Pathway oriented stroid hormone-dependent transcriptome analysis. Establishment of a custom cDNA microarray to study hormone signaling in breast cancer

Para avanzar en el entendimiento de las vías de señalización moleculares involucradas en la progresión de cáncer tumoral, se construyo una plataforma personalizada de cDNAs, la cual contiene genes de vías de señalización representativas para investigar la respuesta dinámica temporal a hormonas (progesterona y estradiol) empleando como modelo la línea celular T47D-MTVL e inhibidores específicos de las vías de señalización. Adicionalmente, se realizó un análisis de los perfiles de expresión de un grupo de tumores de mama encontrando buena correlación con los datos clínico-histopatológicos y mostrando como fenotipos específicos correlacionan con mal pronóstico. Los genes más significativos capaces de discriminar entre los fenotipos de tumor fueron determinados, y probados sobre un nuevo grupo de muestras, asignándolas a los subtipos predichos. El análisis de las vías de señalización de los genes más significativos de cada fenotipo fue realizado para elucidar las vías moleculares más representativas afectadas en cada clase de tumor

Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A

Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5′ of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 3′ splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition

Mutational analysis of progesterone receptor functional domains in stable cell lines delineates sets of genes regulated by different mechanisms

Steroid hormone receptors act directly in the nucleus on the chromatin organization and transcriptional activity of several promoters. Furthermore, they have an indirect effect on cytoplasmic signal transduction pathways, including MAPK, impacting ultimately on gene expression. We are interested in distinguishing between the two modes of action of progesterone receptor (PR) on the control of gene expression and cell proliferation. For this, we have stably expressed, in PR-negative breast cancer cells, tagged forms of the PR isoform B mutated at regions involved either in DNA binding (DNA-binding domain) or in its ability to interact with the estrogen receptor and to activate the c-Src/MAPK/Erk/Msk cascade (estrogen receptor-interacting domain). Both mutants impair PR-mediated activation of a well-understood model promoter in response to progestin, as well as hormone-induced cell proliferation. Additional mutants affecting transactivation activity of PR (activation function 2) or a zinc-finger implicated in dimerization (D-box) have also been tested. Microarrays and gene expression experiments on these cell lines define the subsets of hormone-responsive genes regulated by different modes of action of PR isoform B, as well as genes in which the nuclear and nongenomic pathways cooperate. Correlation between CCND1 expression in the different cell lines and their ability to support cell proliferation confirms CCND1 as a key controller gene. Copyright © 2009 by The Endocrine Society.This work was supported by grants from the Catalan Department for Universities, Research and the Information Society, and the Spanish Ministry of Science and Technology and Fondo Europeo de desarrollo regional (SAF2002-03320, BFU2008-00359). A.J. was recipient of a ‘Ramón y Cajal’ appointment from the Spanish Ministry of Science and Technology. I.Q. was recipient of a Formación personal universitario predoctoral fellowship from the Spanish Ministry of Education. L.M-A. was recipient of a predoctoral fellowship funded by Fundación para la investigación y prevención del SIDA en EspañaPeer Reviewe

Mutations primarily alter the inclusion of alternatively spliced exons

Genetic analyses and systematic mutagenesis have revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, consistent with the concept that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between genome sequence variation and exon inclusion across the transcriptome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans. Therefore, mutations that affect splicing are not evenly distributed across primary transcripts but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.We thank Yamile Márquez and Manuel Irimia for identifying PSMD14 exon 11 as a constitutive exon whose inclusion levels are conserved across many vertebrate species. Work in B.L.’s is supported by a European Research Council (ERC) Consolidator grant (616434), the Spanish Ministry of Economy and Competitiveness (BFU2017-89488-P and SEV-2012-0208), the AXA Research Fund, the Bettencourt Schueller Foundation, Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, SGR-831), the EMBL Partnership, and the CERCA Program/Generalitat de Catalunya. P.B.-C. was funded in part by a Severo Ochoa PhD fellowship. Work in J.V.’s laboratory is supported by Fundación Botín, Banco de Santander through its Santander Universities Global Division, ERC AdvG 670146, AGAUR, Spanish Ministry of Economy and Competitiveness (BFU 2014-005153, BFU 2017 89308-P, and SEV-2012-0208), the EMBL Partnership, and the CERCA program/Generalitat de Catalunya

Genotoxic stress inhibits Ewing sarcoma cell growth by modulating alternative pre-mRNA processing of the RNA helicaseDHX9

Alternative splicing plays a key role in the DNA damage response and in cancer. Ewing Sarcomas (ES) are aggressive tumors caused by different chromosomal translocations that yield in-frame fusion proteins driving transformation. RNA profiling reveals genes differentially regulated by UV light irradiation in two ES cell lines exhibiting different sensitivity to genotoxic stress. In particular, irradiation induces a new isoform of the RNA helicase DHX9 in the more sensitive SK-N-MC cells, which is targeted to nonsense-mediated decay (NMD), causing its downregulation. DHX9 protein forms a complex with RNA polymerase II (RNAPII) and EWS-FLI1 to enhance transcription. Silencing of DHX9 in ES cells sensitizes them to UV treatment and impairs recruitment of EWS-FLI1 to target genes, whereas DHX9 overexpression protects ES cells from genotoxic stress. Mechanistically, we found that UV light irradiation leads to enhanced phosphorylation and decreased processivity of RNAPII in SK-N-MC cells, which in turn causes inclusion of DHX9 exon 6A. A similar effect on DHX9 splicing was also elicited by treatment with the chemotherapeutic drug etoposide, indicating a more general mechanism of regulation in response to DNA damage. Our data identify a new NMD-linked splicing event in DHX9 with impact on EWS-FLI1 oncogenic activity and ES cell viability

