RESUMO
BACKGROUND: Abnormalities in ureteric bud (UB) branching morphogenesis lead to congenital anomalies of the kidney and reduced nephron numbers associated with chronic kidney disease (CKD) and hypertension. Previous studies showed that the epithelial fibroblast growth factor receptor 2 (Fgfr2) IIIb splice variant supports ureteric morphogenesis in response to ligands from the metanephric mesenchyme during renal organogenesis. The epithelial-specific splicing regulator Esrp1 is required for expression of Fgfr2-IIIb and other epithelial-specific splice variants. Our objective was to determine whether Esrp1 is required for normal kidney development. RESULTS: Ablation of Esrp1 in mice, alone or together with its paralog Esrp2, was associated with reduced kidney size and increased incidence of renal aplasia. Three-dimensional imaging showed that embryonic Esrp1 knockout (KO) kidneys had fewer ureteric tips and reduced nephron numbers. Analysis of alternative splicing in Esrp-null ureteric epithelial cells by RNA-Seq confirmed a splicing switch in Fgfr2 as well as numerous other transcripts. CONCLUSIONS: Our findings reveal that Esrp1-regulated splicing in ureteric epithelial cells plays an important role in renal development. Defects in Esrp1 KO kidneys likely reflect reduced and/or absent ureteric branching, leading to decreased nephron induction secondary to incorrect Fgfr2 splicing and other splicing alterations. Developmental Dynamics 245:991-1000, 2016. © 2016 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
Assuntos
Proteínas de Ligação a RNA/metabolismo , Ureter/citologia , Ureter/metabolismo , Animais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Rim/citologia , Rim/embriologia , Masculino , Camundongos , Camundongos Knockout , Néfrons/citologia , Néfrons/metabolismo , Splicing de RNA/genética , Splicing de RNA/fisiologia , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Alternative splicing of the oncogene MDM2 is a phenomenon that occurs in cells in response to genotoxic stress and is also a hallmark of several cancer types with important implications in carcinogenesis. However, the mechanisms regulating this splicing event remain unclear. Previously, we uncovered the importance of intron 11 in MDM2 that affects the splicing of a damage-responsive MDM2 minigene. Here, we have identified discrete cis regulatory elements within intron 11 and report the binding of FUBP1 (Far Upstream element-Binding Protein 1) to these elements and the role it plays in MDM2 splicing. Best known for its oncogenic role as a transcription factor in the context of c-MYC, FUBP1 was recently described as a splicing regulator with splicing repressive functions. In the case of MDM2, we describe FUBP1 as a positive splicing regulatory factor. We observed that blocking the function of FUBP1 in in vitro splicing reactions caused a decrease in splicing efficiency of the introns of the MDM2 minigene. Moreover, knockdown of FUBP1 in cells induced the formation of MDM2-ALT1, a stress-induced splice variant of MDM2, even under normal conditions. These results indicate that FUBP1 is also a strong positive splicing regulator that facilitates efficient splicing of the MDM2 pre-mRNA by binding its introns. These findings are the first report describing the regulation of alternative splicing of MDM2 mediated by the oncogenic factor FUBP1.
Assuntos
Processamento Alternativo/fisiologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Íntrons/fisiologia , Proteínas Nucleares/biossíntese , Precursores de RNA/metabolismo , Fatores de Transcrição/biossíntese , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Precursores de RNA/genética , Proteínas de Ligação a RNA , Transativadores , Fatores de Transcrição/genéticaRESUMO
Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease associated with low levels of the essential survival motor neuron (SMN) protein. Reduced levels of SMN is due to the loss of the SMN1 gene and inefficient splicing of the SMN2 gene caused by a C>T mutation in exon 7. Global analysis of the severe SMNΔ7 SMA mouse model revealed altered splicing and increased levels of the hypoxia-inducible transcript, Hif3alpha, at late stages of disease progression. Severe SMA patients also develop respiratory deficiency during disease progression. We sought to evaluate whether hypoxia was capable of altering SMN2 exon 7 splicing and whether increased oxygenation could modulate disease in a severe SMA mouse model. Hypoxia treatment in cell culture increased SMN2 exon 7 skipping and reduced SMN protein levels. Concordantly, the treatment of SMNΔ7 mice with hyperoxia treatment increased the inclusion of SMN2 exon 7 in skeletal muscles and resulted in improved motor function. Transfection splicing assays of SMN minigenes under hypoxia revealed that hypoxia-induced skipping is dependent on poor exon definition due to the SMN2 C>T mutation and suboptimal 5' splice site. Hypoxia treatment in cell culture led to increased hnRNP A1 and Sam68 levels. Mutation of hnRNP A1-binding sites prevented hypoxia-induced skipping of SMN exon 7 and was found to bind both hnRNP A1 and Sam68. These results implicate hypoxic stress as a modulator of SMN2 exon 7 splicing in disease progression and a coordinated regulation by hnRNP A1 and Sam68 as modifiers of hypoxia-induced skipping of SMN exon 7.
