Myotonic Dystrophy Type I (DM1) is a multi-systemic genetic neuromuscular degenerative disease, has a prevalence in most populations of about 1:8000 and is caused by the nuclear retention of pathogenically expanded DMPK mRNA. A previous DM1 RNAi-kinome screen in our lab has identified kinases that reduced both count and area of DMPK mRNA foci in vitro. One such discovered kinase is PACT, which has showed to decrease foci count and area in DM1 fibroblasts by 30-50%. This study explored PACT as well as binding partner DICER involved in cellular RNA processing machinery, to highlight potential therapeutic targets in DM1. DM1 fibroblasts treated with PACT siRNA showed a non-significant trend of upregulation in MBNL1 mRNA and protein expression. PACT knockdown also showed trend of missplicing normalization in SERCA-1, more prominently seen in DM1-2000 human fibroblasts, whereas IR (insulin receptor) splicing remained unaffected. On the other hand, DICER knockdown did not have profound affect on foci integrity as well as MBNL1 RNA and protein xpressions in DM1 fibroblasts. SERCA-1 splicing in DICER siRNA treated samples also remained unchanged. We report here our findings in pursuit of potential therapeutic targets for the treatment of DM1.

[ES] En plantas, el silenciamiento por RNA constituye un potente mecanismo de defensa frente a virus. Los RNA virales de doble cadena activan este tipo de procesos y son digeridos por las enzimas DCL (Dicer-like), cuya acción genera pequeños RNA (sRNA) de entre 20 y 24 nt. Estos sRNA promueven la degradación de RNA de secuencia complementaria a través de un complejo multiproteico conocido como RISC (RNA-induced silencing complex), cuya molécula efectora es una proteína Argonauta (AGO). Con el fin de evadir esta barrera defensiva del huésped, la mayoría de los virus de plantas codifican supresores del silenciamiento por RNA (VSR), cuyos mecanismos de acción son diversos y en muchos casos no se comprenden del todo. Aunque todas las etapas de la ruta de silenciamiento pueden ser inhibidas por los VSR, los sRNA y las proteínas AGO parecen ser las dianas más frecuentes. Se ha postulado que motivos GW/WG podrían ser fundamentales para la actividad de algunos VSR, al intervenir en la interacción con proteínas AGO. En este trabajo se ha pretendido seguir profundizando en el estudio de la respuesta antiviral en plantas y de los mecanismos de acción de los supresores de silenciamiento. El primer objetivo abordado ha sido identificar el VSR codificado por el virus del arabesco del Pelargonium (Pelargonium line pattern virus, PLPV), un miembro del género Pelarspovirus dentro de la amplia familia Tombusviridae. Los resultados han mostrado que la proteína de cubierta del virus (p37) es capaz de inhibir de manera eficiente el silenciamiento inducido por RNA. La generación de un batería de variantes capaces e incapaces de actuar como VSR y/o de empaquetar el RNA viral mediante mutagénesis dirigida de distintos motivos de la proteína, incluido un motivo GW que está conservado en ortólogos, nos han permitido conocer que: (i) tanto la función de supresión del silenciamiento como la función de encapsidación son esenciales para que el PLPV alcance una infección sistémica y (ii) p37, a pesar de contener un motivo GW funcional e interaccionar con distintas AGO, emplea el secuestro de sRNA como estrategia principal para inhibir el silenciamiento. A pesar de que ambas funciones conocidas de p37 deben ser llevadas a cabo esencialmente en el citoplasma, esta proteína se localiza en citoplasma y núcleo, con gran acumulación en nucleolo, por lo que nos planteamos como segundo objetivo en este trabajo profundizar acerca de la localización nucleolar de p37. Además de mapear la región de la proteína que contiene la señal de localización nucleolar (NoLS) en los primeros 45 aminoácidos de la molécula, también hemos observado que p37 interacciona con diferentes miembros de la familia de las importinas ¿, adaptadores moleculares del transporte nucleocitoplasmático, y que esta interacción es esencial para la localización nucleolar de la proteína. Adicionalmente, la