Les carcinomes spinocellulaires cutanés (cSCCs) sont le deuxième type de cancer par ordre de fréquence et sont responsables de 25% des décès dus aux cancers de la peau. Il est donc essentiel de caractériser les mécanismes responsables de la cancérisation de l'épiderme afin de développer de nouveaux traitements. Dans ce contexte, les miRNAs apparaissent comme des cibles de choix pour le développement de futures thérapies anti-tumorales. Toutefois, leur implication dans la physiopathologie des cSCCs est encore peu documentée. Au cours de cette étude, j’ai identifié, par séquençage à haut débit, 112 miRNAs dont l’expression est altérée au cours du développement tumoral dans un modèle murin de carcinogénèse chimique cutanée. J’ai ensuite focalisé mon attention sur le cluster miR-193b/365a et sur miR-708 dont les niveaux diminuent au cours de la progression tumorale, suggérant des fonctions de suppresseurs de tumeur. En accord avec cette hypothèse, l’expression ectopique de ces miRNAs inhibe la prolifération, la survie et la migration de cellules tumorales, alors que le blocage de leur action par des anti-sens stimule ces fonctions cellulaires dans des kératinocytes normaux. L’association d’approches in silico et d’analyses du transcriptome de cellules de cSCC sur-exprimant ces miRNAs m’a permis d’identifier leurs gènes cibles potentiels. J’ai validé KRAS et MAX comme cibles communes de miR-193b et miR-365a, et montré par l’utilisation de siRNAs que la répression de ces cibles mime les effets de ces miRNAs. Ces résultats suggérent que le ciblage de ces gènes pourrait médier en partie les effets suppresseurs de tumeur de miR-193b et de miR-365a dans les cSCCs. / Cutaneous squamous cell carcinomas (cSCCs) are the second most common cancer and are responsible for up to 25% of all skin cancer deaths. It is therefore essential to characterize the mechanisms responsible for epidermis carcinogenesis to develop new treatments. In this context, miRNAs appear to be prime targets for the development of future anti-tumor therapies. However, their involvement in the pathophysiology of cSCCs is still poorly documented. In this study, I identified using Small RNA sequencing, 112 miRNAs whose expression is altered during tumor development in a mouse model of cutaneous two-stage chemical carcinogenesis. Then, I focused my attention on the miR-193b/365a cluster and on miR-708, that are down-regulated during tumorigenesis, suggesting tumor suppressor functions. Consistent with this hypothesis, the ectopic expression of these miRNAs inhibit the proliferation, survival and migration of tumor cells, while blocking their action with antisense oligonucleotides stimulates these cellular functions in normal keratinocytes. Combining in silico target-prediction approaches and transcriptome analyzes of cSCC cells over-expressing these miRNAs, I identified their potential target genes. I validated KRAS and MAX as direct targets of miR-193b and miR-365a, and I showed that repression of these genes using siRNAs mimics the effects of these miRNAs. These results suggest that targeting these genes might mediate, at least in part, the tumor suppressor action of miR-193b and miR-365a in cSCCs.

La principale contribution de cette thèse est le développement d'une méthode fiable, robuste, et rapide pour la prédiction des pré-miARNs. Deux objectifs avaient été assignés : efficacité et flexibilité. L'efficacité a été rendue possible au moyen d'un algorithme quadratique. La flexibilité repose sur deux aspects, la nature des données expérimentales et la position taxonomique de l'organisme (en particulier plantes ou animaux). Mirinho accepte en entrée des séquences de génomes complets mais aussi les très nombreuses séquences résultant d'un séquençage massif de type NGS de “RNAseq”. “L'universalité” taxonomique est obtenu par la possibilité de modifier les contraintes sur les tailles de la tige (double hélice) et de la boule terminale. Dans le cas de la prédiction des miARN de plantes la plus grande longueur de leur pré-miARN conduit à des méthodes d'extraction de la structure secondaire en tige-boule moins précises. Mirinho prend en compte ce problème lui permettant de fournir des structures secondaires de pré-miARN plus semblables à celles de miRBase que les autres méthodes disponibles. Mirinho a été utilisé dans le cadre de deux questions biologiques précises l'une concernant des RNAseq l'autre de l'ADN génomique. La première question a conduit au traitement et l'analyse des données RNAseq de Acyrthosiphon