexaploid parental species S. alterniflora and S. maritima, and the allopolyploid S. anglica, which resulted from genome doubling of the F1 hybrid S. x townsendii. Integrative approach based on physiological and molecular analyses highlights that hybridization and genome doubling enhance tolerance to xenobiotics in Spartina. The paternal parent S. maritima exhibits higher sensitivity compared to the maternal parent S. alterniflora. Various transcriptomic analyses were performed, to identify de novo stress responsive transcripts, and to annotate small RNAs (miRNAs, their target genes, and siRNAs) involved in gene expression and transposable element regulations. Differential expression analyses in response to stress allowed us to develop a putative miRNA regulatory network (miRNA/target genes) in response to PAH, functionally validated in Arabidopsis as heterologous system. An exploratory profiling of Spartina rhizosphere microbiome exposed to phenanthrene was also performed to characterize environmental degradation abilities, in the perspective of optimizing green remediation strategies.
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Caractérisation des petits ARN régulateurs impliqués dans la formation des cellules géantes induites par les nématodes phytoparasites du genre Meloidogyne / Characterization of small regulatory RNA involved in the development of giant cells induced by plant parasitic nematodes of the genus MeloidogyneMedina, Clémence 03 July 2017 (has links)
Les nématodes à galles du genre Meloidogyne sont des parasites obligatoires des plantes capables d’infecter un large panel de plantes d’intérêt agronomique. Ces parasites ont la capacité d’induire la différenciation de cinq à sept cellules racinaires en cellules géantes, hypertrophiées, métaboliquement actives et multinucléées. Ces cellules géantes constituent le site nourricier indispensable au nématode, et sur lequel il va s’alimenter jusqu’à sa reproduction. Le développement de ces cellules entraine une déformation racinaire appelée « galle » qui va perturber l’absorption de nutriments de la plante et l’affaiblir. Des études transcriptomiques ont montré qu’une vaste reprogrammation transcriptionnelle a lieu lors de la formation de la galle. Cette thèse vise à caractériser le rôle des petits ARN, des ARN non codants, au cours de la formation des galles induites par M. incognita. Les petits ARN non codants sont des régulateurs clés de l’expression génique et comprennent deux grandes familles : les microARN (miARN) et les petits ARN interférents (siARN). Pour cela, les petits ARN de racines de la plante modèle Arabidopsis thaliana saines et infectées par le nématode à galle M. incognita ont été caractérisés par séquençage haut débit à 7 et 14 jours après infection, deux stades importants du développement des cellules géantes. Cette étude a permis d’identifier 24 miARN d’Arabidopsis différentiellement exprimés dans les galles en comparaison aux racines non infectées. L’analyse fonctionnelle de ces miARN a permis de valider le profil d’expression dans les galles de cinq miARN et de démontrer le rôle de miR159 dans la réponse de la plante à M. incognita. De plus, une approche pangénomique a été réalisée afin d’identifier les gènes susceptibles d’être régulés par les siARN lors de l’interaction. En conclusion, ce travail a contribué à démontrer d’une part l’implication des miARN dans l’interaction plante - nématode à galles et a permis l’identification des gènes potentiellement régulés par les siARN lors de l’interaction et impliqués dans la formation des cellules géantes induites par les nématodes à galles. / Root-Knot-Nematodes are obligate plant parasites able to infect a large panel of cultivated plants. These parasites have the ability to induce redifferentiation of five to seven root cells into specialized giant cells, hypertrophied, multinucleated and metabolically overactive. These giant cells form the feeding site upon which nematodes feed continuously until reproduction. Giant cells development leads to a root deformation, named gall, which disturbs plant nutrients absorption causing its weakening. Transcriptomic studies showed that a huge transcriptional reprogramming occurs during gall development. This project aims to characterize the role of small RNAs, non-coding RNAs, during gall development induced by M. incognita. Small non-coding RNAs are key regulators of gene expression and include two major families: microRNAs (miRNAs) and small interfering RNAs (siRNAs). Thus, small RNAs from roots of the model plant Arabidopsis thaliana healthy or infected by M. incognita were characterized by Next Generation Sequencing at 7 and 14 days after infection, two important stages of gall development. This study led to the identification of 24 plant microRNAs differentially expressed in galls compared to uninfected roots. Functional analysis of these miRNAs validated the expression pattern in galls of five miRNAs and demonstrated the role of miR159 in the plant response to M. incognita. In addition, a genome-wide approach was used to identify genes that could be regulated by siRNAs during the interaction. In conclusion, this work contributed todemonstrate, on one hand, the involvement of microRNAs in the plant - RKN interaction and allowed the identification of genes potentially regulated by small interfering and involved in the formation of giant cells induced by root-knot nematodes.