Assuntos
Processamento Alternativo , Hipóxia/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Éxons , Humanos , Hipóxia/metabolismo , Camundongos , Camundongos Knockout , Atividade Motora , Músculo Esquelético/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/fisiopatologia , Oxigênio/metabolismo , Mutação Puntual , Índice de Gravidade de Doença , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
The epithelial to mesenchymal transition (EMT) and reverse mesenchymal to epithelial transition (MET) are developmentally conserved processes that are essential for patterning of developing embryos and organs. The EMT/MET are further utilized in wound healing, but they can also be hijacked by cancer cells to promote tumor progression and metastasis. The molecular pathways governing these processes have historically focused on the transcriptional regulation and networks that control them. Indeed, global profiling of transcriptional changes has provided a wealth of information into how these networks are regulated, the downstream targets, and functional consequence of alterations to the global transcriptome. However, recent evidence has revealed that the posttranscriptional landscape of the cell is also dramatically altered during the EMT/MET and contributes to changes in cell behavior and phenotypes. While studies of this aspect of EMT biology are still in their infancy, recent progress has been achieved by the identification of several RNA binding proteins (RBPs) that regulate splicing, polyadenylation, mRNA stability, and translational control during EMT. This chapter focuses on the global impact of RBPs that regulate mRNA maturation as well as outlines the functional impact of several key posttranscriptional changes during the EMT. The growing evidence of RBP involvement in the cellular transformation during EMT underscores that a coordinated regulation of both transcriptional and posttranscriptional changes is essential for EMT. Furthermore, new discoveries into these events will paint a more detailed picture of the transcriptome during the EMT/MET and provide novel molecular targets for treatment of human diseases.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Genoma Humano/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , Proteínas de Ligação a RNA , Transcriptoma/fisiologia , Animais , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla/métodos , Humanos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Proximal spinal muscular atrophy (SMA) is a neurodegenerative disease caused by low levels of the survival motor neuron (SMN) protein. In humans, SMN1 and SMN2 encode the SMN protein. In SMA patients, the SMN1 gene is lost and the remaining SMN2 gene only partially compensates. Mediated by a C>T nucleotide transition in SMN2, the inefficient recognition of exon 7 by the splicing machinery results in low levels of SMN. Because the SMN2 gene is capable of expressing SMN protein, correction of SMN2 splicing is an attractive therapeutic option. Although current mouse models of SMA characterized by Smn knock-out alleles in combination with SMN2 transgenes adequately model the disease phenotype, their complex genetics and short lifespan have hindered the development and testing of therapies aimed at SMN2 splicing correction. Here we show that the mouse and human minigenes are regulated similarly by conserved elements within in exon 7 and its downstream intron. Importantly, the C>T mutation is sufficient to induce exon 7 skipping in the mouse minigene as in the human SMN2. When the mouse Smn gene was humanized to carry the C>T mutation, keeping it under the control of the endogenous promoter, and in the natural genomic context, the resulting mice exhibit exon 7 skipping and mild adult onset SMA characterized by muscle weakness, decreased activity and an alteration of the muscle fibers size. This Smn C>T mouse represents a new model for an adult onset form of SMA (type III/IV) also know as the Kugelberg-Welander disease.
Assuntos
Éxons , Mimetismo Molecular , Atrofia Muscular Espinal/genética , Splicing de RNA , Animais , Sequência de Bases , Modelos Animais de Doenças , Humanos , Íntrons , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fenótipo , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Mouse models of human disease are an important tool for studying disease mechanism and manifestation in a way that is physiologically relevant. Spinal muscular atrophy (SMA) is a neurodegenerative disease that is caused by deletion or mutation of the survival motor neuron gene (SMN1). The SMA disease is present in a spectrum of disease severities ranging from infant mortality, in the most severe cases, to minor motor impairment, in the mildest cases. The variability of disease severity inversely correlates with the copy number, and thus expression of a second, partially functional survival motor neuron gene, SMN2. Correspondingly, a plethora of mouse models has been developed to mimic these different types of SMA. These models express a range of SMN protein levels and extensively cover the severe and mild types of SMA, with neurological and physiological manifestation of disease supporting the relevance of these models. The SMA models provide a strong background for studying SMA and have already shown to be useful in pre-clinical therapeutic studies. The purpose of this review is to succinctly summarize the genetic and disease characteristic of the SMA mouse models and to highlight their use for therapeutic testing.