pisum, le puceron du pois. L'objectif était d'identifier les miARN qui sont différentiellement exprimés au cours des quatre stades de développement de cette espèce et sont donc des candidats à la régulation des gènes au cours du développement. Pour cette analyse, nous avons développé un pipeline, appelé MirinhoPipe. La deuxieme question a permis d'aborder les problèmes liés à la prévision et l'analyse des ARN non-codants (ARNnc) dans la bactérie Mycoplasma hyopneumoniae. Alvinho a été développé pour la prédiction de cibles des miRNA autour d'une segmentation d'une séquence numérique et de la détection de la conservation des séquences entre ncRNA utilisant un graphe k-partite. Nous avons finalement abordé un problème lié à la recherche de motifs conservés dans un ensemble de séquences et pouvant ainsi correspondre à des éléments fonctionnels / The main contribution of this thesis is the development of a reliable, robust, and much faster method for the prediction of pre-miRNAs. With this method, we aimed mainly at two goals: efficiency and flexibility. Efficiency was made possible by means of a quadratic algorithm. Flexibility relies on two aspects, the input type and the organism clade. Mirinho can receive as input both a genome sequence and small RNA sequencing (sRNA-seq) data of both animal and plant species. To change from one clade to another, it suffices to change the lengths of the stem-arms and of the terminal loop. Concerning the prediction of plant miRNAs, because their pre-miRNAs are longer, the methods for extracting the hairpin secondary structure are not as accurate as for shorter sequences. With Mirinho, we also addressed this problem, which enabled to provide pre-miRNA secondary structures more similar to the ones in miRBase than the other available methods. Mirinho served as the basis to two other issues we addressed. The first issue led to the treatment and analysis of sRNA-seq data of Acyrthosiphon pisum, the pea aphid. The goal was to identify the miRNAs that are expressed during the four developmental stages of this species, allowing further biological conclusions concerning the regulatory system of such an organism. For this analysis, we developed a whole pipeline, called MirinhoPipe, at the end of which Mirinho was aggregated. We then moved on to the second issue, that involved problems related to the prediction and analysis of non-coding RNAs (ncRNAs) in the bacterium Mycoplasma hyopneumoniae. A method, called Alvinho, was thus developed for the prediction of targets in this bacterium, together with a pipeline for the segmentation of a numerical sequence and detection of conservation among ncRNA sequences using a kpartite graph. We finally addressed a problem related to motifs, that is to patterns, that may be composed of one or more parts, that appear conserved in a set of sequences and may correspond to functional elements.

No description available.

No description available.

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Tesis por compendio / [ES] Los pequeños ARNs artificiales (art-sRNAs) son moléculas de ARN de simple cadena, de 21 nucleótidos (nt), diseñados computacionalmente para silenciar genes de la planta con alta eficacia y especificidad. Los art-sRNAs se clasifican en dos grupos: micro ARNs artificiales (amiRNAs) y pequeños ARNs interferentes sintéticos que actúan en trans (syn-tasiRNAs). A pesar de que los art-sRNAs se usan frecuentemente para silenciar genes endógenos y exógenos en plantas, todavía hay algunos aspectos desconocidos que limitan sus aplicaciones. Uno de ellos es su capacidad de movimiento, dado que se desconoce si son capaces de moverse y silenciar genes endógenos en tejidos distintos a los de origen. Por tanto, el uso de art-sRNAs para inducir silenciamiento génico a nivel de toda la planta, habitualmente implica la generación de plantas transgénicas que expresen los precursores completos de art-sRNAs, limitando su versatilidad como herramienta biotecnológica para genómica funcional y mejora de cultivos. En esta tesis, hemos investigado en primer lugar la capacidad de silenciamiento sistémico de los art-sRNAs y, posteriormente, hemos diseñado precursores mínimos de art-sRNAs para facilitar su expresión no transgénica a partir de virus y en toda la planta. En primer lugar, estudiamos la capacidad de silenciamiento local y sistémico de art-sRNAs dirigidos contra la subunidad CHLI de la quelatasa de magnesio, codificada por el gen SULPHUR (NbSu), mediante ensayos de expresión transitoria en Nicotiana