Rozprawy doktorskie na temat „Antisense RNA”

Antisense RNA (asRNA) strategies are identified as an effective and specific method for gene down-regulation at the post-transcriptional level. In this study, the major purpose is to find a correlation between the expression level and minimum free energy to enable the design of specific asRNA fragments. The thermodynamics of asRNA and mRNA hybridization were computed based on the fluorescent protein reporter genes. Three different fluorescent proteins (i) green fluorescent protein (GFP), (ii) cyan fluorescent protein (CFP) and (iii) yellow fluorescent protein (YFP) were used as reporters. Each fluorescent protein was cloned into the common pUC19 vector. The asRNA fragments were randomly amplified and the resulted antisense DNA fragments were inserted into the constructed plasmid under the control of an additional inducible plac promoter and terminator. The expression levels of fluorescent reporter protein were determined in real time by plate reader. Different results have been observed according to the fluorescent protein and the antisense fragment sequence. The CFP expression level was decreased by 50 to 78% compared to the control. However, with the GFP, the down-regulation did not exceed 30% for the different constructs used. For certain constructs, the effect was the opposite of expected and the expression level was increased. In addition, the YFP showed a weak signal compared to growth media, therefore the expression level was hard to be defined. Based on these results, a thermodynamic model to describe the relationship between the particular asRNA used and the observed expression level of the fluorescent reporter was developed. The minimum free energy and binding percentage of asRNA-mRNA complex were computed by NUPACK software. The expression level was drawn as a function of the minimum free energy. The results showed a weak correlation, but linear trends were observed for low energy values and low expression levels the CFP gene. The linear aspect is not verified for higher energy values. These findings suggest that the lower the energy is, the more stable is the complex asRNA-mRNA and therefore more reduction of the expression is obtained. Meanwhile, the non-linearity involves that there are other parameters to be investigated to improve the mathematical correlation. This model is expected to offer the chance to "fine-tune" asRNA effectiveness and subsequently modulate gene expression and redirect metabolic pathways toward the desired component. In addition, the investigation of the localization of antisense binding indicates that there are some regions that favors the hybridization and promote hence the down-regulation mechanisms.
Master of Science

Au cours de la dernière décennie, les avancées du séquençage à haut débit ont permis de caractériser un grand nombre d’ARN non codant et d’établir l’existence de transcrits “antisens” pour de nombreux gènes chez les mammifères. Cependant leur rôle dans le contrôle de l’expression des gènes “sens” auxquels ils sont associés est encore très mal connu. Mes travaux ont porté sur la caractérisation de certains aspects du mécanisme d’action des longs ARN non codants. Ils reposent sur le développement d’une approche de constructions indicatrices fluorescentes dont l’expression est suivie par cytométrie en flux en présence ou non d’ARN “antisens”. Cette approche a le potentiel de mettre en évidence une régulation même si elle n’est présente que dans une sous population cellulaire. Une première série d’expériences a été réalisée en expression transitoire pour bénéficier d’un contexte chromatinien simplifié. Mais dans ce cas les silencing observés sont aussi actifs sur une construction contrôle, indiquant la mise en place d’une réponse non spécifique de séquence qui évoque la réponse de type interféron. Cependant, l’expression globale des gènes cellulaire n’est pas significativement affectée, indiquant une certaine spécificité de la réponse. Parmi les voies testées ni la kinase PKR, ni la RNaseL ou la voie de l’interférence par l’ARN ne peuvent rendre compte du silencing observé. Une des caractéristiques de cette régulation est qu’elle n’affecte pas les gènes intégrés au génome mais uniquement les gènes exprimés à partir d’une construction épisomale ce qui évoque des caractéristiques souhaitables pour un mécanisme antiviral. Cependant l’ampleur de cette réponse non spécifique empêche toute étude plus approfondie d’un mécanisme spécifique s’il existe. Mes travaux se sont alors portés sur l’étude de ces mêmes constructions en clone stable dans deux contextes différents pour l’expression de l’ARN antisens ; en cis ou en trans. Dans le cas de l’expression en trans, un ARN antisens sans séquence régulatrice particulière ne permet pas la mise en place d’un silencing. Cette observation est en accord avec le faible nombre de longs ARN antisens connus pour agir en trans dans la nature. Par contre l’expression en cis d’un ARN antisens peut conduire à un silencing spécifique. Cette organisation dans laquelle les gènes « sens » et « antisens » sont situés sur le même fragment d’ADN correspond à celle majoritairement observée pour les longs ARNs antisens dans la nature (cisNAT, cis Natural Antisense Transcripts). Cependant, mes travaux montrent que le silencing observé n’est pas stable dans le temps et disparaît dès lors que la transcription antisens cesse, indiquant l’absence d’une mémoire épigénétique. Un tel mode de régulation est compatible avec une interférence transcriptionnelle dans laquelle la transcription et non le produit ARN est la cause du silencing. Par ailleurs, j’ai observé un certain nombre de cas de co-régulation du transcrit sens et antisens ce qui traduit la possibilité d’activer en cis la transcription du gène cible par le promoteur de son ARN antisens. Ce phénomène est probablement facilité par la petite taille de nos constructions, mais cette dualité de réponse est en accord avec l’absence de corrélation (positive ou négative) entre l’expression des gènes et de leur transcrits antisens. L’ensemble de mes travaux montrent la faible capacité d’un ARN antisens à induire un silencing. L’approche développée doit donc permettre de rechercher des co-activateurs du silencing, par exemple en introduisant des sites de recrutement de complexes modificateurs de la chromatine
During the last decade next generation sequencing has allowed the characterization of a large number of non-coding RNA and to establish that a majority of mammalian genes were also transcribed in the opposite orientation. However the functional significance of this antisense transcription is currently unclear.My work focused on the characterization of the regulatory potential of long non-coding RNA. It relied on the use of fluorescent reporter constructs, the expression of which in the presence or absence of antisense RNA is analyzed by flow cytometry. . This approach has the potential to uncover a regulation mechanism even if it takes place only in a subpopulation of cells.A first series of experiments has been realized by transient expression assays in order to benefit from a simplified chromatin context. However in this case the silencing associated with antisense transcripts is also active on control constructs, indicating that at least part of the response is not sequence specific suggesting the involvement of an interferon-type response. However, cellular gene expression is not significantly affected indicating some level of specificity. Among the investigated pathways, neither the PKR kinase, nor RNaseL or RNA interference pathway can account for the observed silencing. One of this regulation attributes is that it does not affect genes integrated in the genome but only genes expressed from episomes, a selectivity which would seem appropriate for an antiviral mechanism. Nevertheless the extent of this non-specific response impedes any further study on a specific mechanism, if it operates.My work then focused on the study of these reporter constructs after integration in the genome, antisense RNA being expressed in cis or in trans.In the case of trans expression, an antisense RNA devoid of any specific regulatory sequence does not allow the setting of a silencing. This observation is consistent with the low number of long antisense RNA known to act in trans in nature.On the other side, the cis expression of an antisense RNA can lead to a specific silencing. This organization in which “sense” and “antisense” genes are located in the same DNA fragment matches with the ones mostly observed for long antisense RNA in nature (cisNAT, cis Natural Antisense Transcripts). However, my work shows that the observed silencing is not stable over time and the effects terminate once antisense transcription stops, which indicates the absence of an epigenetic memory. This mode of regulation is compatible with a transcriptional interference in which transcription – and not its RNA product - is causing the silencing. Besides, I observed a certain number of sense and antisense transcript co-regulation cases highlighting the possibility to activate the transcription of the target gene by the promoter of its antisense RNA. This phenomenon is probably facilitated by the small size of our constructs, but this duality of response is in agreement with the lack of correlation (either positive or negative) between the expression of genes and their antisense transcripts.This study shows the limited capacity of an antisense RNA to induce a silencing. The developed approach should allow the search for silencing co-activators, for instance by introducing chromatin remodeling complexes recruitment sites

