RESUMEN
RNA helicases comprise the largest family of enzymes involved in the metabolism of mRNAs, the processing and fate of which rely on their packaging into messenger ribonucleoprotein particles (mRNPs). In this Review, we describe how the capacity of some RNA helicases to either remodel or lock the composition of mRNP complexes underlies their pleiotropic functions at different steps of the gene expression process. We illustrate the roles of RNA helicases in coordinating gene expression steps and programmes, and propose that RNA helicases function as molecular drivers and guides of the progression of their mRNA substrates from one RNA-processing factory to another, to a productive mRNA pool that leads to protein synthesis or to unproductive mRNA pools that are stored or degraded.
Asunto(s)
Regulación de la Expresión Génica , ARN Helicasas/fisiología , Animales , Expresión Génica , Humanos , Empalme del ARN , Transporte de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The NF-κB protein p65/RelA plays a pivotal role in coordinating gene expression in response to diverse stimuli, including viral infections. At the chromatin level, p65/RelA regulates gene transcription and alternative splicing through promoter enrichment and genomic exon occupancy, respectively. The intricate ways in which p65/RelA simultaneously governs these functions across various genes remain to be fully elucidated. In this study, we employed the HTLV-1 Tax oncoprotein, a potent activator of NF-κB, to investigate its influence on the three-dimensional organization of the genome, a key factor in gene regulation. We discovered that Tax restructures the 3D genomic landscape, bringing together genes based on their regulation and splicing patterns. Notably, we found that the Tax-induced gene-gene contact between the two master genes NFKBIA and RELA is associated with their respective changes in gene expression and alternative splicing. Through dCas9-mediated approaches, we demonstrated that NFKBIA-RELA interaction is required for alternative splicing regulation and is caused by an intragenic enrichment of p65/RelA on RELA. Our findings shed light on new regulatory mechanisms upon HTLV-1 Tax and underscore the integral role of p65/RelA in coordinated regulation of NF-κB-responsive genes at both transcriptional and splicing levels in the context of the 3D genome.
The NF-κB pathway is essential for coordinating gene expression in response to various stimuli, including viral infections. Most studies have focused on the role of NF-κB in transcriptional regulation. In the present study, the impact of the potent NF-κB activator HTLV-1 Tax oncoprotein on the three-dimensional organization of the genome was investigated. Tax-mediated NF-κB activation was found to restructure the 3D genomic landscape in cells and to bring genes together in multigene complexes that are coordinately regulated either transcriptionally or through alternative splicing by NF-κB. Induced coordinate changes in transcription and alternative splicing included the two master genes of NF-κB pathway NFKBIA and RELA. The findings have significant implications for understanding cell fate determination and disease development associated with HTLV-1 infection, as well as chronic NF-κB activation in various human inflammatory diseases and cancer.
Asunto(s)
Ensamble y Desensamble de Cromatina , Regulación de la Expresión Génica , Subunidad p50 de NF-kappa B , Empalme Alternativo/genética , Ensamble y Desensamble de Cromatina/genética , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Activación Transcripcional , Humanos , Subunidad p50 de NF-kappa B/metabolismoRESUMEN
BACKGROUND & AIMS: Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. METHODS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in in vitro-infected hepatocytes and in chronically infected patients. RESULTS: The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.
RESUMEN
Muscleblind-like splicing regulators (MBNLs) activate or repress the inclusion of alternative splicing (AS) events, enabling the developmental transition of fetal mRNA splicing isoforms to their adult forms. Herein, we sought to elaborate the mechanism by which MBNLs mediate AS related to biological processes. We evaluated the functional role of DEAD-box (DDX) RNA helicases, DDX5 and DDX17 in MBNL-dependent AS regulation. Whole-transcriptome analysis and validation approaches revealed a handful of MBNLs-dependent AS events to be affected by DDX5 and DDX17 in mostly an opposite manner. The opposite expression patterns of these two groups of factors during muscle development and coordination of fetal-to-adult splicing transition indicate the importance of these proteins at early stages of development. The identified pathways of how the helicases modulate MBNL splicing activity include DDX5 and DDX17-dependent changes in the ratio of MBNL splicing isoforms and most likely changes in accessibility of MBNL-binding sites. Another pathway involves the mode of action of the helicases independent of MBNL activity. These findings lead to a deeper understanding of the network of interdependencies between RNA-binding proteins and constitute a valuable element in the discussion on developmental homeostasis and pathological states in which the studied protein factors play a significant role.