benthamiana. Tanto amiRNAs como syn-tasiRNAs indujeron el silenciamiento sistémico de NbSu, para lo cual se requieren altos niveles de expresión cerca del peciolo. Esta actividad sistémica está facilitada por el movimiento vascular de art-sRNAs de doble cadena y 21 nt a través de la planta, permitiendo silenciar genes en tejidos distintos a los de origen. Este resultado pone de manifiesto el potencial biotecnológico de expresar localmente art-sRNAs que sean capaces de moverse a tejidos distales de la planta y desencadenar un silenciamiento génico altamente específico. En segundo lugar, nos centramos en diseñar precursores de tamaño mínimo derivados de las moléculas precursoras endógenas de Arabidopsis thaliana (Arabidopsis): MIR390a (521 nt) y TAS1c (1011 nt). Evaluamos la eficacia de silenciamiento de una colección de construcciones que contenían versiones acortadas de los precursores AtMIR390a y AtTAS1c mediante expresión transitoria y estable en N. benthamiana y Arabidopsis, respectivamente. Observamos que, tanto amiRNAs como syn-tasiRNAs, son altamente eficientes y se procesan de forma precisa cuando se producen a partir de precursores mínimos de solo 89 y 54 nt respectivamente. Además, comprobamos que cuando se expresan a partir de un vector viral de ARN, como el virus X de la patata, solo se producen art-sRNAs auténticos a partir de precursores mínimos, y no de los de longitud completa, lo que da lugar al silenciamiento eficiente de genes endógenos en N. benthamiana. Asimismo, pudimos inducir silenciamiento génico mediante art-sRNAs expresados a partir de un virus, de forma no transgénica y escalable, mediante la pulverización sobre plantas de extractos infecciosos que contenían los vectores modificados. Estos resultados muestran que la longitud de los precursores de art-sRNAs puede ser acortada de forma significativa sin comprometer la eficacia del silenciamiento inducido, y que los precursores mínimos ofrecen una ventaja biotecnológica sobre los precursores de longitud completa cuando se expresan a partir de vectores virales. / [CA] Els xicotets ARNs artificials (art-sRNAs) són molècules d'ARN de simple cadena de 21 nucleòtids (nt), dissenyats computacionalment per a silenciar gens de plantes amb alta eficàcia i especificitat. Els art-sRNAs es classifiquen en dos tipus: microARNs artificials (amiRNAs) i xicotets ARNs interferents sintètics que actuen en trans (syn-tasiRNAs). Encara que els art-sRNAs són àmpliament utilitzats per a silenciar tant gens endògens com exògens en plantes, hi ha alguns aspectes desconeguts que limiten les seues aplicacions. Un d'ells és la seua capacitat de moviment, ja que encara no s¿ha establit si poden desplaçar-se i silenciar gens endògens en teixits diferents del d'origen. Conseqüentment, aconseguir un silenciament gènic en tota la planta mediat per art-sRNAs normalment requereix la producció de plantes transgènices que expressen els precursors complets dels art-sRNAs, la qual cosa restringeix el seu ús versàtil com a eina biotecnològica per a la genòmica funcional i la millora de cultius. En aquesta tesi, primer vam investigar la capacitat de silenciament sistèmic dels art-sRNAs, i després vam dissenyar precursors mínims d'art-sRNAs per a facilitar la seua aplicació de manera no transgènica a tota la planta, expressant els art-sRNAs a partir de vectors virals. Primer, vam estudiar la capacitat de silenciament local i sistèmic dels art-sRNAs, dirigits contra el gen SULPHUR (NbSu) ,que codifica per a una subunitat de la quelatasa de magnesi, assajada en Nicotiana benthamiana mitjançant l'expressió transitòria. Tant amiRNAs com syn-tasiRNAs van produir silenciament sistèmic de NbSu, amb una alta expressió d'art-sRNAs prop del pecíol de la fulla. El moviment vascular dels dúplexs d'art-sRNAs de 21 nt va facilitar aquesta activitat sistèmica a través de la planta, permetent el silenciament d'aquest gen en teixits distals als quals els art-sRNAs han sigut produïts. Aquest resultat destaca el gran potencial biotecnològic dels art-sRNAs expressats localment, capaços de moure's a grans distancies i amb molta especificitat, desencadenant el silenciament gènic a tota la planta. En segon lloc, ens vam centrar en dissenyar precursors d'amiRNAs i syn-tasiRNAs del mínim tamany possible, derivats dels precursors endògens MIR390a (521 nt) i TAS1c (1011 nt) d' Arabidopsis thaliana, respectivament. Vam avaluar l'eficàcia de silenciament d'una col·lecció de construccions que incloïen les versions acurtades dels precursors AtMIR390a i AtTAS1c, fent