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

Expression of Type four pili is important for colonization and virulence in Neisseria meningitidis, which is a major causative agent of bacterial meningitis and septicaemia. Pili mediate adhesion, twitching motility, DNA uptake, and can be subject to phase and antigenic variation (Av). Pilin expression and Av may be modulated in response to environmental cues; however, the mechanisms of regulation are still unclear. This work demonstrates the identification of a novel cis-encoded RNA on the antisense (AS) strand of pilE, which encodes the major pilin subunit. The AS promoter is conserved in different N. meningitidis isolates, suggesting that the AS RNA may play an important role N. meningitidis biology. By Northern blot and strand-specific qRT-PCR, the AS transcript was shown to be expressed during specific conditions, namely after overnight growth and in response to salt stress. The AS RNA was found to encompass sequences antisense to the entire pilE coding sequence and 5' untranslated region, and extends to a promoter upstream of pilE that drives expression of a G4 small RNA that is important for pilin Av. Noncoding RNAs are increasingly recognized as important regulators of gene expression in bacteria. AS RNAs often modulate expression of the sense mRNA, however in this study, overexpression or deletion of the AS RNA had no significant effect on pilE transcript or pilin levels despite its long stretch of complementarity to the pilE mRNA. Potential trans targets were also investigated by performing RNAseq analysis to identify differentially expressed genes in the absence of the AS RNA. Expression of the AS RNA was found to reduce the frequency of pilE variation. The possible influence of the AS RNA on G4 small RNA transcription was investigated by examining its effect on the levels of G4 small RNA and RNA:DNA hybrids. Although technical issues prevented us from obtaining definitive results, our findings suggest the AS RNA forms RNA:DNA hybrids, adding an additional layer of complexity of molecular processes in the pilE locus of N. meningitidis.

Duchenne muscular dystrophy (DMD), the most common lethal neuromuscular disease in childhood, arises from protein-truncating mutations in the dystrophin gene. A deficiency in dystrophin leads to loss of the dystrophin associated protein complex (DAPC), which in turn, renders muscle fibres vulnerable to injury, and eventually leads to muscle loss, necrosis and fibrosis. Although, the dystrophin gene was identified nearly two decades ago, and extensive research has been directed at finding a therapy for DMD, to date, there is still no effective treatment available. One promising molecular approach to treat DNID is antisense oligomer (AO) induced splice intervention. AOs were most widely used to induce RNaseH-mediated gene transcript degradation, however, the development of different backbone chemistries heralds a new generation of AOs that can modify gene transcript splicing patterns. Application of AOs to the dystrophin pre-mRNA to influence exon selection and induce shortened, in-frame dystrophin isoforms is being vigorously pursued. The majority of the work presented here explores the concept of personalised therapies for DMD, whereby oligomers are designed to specifically target individual mutations. The importance of AO-optimisation to obtain AOs capable of inducing efficient dual exon skipping in an established animal model of muscular dystrophy (4CV mouse), which carries a DMD-causing mutation in exon 53, is demonstrated. Removal of both exons 52 and 53 was required to by-pass the mutation, maintain the reading frame and restore dystrophin expression. One of the major challenges of AO-induced splice intervention for therapeutic purposes will be the design and development of clinically relevant oligomers for many different mutations. Various models, including cells transfected with artificial constructs and mice carrying a human dystrophin transgene, have been proposed as tools to facilitate oligomer design for splice manipulation. This thesis investigates the relevance of using mouse models to design AOs for human application, and also explores the use of cultured human myoblasts, from both unaffected humans and a DMD patient, as a means of establishing the most effective therapeutic compound. In addition to induction of exon skipping, the applicability of AOs to promote exon inclusion, by masking possible intronic silencing motifs of survival motor neuron (SMN) pre-mRNA in cultured fibroblasts from a spinal muscular atrophy (SMA) patient, is investigated. This study provides additional information about a novel oligomer target site that could be used in combination with previously identified splice silencing motifs for a molecular therapeutic approach to SMA, and may perhaps open up new avenues of treatment for other genetic disorders, where oIigomers could be used to induce exon inclusion.

RNA is a highly versatile molecule with functions that span from being a messenger in the transfer from DNA to protein, a catalytic molecule important for key processes in the cell to a regulator of gene expression. The post-genomic era and the use of new techniques to sequence RNAs have dramatically increased the number of regulatory RNAs during the last decade. Many of these are antisense RNAs, as for example the miRNA in eukaryotes and most sRNAs in bacteria. Antisense RNAs bind to specific targets by basepairing and thereby regulate their expression. A major step towards an understanding of the biological role of a miRNA or an sRNA is taken when one identifies which target it regulates. We have used RNA libraries to study the RNA interference pathway during development in the unicellular model organism Dictyostelium discoideum. We have also, by combining computational and experimental methods, discovered the first miRNAs in this organism and shown that they have different expression profiles during development. In parallel, we have developed a novel approach to predict targets for sRNAs in bacteria and used it to discover sRNA/target RNA interactions in the model organism Escherichia coli. We have found evidence for, and further characterized, three of these predicted sRNA/target interactions. For instance, the sRNA MicA is important for regulation of the outer membrane protein OmpA, the sRNAs OmrA and OmrB regulate the transcription factor CsgD, which is important in the sessile lifestyle of E. coli, and MicF regulates its own expression in a feed forward loop via the regulatory protein Lrp. In conclusion, we have discovered novel antisense RNAs, e.g. miRNAs in D. discoideum, developed an approach to identify targets for antisense RNAs, i.e. a target prediction program for sRNAs in bacteria, and verified and characterized some of the predicted antisense RNA interactions.

Two vector systems were developed expressing antisense RNA (of two different sizes) complementary to three target regions in the 5' leader/LTR of HIV-1: the R (TAR) region, the primer binding site and the splice donor-packaging signal (ψ) region. After cloning these regions into an expression vector, pcDNA3, designed to express these sequences at high levels from the CMV IE promoter, stable constitutive expression in a T cell line was demonstrated by RT-PCR. After directly challenging these cell lines with HIV-1 at various doses cell lines expressing antisense RNA targeting the ψ-region showed significant inhibition of replication compared to cell lines expressing the same sequence in sense. A second, co-transfection assay using COS-1 cells was also developed to assess the antiviral capabilities of these vectors. Both sequences targeting the ψ-region and one sequence complementary to the TAR region inhibited expression of viral protein; furthermore analyses of relative levels of cellular and viral RNA from these assays suggested that each of these antisense molecules was exerting its effect at an early stage in the transcription-translation pathway. CXCR4, an important co-receptor for HIV was also targeted for down regulation by antisense RNA. Inducible expression of a sequence complementary to a region of this gene using the 'tet-on' system was accomplished leading to down-regulation of surface CXCR4 expression in T cells. Down-regulation appeared to result in reduced replication of HIV-1 in these cells. Genetic strategies for inhibiting HIV replication may provide an alternative and complementary approach to chemotherapy for infected individuals.