Assuntos
Modelos Animais de Doenças , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/terapia , Animais , Camundongos , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Proximal spinal muscular atrophy (SMA) is caused by low levels of the SMN protein, encoded by the Survival Motor Neuron genes (SMN1 and SMN2). Mouse models of SMA can be rescued by increased SMN expression, but the timing of SMN replacement for complete rescue is unknown. Studies in zebrafish predict restoration of SMN function during embryogenesis may be important for axonal pathfinding, while the mouse models and normal human disease progression suggest that post-natal treatment may be sufficient for amelioration of disease. To evaluate the timing for SMN replacement, we have generated a stably integrated Cre-inducible SMN mouse in which expression of full-length SMN2 occurs after tamoxifen administration. Our temporally inducible SMN transgene is able to express SMN in embryonic, neonatal, and weanling mice and as such can be utilized in severe and mild SMA mouse models to identify the therapeutic window for SMN replacement.
Assuntos
Modelos Animais de Doenças , Atrofia Muscular Espinal/fisiopatologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Tamoxifeno/administração & dosagem , Animais , Linhagem Celular , Clonagem Molecular , Cruzamentos Genéticos , Éxons , Feminino , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Transgenes/genéticaRESUMO
Alternative splicing plays a fundamental role in generating proteome diversity and is critical in regulation of eukaryotic gene expression. It is estimated that 50% of disease-causing mutations alter splicing efficiency and/or patterns of splicing. An alternatively spliced form of murine double-minute 2, MDM2-ALT1, is associated with pediatric rhabdomyosarcoma (RMS) at high frequency in primary human tumors and RMS cell lines. We have identified that this isoform can be induced in response to specific types of stress (UV and cisplatin). However, the mechanism of alternative splicing of MDM2 in human cancer is unknown. Using UV and cisplatin to model alternative splicing of the MDM2 gene, we have developed a damage-inducible in vitro splicing system. This system employs an MDM2 minigene that mimics the damage-induced alternative splicing observed in vivo. Using this in vitro splicing system, we have shown that conserved intronic sequences in intron 11 of MDM2 are required for normal splicing. Furthermore, we showed that these intronic elements are also required for the regulated damage-induced alternative splicing of MDM2. The use of this novel damage-inducible system will allow for the systematic identification of regulatory elements and factors involved in the splicing regulation of the MDM2 gene in response to stress. This study has implications for identification of novel intervention points for development of future therapeutics for rhabdomyosarcoma.
Assuntos
Processamento Alternativo , Sequência Conservada , Íntrons , Proteínas Proto-Oncogênicas c-mdm2/genética , Estresse Fisiológico/genética , Animais , Cisplatino/toxicidade , Camundongos , Raios Ultravioleta/efeitos adversosRESUMO
Alternative splicing contributes to gene expression dynamics in many tissues, yet its role in auditory development remains unclear. We performed whole-exome sequencing in individuals with sensorineural hearing loss (SNHL) and identified pathogenic mutations in Epithelial Splicing-Regulatory Protein 1 (ESRP1). Patient-derived induced pluripotent stem cells showed alternative splicing defects that were restored upon repair of an ESRP1 mutant allele. To determine how ESRP1 mutations cause hearing loss, we evaluated Esrp1-/- mouse embryos and uncovered alterations in cochlear morphogenesis, auditory hair cell differentiation, and cell fate specification. Transcriptome analysis revealed impaired expression and splicing of genes with essential roles in cochlea development and auditory function. Aberrant splicing of Fgfr2 blocked stria vascularis formation due to erroneous ligand usage, which was corrected by reducing Fgf9 gene dosage. These findings implicate mutations in ESRP1 as a cause of SNHL and demonstrate the complex interplay between alternative splicing, inner ear development, and auditory function.
Assuntos
Processamento Alternativo/genética , Cóclea/embriologia , Perda Auditiva/genética , Mutação/genética , Proteínas de Ligação a RNA/genética , Animais , Diferenciação Celular/genética , Cóclea/metabolismo , Camundongos KnockoutRESUMO
Alternative splicing (AS) plays a critical role in cell fate transitions, development, and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS, including a splicing program that is unique to transgene-independent induced pluripotent stem cells (iPSCs). Changes in the expression of AS factors Zcchc24, Esrp1, Mbnl1/2, and Rbm47 were demonstrated to contribute to phase-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA-binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1. These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency.
