RESUMEN
Autism spectrum disorder (ASD) affects 1 in 44 children. Chromatin regulatory proteins are overrepresented among genes that contain high risk variants in ASD. Disruption of the chromatin environment leads to widespread dysregulation of gene expression, which is traditionally thought of as a mechanism of disease pathogenesis associated with ASD. Alternatively, alterations in chromatin dynamics could also lead to dysregulation of alternative splicing, which is understudied as a mechanism of ASD pathogenesis. The anticonvulsant valproic acid (VPA) is a well-known environmental risk factor for ASD that acts as a class I histone deacetylase inhibitor. However, the precise molecular mechanisms underlying defects in human neuronal development associated with exposure to VPA are understudied. To dissect how VPA exposure and subsequent chromatin hyperacetylation influence molecular signatures involved in ASD pathogenesis, we conducted RNA sequencing (RNA-seq) in human cortical neurons that were treated with VPA. We observed that differentially expressed genes (DEGs) were enriched for mRNA splicing, mRNA processing, histone modification and metabolism related gene sets. Furthermore, we observed widespread increases in the number and the type of alternative splicing events. Analysis of differential transcript usage (DTU) showed that exposure to VPA induces extensive alterations in transcript isoform usage across neurodevelopmentally important genes. Finally, we find that DEGs and genes that display DTU overlap with known ASD-risk genes. Altogether, these findings suggest that, in addition to differential gene expression, changes in alternative splicing correlated with alterations in the chromatin environment could act as an additional mechanism of disease in ASD.
Asunto(s)
Trastorno del Espectro Autista , Efectos Tardíos de la Exposición Prenatal , Niño , Humanos , Animales , Femenino , Trastorno del Espectro Autista/etiología , Cromatina/genética , Empalme Alternativo/genética , Ácido Valproico/efectos adversos , ARN Mensajero/metabolismo , Modelos Animales de EnfermedadRESUMEN
Chronic environmental stress can profoundly impact cell and body function. Although the underlying mechanisms are poorly understood, epigenetics has emerged as a key link between environment and health. The genomic effects of stress are thought to be mediated by the action of glucocorticoid stress hormones, primarily cortisol in humans, which act via the glucocorticoid receptor (GR). To dissect how chronic stress-driven GR activation influences epigenetic and cell states, human fibroblasts underwent prolonged exposure to physiological stress levels of cortisol and/or a selective GR antagonist. Cortisol was found to drive robust changes in cell proliferation, migration, and morphology, which were abrogated by concomitant GR blockade. The GR-driven cell phenotypes were accompanied by widespread, yet genomic context-dependent, changes in DNA methylation and mRNA expression, including gene loci with known roles in cell proliferation and migration. These findings provide insights into how chronic stress-driven functional epigenomic patterns become established to shape key cell phenotypes.
RESUMEN
A recent crystal structure of a ribosome complex undergoing partial translocation in the absence of elongation factor EF-G showed disruption of codon-anticodon pairing and slippage of the reading frame by -1, directly implicating EF-G in preservation of the translational reading frame. Among mutations identified in a random screen for dominant-lethal mutations of EF-G were a cluster of six that map to the tip of domain IV, which has been shown to contact the codon-anticodon duplex in trapped translocation intermediates. In vitro synthesis of a full-length protein using these mutant EF-Gs revealed dramatically increased -1 frameshifting, providing new evidence for a role for domain IV of EF-G in maintaining the reading frame. These mutations also caused decreased rates of mRNA translocation and rotational movement of the head and body domains of the 30S ribosomal subunit during translocation. Our results are in general agreement with recent findings from Rodnina and coworkers based on in vitro translation of an oligopeptide using EF-Gs containing mutations at two positions in domain IV, who found an inverse correlation between the degree of frameshifting and rates of translocation. Four of our six mutations are substitutions at positions that interact with the translocating tRNA, in each case contacting the RNA backbone of the anticodon loop. We suggest that EF-G helps to preserve the translational reading frame by preventing uncoupled movement of the tRNA through these contacts; a further possibility is that these interactions may stabilize a conformation of the anticodon that favors base-pairing with its codon.