anulación de la localización nucleolar de p37 mediante el silenciamiento de importinas ¿ ha correlacionado con una disminución de acumulación del virus, lo que sugiere que dicha localización es ventajosa para la infección viral. Por último, para intentar conocer más datos acerca de las actividades de la ruta de silenciamiento que están implicados en la defensa frente al PLPV, analizamos la infección viral en líneas transgénicas de N. benthamiana con la expresión o actividad distintos componentes de la ruta comprometida. Los resultados han mostrado que DCL4 y, en menor medida, DCL2 afectan a la infección viral y que ambas tienen un efecto aditivo, tal y como se ha descrito en diversas interacciones virus-planta. Adicionalmente, AGO2 se ha revelado como un factor clave en la respuesta frente al PLPV, ampliando el número de virus que están afectados por esta endonucleasa particular. En conjunto, los resultados obtenidos muestran que tanto el procesamiento de dsRNA mediado por enzimas DCL como el corte de RNA mediado AGO, contribuyen a la defen / [CAT] En plantes, el silenciament per RNA constitueixen un potent mecanisme de defensa davant virus. Els RNA virals de doble cadena activen aquest tipus de processos, i són digerits per els enzims DCL (Dicer-like), donant lloc a xicotets RNA (sRNA) d'entre 20 i 24 nt. Estos sRNA promouen la degradació de RNA de seqüència complementària a través d'un complex multiproteic conegut com RISC (RNA-induced silencing complex), la molècula efectora del qual és una proteïna Argonauta (AGO). Per tal d'evadir aquesta barrera defensiva de l'hoste, la majoria dels virus de plantes codifiquen supressors del silenciament per RNA (VSR), els mecanismes d'acció dels quals són diversos i en molts casos no es comprenen del tot. Encara que totes les etapes de la ruta poden ser inhibides, els sRNA i les proteïnes AGO semblen ser les dianes més freqüents. S'ha postulat que els motius GW/WG podrien ser fonamentals per a l'activitat d'alguns VSR, en intervindre en la interacció amb proteïnes AGO. En aquest treball s'ha pretés continuar aprofundint en l'estudi de la resposta antiviral en plantes i dels mecanismes d'acció dels supressors de silenciament. El primer objectiu abordat ha sigut identificar l'VSR codificat pel virus de l'arabesc del Pelargonium (Pelargonium line pattern virus, PLPV), un membre del gènere Pelarspovirus dins de l'amplia família Tombusviridae. Els resultats han mostrat que la proteïna de coberta del virus (p37) és capaç d'inhibir de manera eficient el silenciament induït per RNA. La generació d'una bateria de variants capaces i incapaces d'actuar com VSR i/o d'empaquetar l'RNA viral mitjançant la mutagènesi dirigida de diferents motius de la proteïna, inclòs un motiu GW que està conservat en ortòlegs, ens ha permés conèixer que: (i) tant la funció de supressió del silenciament com la funció d'encapsidació són essencials per a que el PLPV aconseguisca una infecció sistèmica i (ii) p37, malgrat contindre un motiu GW funcional i interaccionar amb diferents AGO, empra el segrest de sRNA com a estratègia principal per a inhibir el silenciament. Malgrat que ambdues funcions conegudes de p37 han de ser dutes a terme essencialment en el citoplasma, esta proteïna localitza en citoplasma i nucli, amb gran acumulació en nuclèol, per la qual cosa ens hem plantejat com a segon objectiu en este treball aprofundir sobre la localització nucleolar de p37. Ademés de mapejar la senyal de localització nucleolar (NoLS) en els primers 45 aminoàcids de la molècula, també hem observat que p37 interacciona amb diferents membres de la família de les importines ¿, i que aquesta interacció és essencial per a la localització nucleolar de la proteïna. A més, l'anul¿lació de la localització nucleolar de p37 mitjançant el silenciament d'importines ¿ s'ha correlacionat amb una disminució d'acumulació del virus, la qual cosa suggereix que aquesta localització és avantatjosa per a la infecció viral. Finalment, per a intentar conéixer més dades sobre les activitats de la ruta de silenciament que estan implicats en la defensa front al