Assuntos
Processamento Alternativo/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Reprogramação Celular , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de TempoRESUMO
The epithelial-to-mesenchymal transition (EMT) is an essential biological process during embryonic development that is also implicated in cancer metastasis. While the transcriptional regulation of EMT has been well studied, the role of alternative splicing (AS) regulation in EMT remains relatively uncharacterized. We previously showed that the epithelial cell-type-specific proteins epithelial splicing regulatory proteins 1 (ESRP1) and ESRP2 are important for the regulation of many AS events that are altered during EMT. However, the contributions of the ESRPs and other splicing regulators to the AS regulatory network in EMT require further investigation. Here, we used a robust in vitro EMT model to comprehensively characterize splicing switches during EMT in a temporal manner. These investigations revealed that the ESRPs are the major regulators of some but not all AS events during EMT. We determined that the splicing factor RBM47 is downregulated during EMT and also regulates numerous transcripts that switch splicing during EMT. We also determined that Quaking (QKI) broadly promotes mesenchymal splicing patterns. Our study highlights the broad role of posttranscriptional regulation during the EMT and the important role of combinatorial regulation by different splicing factors to fine tune gene expression programs during these physiological and developmental transitions.
Assuntos
Processamento Alternativo , Transição Epitelial-Mesenquimal , Redes Reguladoras de Genes , Neoplasias/genética , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Modelos Biológicos , Proteínas de Ligação a RNA/genética , Análise de Sequência de RNARESUMO
Tissue- and cell-type-specific regulators of alternative splicing (AS) are essential components of posttranscriptional gene regulation, necessary for normal cellular function, patterning, and development. Mice with ablation of Epithelial splicing regulatory protein (Esrp1) develop cleft lip and palate. Loss of both Esrp1 and its paralog Esrp2 results in widespread developmental defects with broad implications to human disease. Deletion of the Esrps in the epidermis revealed their requirement for establishing a proper skin barrier, a primary function of epithelial cells comprising the epidermis. We profiled the global Esrp-mediated splicing regulatory program in epidermis, which revealed large-scale programs of epithelial cell-type-specific splicing required for epithelial cell functions. These mice represent a valuable model for evaluating the essential role for AS in development and function of epithelial cells, which play essential roles in tissue homeostasis in numerous organs, and provide a genetic tool to evaluate important functional properties of epithelial-specific splice variants in vivo.
Assuntos
Estruturas Animais/embriologia , Células Epiteliais/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/genéticaRESUMO
Although major genetic networks controlling early liver specification and morphogenesis are known, the mechanisms responsible for postnatal hepatic maturation are poorly understood. Here we employ global analyses of the mouse liver transcriptome to demonstrate that postnatal remodelling of the liver is accompanied by large-scale transcriptional and post-transcriptional transitions that are cell-type-specific and temporally coordinated. Combining detailed expression analyses with gain- and loss-of-function studies, we identify epithelial splicing regulatory protein 2 (ESRP2) as a conserved regulatory factor that controls the neonatal-to-adult switch of â¼20% of splice isoforms in mouse and human hepatocytes. The normal shift in splicing coincides tightly with dramatic postnatal induction of ESRP2 in hepatocytes. We further demonstrate that forced expression of ESRP2 in immature mouse and human hepatocytes is sufficient to drive a reciprocal shift in splicing and causes various physiological abnormalities. These findings define a direct role for ESRP2 in the generation of conserved repertoires of adult splice isoforms that facilitate terminal differentiation and maturation of hepatocytes.
Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/metabolismo , Fígado/metabolismo , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Animais , Western Blotting , Diferenciação Celular , Humanos , Imuno-Histoquímica , Hibridização In Situ , Fígado/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Processamento Alternativo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Dano ao DNA , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Mapas de Interação de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , Ativação TranscricionalRESUMO
Proximal spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of the survival motor neuron (SMN) protein. The reduced SMN levels are due to loss of the survival motor neuron-1 (SMN1) gene. Humans carry a nearly identical SMN2 gene that generates a truncated protein, due to a C to T nucleotide alteration in exon 7 that leads to inefficient RNA splicing of exon 7. This exclusion of SMN exon 7 is central to the onset of the SMA disease, however, this offers a unique therapeutic intervention in which corrective splicing of the SMN2 gene would restore SMN function. Exon 7 splicing is regulated by a number of exonic and intronic splicing regulatory sequences and trans-factors that bind them. A better understanding of the way SMN pre-mRNA is spliced has lead to the development of targeted therapies aimed at correcting SMN2 splicing. As therapeutics targeted toward correction of SMN2 splicing continue to be developed available SMA mouse models can be utilized in validating their potential in disease treatment.