La voie Hippo est une voie conservée impliquée dans la croissance des tissus et la suppression de tumeurs. Des études ont démontré son implication dans le développement des cancers chez l'homme. Cette cascade contrôle l'activité du co-activateur transcriptionnel Yorkie (Yki) chez la drosophile et de la protéine YAP (Yes Associated Protein) chez les mammifères. En raison de l'épissage alternatif de leur transcrits, les protéines Yki et YAP existent sous deux isoformes contenant un domaine WW (Yki1/YAP1) ou deux (Yki2/YAP2). Puisque les domaines WW sont essentiels pour l’interaction avec des partenaires spécifiques, l’inclusion alternative de ce domaine dans la protéine Yki/YAP peut remodeler leur réseau d’interaction et donc leur activité. La régulation et les conséquences fonctionnelles de l’épissage alternatif de yki / YAP in vivo sont inconnues.Dans le cadre de ce doctorat, nous avons constaté que la déplétion du facteur d’épissage B52 chez la drosophile réduit l’inclusion de l’exon alternatif dans l’ARNm de yki et favorise l’expression de l’isoforme Yki1 aux dépens de l’isoforme Yki2. La déplétion en B52 dans l'aile réduit la croissance et l'activité de Yki. Nous montrons que l'isoforme Yki1 est une version atténuée de la protéine Yki qui peut entrer en concurrence avec l'isoforme Yki2 dans le noyau. Pour déterminer le rôle de l’épissage alternatif de yki in vivo et l'importance de l'isoforme courte Yki1, nous avons abrogé cet épissage en utilisant la technologie CRISPR/Cas9 et avons créé des mouches capables d'exprimer uniquement l'isoforme Yki2. Ces mouches yki2only sont viables mais présentent un phénotype aléatoire d’ailes asymétriques. Cette augmentation de l'«asymétrie fluctuante», qui traduit une déviation par rapport au développement normal, suggère que l’épissage alternatif de yki est crucial pour la stabilité développementale. Ces résultats mettent en évidence un nouveau niveau de modulation de la voie Hippo via l’épissage alternatif de yki.L'inclusion alternative du deuxième domaine WW est une caractéristique conservée entre Yki et YAP. Cela conforte l'idée que les isoformes Yki1 et YAP1 ont une fonction importante in vivo et que l'épissage alternatif de yki/YAP est un mécanisme conservé de contrôle de la voie Hippo. Cette étude ouvre de nouvelles perspectives pour la modulation de la voie Hippo dans les cellules cancéreuses en modifiant l’épissage alternatif de YAP. / The Hippo pathway is a conserved pathway involved in tissue growth and tumor suppression. Studies have demonstrated its significance in the development of human cancers. This cascade controls the activity of the transcription co-activator Yorkie (Yki) in flies and Yes-associated protein (YAP) in mammals. Due to Alternative Splicing (AS), both Yki and YAP proteins exist as two isoforms containing one (Yki1/YAP1) or two (Yki2/YAP2) WW domains. Since WW domains are essential for interaction with specific partners, the alternative inclusion of this domain in Yki/YAP protein may remodel their interaction network and therefore their activity. The regulation and functional consequences of AS of yki/YAP in vivo are unknown.In this Ph.D. project, we identified that depletion of splicing factor B52 in Drosophila lowers inclusion of the alternative exon in yki mRNAs and favors the expression of Yki1 isoform at the expense of the Yki2 isoform. B52 depletion in the wing reduces growth and Yki activity. We demonstrate that Yki1 isoform is an attenuated version of Yki protein that can compete with Yki2 isoform in the nucleus. To ascertain the role of yki AS in vivo and the importance of short isoform Yki1, we abrogated this splicing by using CRISPR/Cas9 technology and created flies that can express Yki2 isoform only. yki2only flies are viable but display a random phenotype of asymmetric wing size. This rise in “fluctuating asymmetry” that is the consequence of subtle deviation from normal development, suggests that AS of yki is crucial for the development robustness. Taking together, these results highlight a new layer of modulation of Hippo pathway via AS of yki.Alternative inclusion of the second WW domain is a conserved feature between Yki and YAP. This further supports the idea that Yki1 and YAP1 isoforms have an important function in vivo and that AS of yki/YAP is a conserved mechanism of control of the Hippo pathway. This study opens up new perspectives for modulation of the Hippo pathway in cancer cells by altering YAP AS.