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Genome-wide Analysis of F1 Hybrids to Determine the Initiation of Epigenetic Silencing in MaizeYang, Diya 08 January 2021 (has links)
No description available.
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Tnt1 retrotransposon expression and ethylene phytohormone interplay mediates tobacco (Nicotiana tabacum) defense responses / A dinâmica entre a expressão do retrotransposon Tnt1 e o fitormônio etileno envolvida nas respostas de defesa em tabaco (Nicotiana tabacum)Quintanilha, Danielle Maluf 10 October 2014 (has links)
Tnt1 is a transcriptionally active LTR-retrotransposon, present in over 600 copies in the Nicotiana tabacum genome. Under normal growth conditions, Tnt1 expression is limited to basal levels, but its expression is further induced under biotic and abiotic stresses. Transgenic tobacco plants (HP plants) expressing a Tnt1 reverse transcriptase hairpin were generated. These showed pleiotropic phenotypes such as cell death spots on the leaves and callose deposition and other severe abnormal development in aerial and underground portions. RNA sequencing of leaves with cell death spots revealed a rewiring of transcriptional regulatory networks related to stress responses exclusive to HPs. Among the positively modulated genes were ethylene synthesis and response cascade genes. The objective of the present work was to unravel the relation observed between Tnt1 and ethylene, generating a model. The results obtained suggest that HP seedlings and plants have increased ethylene synthesis when compared to the wildtype. Folding prediction of Tnt1 messenger RNA allowed the identification of ethylene-responsive sequences in putative stem loop locations. Thus it is possible that Tnt1 expression can produce small RNAs targeted to sequences present in the Tnt1 retrotransposon itself as well as at the promoter region of other ethylene responsive genes. Quantification of the expression of Tnt1 and ethylene related genes revealed \"phase opposition\" expression kinetics in the HPs, which led us to hypothesize that there might be an antagonistic relationship between the expression of Tnt1 and the expression of ethylene responsive genes involved in plant defense responses. Our findings suggest that Tnt1 could generate sRNAs that exerts transcriptional control over itself as well as other genes. Our model establishes a completely new biological role for a retrotransposon: Tnt1 would provide feedback control to ethylene-mediated gene regulation in tobacco defense responses, bringing the system back to a homeostatic condition and turning the defense responses down. / Tnt1 é um retrotransposon com LTR transcricionalmente ativo, e está presente em mais de 600 cópias no genoma de Nicotiana tabacum. Em condições normais de crescimento Tnt1 é expresso em níveis basais. No entanto, sua expressão é induzida pelo estímulo de estresses bióticos e abióticos. Plantas de tabaco transgênicas (chamadas de HP) expressando um grampo da transcriptase reversa de Tnt1 foram geradas. Estas apresentaram fenótipos como: pontos de morte celular e deposição de calose nas folhas e severas anomalias de desenvolvimento severas nas porções aérea e radicular das plantas. Sequenciamento de RNA de folhas com os pontos de morte celular revelou uma reorganização de redes de regulação transcricional relacionadas a resposta a estresses. Essas novas redes surgiram exclusivamente nas plantas HP. Entre os genes modulados positivamente estavam genes de síntese e de resposta ao etileno. O presente trabalho teve como objetivo elucidar a relação observada entre Tnt1 e o fitormônio etileno gerando um modelo de atuação. Os resultados obtidos permitiram demonstrar que plântulas e plantas HP adultas tem um aumento na síntese de etileno quando comparadas à selvagem. A predição do dobramento do RNA mensageiro de Tnt1 permitiu a identificação de sequências responsivas ao etileno localizadas em posição potencial para formar grampos. Desta forma, é possível que a expressão de Tnt1 leve à produção de pequenos RNAs que tem como alvo sequências responsivas a etileno presentes tanto no próprio elemento quanto em regiões promotoras de outros genes. A quantificação da expressão de Tnt1 versus genes relacionados ao etileno revelou um padrão em \"oposição de fase\" nas HPs, o que nos levou a hipotetizar que talvez ocorra uma relação antagonista entre a expressão de Tnt1 e a expressão de genes responsivos ao etileno envolvidos em respostas de defesa vegetais. Nossos resultados sugerem que Tnt1 pode gerar pequenos RNAs que exercem controle transcricional sobre Tnt1 e outros genes endógenos. Nosso modelo estabelece um novo papel biológico para um retrotransposon: Tnt1 agiria como um modulador da indução de genes mediada por etileno nas respostas de defesa de tabaco, trazendo o sistema de volta à condição homeostática e encerrando as respostas de defesa.