Streptococcus pyogenes is a re-emerging pathogen that produces superficial and life threatening invasive diseases. One important virulence factor in S. pyogenes is hyaluronic acid capsule which has been shown to increase expression at sub-body temperatures in certain strains. This study showed that thermoregulation is common in invasive clinical isolates. Regulation was shown to occur independent of the CovRS two-component regulator in a post transcription manner and before protein level regulation. The endoribonuclease, CvfA, was also confirmed to be required for capsule thermoregulation. The search for a regulator lead to the discovery of the 1st antisense RNAs in S. pyogenes found opposite the capsule synthesis genes. Its role if any in capsule has not been discovered. Finally, a group of sRNAs were characterized adding to the knowledge of this layer of regulation in S. pyogenes.

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.

Thesis (M.S.) -- University of Maryland, College Park, 2003.
Thesis research directed by: Dept. of Chemical Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

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.

Em procariotos, RNAs antisense (asRNAs) constituem a classe de RNAs não codificantes (ncRNAs) mais numerosa detectada por métodos de avaliação de transcritoma em larga escala. Apesar da grande abundância, pouco se sabe sobre mecanismos regulatórios e aspectos da conservação evolutiva dessas moléculas, principalmente em arquéias, onde o mecanismo de degradação de RNAs dupla fita (dsRNAs) é um fenômeno pouco conhecido. No presente estudo, utilizando dados de dRNA-seq, identificamos 1626 inícios de transcrição primários antisense (aTSSs) no genoma de Halobacterium salinarum NRC-1, importante organismo modelo para estudos de regulação gênica no domínio Archaea. Integrando dados de expressão gênica obtidos a partir de 18 bibliotecas de RNA-seq paired-end, anotamos 846 asRNAs a partir dos aTSSs mapeados. Encontramos asRNAs em ~21% dos genes anotados, alguns desses relacionados a importantes características desse organismo como: codificadores de proteínas que constituem vesículas de gás e da proteína bacteriorodopsina, além de vários genes relacionados a maquinaria de tradução e transposases. Além desses, encontramos asRNAs em genes pertencentes a sistemas de toxinas-antitoxinas do tipo II e utilizando dados públicos de dRNA-seq, evidenciamos que esse é um fenômeno que ocorre em bactérias e arquéias. A interação de um ncRNA com seu RNA alvo pode ser dependente de proteínas, em arquéias, a proteína LSm é uma chaperona de RNA homóloga a Hfq de bactérias, implicada no controle pós-transcricional. Utilizamos dados de RIP-seq de RNAs imunoprecipitados com LSm e identificamos 91 asRNAs interagindo com essa proteína, para 81 desses, o mRNA do gene sense também foi encontrado interagindo. Buscando por aTSSs presentes nas mesmas regiões de genes ortólogos, identificamos 160 aTSSs que dão origem a asRNAs em H. salinarum possivelmente conservados em Haloferax volcanii. A expressão dos asRNAs anotados foi avaliada ao longo de uma curva de crescimento e em uma linhagem knockout de um gene que codifica uma RNase R, possível degradadora de dsRNAs em arquéias. Encontramos um total de 144 asRNAs diferencialmente expressos ao longo da curva de crescimento, para 56 desses o gene sense também está diferencialmente expresso, caracterizando possíveis mecanismos de regulação em cis por esses RNAs. Na linhagem knockout, encontramos cinco asRNAs diferencialmente expressos e apenas para um desses o gene sense também está diferencialmente expresso, resultado que não nos permitiu inferir um possível papel de degradação de dsRNAs da RNAse R em H. salinarum NRC-1. Nesse trabalho apresentamos um mapeamento completo do transcritoma antisense primário de H. salinarum NRC-1 com resultados que consistem em um importante passo na direção da compreensão do envolvimento da transcrição antisense na regulação gênica pós-transcricional desse organismo modelo do terceiro domínio da vida.
Antisense RNAs (asRNAs) constitute the most numerous class of non-coding RNAs (ncRNAs) detected by transcriptome highthroughput methods in prokaryotes. Despite this abundance, little is known about regulatory mechanisms and evolutionary aspects of these molecules, mainly in archaea, where the mechanism of double-strand RNA (dsRNA) degradation remains poorly understood. In this study, using dRNA-seq data, we identified 1626 antisense transcription start sites (aTSSs) in the genome of Halobacterium salinarum NRC-1, an important model organism for gene expression regulation studies in Archaea. By integrating gene expression data from 18 RNA-seq paired-end libraries, we were able to annotate 846 asRNAs from mapped aTSSs. We found asRNAs in ~21% of annotated genes including genes related to important characteristics of this organism, such as: gas vesicle proteins, bacteriorhodopsin, translation machinery and transposases. We also found asRNAs in type II toxin-antitoxin systems and using public dRNA-seq data, we show evidences that this phenomenon might be conserved in archaea and bacteria. The interaction of a ncRNA with its target may depend on intermediary proteins action. In archaea, the LSm protein is a RNA chaperone homologous to bacterial Hfq, involved in post-transcriptional regulation. We used RIP-seq data from RNAs immunoprecipitated with LSm and identified 91 asRNAs interacting with this protein, for 81 of these the mRNA of the sense gene is also interacting. We searched for aTSSs present in the same region of orthologous genes in the Haloferax volcanii. We found 160 aTSSs that originated asRNAs in H. salinarum NRC-1 that might be conserved in this two archaea. The expression of annotated asRNAs was analyzed over a growth curve and in a knockout strain for RNase R gene. We found 144 asRNA differentially expressed over the growth curve, for 56 of these the sense gene was also differentially expressed, characterizing possible cis regulators asRNAs. In the knockout strain we found five differentially expressed asRNAs and only one asRNA/gene pair, this result does not allow us to infer a dsRNA degradation in vivo activity for this RNase in H. salinarum NRC- 1. This work contributes to the discovery of the antisense transcriptome in H. salinarum NRC- 1 a relevant step to uncover the post-transcriptional gene regulatory network in this archaeon.