Distinct regulatory programs establish widespread sex-specific alternative splicing in Drosophila melanogaster

In Drosophila melanogaster, female-specific expression of Sex-lethal (SXL) and Transformer (TRA) proteins controls sex-specific alternative splicing and/or translation of a handful of regulatory genes responsible for sexual differentiation and behavior. Recent findings in 2009 by Telonis-Scott et al. document widespread sex-biased alternative splicing in fruitflies, including instances of tissue-restricted sex-specific splicing. Here we report results arguing that some of these novel sex-specific splicing events are regulated by mechanisms distinct from those established by female-specific expression of SXL and TRA. Bioinformatic analysis of SXL/TRA binding sites, experimental analysis of sex-specific splicing in S2 and Kc cells lines and of the effects of SXL knockdown in Kc cells indicate that SXL-dependent and SXL-independent regulatory mechanisms coexist within the same cell. Additional determinants of sex-specific splicing can be provided by sex-specific differences in the expression of RNA binding proteins, including Hrp40/Squid. We report that sex-specific alternative splicing of the gene hrp40/squid leads to sex-specific differences in the levels of this hnRNP protein. The significant overlap between sex-regulated alternative splicing changes and those induced by knockdown of hrp40/squid and the presence of related sequence motifs enriched near subsets of Hrp40/Squid-regulated and sex-regulated splice sites indicate that this protein contributes to sex-specific splicing regulation. A significant fraction of sex-specific splicing differences are absent in germline-less tudor mutant flies. Intriguingly, these include alternative splicing events that are differentially spliced in tissues distant from the germline. Collectively, our results reveal that distinct genetic programs control widespread sex-specific splicing in Drosophila melanogaster

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)

Distinct regulatory programs establish widespread sex-specific alternative splicing in Drosophila melanogaster

In Drosophila melanogaster, female-specific expression of Sex-lethal (SXL) and Transformer (TRA) proteins controls sex-specific alternative splicing and/or translation of a handful of regulatory genes responsible for sexual differentiation and behavior. Recent findings in 2009 by Telonis-Scott et al. document widespread sex-biased alternative splicing in fruitflies, including instances of tissue-restricted sex-specific splicing. Here we report results arguing that some of these novel sex-specific splicing events are regulated by mechanisms distinct from those established by female-specific expression of SXL and TRA. Bioinformatic analysis of SXL/TRA binding sites, experimental analysis of sex-specific splicing in S2 and Kc cells lines and of the effects of SXL knockdown in Kc cells indicate that SXL-dependent and SXL-independent regulatory mechanisms coexist within the same cell. Additional determinants of sex-specific splicing can be provided by sex-specific differences in the expression of RNA binding proteins, including Hrp40/Squid. We report that sex-specific alternative splicing of the gene hrp40/squid leads to sex-specific differences in the levels of this hnRNP protein. The significant overlap between sex-regulated alternative splicing changes and those induced by knockdown of hrp40/squid and the presence of related sequence motifs enriched near subsets of Hrp40/Squid-regulated and sex-regulated splice sites indicate that this protein contributes to sex-specific splicing regulation. A significant fraction of sex-specific splicing differences are absent in germline-less tudor mutant flies. Intriguingly, these include alternative splicing events that are differentially spliced in tissues distant from the germline. Collectively, our results reveal that distinct genetic programs control widespread sex-specific splicing in Drosophila melanogaster

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)

Analysis dataset for the paper "Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks"

This dataset contains additional files related to the paper<br><br>E. Sebestyén*, B. Singh*, B. Miñana, A. Pagès, F. Mateo, M. A. Pujana, J. Valcárcel, E. Eyras (2016) Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks. <i>Genome Res</i>, <b>26</b>: 732-744, doi:10.1101/gr.199935.115<br><br>It contains the following tar.gz archives:<br><br><b>GR-Sebestyen-2016-TCGA-correlations.tgz</b> contains the Spearman correlation of the alternative splicing event PSI values with the expression z-score of the differentially expressed RBPs in a particular tumor type.<br><br><b>GR-Sebestyen-2016-TCGA-deltapsi.tgz</b> contains the differential splicing analysis results of the events between the tumor and normal conditions in a particular tumor type.<br><br><b>GR-Sebestyen-2016-TCGA-diffexp.tgz</b> contains the differential expression analysis results of all genes between the tumor and normal conditions in a particular tumor type.<br> <br><b>GR-Sebestyen-2016-TCGA-motif.tgz</b> contains the fasta sequence of all event types, and the number of RNAcompete motifs found in the events using FIMO.<br><br><b>GR-Sebestyen-2016-TCGA-psi.tgz</b> contains the PSI values of all events in all samples processed in a particular tumor type.<br><br><b>GR-Sebestyen-2016-TCGA-tpm.tgz</b> contains the TPM values of all isoforms in all samples processed in a particular tumor type.  <br><br><b>GR-Sebestyen-2016-TCGA-zscore.tgz</b> contains the expression z-score of all genes in all samples processed in a particular tumor type.<br><br><b>GR-Sebestyen-2016-TCGA-fimo.tgz</b> contains the original RNAcompete FIMO results for all event types.<br><br>For details on data generation, see the Genome Research paper. <br><br>The data presented here are based upon data generated by the TCGA Research Network: <strong>http://cancergenome.nih.gov</strong><br><br>If you reuse the data, please cite the Genome Research paper.<br