servir expressió transitòria en N.benthamiana i expressió estable en A. thaliana. Vam trobar que es poden produir amiRNAs i syn-tasiRNAs altament efectius i correctament processats a partir de precursors mínims de només 89 i 54 nt respectivament. A més, vam observar que art-sRNAs autèntics es produeixen a partir dels precursors mínims i no utilitzant els precursors complets quan s'expressen des d'un vector viral basat en ARN, com el virus X de la creïlla, resultant en un silenciament eficient de gens endògens de N. benthamina. A més, el silenciament de gens mediat per art-sRNAs expressats a partir d'un vector viral es va a aconseguir amb èxit sense l'ús de transgènics i d'una manera escalable, utilitzant un esprai amb extractes crus infecciosos que contenen els vectors virals modificats. Aquests resultats manifesten que la longitud dels precursors d'art-sRNAs pot ser reduïda significantment sense comprometre l'eficàcia de silenciament, i que aquests precursors mínims ofereixen un avantatge biotecnològic sobre els precursors complets quan s'expressen a partir de vectors virals. / [EN] Artificial small RNAs (art-sRNAs) are 21-nucleotide (nt) single-stranded RNA molecules computationally designed to silence genes with high efficacy and specificity. Art-sRNAs are classified into two main types: artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). Although art-sRNAs are widely used to silence both endogenous and exogenous plant genes, there are still some unknown aspects limiting their applications. One of them is their movement capacity, since it has not been established whether they can move and silence endogenous genes in tissues other than where they are originally expressed. Consequently, achieving whole-plant gene silencing mediated by art-sRNAs, typically requires the generation of transgenic plants expressing full-length art-sRNA precursors, restricting their use as versatile biotechnological tools for functional genomics and crop improvement. In this thesis, we first investigated the systemic silencing capacity of art-sRNAs, and then engineered minimal art-sRNA precursors to facilitate transgene-free, virus-based expression of art-sRNAs at the whole-plant level. Firstly, we studied the local and systemic silencing capacity of art-sRNAs targeting the magnesium chelatase subunit CHLI-encoding SULPHUR (NbSu) gene, using transient expression assays in Nicotiana benthamiana. Both amiRNAs and syn-tasiRNAs were found to induce systemic silencing of NbSu, which required high art-sRNA expression near the leaf petiole. This systemic activity was facilitated by the vascular movement of 21-nt art-sRNA duplexes throughout the plant, allowing the silencing of plant genes in tissues different from where art-sRNAs are produced. This result highlights the biotechnological potential of locally expressed art-sRNAs to move long distances and trigger highly specific gene silencing throughout the plant. Secondly, we focused on engineering amiRNA and syn-tasiRNA precursors of minimal size and derived from the endogenous Arabidopsis thaliana (Arabidopsis) MIR390a (521 nt) and TAS1c (1011 nt) precursors, respectively. We evaluated the silencing efficacy of a collection of constructs containing shortened versions of the AtMIR390a and AtTAS1c precursors, through both transient and stable expression in N. benthamiana and Arabidopsis, respectively. We found that highly effective and accurately processed amiRNAs and syn-tasiRNAs can be produced from minimal precursors of only 89 and 54 nt, respectively. Moreover, we observed that authentic art-sRNAs are produced from minimal -but not from full-length precursors- when expressed from an RNA-based viral vector, such as potato virus X, resulting in efficient silencing of endogenous genes in N. benthamiana. Furthermore, virus-induced gene silencing mediated by art-sRNAs was successfully achieved in a transgene-free and scalable manner by spraying plants with infectious crude extracts containing the modified viral vectors. These results reveal that the length of art-sRNA precursors can be significantly shortened without compromising silencing efficacy, and that minimal precursors offer a biotechnological advantage over full-length precursors when expressed from viral vectors. / This work was supported by grants or fellowships from Ministerio de Ciencia y Universidades (MCU, Spain), Agencia Estatal de Investigación (AEI, Spain) and Fondo Europeo de Desarrollo Regional (FEDER, European Union) [RTI2018-095118-A-100, PID2021-122186OB-100 and RYC-2017-21648 to A.C., and PRE2019-088439 and PRE2022-102565 to A.E.C. and J.L.-G., respectively], NextGenerationEU/PRTR (European Union) [CNS2022-135107 to A.C.], from Consejo Superior de Investigaciones Científicas (CSIC, Spain) [JAEINT_21_00860 to A.P.-E.] and from European Commission [Erasmus+ Grant Agreement 2020-1-DE01-KA103-005653 to A.P. / González Cisneros, AE. (2024). Artificial Small RNAs in Plants: Characterization of Systemic Activity, and Engineering Minimal Precursors for Transgene-Free, Virus-Based Expression [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/213397 / Compendio