L’ARN est sans aucun doute la molécule biologique la plus versatile qui soit. Tout comme l’ADN, il peut contenir et transmettre de l’information génétique. Tout comme les protéines, il peut accomplir une multitude de fonctions biologiques. De plus, son rôle le plus connu demeure celui d’intermédiaire entre l’ADN et les protéines. L’ARN est donc au cœur d’un bon nombre de processus biologiques. Ceci lui confère un immense potentiel thérapeutique qui jusqu’à présent demeure largement inexploité. Pour accomplir ses fonctions, l’ARN doit adopter une structure tridimensionnelle précise qui est dépendante à la fois de sa séquence et de son environnement. Ainsi, en modifiant la structure d’un ARN, il est possible d’en moduler sa fonction. C’est l’objectif global des travaux présentés dans cette thèse. Pour y parvenir, de courts oligonucléotides antisens (OA) ont été utilisés. Cette stratégie revêt plusieurs avantages. Comme les OA s’apparient à leur cible en formant des paires de bases Watson-Crick, ils offrent une grande spécificité et leur design est facile. De plus, en se fiant aux données structurales et aux logiciels de prédictions de structures des ARN, on peut aisément identifier les régions à cibler avec les OA. Enfin, cette technique est versatile puisqu’on peut cibler différents motifs d’ARN. La première cible a été le ribozyme du virus de l’hépatite D. Cet ARN, qui catalyse une réaction d’auto-coupure, a été modifié afin que son activité devienne dépendante à la liaison d’OA. Plusieurs modules ont ainsi été créés et combinés afin d’obtenir des ribozymes qui répondaient à la présence d’un ou plusieurs OA. En insérant ces interrupteurs moléculaires dans les régions non traduites d’un ARNm, nous avons ainsi modulé l’expression de ce gène avec les OA. Cet outil a des applications intéressantes pour la régulation de gènes en biologie synthétique. Un autre motif ciblé a été le G-quadruplex (G4). Cette structure non canonique exerce de nombreuses fonctions biologiques et représente donc une cible thérapeutique intéressante. Lorsque présent dans la région 5’ non traduite d’un ARNm, le G4 mène généralement à une diminution de la traduction. En utilisant des OA qui empêchent la formation du G4, nous avons été en mesure d’augmenter la traduction du gène ciblé. De plus, il a été possible de développer des OA qui favorisent la formation d’un G4 dans le but de diminuer l’expression de la cible. Finalement, dans le dernier chapitre de cette thèse, il est démontré que les G4 présents dans les microARN primaires influencent leur maturation en microARN matures. Des OA ciblant ces G4 ont été utilisés afin de favoriser la maturation de microARN suppresseurs de tumeurs, ce qui présente un potentiel thérapeutique intéressant. En bref, les travaux présentés dans cette thèse démontrent clairement que les OA sont un outil de choix pour cibler et modifier la structure de motifs d’ARN spécifiques.
Abstract : RNA is a versatile biological molecule. Like DNA, it can contain and transmit genetic information. Like proteins, it can accomplish multiple biological functions. Also, its most known role remains that of intermediary between DNA and proteins. RNA is thus a key player in many biological processes. This gives it an immense therapeutic potential which remains largely untapped. To fulfill its functions, RNA must adopt a precise threedimensional structure that is dependent on both its sequence and its environment. Thus, by modifying the structure of an RNA, it is possible to modulate its function. This is the overall objective of the work presented in this thesis. To achieve this, small antisense oligonucleotides (ASO) have been used. This strategy has several advantages. As ASO bind their target with Watson-Crick base pairs, they offer great specificity and their design is easy. Moreover, reliance on structural data and RNA structure prediction softwares makes it easy to identify the regions to be targeted with ASO. Finally, this technique is versatile since it is possible to target different RNA motifs. The first target was the HDV self-cleaving motif. This RNA, which catalyzes a self-cleaving reaction, has been modified so that its activity became dependent on the binding of ASO. Several modules were thus created and combined in order to obtain ribozymes which responded to the presence of one or more ASO. By inserting these molecular switches into an mRNA’s UTR, the expression of this gene was modulated with the ASO. This has interesting applications for the regulation of genes in synthetic biology. Another target motif was the G-quadruplex (G4). This non-canonical structure exerts many biological functions and therefore represents an interesting therapeutic target. When present in the mRNA’s 5’UTR, G4 generally lead to a decrease in translation. Using ASO that prevent G4 formation, we were able to increase the translation of the target gene. In addition, it has been possible to develop ASO which promote the formation of a G4 in order to decrease the expression of the target. Finally, in the last chapter of this thesis, it is demonstrated that the G4 present in the primary microRNAs influence their maturation in mature microRNAs. ASO targeting these G4 have been used in order to promote the maturation of tumor suppressor microRNAs, which has an interesting therapeutic potential. The work presented in this thesis clearly demonstrates that ASO are ideal for targeting and altering the structure of specific RNA motifs.

An increasing number of RNA targeted gene repression mechanisms are being discovered operating in mammalian cells. Antisense-RNA mediated silencing of CpG island-associated genes is not limited to imprinting and X inactivation, but can occur at autosomal non-imprinted loci leading to disease. An antisense silencing phenomenon observed in a patient with an inherited form of anaemia was recently recreated in differentiating mouse embryonic stem cells, demonstrating that the silencing and methylation of the oppositely transcribed tissue specific alpha globin CpG island exclusively occurs in the presence of antisense RNA. The aim of this thesis is to use differentiating mouse embryonic stem cells to further characterise this silencing mechanism through analysis of the histone modifications associated with repression of the alpha globin gene upon differentiation, and to further define the developmental period in which repression is established. Also, the requirement for antisense RNA transcription through the alpha globin gene is examined through insertion of a transcriptional terminator to truncate antisense RNA transcription. The gene silencing mechanism is tested with other genes in place of the alpha globin gene to investigate whether repression is limited to the alpha globin gene or is also applicable to other tissue specific or ubiquitously expressed genes. Analysis of the tissue specific gene MYOD and the ubiquitously expressed gene UBC demonstrated that the silencing mechanism could effect other genes in a similar manner, though not all genes were susceptible to repression as the ubiquitously expressed gene ACTB remained unsilenced by antisense RNA expression. These observations establish that this silencing mechanism can play a more general role in repression, thereby suggesting its potential role as a constituent of the multivariate repressive pathways within the mammalian cell.

Synthetic antisense oligoribonucleotides can be used to modulate gene splicing by masking key motifs on the pre-mRNA required for spliceosome assembly. Yet, intracellular expression of oligoribonucleotides generates only a transitory effect whereas stable delivery of antisense sequences can be achieved by linking them to chimeric small RNAs delivered and expressed by viral vectors. In the murine model of Duchenne Muscular Dystrophy a chimeric U7 snRNA (U7Dtex23) induces skipping of the mutated exon 23 and restores the Dystrophin mRNA reading frame. The main limitation of this approach remains the large amount of snRNA vector needed to be produced and administered to patients. To optimize this system we used self-complementary AAV vectors (scAAV) to express the U7snRNA shuttles. ScAAV vectors were tested in mouse myoblast cultures and we observed an increase in U7Dtex23 expression and in dystrophin exon 23 skipping compared to single-stranded AAV, highlighting the potential for this strategy to reduce the vector dose. Alternatively, we have used a muscle and heart-specific enhancer (MHCK) to drive the expression of U7Dtex23 cassettes delivered with AAV vectors and our results showed that MHCK improves chimeric U7snRNA expression and increases dystrophin exon 23 skipping in vitro and in vivo. However, additional U7snRNA species were produced following gene transfer, pointing at a possible limitation of the cellular processing machinery capability with saturating levels of U7 shuttles. We have also explored the possibility of using small nucleolar (sno) RNAs as novel molecular platforms for antisense delivery. We replaced the original antisense of MBII-52 snoRNA with the Dtex23 sequence and observed low levels of exon 23 skipping in AAV-transduced myotubes. While our observation validates the approach, the efficiency of skipping is still considerably lower than with the U7snRNA cassette. As a last approach, we engineered the human C/D box U24snoRNA to specifically target the methylation of an adenosine branch point in a luciferase reporter pre-mRNA in order to induce the skipping of the downstream exon. We were not able to observe any modulation of splicing using this strategy.

A plasmid (pGEM-SD) was constructed in which the Shine-Dalgarno sequence had been deleted from pGEM-7zf(+) plasmid to generate a vector for regulated expression of antisense RNA. The binding of antisense RNA to mRNA provides a potent mechanism by which specific transcripts can be translationally inactivated. Although part of the lac operator sequence was deleted in pGEM-SD plasmid, it was proven that mRNA still can be induced under the control of lac promoter. Recombinant plasmids were generated by ligating bacterial genornic DNA into pGEM-SD plasmid, but the orientation of the inserted gene with respect to the lac promoter has not been determined. These preliminary results show that gene expression can be turned on and off via the pGEM-SD vector. Future experiments will screen antisense recombinant clones for those which would inhibit specific gene products by plating on different selective indicator media both with and without the inducer IPTG. This will allow investigation of the biological functions associated with genomic DNA sequences by means of gene mapping with antisense RNA.

A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems. In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application. AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate. Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).

