Large-scale study of RNA processing alterations in multiple cancers

RNA processing and their alterations are determinant to understand normal and disease cell phenotypes. In particular, specific alterations in the RNA processing of genes has been linked to widely accepted cancer hallmarks. With the availability of large-scale genomic and transcriptomic data for multiple cancer types, it is now possible to address ambitious questions such as obtaining a global view of alterations in RNA processing specific to each cancer type as well as in common across all types. The first objective of this thesis is to obtain a global view of RNA processing alterations across different tumor types along with alterations with respect to RNA binding proteins (trans-component), their tumor-type specificity, differential expression, mutations, copy number variation and whether these alterations result in differential splicing. Using data for more than 4000 patients from 11 tumor types, we provide the link between alterations of RNA binding proteins and splicing changes across multiple tumor types. Second objective moves one step further and explores in detail the RNA-processing alterations with respect to mutations on RNA regulatory sequences (cis-components). Using whole genome sequencing data for more than 1000 cancer patients, we thoroughly study the sequence of entire genes and report significantly mutated short regions in coding and non-coding parts of genes that are moreover enriched in RNA putative RNA regulatory sites, including regions deep into the introns. The recurrence of some of the mutations in non-coding regions is comparable to some of already known driver genes in coding regions. We further analyze the impact of these mutations at the RNA level by using RNA sequencing from the same samples. This work proposes a novel and powerful strategy to study mutations in cancer to identify novel oncogenic mechanisms. In addition, we share the immense amount of data generated in these analyses so that other researchers can study them in detail and validate them experimentally.El procesamiento del ARN y sus alteraciones son determinantes para entender el fenotipo de las células en condiciones normales y de enfermedad. En particular, alteraciones en el procesamiento de ARN de determinados genes se han vinculado a características distintivas del cáncer ampliamente aceptadas. Con la disponibilidad de datos genómicos y transcriptómicos a gran escala paramúltiples tipos de cáncer, es posible abordar cuestiones ambiciosas como la obtención de una visión global de las alteraciones en el procesamiento de ARN que son específicas para cada tipo de cáncer, así como de aquellas las comunes a varios tipos. El primer objetivo de esta tesis es obtener una visión global de las alteraciones del procesamiento de ARN en diferentes tipos de tumores, así como de las alteraciones en las proteínas de unión a ARN (componente trans), y si dichas alteraciones resultan en un procesamiento diferencial del RNA. Utilizando datos de más de 4000 pacientes para 11 tipos de tumores, establecemos la relación entre las alteraciones de las proteínas de unión a ARN y cambios de splicing en múltiples tipos de tumores. El segundo objetivo va un paso más allá y explora en detalle las alteraciones del procesamiento de ARN con respecto a mutaciones en las secuencias reguladoras del ARN (componente cis). Utilizando datos de genomas completos para más de 1000 pacientes, estudiamos a fondo la secuencia de genes para identificar regiones cortas significativamente mutadas en partes codificantes y no codificantes por proteína, y que además están enriquecidas en posibles sitios reguladores del ARN, incluyendo regiones intrónicas profundas. La recurrencia de las mutaciones en algunas regiones no codificantes es comparable a la de algunos genes drivers de cáncer conocidos. Además, analizamos el impacto de estas mutaciones a nivel del ARN mediante el uso de datos de secuenciación de ARN de las mismas muestras. Este trabajo propone una estrategia novedosa y potente para estudiar las mutaciones en cáncer con el fin de identificar nuevos mecanismos oncogénicos. Además, compartimos la inmensa cantidad de datos generados en estos análisis para que otros investigadores los puedan estudiar en detalle y validarlos experimentalmente

The role of alternative splicing in cancer

The functional capacity of cells is defined by the transcriptome. Many recent studies have identified variations in the transcriptome of tumors due to alternative splicing changes, as well as mutations in splicing factors and regulatory signals in most tumor types. Some of these alterations have been linked to tumor progression, metastasis, therapy resistance, and other oncogenic processes. Here, we describe the different mechanisms that drive splicing changes in tumors and their impact in cancer. Motivated by the current evidence, we propose a model whereby a subset of the splicing patterns contributes to the definition of specific tumor phenotypes, and may hold potential for the development of novel clinical biomarkers and therapeutic approaches.This work was supported by the MINECO and FEDER (BIO2014–52566-R) and AGAUR (SGR2014–1121)