PLPV, hem analitzat la infecció viral en línies transgèniques de N. benthamiana amb l'expressió o activitat diferents components de la ruta compromesa. Els resultats han mostrat que DCL4 i, en menor mesura, DCL2 afecten la infecció viral i que ambdues tenen un efecte additiu, tal com s'ha descrit en diverses interacciones virus-planta. Addicionalment, AGO2 s'ha revelat com un factor clau en la resposta front al PLPV, ampliant el nombre de virus que estan afectats per aquesta endonucleasa particular. En conjunt, els resultats obtinguts mostren que tant el processament de dsRNA mediat per enzims DCL com el tall d'RNA mediat AGO, contribueixen a la defensa de N. benthamiana front al PLPV. / [EN] n plants, RNA silencing functions as a potent antiviral mechanism. Viral-derived double-stranded RNAs trigger this type of processes, being digested by DCL (Dicer-like) enzymes into virus-derived small RNAs (vsRNAs) of 20-24 nt. These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. To counteract this host defence response, most plant viruses encode suppressors of RNA silencing (VSRs), whose mechanisms of action are diverse and often not well understood. Though virtually all stages of the antiviral silencing pathway can be blocked by VSRs, sRNAs and AGO proteins seem to be the most common targets. It has been postulated that GW/WG motifs could be fundamental for the activity of some VSRs by directing interaction with AGOs. In this work, we have pursued to get further insights into the antiviral silencing in plants and the mechanisms of action of silencing suppressors. The first objective has been to identify the VSR encoded by Pelargonium line pattern virus (PLPV), a member of the genus Pelarspovirus within family Tombusviridae. The results have shown that the viral coat protein (p37) is able to efficiently inhibit RNA silencing. Generation of suppressor-competent and incompetent molecules and uncoupling of the RSS and particle assembly capacities through site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed us to know that: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic PLPV infection and, (ii) p37, even though it has a functional GW motif and interacts with different AGOs, inhibits silencing most likely through vsRNA sequestration. Despite both p37 functions have to be executed essentially in the cytoplasm, this protein localizes in cytoplasm and nucleus, with high accumulation at the nucleolus, so the second objective of this work has been to gain further insights into the nucleolar localization of p37. Besides mapping the protein region containing the nucleolar localization signal (NoLS) in the first 45 amino acids of the molecule, we have found that p37 interacts with distinct members of the importin ¿ family, main cellular transporters for nucleo-cytoplasmic traffic of proteins, and that these interactions are crucial for nucleolar targeting of p37. In addition, impairment of p37 nucleolar localization through down-regulation of importin ¿ expression has been correlated with a reduction of viral accumulation, suggesting that sorting of the protein to the major subnuclear compartment is advantageous for the infection process. Finally, in order to obtain information on which activities of RNA silencing pathway are involved in the defense against PLPV, we analyzed the viral infection in N. benthamiana transgenic lines with the functions of distinct components of the pathway impaired. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection and that they have additive effects, in agreement with that observed in other plant-virus interactions. Additionally, AGO2 was found to be a key factor in the defense against PLPV, extending the number of viruses that are affected by this particular slicer. Altogether, the results supported that both dicing and slicing activities participate in the defense of N. benthamiana against PLPV. / Pérez Cañamás, M. (2019). Análisis de la interacción del virus del arabesco del Pelargonium con la ruta de silenciamiento por RNA del huésped [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/122297 / TESIS