Cyanobacteria are supposed to have been the first organisms able to evolve oxygen by photosynthesis, the process that uses solar light as energy source to fix CO2 into carbohydrate molecules. Historically, they have been and are a model system for studying fundamental processes including and relating to photosynthesis. In more recent years cyanobacteria acquired interest also in biotechnology, in particular as an alternative energy resource, for instance in ethanol or hydrogen production, and as a source for the production of molecules useful in the pharmaceutical industry. Moreover cyanobacteria are interesting host organisms for proteins expression, in particular for membrane proteins. Indeed, the abundant internal membrane system (the thylakoids) typical of cyanobacteria would provide the proper compartment to harbour these kind of proteins, permitting higher production of these proteins. Synechocystis sp. PCC 6803 is nowadays one of the most extensively studied species among all cyanobacteria and is considered a model organism for the study of photosynthesis. Some of the main reason are that it is spontaneously transformable, it can easily incorporate exogenous DNA into its genome by homologous recombination and it is able to grow both photoautotrophically and heterotrophycally. However, the lack of handy episomal elements for cloning and the presence of 10-12 copies of the chromosome per cell have hampered its use so far. The aim of the present study is to develop a Synechocystis strain suitable for a simplified use of DNA recombinant techniques within this organism and for the inducible expression of proteins. The genetic tool consists in a two component system: i) a new Synechocystis strain, called “T7-cyano”, hosting the highly processive RNA polymerase from the bacteriophage T7 under the control of different inducible promoters; ii) vectors, called pSEV (Synechocystis Expression Vectors), for stable introduction of a DNA fragment of interest under the control of the T7 promoter. The system resembles therefore the BL21 strain of Escherichia coli: induction of the T7 RNA polymerase will lead to transcription of the heterologous sequence in a controlled manner. The present work describes the design and the early development of the T7-cyano system. After a brief introduction on cyanobacteria (chapter one), the second chapter describes the design of the new cyanobacterial tool and the construction of a vector, called Transformation Vector, for the stable integration of the T7 RNA polymerase gene in the genome of Synechocystis sp. PCC 6803. In this same chapter, the construction of the second component of the system is also illustrated. This is represented by three different expression vectors having different applications: pSEV1, for Strep-tagged fusion proteins expression; pSEV2, for recombinant expression of endogenous or heterologous proteins; pSAS, for the synthesis of RNA molecules in antisense. The development of the T7-cyano strain is reported in the third chapter. Three different inducible promoters are chosen to control the expression of the T7 RNA polymerase gene: Pzia, a zinc-inducible endogenous promoter, Pnrs, an endogenous nickel-inducible one and variant of the E. coli promoter Plac. By the developed Transformation Vector the T7 RNA polymerase gene is homoplasmically inserted in the genome of the cyanobacterium. The production of the bacteriophage polymerase in the T7-cyano strain is successfully confirmed by immunoblotting upon induction of the Pzia and Pnrs promoters. The system is tested in the expression of three different heterologous proteins (chapter four): the eGFP fluorescence protein; HydA, a [FeFe]-hydrogenase from Chlamydomonas reinhardtii previously expressed in Synechocystis sp. PCC 6803; and a Baeyer-Villiger monooxygenase, BVMO, from the algae Cyanidioschyzon merolae, previously expressed in Escherichia coli. The sequences of the chosen protein coding genes are cloned in one of the expression vectors developed, pSEV1 and pSEV2, and the obtained vectors are used to transform the T7-cyano strains. In preliminary induction experiments transcript analysis confirms transcription of the heterologous genes by the T7 RNA polymerase. Moreover by western blot, the BVMO enzyme is successfully confirmed expressed in T7-cyano. However the promoters Pzia and Pnrs are found both leaky and the low amount of the BVMO enzyme produced suggests translation inefficiency. In the last chapter the system is proposed for the inducible synthesis of antisense RNAs. Synechocystis sp. PCC 6803, in fact, possesses a significant number of regulatory RNAs transcribed in a wide range of environmental changes and stress conditions. To date, however, only few of them have been characterized and new genetic tools are required for the study and identification of these small RNA molecules. In addition, an interesting application would be the controlled knockdown of endogenous genes. To test the T7-cyano system in the synthesis of antisense RNAs, the characterized IsrR, known to down-regulate the iron-starvation responding gene isiA, is chosen. In addition an antisense is designed against the 5’ UTR of the same isiA gene. The experiments were performed at the Department of Biochemistry of the University of Turku (Finland) at the Molecular Plant Biology group headed by Prof. Eva-Mari Aro, one of the main experts in the study of photosynthetic organisms. Preliminary experiments shows a knockdown phenotype in induced cells, however more experiments are necessary to confirm the silencing of the gene by the synthesis of antisense RNAs in T7-cyano. The present work gives a general view of the new system showing, for the first time, the capability of Synechocystis sp. PCC 6803 to produce the T7 RNA polymerase that in turn is able to transcribe the sequence downstream the T7 promoter. The work also pointed out the two major problematic issues found within the system that are the leakiness of the promoters, Pzia and Pnrs, and a translational inefficiency in proteins expression
Si ritiene che i cianobatteri siano stati i primi organismi in grado di produrre ossigeno attraverso la fotosintesi, il processo chimico che utilizza la luce solare come fonte di energia per fissare la CO2 in molecole di carboidrati. I cianobatteri sono storicamente considerati un sistema modello per lo studio dei processi fotosintetici. Più recentemente questi organismi hanno acquisito un particolare interesse anche in ambito biotecnologico, in particolare come fonte alternativa di energia, per esempio nella produzione di bioetanolo o bioidrogeno, e nella produzione di molecole utili per l'€™industria farmaceutica. I cianobatteri, inoltre, sono interessanti organismi da usare come ospiti per l'espressione di proteine, in particolare per quelle di membrana. Infatti, il loro abbondante sistema membranale interno (i tilacoidi) dovrebbe fornire lo spazio adeguato per la localizzazione di questo tipo di proteine e permettere quindi una maggiore produzione delle stesse. Synechocystis sp. PCC 6803 è a tutt'€™oggi uno dei ceppi cianobatterici più studiati ed è considerato un organismo modello per lo studio della fotosintesi. Le ragioni principali di questo suo ampio utilizzo risiedono in alcune sue caratteristiche: è spontaneamente trasformabile, puù facilmente incorporare DNA esogeno nel suo genoma mediante ricombinazione omologa ed è in grado di crescere sia in fotoautotrofia sia in eterotrofia. Tuttavia, la mancanza di elementi episomali maneggevoli anche per il clonaggio e la presenza di 10-12 copie del genoma per cellula batterica hanno finora ostacolato un suo più vasto sfruttamento. Lo scopo di questo studio è di sviluppare un ceppo di Synechocystis sp. PCC 6803 che semplifichi l'€™utilizzo delle tecniche del DNA ricombinante in questo organismo e che possa essere utilizzato per l'espressione inducibile di proteine. Il tool consiste in un sistema a due componenti: i) un nuovo ceppo di Synechocystis sp. PCC 6803, chiamato "T7-cyano", che ospita il gene codificante la T7 RNA polimerasi sotto il controllo di diversi promotori inducibili; ii) un set di vettori, chiamati pSEV (Synechocystis Expression Vectors), per l'introduzione stabile nel genoma di un frammento di DNA di interesse sotto il controllo del promotore T7. Il sistema è analogo al ceppo BL21 di Escherichia coli: l'€™induzione della T7 RNA polimerasi conduce alla trascrizione della sequenza eterologa in modo controllato. Il presente lavoro descrive il disegno sperimentale del sistema T7-cyano e i risultati preliminari conseguenti alla sua induzione. Dopo una breve introduzione sui cianobatteri (capitolo uno), nel secondo capitolo è descritto il nuovo tool cianobatterico e la costruzione di un vettore, Transformation Vector, per l'integrazione stabile del gene codificante la T7 RNA polimerasi nel genoma di Synechocystis. In questo stesso capitolo, è inoltre illustrata la costruzione della seconda componente del sistema. Questa è rappresentata da tre diversi vettori di espressione aventi diverse applicazioni: pSEV1, per l'espressione di proteine in fusione con uno Strep-tag; pSEV2, per l'espressione di proteine ricombinanti endogene o eterologhe; pSAS, per la sintesi di molecole di RNA in antisenso. La creazione e lo sviluppo del ceppo T7-cyano è riportata nel terzo capitolo. Tre diversi promotori inducibili sono selezionati per controllare l'espressione della T7 RNA polimerasi. I promotori scelti sono: Pzia, un promotore endogeno inducibile dallo zinco, Pnrs, un altro promotore endogeno inducibile da nickel e una variante del promotore Plac di Escherichia coli. Utilizzando il Transformation Vector il gene della T7 RNA polimerasi è inserito in omoplasmia nel genoma del cianobatterio. La produzione della T7 RNA polimerasi è, per la prima volta in Synechocystis, mostrata mediante analisi di immunoblotting nei ceppi con i promotori Pzia e Pnrs. Il sistema è quindi testato nell'espressione di tre differenti proteine eterologhe (capitolo quattro): la proteina fluorescente eGFP; HydA, una [FeFe]-idrogenasi proveniente dall'alga Chlamydomonas reinhardtii precedentemente espressa in Synechocystis sp. PCC 6803; e una Baeyer-Villiger monoossigenasi (BVMO) dell'alga Cyanidioschyzon merolae, già precedentemente espressa in Escherichia coli. Le sequenze codificanti le tre proteine prescelte sono clonate in uno dei vettori di espressione sviluppati, pSEV1 e pSEV2, e i vettori ottenuti sono utilizzati per trasformare i ceppi di T7-cyano. In esperimenti preliminari di induzione è confermata la trascrizione dei geni eterologhi ad opera della T7 RNA polimerasi. Inoltre attraverso immunoblotting è stata con successo rilevata l'espressione dell'enzima BVMO. Tuttavia i promotori Pzia e Pnrs, sia mediante l'analisi dei trascritti che immunoblotting, sono risultati leaky. Inoltre la bassa quantità dell'enzima BVMO prodotta suggerisce una inefficiente traduzione del gene esogeno. Nell'ultimo capitolo il sistema T7-cyano è proposto per la sintesi inducibile di RNA antisenso. Synechocystis sp. PCC 6803, infatti, possiede un numero elevato di piccoli RNA regolatori trascritti in diverse condizioni ambientali e di stress, tuttavia solo pochi RNA antisenso sono stati caratterizzati tutt'oggi. Il nuovo sistema puù quindi essere un utile tool per lo studio e la caratterizzazione di queste piccoli RNA. Inoltre una interessante applicazione sarebbe il knockdown controllato di geni endogeni. Per testare il sistema T7-cyano nella sintesi di RNA antisenso, viene scelto IsrR, uno dei pochi RNA antisenso già caratterizzato in Synechocystis sp. PCC 6803, noto down-regolatore del gene IsiA attivato in condizioni di carenza di ferro. In aggiunta è disegnato un secondo RNA antisenso contro il 5'UTR dello stesso gene IsiA. Gli esperimenti preliminari di induzione sono stati svolti presso il dipartimento di biochimica della University of Turku (Finland) nel gruppo di ricerca Molecular Plant Biology guidato dalla Prof. Eva-Mari Aro, esperta nello studio degli organismi fotosintetici. I primi risultati mostrano un fenotipo knockdown nei ceppi indotti, tuttavia maggiori esperimenti sono necessari per confermare il silenziamento del gene attraverso la sintesi di antisenso nei T7-cyano. Il lavoro di questa tesi fornisce una visione di insieme del nuovo sistema mostrando, per la prima volta, la capacità di Synechocystis sp. PCC 6803 di produrre la T7 RNA polimerasi che a sua volta è in grado di trascrivere il gene clonato a valle del promotore T7. Il lavoro ha inoltre evidenziato le due maggiori problematiche del sistema ovvero la leakiness dei promotori Pzia e Pnrs e una inefficiente traduzione del gene eterologo