Asunto(s)
Empalme Alternativo , ARN Helicasas , Empalme Alternativo/genética , ARN Helicasas/genética , Empalme del ARN , Isoformas de Proteínas/genética , Sitios de Unión/genéticaRESUMEN
DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspects of gene expression, especially transcription and splicing, through incompletely understood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted human cells confirmed the large impact of these RNA helicases on splicing and revealed a widespread deregulation of 3' end processing. In silico analyses and experiments in cultured cells showed the binding and functional contribution of the genome organizing factor CTCF to chromatin sites at or near a subset of DDX5/DDX17-dependent exons that are characterized by a high GC content and a high density of RNA Polymerase II. We propose the existence of an RNA helicase-dependent relationship between CTCF and the dynamics of transcription across DNA and/or RNA structured regions, that contributes to the processing of internal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loops spatially connect RNA helicase-regulated exons with their cognate promoter, and we provide the first direct evidence that de novo gene looping modifies alternative splicing and polyadenylation. Overall our findings uncover the impact of DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.
Asunto(s)
ARN Helicasas DEAD-box , ARN , Humanos , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Helicasas DEAD-box/metabolismo , Empalme Alternativo , Cromatina/genéticaRESUMEN
The inclusion of exons during the splicing process depends on the binding of splicing factors to short low-complexity regulatory sequences. The relationship between exonic splicing regulatory sequences and coding sequences is still poorly understood. We demonstrate that exons that are coregulated by any given splicing factor share a similar nucleotide composition bias and preferentially code for amino acids with similar physicochemical properties because of the nonrandomness of the genetic code. Indeed, amino acids sharing similar physicochemical properties correspond to codons that have the same nucleotide composition bias. In particular, we uncover that the TRA2A and TRA2B splicing factors that bind to adenine-rich motifs promote the inclusion of adenine-rich exons coding preferentially for hydrophilic amino acids that correspond to adenine-rich codons. SRSF2 that binds guanine/cytosine-rich motifs promotes the inclusion of GC-rich exons coding preferentially for small amino acids, whereas SRSF3 that binds cytosine-rich motifs promotes the inclusion of exons coding preferentially for uncharged amino acids, like serine and threonine that can be phosphorylated. Finally, coregulated exons encoding amino acids with similar physicochemical properties correspond to specific protein features. In conclusion, the regulation of an exon by a splicing factor that relies on the affinity of this factor for specific nucleotide(s) is tightly interconnected with the exon-encoded physicochemical properties. We therefore uncover an unanticipated bidirectional interplay between the splicing regulatory process and its biological functional outcome.
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Empalme Alternativo , Exones/genética , Sitios de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Aminoácidos/química , Composición de Base/genética , Línea Celular , Código Genético , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Intrones/genética , Motivos de Nucleótidos/genética , Análisis de Secuencia de Proteína , Análisis de Secuencia de ARN , Factores de Empalme Serina-Arginina/metabolismoRESUMEN
Caspase-4, the cytosolic LPS sensor, and gasdermin D, its downstream effector, constitute the non-canonical inflammasome, which drives inflammatory responses during Gram-negative bacterial infections. It remains unclear whether other proteins regulate cytosolic LPS sensing, particularly in human cells. Here, we conduct a genome-wide CRISPR/Cas9 screen in a human monocyte cell line to identify genes controlling cytosolic LPS-mediated pyroptosis. We find that the transcription factor, IRF2, is required for pyroptosis following cytosolic LPS delivery and functions by directly regulating caspase-4 levels in human monocytes and iPSC-derived monocytes. CASP4, GSDMD, and IRF2 are the only genes identified with high significance in this screen highlighting the simplicity of the non-canonical inflammasome. Upon IFN-γ priming, IRF1 induction compensates IRF2 deficiency, leading to robust caspase-4 expression. Deficiency in IRF2 results in dampened inflammasome responses upon infection with Gram-negative bacteria. This study emphasizes the central role of IRF family members as specific regulators of the non-canonical inflammasome.
