RESUMO
Alternative splicing of Fas/CD95 exon 6 generates either a membrane-bound receptor that promotes, or a soluble isoform that inhibits, apoptosis. Using an automatized genome-wide siRNA screening for alternative splicing regulators of endogenous transcripts in mammalian cells, we identified 200 genes whose knockdown modulates the ratio between Fas/CD95 isoforms. These include classical splicing regulators; core spliceosome components; and factors implicated in transcription and chromatin remodeling, RNA transport, intracellular signaling, and metabolic control. Coherent effects of genes involved in iron homeostasis and pharmacological modulation of iron levels revealed a link between intracellular iron and Fas/CD95 exon 6 inclusion. A splicing regulatory network linked iron levels with reduced activity of the Zinc-finger-containing splicing regulator SRSF7, and in vivo and in vitro assays revealed that iron inhibits SRSF7 RNA binding. Our results uncover numerous links between cellular pathways and RNA processing and a mechanism by which iron homeostasis can influence alternative splicing.
Assuntos
Processamento Alternativo , Redes Reguladoras de Genes , Genoma , Ferro/metabolismo , Spliceossomos/metabolismo , Receptor fas/genética , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Desferroxamina/farmacologia , Éxons , Estudo de Associação Genômica Ampla , Células HeLa , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Íntrons , Ferro/farmacologia , Quelantes de Ferro/farmacologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Anotação de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina , Spliceossomos/química , Spliceossomos/efeitos dos fármacos , Receptor fas/antagonistas & inibidores , Receptor fas/metabolismoRESUMO
Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation.
Assuntos
Processamento Alternativo , Redes Reguladoras de Genes , Ribonucleoproteínas/metabolismo , Spliceossomos/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação , Proliferação de Células , Éxons , Células HeLa , Humanos , Íntrons , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Componente Principal , Ligação Proteica , Mapeamento de Interação de Proteínas , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/genética , Spliceossomos/química , Fator de Processamento U2AFRESUMO
Interindividual variability is an inherent characteristic of biological systems. Whereas the underlying molecular sources of interindividual variability remain poorly understood, recent work by Ecker et al. (Ecker S, Chen L, Pancaldi V, Bagger FO, et al. 2017. Genome Biol 18: 18.) sheds light on the characterization of this phenomenon in a complex biological scenario. By combining data from the BLUEPRINT Epigenome Project with a novel analytical approach, these authors were able to measure the degree of transcriptional and epigenetic variability across a wide panel of samples and types of immune cell. Interestingly, neutrophils displayed increased variability compared to monocytes and T cells, which may be related to the crucial role of the former as an initial mediator of immune responses. Here we review recent literature in this area, and discuss some important issues raised by these innovative analyses. Furthermore, we summarize other potential sources of epigenetic variability, such as epigenetic drift and the epigenetic clock, as well as the current ongoing direction of the field.
Assuntos
Epigênese Genética/genética , Animais , Epigenômica/métodos , Humanos , Monócitos/fisiologia , Neutrófilos/fisiologia , Linfócitos T/fisiologia , Transcrição Gênica/genéticaRESUMO
The OLR1 gene encodes the oxidized low-density lipoprotein receptor (LOX-1), which is responsible for the cellular uptake of oxidized LDL (Ox-LDL), foam cell formation in atheroma plaques and atherosclerotic plaque rupture. Alternative splicing (AS) of OLR1 exon 5 generates two protein isoforms with antagonistic functions in Ox-LDL uptake. Previous work identified six single nucleotide polymorphisms (SNPs) in linkage disequilibrium that influence the inclusion levels of OLR1 exon 5 and correlate with the risk of cardiovascular disease. Here we use minigenes to recapitulate the effects of two allelic series (Low- and High-Risk) on OLR1 AS and identify one SNP in intron 4 (rs3736234) as the main contributor to the differences in exon 5 inclusion, while the other SNPs in the allelic series attenuate the drastic effects of this key SNP. Bioinformatic, proteomic, mutational and functional high-throughput analyses allowed us to define regulatory sequence motifs and identify SR protein family members (SRSF1, SRSF2) and HMGA1 as factors involved in the regulation of OLR1 AS. Our results suggest that antagonism between SRSF1 and SRSF2/HMGA1, and differential recognition of their regulatory motifs depending on the identity of the rs3736234 polymorphism, influence OLR1 exon 5 inclusion and the efficiency of Ox-LDL uptake, with potential implications for atherosclerosis and coronary disease.
Assuntos
Processamento Alternativo , Proteína HMGA1a/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Receptores Depuradores Classe E/genética , Biologia Computacional/métodos , Doença das Coronárias/genética , Doença das Coronárias/metabolismo , Predisposição Genética para Doença , Proteína HMGA1a/genética , Humanos , Íntrons , Desequilíbrio de Ligação , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Ribonucleico , Ribonucleoproteínas/genética , Receptores Depuradores Classe E/metabolismo , Fatores de Processamento de Serina-ArgininaAssuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Doenças em Gêmeos , Células-Tronco Hematopoéticas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transativadores/genética , Gêmeos Monozigóticos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem da Célula/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 19/genética , Progressão da Doença , Doenças em Gêmeos/genética , Doenças em Gêmeos/patologia , Células-Tronco Hematopoéticas/patologia , Humanos , Lactente , Técnicas de Diagnóstico Molecular , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transativadores/metabolismo , Translocação GenéticaRESUMO
AIM: Epigenetic regulation plays an important role in cellular development and differentiation. A detailed map of the DNA methylation dynamics that occur during cell differentiation would contribute to decipher the molecular networks governing cell fate commitment. METHODS: Illumina MethylationEPIC BeadChip platform was used to describe the genome-wide DNA methylation changes observed throughout hematopoietic maturation by analyzing multiple myeloid and lymphoid hematopoietic cell types. RESULTS: We identified a plethora of DNA methylation changes that occur during human hematopoietic differentiation. We observed that T lymphocytes display substantial enhancement of de novo CpG hypermethylation as compared with other hematopoietic cell populations. T-cell-specific hypermethylated regions were strongly associated with open chromatin marks and enhancer elements, as well as binding sites of specific key transcription factors involved in hematopoietic differentiation, such as PU.1 and TAL1. CONCLUSION: These results provide novel insights into the role of DNA methylation at enhancer elements in T-cell development.