The initial description of genomes organization has consisted in the separation between regulatory and protein-coding DNA stretches. This simple and elegant model has supported the “one region-one function” theory: a genome is a linear arrangement of functional elements interspersed with nonfunctional regions. Recently the advances in transcriptomics technologies have shown that a genomic region can be used for different purposes and that functional elements can co-locate in the same region of the genome.

Friedreich ataxia (FRDA) is a neurodegenerative disorder that is inherited in an autosomal recessive pattern. The most common FRDA mutation is hyperexpansion of a GAA triplet repeat sequence in the first intron of the affected gene, frataxin (FXN), resulting in decreased frataxin protein expression. The hyperexpanded GAA repeats can adopt unusual DNA structures and induce aberrant epigenetic changes leading to heterochromatin mediated gene silencing. Several epigenetic changes, including increased levels of DNA methylation, histone modifications, repressive chromatin formation and elevated levels of non-coding RNA have been reported in FRDA. It has been reported that a novel FXN antisense transcript (FAST-1), is present at higher levels in FRDA patient-derived fibroblasts and its overexpression is associated with the depletion of CTCF, a chromatin insulator protein, and heterochromatin formation involving the critical +1 nucleosome. Previously, characteristics of FAST-1 were investigated in our lab and a full-length FAST-1 transcript containing a poly (A) tail was identified. To investigate any possible effects of FAST-1 on FXN expression, I first overexpressed this FAST-1 transcript in three different non-FRDA cell lines and a consistent decrease of FXN expression was observed in each cell type compared to control cells. I also identified that FAST-1 copy number is positively correlated with increased FAST-1 expression, which in turn is negatively correlated with FXN expression in FAST-1 overexpressing cells. Additionally, we found that FAST-1 overexpression is associated with increased levels of DNA methylation at CpG sites U6 and U11 of the FXN upstream GAA repeat region, together with CTCF depletion and heterochromatin formation at the 5'UTR of the FXN gene. To further investigate the role of FAST-1 in FXN gene silencing, I used a small hairpin RNA (shRNA) strategy to knock down FAST-1 expression in FRDA fibroblast cells. I found that knocking down FAST-1 increases FXN expression, but not to the level of control cells. Lastly, I investigated the pattern of FAST-1 expression and histone modifications at the FXN transgene in our new FRDA mouse model, designated YG8LR. The YG8LR mice showed decreased levels of FXN expression and H3K9ac and increased levels of FAST-1 expression and H3K9me3. Our data suggest that since FAST-1 is associated with FXN gene silencing, inhibition of FAST-1 may be an approach for FRDA therapy.