Asunto(s)
Caspasas Iniciadoras/metabolismo , Factor 2 Regulador del Interferón/metabolismo , Caspasas Iniciadoras/genética , Muerte Celular/efectos de los fármacos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Humanos , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Factor 2 Regulador del Interferón/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Células U937RESUMEN
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
The Repressor Element 1-silencing transcription factor (REST) represses a number of neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulators of DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.
Asunto(s)
ARN Helicasas DEAD-box/genética , Perfilación de la Expresión Génica , MicroARNs/genética , Proteínas Represoras/genética , Línea Celular Tumoral , ARN Helicasas DEAD-box/metabolismo , Humanos , Células MCF-7 , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Proteínas Represoras/metabolismoRESUMEN
NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.
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Empalme Alternativo , Apoptosis , Neoplasias/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular Tumoral , Núcleo Celular/enzimología , Supervivencia Celular , Humanos , Quinasas Relacionadas con NIMA , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/análisis , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina , Proteína bcl-X/genéticaRESUMEN
Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2ß (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2ß is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2ß binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2ß regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2ß binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2ß protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2ß. Versions of Tra2ß lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2ß protein.
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Desarrollo Embrionario/genética , Exones/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo/genética , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Secuencia de Bases , Sitios de Unión , Encéfalo/anomalías , Proteínas de Ciclo Celular , Diferenciación Celular , Roturas del ADN de Doble Cadena , Evolución Molecular , Células Germinativas/citología , Masculino , Meiosis/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Factores de Empalme Serina-Arginina , Espermatogénesis/genéticaRESUMEN
The cellular mechanisms underlying axonal morphogenesis are essential to the formation of functional neuronal networks. We previously identified the autism-linked kinase NUAK1 as a central regulator of axon branching through the control of mitochondria trafficking. However, (1) the relationship between mitochondrial position, function and axon branching and (2) the downstream effectors whereby NUAK1 regulates axon branching remain unknown. Here, we report that mitochondria recruitment to synaptic boutons supports collateral branches stabilization rather than formation in mouse cortical neurons. NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration, and upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Finally, we found that NUAK1 regulates axon branching through the mitochondria-targeted microprotein BRAWNIN. Our results demonstrate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondrial distribution and metabolic activity.
Asunto(s)
Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP , Animales , Ratones , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Axones/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismoRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterized by many features of premature aging. Most cases of HGPS are due to a heterozygous silent mutation (c.1824C>T; p.Gly608Gly) that enhances the use of an internal 5' splice site (5'SS) in exon 11 of the LMNA pre-mRNA and leads to the production of a truncated protein (progerin) with a dominant negative effect. Here we show that HGPS mutation changes the accessibility of the 5'SS of LMNA exon 11 which is sequestered in a conserved RNA structure. Our results also reveal a regulatory role of a subset of serine-arginine (SR)-rich proteins, including serine-arginine rich splicing factor 1 (SRSF1) and SRSF6, on utilization of the 5'SS leading to lamin A or progerin production and a modulation of this regulation in the presence of the c.1824C>T mutation is shown directly on HGPS patient cells. Mutant mice carrying the equivalent mutation in the LMNA gene (c.1827C>T) also accumulate progerin and phenocopy the main cellular alterations and clinical defects of HGPS patients. RNAi-induced depletion of SRSF1 in the HGPS-like mouse embryonic fibroblasts (MEFs) allowed progerin reduction and dysmorphic nuclei phenotype correction, whereas SRSF6 depletion aggravated the HGPS-like MEF's phenotype. We demonstrate that changes in the splicing ratio between lamin A and progerin are key factors for lifespan since heterozygous mice harboring the mutation lived longer than homozygous littermates but less than the wild-type. Genetic and biochemical data together favor the view that physiological progerin production is under tight control of a conserved splicing mechanism to avoid precocious aging.