Asunto(s)
Escherichia coli/genética , Sistema de Lectura Ribosómico , Mutación , Extensión de la Cadena Peptídica de Translación , Factor G de Elongación Peptídica/genética , Ribosomas/genética , Anticodón/química , Anticodón/metabolismo , Sitios de Unión , Codón/química , Codón/metabolismo , Escherichia coli/metabolismo , Histidina/genética , Histidina/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Factor G de Elongación Peptídica/química , Factor G de Elongación Peptídica/metabolismo , Unión Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , ARN Mensajero , ARN de Transferencia , Sistemas de Lectura , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/metabolismoRESUMEN
Psychosocial stress, especially when chronic or excessive, can increase disease risk and accelerate biological aging. Although the underlying mechanisms are unclear, in vivo studies have associated exposure to stress and glucocorticoid stress hormones with shorter telomere length. However, the extent to which prolonged glucocorticoid exposure can shorten telomeres in controlled experimental settings remains unknown. Using a well-characterized cell line of human fibroblasts that undergo gradual telomere shortening during serial passaging in culture, we show that prolonged exposure (up to 51 days) to either naturalistic levels of the human endogenous glucocorticoid cortisol or the more potent synthetic glucocorticoid dexamethasone is not sufficient to accelerate telomere shortening. While our findings await extension in other cell types and biological contexts, they indicate that the in vivo association of psychosocial stress with telomere shortening is unlikely to be mediated by a direct and universal glucocorticoid effect on telomere length.
Asunto(s)
Dexametasona/farmacología , Fibroblastos/efectos de los fármacos , Hidrocortisona/farmacología , Acortamiento del Telómero/efectos de los fármacos , Línea Celular , Senescencia Celular/efectos de los fármacos , Fibroblastos/ultraestructura , Humanos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Estrés Psicológico , Proteínas de Unión a Tacrolimus/biosíntesis , Proteínas de Unión a Tacrolimus/genética , Telómero/efectos de los fármacos , Telómero/ultraestructura , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In the eukaryotic cell, spliceosomes assemble onto pre-mRNA cotranscriptionally. Spliceosome assembly takes place in the context of the chromatin environment, suggesting that the state of the chromatin may affect splicing. The molecular details and mechanisms through which chromatin affects splicing, however, are still unclear. Here, we show a role for the histone methyltransferase Set2 and its histone modification, H3K36 methylation, in pre-mRNA splicing through high-throughput sequencing. Moreover, the effect of H3K36 methylation on pre-mRNA splicing is mediated through the chromodomain protein Eaf3. We find that Eaf3 is recruited to intron-containing genes and that Eaf3 interacts with the splicing factor Prp45. Eaf3 acts with Prp45 and Prp19 after formation of the precatalytic B complex around the time of splicing activation, thus revealing the step in splicing that is regulated by H3K36 methylation. These studies support a model whereby H3K36 facilitates recruitment of an "adapter protein" to support efficient, constitutive splicing.
Asunto(s)
Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Transcripción Genética , Acetiltransferasas/genética , Histonas/genética , Metilación , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Empalmosomas/genéticaRESUMEN
In a recent issue of Molecular Cell, Boehm et al. (2018), Blazquez et al. (2018), and Gonatopoulos-Pournatzis et al. (2018) uncover novel mechanisms by which the cell regulates splicing of cryptic splice sites and microexons.
Asunto(s)
Sitios de Empalme de ARN , Transcriptoma , Núcleo Celular , Exones , Empalme del ARNRESUMEN
GPIHBP1, a glycosylphosphatidylinositol-anchored glycoprotein of microvascular endothelial cells, binds lipoprotein lipase (LPL) within the interstitial spaces and transports it across endothelial cells to the capillary lumen. The ability of GPIHBP1 to bind LPL depends on the Ly6 domain, a three-fingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Here, we report a patient with severe hypertriglyceridemia who was homozygous for a GPIHBP1 point mutation that converted a serine in the GPIHBP1 Ly6 domain (Ser-107) to a cysteine. Two hypertriglyceridemic siblings were homozygous for the same mutation. All three homozygotes had very low levels of LPL in the preheparin plasma. We suspected that the extra cysteine in GPIHBP1-S107C might prevent the trafficking of the protein to the cell surface, but this was not the case. However, nearly all of the GPIHBP1-S107C on the cell surface was in the form of disulfide-linked dimers and multimers, whereas wild-type GPIHBP1 was predominantly monomeric. An insect cell GPIHBP1 expression system confirmed the propensity of GPIHBP1-S107C to form disulfide-linked dimers and to form multimers. Functional studies showed that only GPIHBP1 monomers bind LPL. In keeping with that finding, there was no binding of LPL to GPIHBP1-S107C in either cell-based or cell-free binding assays. We conclude that an extra cysteine in the GPIHBP1 Ly6 motif results in multimerization of GPIHBP1, defective LPL binding, and severe hypertriglyceridemia.