We previously reported that the CNGB1 locus encoding the rod photoreceptor cGMP-gated channel β-subunit is complex, comprising non- overlapping transcription units that give rise to at least six transcripts (Ardell, M.D., Aragon, I., Oliveira, L., Porche, G.E., Burke, E., Pittler, S.J., 1996. The beta subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit. FEBS Lett. 389, 213- 218). To further understand the transcriptional regulation of this extraordinarily complex locus, and to develop a screen for defects in the gene in patients with hereditary disease, we determined its genomic organization and DNA sequence. The CNGB1 locus consists of 33 exons, which span approximately 100 kb of genomic DNA on chromosome 16. The β-subunit comprises two domains, an N-terminal glutamic acid-rich segment (GARP), and a C-terminal channel-like portion. Two additional exons encoding a short GARP transcript and a truncated channel-like transcript have been identified. A major transcription start point was identified 79 bp upstream of the initiator ATG. To begin analysis of the basis for the generation of multiple transcripts, and to identify promoters driving expression in retina, approximately 2.5 kb of the upstream region were sequenced. Putative cis- elements, which can bind the retina-specific transcription factors Crx and Erx, were found immediately upstream of the transcription start point, and may be important for gene expression in this tissue. From our analysis, a model is reported to account for at least four of the retinal transcripts.

Indiana University-Purdue University Indianapolis (IUPUI) / Several experimental methods has been developed for the study of the central dogma since late 20th century. Protein mass spectrometry and next generation sequencing (including DNA-Seq and RNA-Seq) forms a triangle of experimental methods, corresponding to the three vertices of the central dogma, i.e., DNA, RNA and protein. Numerous RNA sequencing and protein mass spectrometry experiments has been carried out in attempt to understand how the expression change of known genes affect biological functions in various of organisms, however, it has been once overlooked that the result data of these experiments are in fact holograms which also reveals other delicate biological mechanisms, such as RNA splicing and the expression of long non-coding RNAs. In this dissertation, we carried out five studies based on high-throughput sequencing data, in an attempt to understand how RNA splicing and differential expression of long non-coding RNAs is associated biological functions. In the first two studies, we identified and characterized 197 stimulant induced and 477 developmentally regulated alternative splicing events from RNA sequencing data. In the third study, we introduced a method for identifying novel alternative splicing events that were never documented. In the fourth study, we introduced a method for identifying known and novel RNA splicing junctions from protein mass spectrometry data. In the fifth study, we introduced a method for identifying long non-coding RNAs from poly-A selected RNA sequencing data. Taking advantage of these methods, we turned RNA sequencing and protein mass spectrometry data into an information gold mine of splicing and long non-coding RNA activities. / 2019-05-06

[ES] Uno de los aspectos más apasionantes de la transcripción es la plasticidad transcriptómica y proteómica mediada por los procesos de regulación post-transcripcional (PTR). Los mecanismos PTR como el splicing alternativo (AS) y la poliadenilación alternativa (APA) han emergido como procesos estrechamente regulados que juegan un papel clave en la generación de la complejidad transcriptómica y están asociados con la coordinación de la diferenciación celular o el desarrollo de tejidos. Sin embargo nuestro conocimiento sobre cómo estos mecanismos regulan las propiedades de los productos resultantes para definir el fenotipo es aún muy reducido. La cantidad de variantes existentes y el amplio rango de posibles consecuencias funcionales, hacen su validación funcional una tarea impracticable si se realiza caso por caso. Además, la falta de herramientas para la evaluación funcional orientada a isoformas ha provocado que gran parte del trabajo computacional haya empleado pipelines ad-hoc aplicadas