Development of a bioinformatics approach for the functional analysis of alternative splicing

[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 no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124974TESI

Analysis of Differentially Expressed MicroRNAs in Serum and Lung Tissues from Individuals with Severe Asthma Treated with Oral Glucocorticoids

Biomarker; Individuals with severe asthma; Oral corticosteroidsBiomarcador; Persones amb asma greu; Corticosteroides oralsBiomarcador; Personas con asma grave; Corticosteroides oralesNowadays, microRNAs (miRNAs) are increasingly used as biomarkers due to their potential contribution to the diagnosis and targeted treatment of a range of diseases. The aim of the study was to analyze the miRNA expression profiles in serum and lung tissue from patients with severe asthma treated with oral corticosteroids (OCS) and those without OCS treatment. For this purpose, serum and lung tissue miRNAs of OCS and non-OCS asthmatic individuals were evaluated by miRNAs-Seq, and subsequently miRNA validation was performed using RT-qPCR. Additionally, pathway enrichment analysis of deregulated miRNAs was conducted. We observed altered expression by the next-generation sequencing (NGS) of 11 miRNAs in serum, of which five (hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) were validated by RT-qPCR, and three miRNAs in lung tissue (hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a). The best multivariate logistic regression model to differentiate individuals with severe asthma, treated and untreated with OCS, was to combine the serum miRNAs hsa-miR-221-5p and hsa-miR-769-5p. Expression of hsa-miR-148b-3p and hsa-miR-221-5p correlated with FEV1/FVC (%) and these altered miRNAs act in key signaling pathways for asthma disease and the regulated expression of some genes (FOXO3, PTEN, and MAPK3) involved in these pathways. In conclusion, there are miRNA profiles differentially expressed in OCS-treated individuals with asthma and could be used as biomarkers of OCS treatment.This work was supported by ISCIII—Instituto de Salud Carlos III, FIS (Fondo de Investigación Sanitaria—Spanish Health Research Fund) grants PI18/00167, PI21/00896, and FI19/00067; Ciber de Enfermedades Respiratorias (CIBERES); RTC-2017-6501-1 (Ministerio de Ciencia, Innovación y Universidades), a Carlos III Institute of Health Initiative; and FEDER funds (Fondo Europeo de Desarrollo Regional)

Analysis of Differentially Expressed MicroRNAs in Serum and Lung Tissues from Individuals with Severe Asthma Treated with Oral Glucocorticoids

Nowadays, microRNAs (miRNAs) are increasingly used as biomarkers due to their potential contribution to the diagnosis and targeted treatment of a range of diseases. The aim of the study was to analyze the miRNA expression profiles in serum and lung tissue from patients with severe asthma treated with oral corticosteroids (OCS) and those without OCS treatment. For this purpose, serum and lung tissue miRNAs of OCS and non-OCS asthmatic individuals were evaluated by miRNAs-Seq, and subsequently miRNA validation was performed using RT-qPCR. Additionally, pathway enrichment analysis of deregulated miRNAs was conducted. We observed altered expression by the next-generation sequencing (NGS) of 11 miRNAs in serum, of which five (hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) were validated by RT-qPCR, and three miRNAs in lung tissue (hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a). The best multivariate logistic regression model to differentiate individuals with severe asthma, treated and untreated with OCS, was to combine the serum miRNAs hsa-miR-221-5p and hsa-miR-769-5p. Expression of hsa-miR-148b-3p and hsa-miR-221-5p correlated with FEV/FVC (%) and these altered miRNAs act in key signaling pathways for asthma disease and the regulated expression of some genes (FOXO3, PTEN, and MAPK3) involved in these pathways. In conclusion, there are miRNA profiles differentially expressed in OCS-treated individuals with asthma and could be used as biomarkers of OCS treatment

Analysis of Differentially Expressed MicroRNAs in Serum and Lung Tissues from Individuals with Severe Asthma Treated with Oral Glucocorticoids

Nowadays, microRNAs (miRNAs) are increasingly used as biomarkers due to their potential contribution to the diagnosis and targeted treatment of a range of diseases. The aim of the study was to analyze the miRNA expression profiles in serum and lung tissue from patients with severe asthma treated with oral corticosteroids (OCS) and those without OCS treatment. For this purpose, serum and lung tissue miRNAs of OCS and non-OCS asthmatic individuals were evaluated by miRNAs-Seq, and subsequently miRNA validation was performed using RT-qPCR. Additionally, pathway enrichment analysis of deregulated miRNAs was conducted. We observed altered expression by the next-generation sequencing (NGS) of 11 miRNAs in serum, of which five (hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) were validated by RT-qPCR, and three miRNAs in lung tissue (hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a). The best multivariate logistic regression model to differentiate individuals with severe asthma, treated and untreated with OCS, was to combine the serum miRNAs hsa-miR-221-5p and hsa-miR-769-5p. Expression of hsa-miR-148b-3p and hsa-miR-221-5p correlated with FEV1/FVC (%) and these altered miRNAs act in key signaling pathways for asthma disease and the regulated expression of some genes (FOXO3, PTEN, and MAPK3) involved in these pathways. In conclusion, there are miRNA profiles differentially expressed in OCS-treated individuals with asthma and could be used as biomarkers of OCS treatment

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present