RESUMEN
Transcriptomic genome-wide analyses demonstrate massive variation of alternative splicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing variation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named "Exon Ontology," based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encoded protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applying this strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splicing. We also show that Exon Ontology helps to unravel biological processes that depend on suites of coregulated alternative exons, as we uncovered a role of epithelial cell-enriched splicing factors in the AKT signaling pathway and of mesenchymal cell-enriched splicing factors in driving splicing events impacting on autophagy. Freely available on the web, Exon Ontology is the first computational resource that allows getting a quick insight into the protein features encoded by alternative exons and investigating whether coregulated exons contain the same biological information.
Asunto(s)
Empalme Alternativo , Exones , Perfilación de la Expresión Génica/métodos , Genoma Humano , Anotación de Secuencia Molecular/métodos , Transcriptoma , Autofagia , Línea Celular Tumoral , Ontología de Genes , Estudio de Asociación del Genoma Completo , Humanos , Células MCF-7 , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Transducción de Señal , Programas InformáticosRESUMEN
Alternative 3'-terminal exons, which use intronic polyadenylation sites, are generally less conserved and expressed at lower levels than the last exon of genes. Here we discover a class of human genes, in which the last exon appeared recently during evolution, and the major gene product uses an alternative 3'-terminal exon corresponding to the ancestral last exon of the gene. This novel class of alternative 3'-terminal exons are downregulated on a large scale by doxorubicin, a cytostatic drug targeting topoisomerase II, and play a role in cell cycle regulation, including centromere-kinetochore assembly. The RNA-binding protein HuR/ELAVL1 is a major regulator of this specific set of alternative 3'-terminal exons. HuR binding to the alternative 3'-terminal exon in the pre-messenger RNA promotes its splicing, and is reduced by topoisomerase inhibitors. These findings provide new insights into the evolution, function and molecular regulation of alternative 3'-terminal exons.
Asunto(s)
Exones/genética , Inhibidores de Topoisomerasa/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Centrómero/metabolismo , Inmunoprecipitación de Cromatina , Doxorrubicina/farmacología , Proteína 1 Similar a ELAV/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Cinetocoros/metabolismo , Células MCF-7RESUMEN
The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation that dynamically orchestrate several layers of gene expression.
Asunto(s)
ARN Helicasas DEAD-box/genética , MicroARNs/genética , Empalme Alternativo , Animales , Diferenciación Celular/genética , ARN Helicasas DEAD-box/metabolismo , Regulación hacia Abajo , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal/genética , Exones , Regulación de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Células MCF-7 , Ratones , MicroARNs/biosíntesis , MicroARNs/metabolismo , Mioblastos/enzimología , Mioblastos/metabolismo , Mioblastos/fisiología , Transcripción GenéticaRESUMEN
GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABAA or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Mitofagia/fisiología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Aldehídos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/genética , Beclina-1 , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Daño del ADN , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Metabolismo Energético , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Lisosomas/patología , Potencial de la Membrana Mitocondrial , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Mitofagia/genética , Invasividad Neoplásica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Interferente Pequeño/genética , Proteína Sequestosoma-1 , Ensayo de Tumor de Célula MadreRESUMEN
The GABARAPL1 (GABARAP-LIKE 1) gene was first described as an early estrogen-regulated gene that shares a high sequence homology with GABARAP and is thus a part of the GABARAP family. GABARAPL1, like GABARAP, interacts with the GABAA receptor and tubulin and promotes tubulin polymerization. The GABARAP family members (GABARAP, GABARAPL1 and GABARAPL2) and their close homologs (LC3 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. However, despite these similarities, GABARAPL1 displays a complex regulation that is different from that of other GABARAP family members. Moreover, it presents a regulated tissue expression and is the most highly expressed gene among the family in the central nervous system. In this review article, we will outline the specific functions of this protein and also hypothesize about the roles that GABARAPL1 might have in several important biological processes such as cancer or neurodegenerative diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Regulación de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Cobayas , Humanos , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Tubulina (Proteína)/químicaRESUMEN
Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Anticuerpos/inmunología , Proteínas Adaptadoras Transductoras de Señales/química , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Células HEK293 , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Alineación de SecuenciaRESUMEN
Gabarapl1 (gec1) was first described as an estrogen regulated gene which shares a high sequence homology with the gabarap gene. We previously demonstrated that GABARAPL1, like GABARAP, interacts with the GABAA receptor and tubulin and promotes tubulin polymerization. Previous work has demonstrated that the GABARAP family members (GABARAP, LC3, GATE-16 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. We therefore asked whether GABARAPL1 might also play a role in autophagy. First, we showed that GABARAPL1 is cleaved at glycine 116, a residue which is conserved in other members of the family. We also demonstrated that GABARAPL1 is linked to phospholipids, delipidated by Atg4B, associated with intracellular membranes and accumulated in intracellular vesicles after inhibition of lysosomal activity. Finally, we showed that GABARAPL1 partially colocalizes with LC3 or Lysotracker green in intracellular vesicles. Taken together, our results demonstrate that GABARAPL1 associates with autophagic vesicles.