a sistemas biológicos específicos o simplemente hayan confiado en análisis de enriquecimiento GO, los cuales no son informativos del impacto en las propiedades de las isoformas que hay detrás de la regulación PTR. De hecho, a pesar de las más de sesenta mil publicaciones relativas al AS, muy pocas isoformas se han asociado con propiedades específicas, mientras que el número de nuevas variantes AS/APA con function desconocida crece exponencialmente debido a las técnicas de secuenciación de segunda generación (NGS). Además, y debido a limitaciones técnicas de las NGS para reconstruir la estructura de los transcritos, las tecnologías de secuenciación de tercera generación (TGS) están definiendo una nueva era en la que, por primera vez, es posible conocer la secuencia de elementos estructurales y funcionales en los mRNAs. En esta tesis se han abordado tres propósitos principales para poder avanzar en el estudio funcional de las isoformas. En primer lugar, con las TGS siendo cada vez más utilizadas, la evaluación de la calidad de los transcriptomas \textit{de novo} es esencial para asegurar la fiabilidad de la diversidad transcriptómica encontrada. La falta de análisis de calidad orientados a secuencias largas ha motivado el desarrollo de SQANTI, una pipeline automatizado para la exhaustiva evaluación de TGS transcriptomas. En segundo lugar, la información a nivel de gen de la mayoría de bases de datos funcionales sigue siendo el principal escollo para el estudio de la variabilidad entre isoformas, especialmente en el caso de las isoformas nuevas, en las que las bases de datos estáticas impiden su caracterización. Así, hemos diseñado IsoAnnot, que construye una base de datos de anotaciones funcionales con resolución a nivel de isoformas integrando información diseminada por múltiples bases de datos y métodos de predicción. Finalmente, la indisponibilidad de métodos para estudiar el impacto funcional de la regulación de isoformas, nos ha motivado a desarrollar tappAS, una herramienta dinámica, flexible y diseñada para facilitar el abordaje de este tipo de estudios. Por lo tanto, durante esta tesis hemos desarrollado una infraestructura que resuelve los retos principales del análisis funcional de isoformas, proporcionando un conjunto de nuevos métodos y herramientas que ofrecen una oportunidad única para explorar cómo el fenotipo se especifica post-transcripcionalmente, mediante la alteración de las propiedades funcionales de las isoformas expresadas. La aplicación de nuestro análisis a un doble sistema de diferenciación neuronal en ratón definió el efecto de la regulación de isoformas entre la diferenciación de motoneuronas y oligodendrocitos para múltiples elementos funcionales. Entre ellos, hemos descubierto regiones transmembrana que son diferencialmente incluidas en las isoformas expresadas entre ambos tipos celulares y cuya regulación podría estar contribuyendo al control de / [CA] Un dels aspectes més emocionants de la biologia del transcriptoma és l'adaptabilitat contextual de transcriptomes i proteomes eucariotes mitjançant la regulació post-transcripcional (PTR). Els mecanismes PTR, com el splicing alternatiu (AS) i la poliadenilació alternativa (APA), s'han convertit en processos molt regulats que juguen un paper clau en la generació de la complexitat del transcriptoma i en la coordinació de la diferenciació cel·lular o del desenvolupament de teixits. No obstant això, el nostre coneixement de com aquests mecanismes imprimeixen característiques funcionals diferents al conjunt resultant d'isoformes per definir el fenotip observat és encara escàs. El nombre de variants de PTR i les seues conseqüències potencialment funcionals fa que la validació funcional sigui una tasca poc pràctica si es fa cas per cas. A més, la manca d'enfocaments funcionals orientats a isoformes ha fet que gran part del treballs computacionals per esbrinar qüestions funcionals a nivell de transcriptoma siguen estratègies computacionals ad hoc aplicades a sistemes biològics específics o bé basats en un simple anàlisi d'enriquiment GO, que no aporten informació sobre l'impacte de la PTR sobre les propietats de les isoformes. Així, malgrat les més de 60.000 publicacions existents sobre AS, poques de les isoformes existents s'han associat a propietats específiques, mentre que el