京都大学 / 新制・課程博士 / 博士(情報学) / 甲第23397号 / 情博第766号 / 新制||情||131(附属図書館) / 京都大学大学院情報学研究科知能情報学専攻 / (主査)教授 阿久津 達也, 教授 山本 章博, 教授 鹿島 久嗣 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DFAM

No description available.

Polyadenylation signals in yeast are not very well defined and are believed to be largely degenerate. Here, we present a computational and experimental genome-wide analysis of polyadenylation signals in Schizosaccharomyces pombe (S. pombe), identifying the canonical AATAAA motif as the most frequent and functional signal. RNA-Seq data from cells grown under various physiological conditions were used to map 3’UTRs, which classify as commonly heterogenic. We have shown that many genes have alternative 3’UTRs. Our results are summarised and can be accessed in a user-friendly online database Pomb(A). It has been shown that convergent genes require trans elements, like Cohesin, for efficient transcription termination. We demonstrate that convergent genes lacking Cohesin are generally associated with longer overlapping transcripts. Furthermore, we analysed ChIP-chip data of Rad21 and Mis4 as well as other Cohesin and loading complex subunits and show that regions of Rad21/Mis4 co-localisation are generally associated with highly transcribed genes. They are also cohesive, while sites with Rad21 only are less cohesive. Rad21/Mis4 co-localisation sites are in close proximity to annotated origins of replication, suggesting that cohesive sites may facilitate replication. microRNAs (miRNAs) are well studies in higher eukaryotes and participate on post-transcriptional gene silencing by degrading target mRNA or blocking translation. It is believed that miRNAs do not exist in yeast. We reanalyzed miRNA presence in yeast using recently available small RNA data sets. Potential miRNA genes and targets in S. pombe were computationally predicted based on the described alternative 3’UTR data and further experimentally tested. Dicer is an enzyme, which recognizes long dsRNA substrates and cleaves them into siRNA e↵ector molecules, essential for gene silencing. Dicer has been thought to be a purely cytoplasmic protein. However, we employed ChIP-Seq and dsRNA RNA-Seq data to show that Dicer localises in the nucleus of mammalian cells and associates with the chromatin on numerous loci. Furthermore, we present evidence that Dicer processes long dsRNA into siRNA in the nucleus and the lack of Dicer causes the accumulation of long dsRNA. This consequently induces the interferon response pathway, which ultimately leads to apoptosis and cell death.

Les cancers du sein sont la première cause de mortalité chez les femmes occidentales. Ces tumeurs solides présentent une grande hétérogénéité associée à une résistance aux traitements et un taux de rechute important à long terme. Ainsi, les cliniciens doivent personnaliser la prise en charge des patientes, ce qui est rendu possible par une meilleure connaissance des causes moléculaires qui participent à l'apparition et à la progression des tumeurs mammaires. Pour ce projet, nous avons choisi d'étudier, dans ces cancers, les éventuelles valeurs pronostique et diagnostique de dicer, gène codant pour la ribonucléase clef du mécanisme d'interférence ARN. Il a été montré une implication de Dicer dans la mise en place de l'hétérochromatine de novo des régions péricentromériques. Encore peu étudiée chez l'homme, les données disponibles chez la levure nous ont permis d'envisager que Dicer pourrait être impliquée dans la régulation de la stabilité chromosomique. Nous avons donc testé les valeurs pronostique et diagnostique de dicer (en PCR quantitative et en Tissue Microarray) dans une centaine d'échantillons de tumeurs, dans des lignées cellulaires et dans des modèles de progression tumorale et métastatique. Parallèlement, nous avons étudié les conséquences d'une inhibition de l'expression de dicer sur le cycle cellulaire et sur la réponse à un stress réplicatif. Nos résultats ont montré que l'expression de dicer est un facteur pronostique indépendant de rechutes métastatiques et est associé avec l'expression des récepteurs hormonaux. Par ailleurs, des cellules n'exprimant pas dicer ont montré des dérégulations du cycle cellulaire et de la voie de réponse aux cassures de l'ADN. En conclusion, l'altération de l'expression de dicer pourrait jouer un rôle dans l'apparition de l'instabilité chromosomique des cancers du sein et son analyse pourrait permettre une meilleure prise en charge des patientes à risque pour une rechute métastatique.

MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...