Asunto(s)
Envejecimiento Prematuro/genética , Evolución Molecular , Lamina Tipo A/genética , Empalme del ARN/genética , Animales , Secuencia de Bases , Células Cultivadas , Secuencia Conservada/genética , Exones/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Progeria/genética , Progeria/patología , Isoformas de Proteínas/genética , Precursores de Proteínas/genética , ARN/química , ARN/genética , Sitios de Empalme de ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Factores de Empalme Serina-Arginina , TransfecciónRESUMEN
Tra2ß regulates a number of splicing switches including activation of the human testis-specific exon HIPK3-T in the Homeodomain Interacting Protein Kinase 3 gene. By testing HIPK3-T exons of different intrinsic strengths, we found Tra2ß most efficiently activated splicing inclusion of intrinsically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2ß were required for splicing activation. Bioinformatic searches for splicing enhancers and repressors mapped four physically distinct exonic splicing enhancers (ESEs) within HIPK3-T, each containing the known Tra2ß AGAA-rich binding site. Surprisingly disruption of each single ESE prevented Tra2ß-mediated activation, although single mutated exons could still bind Tra2ß protein by gel shifts and functional splicing analyses. Titration experiments indicate an additive model of HIPK3-T splicing activation, requiring availability of an array of four distinct ESEs to enable splicing activation. To enable this efficient Tra2ß-mediated splicing switch to operate, a closely adjacent downstream and potentially competitive stronger 5'-splice site is actively repressed. Our data indicate that a novel arrangement of multiple mono-specific AGAA-rich ESEs coupled to a weak 5'-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2ß, and co-ordinately regulates HIPK3-T exon splicing inclusion.
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Empalme Alternativo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Exones , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Sitios de Empalme de ARN , Proteínas de Unión al ARN/química , Factores de Empalme Serina-ArgininaRESUMEN
One challenge faced by scientists from the alternative RNA splicing field is to decode the cooperative or antagonistic effects of splicing factors (SFs) to understand and eventually predict splicing outcomes on a genome-wide scale. In this manuscript, we introduce SplicingLore, an open-access database and web resource that help to fill this gap in a straightforward manner. The database contains a collection of RNA-sequencing-derived lists of alternative exons regulated by a total of 75 different SFs. All datasets were processed in a standardized manner, ensuring valid comparisons and correlation analyses. The user can easily retrieve a factor-specific set of differentially included exons from the database or provide a list of exons and search which SF(s) control(s) their inclusion. Our simple workflow is fast and easy to run, and it ensures a reliable calculation of correlation scores between the tested datasets. As a proof of concept, we predicted and experimentally validated a novel functional cooperation between the RNA helicases DDX17 and DDX5 and the heterogeneous nuclear ribonucleoprotein C (HNRNPC) protein. SplicingLore is available at https://splicinglore.ens-lyon.fr/. Database URL: https://splicinglore.ens-lyon.fr/.
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Empalme Alternativo , Empalme del ARN , Humanos , Factores de Empalme de ARN/genética , Empalme del ARN/genética , Genoma , Exones/genéticaRESUMEN
RBMY is a male germline RNA binding protein and potential alternative splicing regulator, but the lack of a convenient biological system has made its cellular functions elusive. We found that human RBMY fused to green fluorescent protein was strictly nuclear in transfected cells, but spatially enriched in areas around nuclear speckles with some components of the exon junction complex (EJC). Human RBMY (hRBMY) and the EJC components Magoh and Y14 also physically interacted but, unlike these two proteins, hRBMY protein did not shuttle to the cytoplasm. In addition, it relocalised into nucleolar caps after inhibition of RNA polymerase II transcription. Protein interactions were also detected between RBMY and splicing factors 9G8 and transformer-2 protein homolog beta (Tra2-beta), mediated by multiple regions of the RBMY protein that contain serine/arginine-rich dipeptides, but not by the single region lacking such dipeptides. These interactions modulated the splicing of several pre-mRNAs regulated by 9G8 and Tra2-beta. Importantly, ectopic expression of hRBMY stimulated the inclusion of a testis-enriched exon from the Acinus gene, whereas 9G8 and Tra2-beta repressed this exon. We propose that hRBMY associates with regions of the nucleus enriched in nascent RNA and participates in the regulation of specific splicing events in the germline by modulating the activity of constitutively expressed splicing factors.