nombre de noves variants AS/APA amb funcions desconegudes i fins i tot inexplorades augmenta de manera exponencial gràcies a la seqüenciació de nova generació (NGS). A causa de les limitacions tècniques del NGS per reconstruir l'estructura dels transcrits, la seqüenciació d'alt rendiment de transcrits de longitud completa mitjançant tecnologies de tercera generació (TGS) obre una nova era en la transcriptòmica, ja que millora la definició dels models genètics i, per primera vegada, permet associar amb precisió esdeveniments funcionals dins de la molècula d'ARN. Aquesta tesi aborda tres grans reptes per a progressar en l'estudi de la funció de les isoformes. En primer lloc, amb l'aparició i la popularitat creixent del TGS, la definició precisa i la caracterització completa dels transcriptomes de novo són essencials per garantir la qualitat de qualsevol conclusió sobre la diversitat del transcriptoma. La manca d'anàlisis de qualitat orientats a lectures llargues va motivar el desenvolupament de SQANTI (https://bitbucket.org/ ConesaLab / sqanti), una estratègia computacional automatitzada per a la caracterització estructural i l'avaluació de la qualitat dels transcriptomes de longitud completa. En segon lloc, els recursos funcionals existents centrats en el gen suposen una gran limitació per a l'estudi extensiu de la variabilitat funcional de les isoformes, especialment en les noves isoformes, que no es poden caracteritzar per bases de dades estàtiques. Per tant, vam dissenyar IsoAnnot, que construeix dinàmicament una base de dades amb anotacions funcionals a nivell d'isoforma, que utilitza com a informació d'entrada les seqüències dels transcrits i integra informació de diverses bases de dades i mètodes de predicció. Finalment, com no hi havia cap mètode per interrogar l'impacte funcional del PTR, vam desenvolupar nous enfocaments i eines fàcils d'utilitzar, com ara tappAS (http://tappas.org/), dissenyada per facilitar als investigadors els estudis funcionals de transcriptoma complet i de regulació d'isoformes en contexts específics. Per tant, aquesta tesi descriu el desenvolupament d'un marc d'anàlisi que aborda els reptes fonamentals de l'anàlisi funcional d'isoformes. Aplicada a un sistema de diferenciació neuronal murina, vam descobrir regions transmembrana específiques d'isoformes, la modulació de les quals per PTR podria contribuir a controlar la dinàmica mitocondrial específica del tipus cel·lular durant la determinació del destí neuronal. / [EN] One of the most exciting aspects of transcriptome biology is the contextual adaptability of eukaryotic transcriptomes and proteomes by post-transcriptional regulation (PTR). PTR mechanisms such as alternative splicing (AS) and alternative polyadenylation (APA) have emerged as tightly regulated processes playing a key role in generating transcriptome complexity and coordinating cell differentiation or tissue development. However, how these mechanisms imprint distinct functional characteristics on the resulting set of isoforms to define the observed phenotype remains poorly understood. The number of PTR variants and their resulting range of potentially functional consequences makes their functional validation an impractical task if done on a case-by-case basis. Besides, the lack of isoform-oriented functional profiling approaches has made that much of the computational work done to elucidate transcriptome-wide functional questions has either involved ad hoc computational pipelines applied to specific biological systems or has relied on simple GO-enrichment analysis that are not informative about the PTR impact on isoform properties. Thus, even though more than 60,000 publications on AS, a few number of existing isoforms have been associated with specific properties while the number of novel AS/APA variants with unknown and even unexplored functions is exponentially increasing thanks to the use of next-generation sequencing (NGS). Due to the technical limitations of NGS to reconstruct the transcript structure, high-throughput sequencing of full-length transcripts using third-generation technologies (TGS) is opening up a new transcriptomics era that enhances the definition of gene models and, for the first time, enables to precisely associate functional events within the RNA molecule. This thesis addresses three