The mechanisms underpinning gene regulation in P. falciparum malaria remain largely elusive, though mounting evidence suggests a major role for epigenetic feedback. Interestingly, long non-(protein)-coding RNAs (lncRNAs) have been found to play a dominant role in initiating and guiding the transcriptional, epigenetic, and post-transcriptional status of specific loci across a broad range of organisms. LncRNAs are uniquely poised to act co-transcriptionally on neighboring loci, and/or to remain physically tethered at their site of origin, and through sequence-specific binding activities can impart temporal and spatial specificity to ubiquitously expressed nuclear protein complexes. Proteins, on the other hand, must be translated in the cytoplasm, and hence lose memory of their transcriptional origins. Encouraged by these features of lncRNAs, we set out to investigate the regulatory capacity of P. falciparum lncRNAs on a genome-wide scale. First, we surveyed transcriptional activity across approximately one quarter of the P. falciparum genome using a custom high-density DNA tiling array. We predicted a set of 60 developmentally regulated intergenic lncRNAs, and found that many of these novel loci neighbored genes involved in parasite survival or virulence pathways. Remarkably, upon further analysis of intergenic lncRNA properties, we discovered a family of twenty-two telomere-associated lncRNAs encoded in the telomere-associated repetitive element (TARE) region of P. falciparum chromosome ends. We found that each lncRNA-TARE was encoded adjacent and divergent to a subtelomeric var virulence gene. Moreover, we found that lncRNA-TARE expression was sharply induced between the parasite DNA replication and cell division cycles, that lncRNA-TARE loci contained numerous transcription factor binding sites only otherwise found in subtelomeric var promoter regions, and that the GC content and evolutionary sequence conservation of lncRNA-TAREs was similar to that of P. falciparum ribosomal RNA. Next, we set out to assemble P. falciparum intergenic lncRNA and antisense RNA transcript structures using state-of-the-art deep sequencing and computational tools. Towards this end, we harvested an unprecedented sample set that finely maps temporal changes across 56 hours of P. falciparum blood stage development, and developed and validated strand-specific, non-polyA-selected RNA sequencing methods. This enabled the annotation of over one thousand high-confidence, bona fide lncRNA transcript models, and their comprehensive global analysis. We discovered an enrichment of negatively correlated, tail-to-tail overlapping sense-antisense transcript pairs, suggesting a conserved role for antisense-mediated transcriptional interference in P. falciparum gene regulation. We also discovered a highly correlated spliced antisense counterpart to a gene required for sexual commitment, that the expression of an intriguing subset of antisense transcripts significantly dropped during parasite invasion, and that lncRNA-TARE and 'sterile' var virulence gene transcription was markedly up-regulated during parasite invasion. Lastly, we predicted over one thousand circular RNAs (circRNAs), and validated six circRNA transcript structures. Importantly, this thesis work represents the first focused investigation of lncRNAs in P. falciparum malaria, with the characterization of a compelling family of telomere-associated lncRNAs and numerous antisense RNAs. The data, methods, and results herein offer exceptional technological advancements coupled with compelling insights into the biology of the devastating human pathogen P. falciparum malaria. It is my hope that this work will facilitate future P. falciparum lncRNA functional studies and the strand-specific profiling of additional P. falciparum samples.

Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs.

In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using in vivo reporter gene fusion expression and different in vitro methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays.

Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn.

Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.

Non-coding RNA functions are emerging in the recent years. In this thesis we describe a Natural Antisense Transcript (NAT) that controls the expression of LEF1 transcriptional factor. This LEF1 NAT is transcribed from a promoter present in the first LEF1 intron and undergoes splicing in mesenchymal cells. In epithelial cells, there is no expression of LEF1 NAT. However, in metastable epithelial cells, LEF1 NAT is transcribed and a significant part of it remains unspliced and, contrarily to the spliced NAT, down-regulates the main LEF1 promoter and LEF1 mRNA and protein expression. Moreover, unspliced NAT also down-regulates cell migration and up-regulates Ecadherin expression. Unspliced LEF1 NAT interacts with LEF1 promoter and physically associates with Polycomb Repressive Complex 2 (PRC2) inducing its binding to the LEF1 promoter and trimethylating Lysine 27 in Histone 3. Spliced LEF1 NAT prevents the binding between unspliced LEF1 NAT and LEF1 promoter, inhibiting LEF1 promoter repression. Thus, these results indicate that LEF1 gene expression is finely controlled by splicing of the LEF1 NAT that, when is not processed, recruits PRC2 to LEF1 promoter to inhibit it.
En els darrers anys, les funcions exercides pels ARN no codificants estan creixent. En aquesta tesi es descriu un Natural Antisense Transcript (NAT) que controla l’expressió del factor de transcripció LEF1. Aquest NAT de LEF1 és transcrit des del promotor que es troba al primer intró de LEF1 i es processa mitjançant splicing en les cèl·lules mesenquimals. En les cèl·lules epitelials no hi ha expressió del NAT de LEF1. No obstant, en les cèl·lules epitelials que inicien la Transició Epiteli-Mesènquima (EMT), una part significativa de NAT no es processa i, contràriament al NAT que ha estat processat, fa baixar l’activitat del principal promotor de LEF1 i disminueix l’expressió de LEF1, a nivell d’ARN i proteïna. A més, el NAT que no ha estat processat també disminueix la migració cel·lular i incrementa l’expressió de l’E-caderina. El NAT de LEF1 interactua amb el promotor de LEF1 i s’uneix físicament amb Polycomb Repressive Complex 2 (PRC2) induint-ne la seva unió al promotor de LEF1 i trimetilant la Lisina 27 de l’Histona 3. El NAT de LEF1 que ha estat processat prevé la unió entre el NAT que no ho ha estat i el promotor de LEF1, prevenint la repressió del promotor de LEF1. Per tant, aquests resultats indiquen que l’expressió de LEF1 està finament controlada pel processament del NAT de LEF1 que, quan no ha patit splicing, recluta PRC2 al promotor de LEF1 per inhibir-lo.

Thanks to continuous technical advances in the sequencing field nowadays we know that most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs) and repetitive sequences, such as Short Interspersed Nuclear Elements (SINEs). A large percentage of ncRNAs is nuclear-enriched with unknown function. LncRNAs may be transcribed in antisense direction and may form sense/antisense pairs by pairing with an mRNA from the opposite strand to regulate chromatin conformation, transcription and mRNA stability. We have identified a nuclear-enriched lncRNA antisense to mouse Ubiquitin Carboxyterminal Hydrolase L1 (Uchl1), a gene expressed in dopaminergic cells and involved in brain function and neurodegenerative diseases. Antisense Uchl1 (AS Uchl1) increases Uchl1 protein synthesis at a post-transcriptional level. AS Uchl1 function is under the control of stress signaling pathways, as mTORC1 inhibition by rapamycin causes an increase in Uchl1 protein that is associated to the shuttling of AS Uchl1 lncRNA from the nucleus to the cytoplasm of dopaminergic cells. AS Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. Moreover, AS Uchl1 activity depends on the presence of a 5’ domain overlapping Uchl1 mRNA and an inverted SINEB2 element embedded along its 3’ sequence. These features are shared by other natural antisense transcripts and among them a lncRNA antisense to Ubiquitously eXpressed Transcript (AS Uxt) increases Uxt protein expression in the presence of stable mRNA level similar to AS Uchl1. These data identified a new functional class of lncRNAs and reveal another layer of gene expression control at the post-transcriptional level. Furthermore, through the replacement of AS Uchl1 5’ overlapping region with an antisense sequence to Green Fluorescence Protein (GFP) we were able to redirect this upregulation of protein synthesis. In fact, the presence of a 5’ overlapping sequence and an embedded inverted SINEB2 element confer to this artificial AS lncRNA to GFP (AS GFP) the capability to induce GFP protein with stable mRNA levels both in cells and in vitro translation assay. Further experiments are needed to set up the in vitro translation assay and to understand the translation enhancement of AS lncRNAs.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: Lorson, Christian L. "December 2008" Includes bibliographical references