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Small RNA and genome interactions in Chlamydomonas reinhardtii recombinantsHessenberger, Daisy Sophia Innes January 2015 (has links)
When conspecific individuals are crossed, the ensuing hybridization creates a spectrum of phenotypes in the resulting offspring. Many of hybrid traits will be additive, similar to the parental phenotypes. In some cases however, transgressive phenotypes are formed, outside the range of that of the parental phenotypes. Transgressive phenotypes can either be restricted to the F1 generation or be heritable throughout the hybrid lineage. While the mechanism behind heritable transgressive phenotyped is yet to be determined, transgressive gene expression is thought to be the root cause of their formation. Epigenetics modifications, heritable variation separate to the DNA code, can alter gene expression, persist through generations, and vary between individuals and over time. This makes them ideal candidates to be involved in the formation of transgressive phenotypes. RNA silencing is an epigenetic mechanism of gene regulation relying on 20Q24nt single stranded small RNAs (sRNAs). Small RNAs, due to their ability to set up persistent epigenetic marks at a locus, have the potential to create heritable transgressive gene expression. For example, when genetic variation from one parental genome presents novel targets to the sRNAs of the other parental genome, new epigenetic marks such as DNA methylation or secondary sRNAs can be created at target sites. In order to understand the potential of small RNAs to influence hybrid phenotype, I designed crossing experiments with Chlamydomonas reinhardtii, choosing this unicellular alga due to the genetic tools available and the haploid nature of its vegetative cells. The specific aim of the experiment was to identify transgressively expressed sRNA populations. Crossing two geographically distinct strains of C. reinhardtii, and sequencing both the genomes and sRNAomes of parents and recombinants, I was able catalogue both genetic and epigenetic variation in the parental strains providing unique insight into the inheritance of small RNAs in this alga. In this thesis, I first compare the genomes of the parental strains, identifying polymorphisms and assessing genetic variation in RNA silencing pathway components. I then describe the sRNA profiles of the parental strains, identifying differentially expressed sRNA loci. I then describe my approach to identifying transgressively expressed sRNA loci in the hybrids. While many sRNA loci in the recombinants exhibit additive sRNA expression, I found multiple transgressively expressed sRNA loci. Using the available bioinformatics tools, I identified potential miRNAs and phased secondary sRNAs within the list of transgressively expressed loci. Target analysis of one of the transgressively expressed miRNAs linked it with the transgressive expression of certain phased loci, suggesting a potential for sRNAs to be able to set up heritable epigenetic marks in recombinant C. reinhardtii cells.
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Role of small RNAs and chromatin in transposable element silencing during global demethylationBerrens, Rebecca V. January 2017 (has links)
DNA methylation entails the addition of a methyl group to the 5-carbon of the cytosine base of the DNA. This modification is important during many biological processes such as imprinting, X-chromosome inactivation, cell differentiation as well as silencing of transposable elements (TEs). DNA methylation is dynamic during early mammalian development, despite being a more static mark in somatic cells. Global hypomethylation is a hallmark of epigenetic reprogramming in mammalian primordial germ cells (PGCs), the early embryo and in naïve embryonic stem cells (ESCs). Genome integrity is crucial during early development, as the germline DNA needs to be protected for future generations. Therefore, epigenetic reprogramming presents a critical phase for TE defence since presumably alternative silencing pathways need to be employed to limit their activity. In this thesis, I investigate the role of small RNAs to control TEs during global waves of DNA demethylation in cellular reprogramming, naïve pluripotency as well as early mammalian development. Following an introduction to the research questions, in chapter 3 I investigate the mechanism of TE regulation in an in vitro model of Dnmt1 deletion in mouse ES cells to recapitulate in vivo epigenetic reprogramming. I find that certain classes of TEs become transcriptionally upregulated and subsequently resilenced by a mechanism independent of DNA methylation. I identify ARGONAUTE 2 (AGO2) bound siRNAs as the prominent mechanism to control certain classes of TEs, while others appear to be regulated by redistribution of repressive histone modifications. In chapter 4, I construct Dicer constitutive and conditional KO ESCs in the background of the Dnmt1f l/f l ESCs using CRISPR-Cas9. I dissect the role of DNA methylation and of DICER dependent small RNAs on transcriptional changes of ESCs. Additionally, I find that DICER dependent small interfering RNAs (siRNAs) re-silence transcriptionally active TE classes. Finally, in chapter 5, I examine the role of small RNAs in TE silencing in different models of global hypomethylation in vivo and in vitro PGCs, during iPSC reprogramming and in a transition from serum to 2i culturing of mouse ESCs.
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