Asunto(s)
Homocigoto , Hiperlipoproteinemia Tipo I , Lipoproteína Lipasa/metabolismo , Mutación Missense , Multimerización de Proteína/genética , Receptores de Lipoproteína , Adulto , Sustitución de Aminoácidos , Línea Celular , Humanos , Hiperlipoproteinemia Tipo I/genética , Hiperlipoproteinemia Tipo I/metabolismo , Hiperlipoproteinemia Tipo I/patología , Lipoproteína Lipasa/genética , Masculino , Unión Proteica/genética , Estructura Terciaria de Proteína , Transporte de Proteínas/genética , Receptores de Lipoproteína/genética , Receptores de Lipoproteína/metabolismoRESUMEN
Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5-25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL-GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Lipoproteína Lipasa/metabolismo , Receptores de Lipoproteína/metabolismo , Triglicéridos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Células CHO , Bovinos , Cricetulus , Expresión Génica , Heparina/metabolismo , Humanos , Lipasa/metabolismo , Lipoproteína Lipasa/genética , Ratones , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptores de Lipoproteína/genética , TransfecciónRESUMEN
The S447X polymorphism in lipoprotein lipase (LPL), which shortens LPL by two amino acids, is associated with low plasma triglyceride levels and reduced risk for coronary heart disease. S447X carriers have higher LPL levels in the pre- and post-heparin plasma, raising the possibility that the S447X polymorphism leads to higher LPL levels within capillaries. One potential explanation for increased amounts of LPL in capillaries would be more avid binding of S447X-LPL to GPIHBP1 (the protein that binds LPL dimers and shuttles them to the capillary lumen). This explanation seems plausible because sequences within the carboxyl terminus of LPL are known to mediate LPL binding to GPIHBP1. To assess the impact of the S447X polymorphism on LPL binding to GPIHBP1, we compared the ability of internally tagged versions of wild-type LPL (WT-LPL) and S447X-LPL to bind to GPIHBP1 in both cell-based and cell-free binding assays. In the cell-based assay, we compared the binding of WT-LPL and S447X-LPL to GPIHBP1 on the surface of cultured cells. This assay revealed no differences in the binding of WT-LPL and S447X-LPL to GPIHBP1. In the cell-free assay, we compared the binding of internally tagged WT-LPL and S447X-LPL to soluble GPIHBP1 immobilized on agarose beads. Again, no differences in the binding of WT-LPL and S447X-LPL to GPIHBP1 were observed. We conclude that increased binding of S447X-LPL to GPIHBP1 is unlikely to be the explanation for more efficient lipolysis and lower plasma triglyceride levels in S447X carriers.
Asunto(s)
Proteínas Inmovilizadas/metabolismo , Lipoproteína Lipasa/metabolismo , Polimorfismo de Nucleótido Simple , Receptores de Lipoproteína/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Triglicéridos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bioensayo , Células CHO , Cricetulus , Expresión Génica , Humanos , Proteínas Inmovilizadas/genética , Metabolismo de los Lípidos , Lipoproteína Lipasa/genética , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Receptores de Lipoproteína/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and -3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the VDAC2 and -3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish VDAC2 (zfVDAC2). Further, we resolved the structure of zfVDAC2 at 2.8 Å resolution, revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of â¼20% in lauryldimethine amine oxide detergent micelles, whereas in lipidic bicelles a higher population of dimeric and higher order oligomers species were observed. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1.