Les récentes études transcriptomiques chez divers organismes ont montré que la transcription des gènes convergents peut produire des ARN messagers (ARNm) chevauchants. Ce phénomène a été analysé dans le contexte de l’interférence par ARN (ARNi) nucléaire, et peu d’information existe quant au destin cytoplasmique des messagers 3’ chevauchants ou leur impact sur l’expression des gènes. Dans ce travail, nous avons abordé les conséquences potentielles de l’interaction entre des paires d’ARNm sens-antisens chez Saccharomyces cerevisiae, un organisme modèle naturellement dépourvu de l’ARNi. Nous avons démontré que les extrémités 3’ complémentaires des ARNm peuvent interagir dans le cytoplasme et moduler la stabilité ainsi que la traduction d’ARNm. Nos résultats sont issus d’une étude détaillée d’une paire d’ARNm convergents, POR1 et OCA2, ensuite généralisée par l’approche de l’ARNi reconstituée chez S. cerevisiae. L’analyse globale a confirmé que dans les cellules sauvages, les paires d’ARNm sens-antisens forment des duplexes d’ARN in vivo et ont un rôle potentiel à moduler l’expression d’ARNm ou de protéines respectifs, dans des différentes conditions de croissance. Nous avons montré que le destin de centaines des messagers convergents est contrôlé par Xrn1, révélant l’importance de cette exoribonucléase 5’-3’ cytoplasmique très conservée dans la régulation post-transcriptionnelle des gènes convergents. Notre travail ouvre donc la perspective de considérer un nouveau mécanisme de l’interaction entre les paires d’ARNm sens-antisens dans le cytoplasme, chez les organismes contenant ou non la voie de l’interférence par ARN
Recent transcriptome analyses have revealed that convergent gene transcription can produce many 3’ overlapping mRNAs in diverse organisms. This phenomenon has been studied in the context of nuclear RNA interference (RNAi) pathway, however little is known about the cytoplasmic fate of 3’ overlapping messengers or their impact on gene expression. In this work, we address the outcomes of interaction between sense-antisense mRNA pairs in Saccharomyces cerevisiae, a model organism naturally devoid of RNAi. We demonstrate that the complementary tails of 3’ overlapping mRNAs can interact in the cytoplasm in a sequence-specific manner and promote post-transcriptional remodeling of mRNA stability and translation. Our findings are based on the detailed analysis of a convergent mRNA pair, POR1 and OCA2, subsequently generalized using the reconstituted RNAi approach in S. cerevisiae. Genome-wide experiments confirm that in wild-type cells, sense-antisense mRNA pairs form RNA duplexes in vivo and thus have potential roles in modulating the respective mRNA or protein levels under different growth conditions. We show that the fate of hundreds of messenger-interacting messengers is controlled by Xrn1, revealing the extent to which this conserved 5’-3’ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. In sum, our work opens a perspective to consider an additional, cytoplasmic mechanism of interaction between sense-antisense mRNA pairs, in both RNAi-positive and negative organisms

Les récentes études transcriptomiques chez divers organismes ont montré que la transcription des gènes convergents peut produire des ARN messagers (ARNm) chevauchants. Ce phénomène a été analysé dans le contexte de l’interférence par ARN (ARNi) nucléaire, et peu d’information existe quant au destin cytoplasmique des messagers 3’ chevauchants ou leur impact sur l’expression des gènes. Dans ce travail, nous avons abordé les conséquences potentielles de l’interaction entre des paires d’ARNm sens-antisens chez Saccharomyces cerevisiae, un organisme modèle naturellement dépourvu de l’ARNi. Nous avons démontré que les extrémités 3’ complémentaires des ARNm peuvent interagir dans le cytoplasme et moduler la stabilité ainsi que la traduction d’ARNm. Nos résultats sont issus d’une étude détaillée d’une paire d’ARNm convergents, POR1 et OCA2, ensuite généralisée par l’approche de l’ARNi reconstituée chez S. cerevisiae. L’analyse globale a confirmé que dans les cellules sauvages, les paires d’ARNm sens-antisens forment des duplexes d’ARN in vivo et ont un rôle potentiel à moduler l’expression d’ARNm ou de protéines respectifs, dans des différentes conditions de croissance. Nous avons montré que le destin de centaines des messagers convergents est contrôlé par Xrn1, révélant l’importance de cette exoribonucléase 5’-3’ cytoplasmique très conservée dans la régulation post-transcriptionnelle des gènes convergents. Notre travail ouvre donc la perspective de considérer un nouveau mécanisme de l’interaction entre les paires d’ARNm sens-antisens dans le cytoplasme, chez les organismes contenant ou non la voie de l’interférence par ARN
Recent transcriptome analyses have revealed that convergent gene transcription can produce many 3’ overlapping mRNAs in diverse organisms. This phenomenon has been studied in the context of nuclear RNA interference (RNAi) pathway, however little is known about the cytoplasmic fate of 3’ overlapping messengers or their impact on gene expression. In this work, we address the outcomes of interaction between sense-antisense mRNA pairs in Saccharomyces cerevisiae, a model organism naturally devoid of RNAi. We demonstrate that the complementary tails of 3’ overlapping mRNAs can interact in the cytoplasm in a sequence-specific manner and promote post-transcriptional remodeling of mRNA stability and translation. Our findings are based on the detailed analysis of a convergent mRNA pair, POR1 and OCA2, subsequently generalized using the reconstituted RNAi approach in S. cerevisiae. Genome-wide experiments confirm that in wild-type cells, sense-antisense mRNA pairs form RNA duplexes in vivo and thus have potential roles in modulating the respective mRNA or protein levels under different growth conditions. We show that the fate of hundreds of messenger-interacting messengers is controlled by Xrn1, revealing the extent to which this conserved 5’-3’ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. In sum, our work opens a perspective to consider an additional, cytoplasmic mechanism of interaction between sense-antisense mRNA pairs, in both RNAi-positive and negative organisms

Murine embryonic stem cells are derived from the inner cell mass of a pre-implantation mouse blastocyst. These cells exhibit dual defining characteristics of indefinite self-renewal and pluripotent differentiation. This thesis sought to explore two different methods of functional genetic analysis of embryonic stem cells, namely antisense gene trapping and RNA interference. Antisense gene trapping in embryonic stem cells involves the sequential genomic integration of two vectors: the first to randomly trap active gene promoters and the second to induce functional antisense gene knockout. Clones were selected according to the sequence identity of the trapped locus. Five such gene-trap clones were characterised (Tsix, Catns, Rex2, Nasp, and Ctbp2) and the analyses of these clones are presented. The use of RNA interference to study embryonic stem cell biology is explored including demonstration of knockdown of the maintenance methyltransferase gene, Dnmt1, that results in significant demethylation of genomic DNA. Antisense gene trapping and RNA interference represent two complementary strategies for the functional genetic analysis of the mammalian cell. In both cases, the goal is to correlate genotype with phenotype, or vice versa, through the bi-allelic knockdown of a target gene. By contrast with one another, while gene trapping has the potential to define novel genes according to their function, RNA interference can only be used for known genes whose functions are predicted from their respective sequences. Intriguingly, both methodologies involve double-stranded RNA suggesting a commonality of underlying molecular mechanism: in the case of antisense gene trapping this is generated endogenously, while in the case of RNA interference it is introduced exogenously in the form of short duplexes. In this manner, double-stranded RNA represents a unifying thread to this thesis.