Die Genexpressionsmaschinerie in Chloroplasten und Mitochondrien und die ihrer prokaryotischen Vorläufer sind konserviert. Innerhalb eines bakteriellen Grundgerüsts entwickelte sich darüber hinaus ein komplexer RNA-Metabolismus. In der vorliegenden Arbeit wird eine neue Klasse kleiner RNAs (15-50nt) mit plastidärem und mitochondrialen Ursprung beschrieben. Diese kurzen RNAs überlappen mit Bindestellen von RNA-bindenden Proteinen, die mRNAs gegen exonukleolytischen Verdau beschützen. Diese stabilisierende Funktion wird vermutlich hauptsächlich von PPR (Pentatricopeptid repeat) Proteinen und verwandten Proteine bewerkstelligt. Die kleinen RNAs repräsentieren dabei minimale nuklease-resistente Bereiche, sogenannte RNA-Bindeprotein footprints. Solche footprints finden sich in fast jedem intergenischen Bereich, der Prozessierung aufweist. Durch transkriptomweite Untersuchungen von kleinen RNAs in Mutanten von RNA-Bindeproteinen konnte für diese eine Reihe von Bindestellen identifiziert werden. Nuklease-resistente kleine RNAs fehlen in entsprechenden Mutanten. Der Vergleich neu identifizierter Ziele einzelner RNA-Bindeproteine führte dabei zu neuen Erkenntnissen über den Mechanismus der RNA-Erkennung durch PPR Proteine. Im Gegensatz zu Plastiden befinden sich kleine RNAs in Mitochondrien überwiegend an den 3‘ Enden von Transkripten, deren Stabilität vermutlich maßgeblich von diesen RNA-Bindeproteinen beeinflusst wird. Für das chloroplastidäre Ribonukleoprotein CP31A konnte gezeigt werden, dass es an der Stabilisierung der ndhF mRNA beteiligt ist. Die Interaktion mit der ndhF mRNA, die eine zentrale Komponente des NDH-Komplexes kodiert, wird dabei über die 3‘ untranslatierte Region vermittelt. Zusätzlich konnte gezeigt werden, dass CP31A die Stabilität einiger antisense Transkripte beeinflusst. Weiterhin wurden zehn neue Cytidin Desaminierungungen durch die Analyse von RNA-Seq Datensätzen in der Modellpflanze Arabidopsis thaliana identifiziert. / Chloroplasts and mitochondria are of endosymbiotic origin. Their basic gene expression machineries are retained from their free-living prokaryotic progenitors. On top of this bacterial scaffold, a number of organelle-specific RNA processing steps evolved. In this thesis, a novel class of organelle-specific short (15-50nt) RNAs is described on a transcriptome-wide scale. The small RNAs are found at binding sites of PPR (Pentatricopeptide repeat) and PPR-like proteins, which protect mRNAs against exonucleolytic decay. The small RNAs represent minimal nuclease resistant RNAs, so called PPR footprints. Small RNAs were identified in almost every intergenic region subjected to intergenic processing. This finding suggests that accumulation of processed transcripts in plastids is mostly due to protection by highly specific RNA-binding proteins. Small RNA sequencing identified a number of nuclease insensitive sites missing in mutants of RNA-binding proteins. Analysis of multiple small RNAs representing target sites of single PPR proteins expands the knowledge of target specificity. In mitochondria, accumulations of small RNAs predicts that at least two thirds of mitochondrial mRNAs are stabilized by RNA-binding proteins binding in their 3’UTR. In sum, small organellar RNAs turned out to be instrumental in elucidating the hitherto enigmatic intercistronic processing of organellar RNAs and allowed novel insights into the function of the dominant family of organellar RNA binding proteins, the PPR proteins. A chloroplast ribonucleoprotein CP31A is shown to be involved in stabilization of an mRNA for a central component of the NDH-complex by interaction with its 3’UTR. In addition, CP31A represents the first factor described that influences the accumulation of chloroplast antisense transcripts. Finally, ten novel plastid C to U RNA-editing sites were identified in the model plant Arabidopsis thaliana, using a novel RNA-Seq based approach.