major challenges to the progression of the study of isoform function. First, with the emergence and increasing popularity of TGS, the accurate definition and comprehensive characterisation of de novo transcriptomes is essential to ensure the quality of any conclusions on transcriptome diversity drawn from these data. The lack of long-read oriented quality aware analysis motivated the development of SQANTI \url{(https://bitbucket.org/ConesaLab/sqanti)}, an automated pipeline for the structural characterization and quality assessment of full-length transcriptomes. Secondly, the gene-centric nature of functional resources remained the major limitation to the extended study of functional isoform variability, especially for novel isoforms, which cannot be characterised by static databases. Thus, we designed IsoAnnot, which dynamically constructs an isoform-resolved rich database of functional annotations by using as input transcript sequences and integrating information disseminated across several databases and prediction methods. Finally, because no methods to interrogate the functional impact of PTR were available, we developed novel approaches and user-friendly tools such as tappAS \url{(http://tappas.org/)}, designed to facilitate researchers the transcriptome-wide functional study of context-specific isoform regulation. Thereby, this thesis describes the development of an analysis framework that tackles the fundamental challenges of the isoform functional analysis by providing a set of novel methods and tools that offer an unique opportunity to explore how the phenotype is specified by altering the functional characteristics of expressed isoforms. Applied to a murine neural differentiation system, our pipeline profiled the effect of isoform regulation on the inclusion of several functional elements within transcripts between motor-neuron and oligodendrocyte differentiation systems and specifically, we discovered isoform-specific transmembrane regions whose modulation by PTR might contribute to control cell type-specific mitochondrial dynamics during neural fate determination. / This work was funded by the following grants: From 2014 to 2018. FPU: Training programme for Academic Staff. Spanish Ministry of Education, FPU2013/02348. From 2016 to 2019. NOVELSEQ: Novel methods for new challenges in the analysis of high-throughput sequencing data. MINECO, BIO2015-1658-R. From 2014 to 2017. DEANN: Developing a European American NGS Network. EU Marie Curie IRSES, GA-612583. / Fuente Lorente, LDL. (2019). Development of a bioinformatics approach for the functional analysis of alternative splicing [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124974 / TESIS

Plants employ sophisticated molecular machineries to fine-tune their responses to growth, developmental, and stress cues. Plants cellular response influences gene expression through regulating processes like transcription and splicing. To increase the genome coding potential and further regulate the expression, pre-mRNA is alternatively spliced. Serine/Arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Recent studies reported only 22 SR proteins encoded in the genome of rice (Oryza sativa), which are classified into 6 subfamilies. Oryza s. SC subfamily 106 kDa (Os-Sc106) locus is homologous to the human SR protein SFSR11 (SRp54). Os-Sc106 contains SR proteins characteristics, and was not included among the rice SR proteins. The clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein 9 (Cas9) system, an RNA-guided endonuclease complex that introduces a double-strand break (DSB) into the DNA. Innovative scientific advances in genome engineering have made CRISPR/Cas9 an excellent system to conduct functional knockout studies of genes in most biological systems including plants. In this study, I targeted the rice Os-Sc106 locus at exon1, and 3 via CRISPR/Cas9 system. Genotyping analyses revealed the recovery of Os-Sc106 mutants including complete functional knockouts such as sf11h-2, sf11h-8, and sf11h-55. Phenotypic analyses show that Os-Sc106 mutants (sf11h-2, 8, 55, and 57) are oversensitive under abiotic stress in comparison to WT plants, suggesting that Os-Sc106 locus encodes a protein that is important for regulating plant stress responses.

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