The role of alternative splicing in cancer

The functional capacity of cells is defined by the transcriptome. Many recent studies have identified variations in the transcriptome of tumors due to alternative splicing changes, as well as mutations in splicing factors and regulatory signals in most tumor types. Some of these alterations have been linked to tumor progression, metastasis, therapy resistance, and other oncogenic processes. Here, we describe the different mechanisms that drive splicing changes in tumors and their impact in cancer. Motivated by the current evidence, we propose a model whereby a subset of the splicing patterns contributes to the definition of specific tumor phenotypes, and may hold potential for the development of novel clinical biomarkers and therapeutic approaches.This work was supported by the MINECO and FEDER (BIO2014–52566-R) and AGAUR (SGR2014–1121)

A semi-supervised approach uncovers thousands of intragenic enhancers differentially activated in human cells

Background. Transcriptional enhancers are generally known to regulate gene transcription from afar. Their activation involves a series of changes in chromatin marks and recruitment of protein factors. These enhancers may also occur inside genes, but how many may be active in human cells and their effects on the regulation of the host gene remains unclear./nResults. We describe a novel semi-supervised method based on the relative enrichment of chromatin signals between 2 conditions to predict active enhancers. We applied this method to the tumoral K562 and the normal GM12878 cell lines to predict enhancers that are differentially active in one cell type. These predictions show enhancer-like properties according to positional distribution, correlation with gene expression and production of enhancer RNAs. Using this model, we predict 10,365 and 9777 intragenic active enhancers in K562 and GM12878, respectively, and relate the differential activation of these enhancers to expression and splicing differences of the host genes./nConclusions. We propose that the activation or silencing of intragenic transcriptional enhancers modulate the regulation of the host gene by means of a local change of the chromatin and the recruitment of enhancer-related factors that may interact with the RNA directly or through the interaction with RNA binding proteins. Predicted enhancers are available at http://regulatorygenomics.upf.edu/Projects/enhancers.html.The authors would like to thank E. Furlong, Y. Barash, B. Blencowe and U. Braunschweig for useful discussions. This work was supported by grants from Plan Nacional I + D (BIO2011-23920) and Consolider (CSD2009-00080) from MINECO (Spanish Government), and by the Sandra Ibarra Foundation for Cancer (FSI 2013). JGV and BS were supported FPI grants from the MINECO (Spanish Government) BES-2009-018064 and BES-2012-052683, respectively

Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks

Alternative splicing is regulated by multiple RNA-binding proteins and influences the expression of most eukaryotic genes. However, the role of this process in human disease, and particularly in cancer, is only starting to be unveiled. We systematically analyzed mutation, copy number, and gene expression patterns of 1348 RNA-binding protein (RBP) genes in 11 solid tumor types, together with alternative splicing changes in these tumors and the enrichment of binding motifs in the alternatively spliced sequences. Our comprehensive study reveals widespread alterations in the expression of RBP genes, as well as novel mutations and copy number variations in association with multiple alternative splicing changes in cancer drivers and oncogenic pathways. Remarkably, the altered splicing patterns in several tumor types recapitulate those of undifferentiated cells. These patterns are predicted to be mainly controlled by MBNL1 and involve multiple cancer drivers, including the mitotic gene NUMA1. We show that NUMA1 alternative splicing induces enhanced cell proliferation and centrosome amplification in nontumorigenic mammary epithelial cells. Our study uncovers novel splicing networks that potentially contribute to cancer development and progression.We thank P. Papasaikas, B. Blencowe, M. Irimia, and Q. Morris for comments and discussions. E.S., B.S., A.P., and E.E. were supported by the Ministerio de Economía y Competitividad (MINECO) and European Commission (FEDER) (BIO2014-52566-R), Consolider RNAREG (CSD2009-00080), by Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (SGR2014-1121), and by the Sandra Ibarra Foundation for Cancer (FSI2013). J.V. and B.M. were supported by Fundación Botín, by Banco de Santander through its Santander Universities Global Division, and by Consolider RNAREG (CSD2009-00080), MINECO, and AGAUR. F.M. and M.A.P. were supported by AECC (Hereditary Cancer), AGAUR (SGR2014-364), the Instituto de Salud Carlos III (ISCIII), the MINECO, and FEDER (PIE13/00022-ONCOPROFILE, PI15/00854, and RTICC RD12/0036/0008)

SUPPA2: fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions

Despite the many approaches to study differential splicing from RNA-seq, many challenges remain unsolved, including computing capacity and sequencing depth requirements. Here we present SUPPA2, a new method that addresses these challenges, and enables streamlined analysis across multiple conditions taking into account biological variability. Using experimental and simulated data, we show that SUPPA2 achieves higher accuracy compared to other methods, especially at low sequencing depth and short read length. We use SUPPA2 to identify novel Transformer2-regulated exons, novel microexons induced during differentiation of bipolar neurons, and novel intron retention events during erythroblast differentiation.This work was supported by the MINECO and FEDER with grants BIO2014-52566-R and BIO2017-85364-R, by AGAUR with grants SGR2014-1121 and SGR2017-1020, by BBSRC (BB/P006612/1), and by Breast Cancer Now (2014NovPR355). GH is a BBSRC-funded PhD student

Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks

Alternative splicing is regulated by multiple RNA-binding proteins and influences the expression of most eukaryotic genes. However, the role of this process in human disease, and particularly in cancer, is only starting to be unveiled. We systematically analyzed mutation, copy number, and gene expression patterns of 1348 RNA-binding protein (RBP) genes in 11 solid tumor types, together with alternative splicing changes in these tumors and the enrichment of binding motifs in the alternatively spliced sequences. Our comprehensive study reveals widespread alterations in the expression of RBP genes, as well as novel mutations and copy number variations in association with multiple alternative splicing changes in cancer drivers and oncogenic pathways. Remarkably, the altered splicing patterns in several tumor types recapitulate those of undifferentiated cells. These patterns are predicted to be mainly controlled by MBNL1 and involve multiple cancer drivers, including the mitotic gene NUMA1. We show that NUMA1 alternative splicing induces enhanced cell proliferation and centrosome amplification in nontumorigenic mammary epithelial cells. Our study uncovers novel splicing networks that potentially contribute to cancer development and progression.We thank P. Papasaikas, B. Blencowe, M. Irimia, and Q. Morris for comments and discussions. E.S., B.S., A.P., and E.E. were supported by the Ministerio de Economía y Competitividad (MINECO) and European Commission (FEDER) (BIO2014-52566-R), Consolider RNAREG (CSD2009-00080), by Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (SGR2014-1121), and by the Sandra Ibarra Foundation for Cancer (FSI2013). J.V. and B.M. were supported by Fundación Botín, by Banco de Santander through its Santander Universities Global Division, and by Consolider RNAREG (CSD2009-00080), MINECO, and AGAUR. F.M. and M.A.P. were supported by AECC (Hereditary Cancer), AGAUR (SGR2014-364), the Instituto de Salud Carlos III (ISCIII), the MINECO, and FEDER (PIE13/00022-ONCOPROFILE, PI15/00854, and RTICC RD12/0036/0008)

IRFinder: assessing the impact of intron retention on mammalian gene expression

Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated.This work was supported by the Agence Nationale de la Recherche (ANR 143683); the National Health and Medical Research Council (grant #1061906, #1080530, #1128175, #1126306). BS and EE were supported by grants BIO2014-52566-R from the MINECO (Spanish Government) and FEDER funds, by AGAUR (2014-SGR1121), and by the Sandra Ibarra Foundation for Cancer (FSI2013)

A quality control portal for sequencing data deposited at the European genome-phenome archive

Since its launch in 2008, the European Genome-Phenome Archive (EGA) has been leading the archiving and distribution of human identifiable genomic data. In this regard, one of the community concerns is the potential usability of the stored data, as of now, data submitters are not mandated to perform any quality control (QC) before uploading their data and associated metadata information. Here, we present a new File QC Portal developed at EGA, along with QC reports performed and created for 1 694 442 files [Fastq, sequence alignment map (SAM)/binary alignment map (BAM)/CRAM and variant call format (VCF)] submitted at EGA. QC reports allow anonymous EGA users to view summary-level information regarding the files within a specific dataset, such as quality of reads, alignment quality, number and type of variants and other features. Researchers benefit from being able to assess the quality of data prior to the data access decision and thereby, increasing the reusability of data (https://ega-archive.org/blog/data-upcycling-powered-by-ega/).Funding: the File QC feature project has received funding from Horizon 2020 ELIXIR-CONVERGE project (grant agreement No 871075), the ELIXIR-FHD-IS and La Caixa Foundation (LCF/PR/CE20/50740008

IRFinder: assessing the impact of intron retention on mammalian gene expression

Intron retention (IR) occurs when an intron is transcribed into pre-mRNA and remains in the final mRNA. We have developed a program and database called IRFinder to accurately detect IR from mRNA sequencing data. Analysis of 2573 samples showed that IR occurs in all tissues analyzed, affects over 80% of all coding genes and is associated with cell differentiation and the cell cycle. Frequently retained introns are enriched for specific RNA binding protein sites and are often retained in clusters in the same gene. IR is associated with lower protein levels and intron-retaining transcripts that escape nonsense-mediated decay are not actively translated.This work was supported by the Agence Nationale de la Recherche (ANR 143683); the National Health and Medical Research Council (grant #1061906, #1080530, #1128175, #1126306). BS and EE were supported by grants BIO2014-52566-R from the MINECO (Spanish Government) and FEDER funds, by AGAUR (2014-SGR1121), and by the Sandra Ibarra Foundation for Cancer (FSI2013)