The increasing demand on healthcare from pregnancies complicated by gestational diabetes (GDM) and obesity is caused in large part by fetal macrosomia (FM). Alterations to the vasculature of the placenta leading to changes to nutrient flux may be more frequent when GDM and obesity occur concomitantly. However, the impact of obesity as an independent comorbidity is poorly understood. The current study sought to characterise structural and functional changes in placenta from pregnancies complicated by GDM and/or obesity and examine the involvement of miRs in this phenomenon, as the phenotype of vascular smooth muscle (VSM) has been documented to be influenced by microRNA (miR) expression. Patients were stratified according to the presence or absence of GDM and/or obesity, which resulted in four groups. Morphometric analysis of CD31 immuno-stained placentas showed that pregnancies complicated by GDM or obesity both had a higher mean sum ratio of the area of the lumen compared to the endothelium. No relationship was found with FM. The ratio increased with maternal body mass index (BMI) in all pregnancies. Immunohistochemistry with a panel of VSM markers suggested an altered phenotype of VSM in pregnancies complicated by GDM and/or obesity. RT-QPCR and immunoblotting showed a higher expression of smooth muscle myosin (SM-MHC), h-caldesmon (HC) and alpha smooth muscle actin (ASMA) in pregnancies complicated by obesity, consistent with a greater contractile capacity. This was most marked when obesity occurred without GDM.Studies were conducted on two miRs, miR-145, which is associated with VSM in many vascular tissues, and the snoRNA-derived species miR-664a-3p, which microarray studies had shown to be higher in placentas from pregnancies complicated by GDM. Dicer and dyskerin, components of the snoRNA-derived miR biogenesis pathway, were increased and reduced respectively in GDM placenta. However, studies in cultured placental villous explants suggested that neither miR species was regulated by glucose, insulin or IGF-I. Placental mesenchymal cells are the developmental precursors of VSM. In primary culture, these cells expressed both miRs. To determine the function of miR-664a-3p, a nucleofection protocol was developed in a fetal mesenchymal cell line, WI38, and applied to first-trimester placental mesenchymal cells. Preliminary proteomic analysis after nucleofection-mediated knockdown of miR-664a-3p suggested a series of novel candidate target proteins for this uncharacterised miR species. Blood vessel structure and VSM phenotype are both altered in pregnancies complicated by GDM and/or obesity. The significance of apparently higher level of contractile proteins with wider vessel lumens in obesity requires further investigation. Translational regulation by miRs including miR-145 and miR-664a-3p is implicated in these alterations. In future, targeted therapies that alter miR levels in the placenta may be useful in control of fetal overgrowth such as FM.

All organisms exist in some sort of symbiosis with their environment. The food we eat, air we breathe, and things we touch all have their own microbiota and we interact with these microbiota on a daily basis. As such, we employ a method of compartmentalization in order to keep foreign entities outside of the protected internal environments of the body. However, as other organisms seek to replicate themselves, they may invade our sterile compartments in order to do so. To protect ourselves from unfettered replication of pathogens or from cellular damage, we have developed a series of receptors and signaling pathways that detect foreign bodies as well as abnormal signals from our own perturbed cells. The downstream effector molecules that these signaling pathways initiate can be toxic and damaging to both pathogen and host, so special care is given to the regulation of these systems. One method of regulation is the production of endogenous small ribonucleic acids that can regulate the expression of various receptors and adaptors in the immune signaling pathways. In this dissertation, I present work that establishes an important protein in small ribonucleic acid regulation, Dicer, as an essential protein for regulating the innate immune response to immuno-stimulatory nucleic acids as well as regulating the productive infection of encephalomyocarditis virus. Depleting Dicer from murine embryonic fibroblasts renders a disparate type I interferon response where nucleic acid stimulation in the Dicer null cells fails to produce an appreciable interferon response while infection with the paramyxovirus, Sendai, induces a more robust interferon response than the wild-type control. Additionally, I show that Dicer plays a vital role in controlling infection by the picornavirus, encephalomyocarditis virus. Encephalomyocarditis virus fails to grow efficiently in Dicer null cells due to the inability for the virus to bind to the outside of the cell, suggesting that Dicer has a role in modulating viral infection by affecting host cellular protein levels. Together, this work identifies Dicer as a key protein in viral innate immunology by regulating both the growth of virus and also the immune response generated by exposure to pathogen associated molecular patterns. Understanding this regulation will be vital for future development of small molecule therapeutics that can either modulate the innate immune response or directly affect viral growth.

No description available.