Asunto(s)
Empalme Alternativo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Unión al ARN/metabolismo , Testículo/metabolismo , Animales , Arginina , Células HeLa , Humanos , Masculino , Ratones , Células 3T3 NIH , Proteínas del Tejido Nervioso/química , Proteínas de Transporte Nucleocitoplasmático/química , Unión Proteica , Ingeniería de Proteínas , Transporte de Proteínas , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/química , Serina , Factores de Empalme Serina-Arginina , Testículo/citología , Activación TranscripcionalRESUMEN
Alternative splicing is regulated in part by variations in the relative concentrations of a variety of factors, including serine/arginine-rich (SR) proteins. The SR protein SC35 self-regulates its expression by stimulating unproductive splicing events in the 3' untranslated region of its own pre-mRNA. Using various minigene constructs containing the terminal retained intron and flanking exons, we identified in the highly conserved last exon a number of exonic splicing enhancer elements responding specifically to SC35, and showed an inverse correlation between affinity of SC35 and enhancer strength. The enhancer region, which is included in a long stem loop, also contains repressor elements, and is recognized by other RNA-binding proteins, notably hnRNP H protein and TAR DNA binding protein (TDP-43). Finally, in vitro and in cellulo experiments indicated that hnRNP H and TDP-43 antagonize the binding of SC35 to the terminal exon and specifically repress the use of SC35 terminal 3' splice site. Our study provides new information about the molecular mechanisms of SC35-mediated splicing activation. It also highlights the existence of a complex network of self- and cross-regulatory mechanisms between splicing regulators, which controls their homeostasis and offers many ways of modulating their concentration in response to the cellular environment.
Asunto(s)
Empalme Alternativo , Intrones , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Proteínas de Unión al ADN/metabolismo , Exones , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Sitios de Empalme de ARN , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Factores de Empalme Serina-ArgininaRESUMEN
The human testis has almost as high a frequency of alternative splicing events as brain. While not as extensively studied as brain, a few candidate testis-specific splicing regulator proteins have been identified, including the nuclear RNA binding proteins RBMY and hnRNP G-T, which are germ cell-specific versions of the somatically expressed hnRNP G protein and are highly conserved in mammals. The splicing activator protein Tra2beta is also highly expressed in the testis and physically interacts with these hnRNP G family proteins. In this study, we identified a novel testis-specific cassette exon TLE4-T within intron 6 of the human transducing-like enhancer of split 4 (TLE4) gene which makes a more transcriptionally repressive TLE4 protein isoform. TLE4-T splicing is normally repressed in somatic cells because of a weak 5' splice site and surrounding splicing-repressive intronic regions. TLE4-T RNA pulls down Tra2beta and hnRNP G proteins which activate its inclusion. The germ cell-specific RBMY and hnRNP G-T proteins were more efficient in stimulating TLE4-T incorporation than somatically expressed hnRNP G protein. Tra2b bound moderately to TLE4-T RNA, but more strongly to upstream sites to potently activate an alternative 3' splice site normally weakly selected in the testis. Co-expression of Tra2beta with either hnRNP G-T or RBMY re-established the normal testis physiological splicing pattern of this exon. Although they can directly bind pre-mRNA sequences around the TLE4-T exon, RBMY and hnRNP G-T function as efficient germ cell-specific splicing co-activators of TLE4-T. Our study indicates a delicate balance between the activity of positive and negative splicing regulators combinatorially controls physiological splicing inclusion of exon TLE4-T and leads to modulation of signalling pathways in the testis. In addition, we identified a high-affinity binding site for hnRNP G-T protein, showing it is also a sequence-specific RNA binding protein.
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Empalme Alternativo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas Nucleares/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Exones , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Here, using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types (neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading, and migration. RNA-dependent toxicity and phenotypes are also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses reveal extensive expression and accumulation of toxic RNA in astrocytes, which result in RNA spliceopathy that is more severe than in neurons. Astrocyte missplicing affects primarily transcripts that regulate cell adhesion, cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Our findings demonstrate that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function.
Asunto(s)
Distrofia Miotónica , Animales , Astrocitos/metabolismo , Humanos , Ratones , Ratones Transgénicos , Distrofia Miotónica/metabolismo , ARN/genética , Proteínas de Unión al ARN/metabolismo , Adherencias TisularesRESUMEN
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a non-coding CTG repeat expansion in the DMPK gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system (CNS) has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease.