RESUMEN
Control over gene expression is exerted, in multiple stages of spermatogenesis, at the post-transcriptional level by RNA binding proteins (RBPs). We identify here an essential role in mammalian spermatogenesis and male fertility for 'RNA binding protein 46' (RBM46). A highly evolutionarily conserved gene, Rbm46 is also essential for fertility in both flies and fish. We found Rbm46 expression was restricted to the mouse germline, detectable in males in the cytoplasm of premeiotic spermatogonia and meiotic spermatocytes. To define its requirement for spermatogenesis, we generated Rbm46 knockout (KO, Rbm46-/-) mice; although male Rbm46-/- mice were viable and appeared grossly normal, they were infertile. Testes from adult Rbm46-/- mice were small, with seminiferous tubules containing only Sertoli cells and few undifferentiated spermatogonia. Using genome-wide unbiased high throughput assays RNA-seq and 'enhanced crosslinking immunoprecipitation' coupled with RNA-seq (eCLIP-seq), we discovered RBM46 could bind, via a U-rich conserved consensus sequence, to a cohort of mRNAs encoding proteins required for completion of differentiation and subsequent meiotic initiation. In summary, our studies support an essential role for RBM46 in regulating target mRNAs during spermatogonia differentiation prior to the commitment to meiosis in mice.
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Proteínas de Unión al ARN/metabolismo , Espermatogénesis , Espermatogonias , Animales , Diferenciación Celular/genética , Masculino , Mamíferos/genética , Meiosis/genética , Ratones , Ratones Noqueados , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Espermatocitos/metabolismo , Espermatogénesis/genética , Espermatogonias/metabolismo , TestículoRESUMEN
Cleft lip is one of the most common human birth defects. However, there remain a limited number of mouse models of cleft lip that can be leveraged to characterize the genes and mechanisms that cause this disorder. Crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, but the basic molecular events mediating this crosstalk remain poorly understood. We previously demonstrated that mice lacking the epithelial-specific splicing factor Esrp1 have fully penetrant bilateral cleft lip and palate. In this study, we further investigated the mechanisms leading to cleft lip as well as cleft palate in both existing and new Esrp1 mutant mouse models. These studies included a detailed transcriptomic analysis of changes in ectoderm and mesenchyme in Esrp1-/- embryos during face formation. We identified altered expression of genes previously implicated in cleft lip and/or palate, including components of multiple signaling pathways. These findings provide the foundation for detailed investigations using Esrp1 mutant disease models to examine gene regulatory networks and pathways that are essential for normal face and palate development - the disruption of which leads to orofacial clefting in human patients.
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Labio Leporino/patología , Fisura del Paladar/patología , Epitelio/patología , Mesodermo/patología , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Empalme Alternativo/genética , Animales , Proliferación Celular , Labio Leporino/embriología , Labio Leporino/genética , Fisura del Paladar/embriología , Fisura del Paladar/genética , Ectodermo/embriología , Ectodermo/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Epitelio/embriología , Cara , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Mesodermo/embriología , Ratones Noqueados , Organogénesis/genética , Hueso Paladar/embriología , Hueso Paladar/patologíaRESUMEN
Irf6 and Esrp1 are important for palate development across vertebrates. In zebrafish, we found that irf6 regulates the expression of esrp1 We detailed overlapping Irf6 and Esrp1/2 expression in mouse orofacial epithelium. In zebrafish, irf6 and esrp1/2 share expression in periderm, frontonasal ectoderm and oral epithelium. Genetic disruption of irf6 and esrp1/2 in zebrafish resulted in cleft of the anterior neurocranium. The esrp1/2 mutant also developed cleft of the mouth opening. Lineage tracing of cranial neural crest cells revealed that the cleft resulted not from migration defect, but from impaired chondrogenesis. Analysis of aberrant cells within the cleft revealed expression of sox10, col1a1 and irf6, and these cells were adjacent to krt4+ and krt5+ cells. Breeding of mouse Irf6; Esrp1; Esrp2 compound mutants suggested genetic interaction, as the triple homozygote and the Irf6; Esrp1 double homozygote were not observed. Further, Irf6 heterozygosity reduced Esrp1/2 cleft severity. These studies highlight the complementary analysis of Irf6 and Esrp1/2 in mouse and zebrafish, and identify a unique aberrant cell population in zebrafish expressing sox10, col1a1 and irf6 Future work characterizing this cell population will yield additional insight into cleft pathogenesis.
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Factores Reguladores del Interferón/genética , Desarrollo Maxilofacial/genética , Morfogénesis/genética , Proteínas de Unión al ARN/genética , Animales , Ectodermo/crecimiento & desarrollo , Ectodermo/metabolismo , Epitelio/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Ratones , Mutación/genética , Cresta Neural/crecimiento & desarrollo , Factores de Transcripción SOXE/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
A major limitation of RNA sequencing (RNA-seq) analysis of alternative splicing is its reliance on high sequencing coverage. We report DARTS (https://github.com/Xinglab/DARTS), a computational framework that integrates deep-learning-based predictions with empirical RNA-seq evidence to infer differential alternative splicing between biological samples. DARTS leverages public RNA-seq big data to provide a knowledge base of splicing regulation via deep learning, thereby helping researchers better characterize alternative splicing using RNA-seq datasets even with modest coverage.
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Aprendizaje Profundo , Empalme del ARN , ARN/análisis , Análisis de Secuencia de ARN , Algoritmos , Empalme Alternativo , Teorema de Bayes , Epigenómica , Exones , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células Hep G2 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células K562 , Modelos Estadísticos , ARN/genética , Reproducibilidad de los Resultados , Procesamiento de Señales Asistido por ComputadorRESUMEN
Using Caenorhabditis elegans as a model system, Norris et al. (2014) define complex combinatorial regulation of alternative splicing at single-neuron resolution and illustrate functional coherence among components of a splicing regulatory network controlled by a neuronal splicing factor.
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Empalme Alternativo/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/genética , Neuronas Colinérgicas/citología , Neuronas GABAérgicas/citología , Proteínas de Unión al ARN/fisiología , Transmisión Sináptica/genética , Sintaxina 1/genética , AnimalesRESUMEN
Non-syndromic cleft lip with or without cleft palate (NS-CL/P) is one of the most common human birth defects and is generally considered a complex trait. Despite numerous loci identified by genome-wide association studies, the effect sizes of common variants are relatively small, with much of the presumed genetic contribution remaining elusive. We report exome-sequencing results in 209 people from 72 multi-affected families with pedigree structures consistent with autosomal-dominant inheritance and variable penetrance. Herein, pathogenic variants are described in four genes encoding components of the p120-catenin complex (CTNND1, PLEKHA7, PLEKHA5) and an epithelial splicing regulator (ESRP2), in addition to the known CL/P-associated gene, CDH1, which encodes E-cadherin. The findings were also validated in a second cohort of 497 people with NS-CL/P, comprising small families and singletons with pathogenic variants in these genes identified in 14% of multi-affected families and 2% of the replication cohort of smaller families. Enriched expression of each gene/protein in human and mouse embryonic oro-palatal epithelia, demonstration of functional impact of CTNND1 and ESRP2 variants, and recapitulation of the CL/P spectrum in Ctnnd1 knockout mice support a causative role in CL/P pathogenesis. These data show that primary defects in regulators of epithelial cell adhesion are the most significant contributors to NS-CL/P identified to date and that inherited and de novo single gene variants explain a substantial proportion of NS-CL/P.
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Cadherinas/genética , Cateninas/genética , Labio Leporino/genética , Fisura del Paladar/genética , Predisposición Genética a la Enfermedad , Mutación/genética , Alelos , Secuencia de Aminoácidos , Animales , Biotinilación , Epitelio/metabolismo , Epitelio/patología , Femenino , Eliminación de Gen , Humanos , Lactante , Recién Nacido , Masculino , Ratones , Hueso Paladar/patología , Linaje , Síndrome , Secuenciación del Exoma , Catenina deltaRESUMEN
RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.
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Regulación de la Expresión Génica/genética , Motivos de Nucleótidos/genética , Proteínas de Unión al ARN/metabolismo , Trastorno Autístico/genética , Secuencia de Bases , Sitios de Unión/genética , Secuencia Conservada/genética , Células Eucariotas/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína/genética , Factores de Empalme de ARN , Estabilidad del ARN/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Metastatic breast cancer is a leading cause of cancer-related deaths in women worldwide. Patients with triple negative breast cancer (TNBCs), a highly aggressive tumor subtype, have a particularly poor prognosis. Multiple reports demonstrate that altered content of the multicopy mitochondrial genome (mtDNA) in primary breast tumors correlates with poor prognosis. We earlier reported that mtDNA copy number reduction in breast cancer cell lines induces an epithelial-mesenchymal transition associated with metastasis. However, it is unknown whether the breast tumor subtypes (TNBC, Luminal and HER2+) differ in the nature and amount of mitochondrial defects and if mitochondrial defects can be used as a marker to identify tumors at risk for metastasis. By analyzing human primary tumors, cell lines and the TCGA dataset, we demonstrate a high degree of variability in mitochondrial defects among the tumor subtypes and TNBCs, in particular, exhibit higher frequency of mitochondrial defects, including reduced mtDNA content, mtDNA sequence imbalance (mtRNR1:ND4), impaired mitochondrial respiration and metabolic switch to glycolysis which is associated with tumorigenicity. We identified that genes involved in maintenance of mitochondrial structural and functional integrity are differentially expressed in TNBCs compared to non-TNBC tumors. Furthermore, we identified a subset of TNBC tumors that contain lower expression of epithelial splicing regulatory protein (ESRP)-1, typical of metastasizing cells. The overall impact of our findings reported here is that mitochondrial heterogeneity among TNBCs can be used to identify TNBC patients at risk of metastasis and the altered metabolism and metabolic genes can be targeted to improve chemotherapeutic response.
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ADN Mitocondrial , Mitocondrias , Proteínas Mitocondriales , Proteínas de Neoplasias , Proteínas de Unión al ARN , Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Femenino , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Cell-type-specific expression of epithelial and mesenchymal isoforms of Fibroblast Growth Factor Receptor 2 (FGFR2) is achieved through tight regulation of mutually exclusive exons IIIb and IIIc, respectively. Using an application of cell-based cDNA expression screening, we identified two paralogous epithelial cell-type-specific RNA-binding proteins that are essential regulators of FGFR2 splicing. Ectopic expression of either protein in cells that express FGFR2-IIIc caused a switch in endogenous FGFR2 splicing to the epithelial isoform. Conversely, knockdown of both factors in cells that express FGFR2-IIIb by RNA interference caused a switch from the epithelial to mesenchymal isoform. These factors also regulate splicing of CD44, p120-Catenin (CTNND1), and hMena (ENAH), three transcripts that undergo changes in splicing during the epithelial-to-mesenchymal transition (EMT). These studies suggest that Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2) are coordinators of an epithelial cell-type-specific splicing program.
Asunto(s)
Empalme Alternativo , Células Epiteliales/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cateninas , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Exones , Regulación de la Expresión Génica , Humanos , Receptores de Hialuranos/metabolismo , Intrones , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Isoformas de Proteínas , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción Genética , Catenina deltaRESUMEN
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.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Uréter/citología , Uréter/metabolismo , Animales , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Riñón/citología , Riñón/embriología , Masculino , Ratones , Ratones Noqueados , Nefronas/citología , Nefronas/metabolismo , Empalme del ARN/genética , Empalme del ARN/fisiología , Proteínas de Unión al ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Human mena (hMENA), a member of the actin cytoskeleton regulators Ena/VASP, is overexpressed in high-risk preneoplastic lesions and in primary breast tumors and has been identified as playing a role in invasiveness and poor prognosis in breast cancers that express HER2. Here we identify a unique isoform, hMENAΔv6, derived from the hMENA alternative splicing program. In an isogenic model of human breast cancer progression, we show that hMENA(11a) is expressed in premalignant cells, whereas hMENAΔv6 expression is restricted to invasive cancer cells. "Reversion" of the malignant phenotype leads to concurrent down-regulation of all hMENA isoforms. In breast cancer cell lines, isoform-specific hMENA overexpression or knockdown revealed that in the absence of hMENA(11a), overexpression of hMENAΔv6 increased cell invasion, whereas overexpression of hMENA(11a) reduced the migratory and invasive ability of these cells. hMENA(11a) splicing was shown to be dependent on the epithelial regulator of splicing 1 (ESRP1), and forced expression of ESRP1 in invasive mesenchymal breast cancer cells caused a phenotypic switch reminiscent of a mesenchymal-to-epithelial transition (MET) characterized by changes in the cytoskeletal architecture, reexpression of hMENA(11a), and a reduction in cell invasion. hMENA-positive primary breast tumors, which are hMENA(11a)-negative, are more frequently E-cadherin low in comparison with tumors expressing hMENA(11a). These data suggest that polarized and growth-arrested cellular architecture correlates with absence of alternative hMENA isoform expression, and that the hMENA splicing program is relevant to malignant progression in invasive disease.
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Empalme Alternativo/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Mesodermo/patología , Proteínas de Microfilamentos/genética , Citoesqueleto de Actina/metabolismo , Antígenos CD , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Clonación Molecular , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Mesodermo/metabolismo , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Invasividad Neoplásica , Fenotipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Transfección , Vimentina/metabolismoRESUMEN
Alternative splicing achieves coordinated changes in post-transcriptional gene expression programmes through the activities of diverse RNA-binding proteins. Epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) are cell-type-specific regulators of transcripts that switch splicing during the epithelial-mesenchymal transition (EMT). To define a comprehensive programme of alternative splicing that is regulated during the EMT, we identified an extensive ESRP-regulated splicing network of hundreds of alternative splicing events within numerous genes with functions in cell-cell adhesion, polarity, and migration. Loss of this global ESRP-regulated epithelial splicing programme induces the phenotypic changes in cell morphology that are observed during the EMT. Components of this splicing signature provide novel molecular markers that can be used to characterize the EMT. Bioinformatics and experimental approaches revealed a high-affinity ESRP-binding motif and a predictive RNA map that governs their activity. This work establishes the ESRPs as coordinators of a complex alternative splicing network that adds an important post-transcriptional layer to the changes in gene expression that underlie epithelial-mesenchymal transitions during development and disease.
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Empalme Alternativo/fisiología , Diferenciación Celular/fisiología , Células Epiteliales/citología , Regulación de la Expresión Génica/fisiología , Mesodermo/citología , Proteínas de Unión al ARN/fisiología , Sitios de Unión/genética , Adhesión Celular/genética , Línea Celular , Movimiento Celular/genética , Polaridad Celular/genética , Biología Computacional , Ensayo de Cambio de Movilidad Electroforética , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Análisis por Micromatrices , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Transporte Vesicular/genéticaRESUMEN
Ultra-deep RNA sequencing has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We develop MATS (multivariate analysis of transcript splicing), a bayesian statistical framework for flexible hypothesis testing of differential alternative splicing patterns on RNA-Seq data. MATS uses a multivariate uniform prior to model the between-sample correlation in exon splicing patterns, and a Markov chain Monte Carlo (MCMC) method coupled with a simulation-based adaptive sampling procedure to calculate the P-value and false discovery rate (FDR) of differential alternative splicing. Importantly, the MATS approach is applicable to almost any type of null hypotheses of interest, providing the flexibility to identify differential alternative splicing events that match a given user-defined pattern. We evaluated the performance of MATS using simulated and real RNA-Seq data sets. In the RNA-Seq analysis of alternative splicing events regulated by the epithelial-specific splicing factor ESRP1, we obtained a high RT-PCR validation rate of 86% for differential exon skipping events with a MATS FDR of <10%. Additionally, over the full list of RT-PCR tested exons, the MATS FDR estimates matched well with the experimental validation rate. Our results demonstrate that MATS is an effective and flexible approach for detecting differential alternative splicing from RNA-Seq data.
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Empalme Alternativo , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN , Teorema de Bayes , Encéfalo/metabolismo , Línea Celular Tumoral , Humanos , Análisis Multivariante , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
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Transición Epitelial-Mesenquimal/fisiología , Genoma Humano/fisiología , Procesamiento Postranscripcional del ARN/fisiología , Proteínas de Unión al ARN , Transcriptoma/fisiología , Animales , Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo/métodos , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The epithelial-to-mesenchymal transition (EMT) is an important developmental process that is also implicated in disease pathophysiology, such as cancer progression and metastasis. A wealth of literature in recent years has identified important transcriptional regulators and large-scale changes in gene expression programs that drive the phenotypic changes that occur during the EMT. However, in the past couple of years it has become apparent that extensive changes in alternative splicing also play a profound role in shaping the changes in cell behavior that characterize the EMT. While long known splicing switches in FGFR2 and p120-catenin provided hints of a larger program of EMT-associated alternative splicing, the recent identification of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) began to reveal this genome-wide post-transcriptional network. Several studies have now demonstrated the truly vast extent of this alternative splicing program. The global switches in splicing associated with the EMT add an important additional layer of post-transcriptional control that works in harmony with transcriptional and epigenetic regulation to effect complex changes in cell shape, polarity, and behavior that mediate transitions between epithelial and mesenchymal cell states. Future challenges include the need to investigate the functional consequences of these splicing switches at both the individual gene as well as systems level.
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Empalme Alternativo , Transición Epitelial-Mesenquimal/genética , Precursores del ARN/genética , ARN Mensajero/metabolismo , Animales , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Isoformas de ARN/genética , Isoformas de ARN/metabolismo , Precursores del ARN/metabolismoRESUMEN
Hepatocyte polyploidy and maturity are critical to acquiring specialized liver functions. Multiple intra- and extracellular factors influence ploidy, but how they cooperate temporally to steer liver polyploidization and maturation or how post-transcriptional mechanisms integrate into these paradigms is unknown. Here, we identified an important regulatory hierarchy in which postnatal activation of Epithelial-Splicing-Regulatory-Protein-2 (ESRP2) stimulates biogenesis of liver-specific microRNA (miR-122), thereby facilitating polyploidization, maturation, and functional competence of hepatocytes. By determining transcriptome-wide protein-RNA interactions in vivo and integrating them with single-cell and bulk hepatocyte RNA-seq datasets, we delineate an ESRP2-driven RNA processing program that drives sequential replacement of fetal-to-adult transcript isoforms. Specifically, ESRP2 binds the primary miR-122 host gene transcript to promote its processing/biogenesis. Combining constitutive and inducible ESRP2 gain- and loss-of-function mice models with miR-122 rescue experiments, we demonstrate that timed activation of ESRP2 augments miR-122-driven program of cytokinesis failure, ensuring proper onset and extent of hepatocyte polyploidization.
RESUMEN
Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators ESRP1 and ESRP2 regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using esrp1/2 mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human ESRP1/2 gene variants. We found that many variants predicted by in silico methods to be pathogenic were functionally benign. Esrp1 also regulates the alternative splicing of Ctnnd1 and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of ctnnd1 is sufficient to rescue morphogenesis of epithelial-derived structures in esrp1/2 zebrafish mutants. Additionally, we identified 13 CTNND1 variants from genome sequencing of OFC cohorts, confirming CTNND1 as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of Esrp-Ctnnd1 acting in the embryonic epithelium to regulate palatogenesis.
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Fisura del Paladar , Isoformas de Proteínas , Proteínas de Unión al ARN , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/embriología , Humanos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Fisura del Paladar/genética , Fisura del Paladar/embriología , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Labio Leporino/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Empalme Alternativo , Línea Celular , MutaciónRESUMEN
Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators ESRP1 and ESRP2 regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using esrp1/2 mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human ESRP1/2 gene variants. We found that many variants predicted by in silico methods to be pathogenic were functionally benign. Esrp1 also regulates the alternative splicing of Ctnnd1 and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of ctnnd1 is sufficient to rescue morphogenesis of epithelial-derived structures in esrp1/2 zebrafish mutants. Additionally, we identified 13 CTNND1 variants from genome sequencing of OFC cohorts, confirming CTNND1 as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of Esrp-Ctnnd1 acting in the embryonic epithelium to regulate palatogenesis.
RESUMEN
The epithelial splicing regulatory proteins, ESRP1 and ESRP2, are essential for mammalian development through the regulation of a global program of alternative splicing of genes involved in the maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1, we performed enhanced crosslinking immunoprecipitation coupled with high-throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to promote either exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3' and 5' untranslated regions (UTRs) of genes involved in epithelial cell function, suggesting that its post-transcriptional functions extend beyond splicing regulation.
RESUMEN
MOTIVATION: The Affymetrix Human Exon Junction Array is a newly designed high-density exon-sensitive microarray for global analysis of alternative splicing. Contrary to the Affymetrix exon 1.0 array, which only contains four probes per exon and no probes for exon-exon junctions, this new junction array averages eight probes per probeset targeting all exons and exon-exon junctions observed in the human mRNA/EST transcripts, representing a significant increase in the probe density for alternative splicing events. Here, we present MADS+, a computational pipeline to detect differential splicing events from the Affymetrix exon junction array data. For each alternative splicing event, MADS+ evaluates the signals of probes targeting competing transcript isoforms to identify exons or splice sites with different levels of transcript inclusion between two sample groups. MADS+ is used routinely in our analysis of Affymetrix exon junction arrays and has a high accuracy in detecting differential splicing events. For example, in a study of the novel epithelial-specific splicing regulator ESRP1, MADS+ detects hundreds of exons whose inclusion levels are dependent on ESRP1, with a RT-PCR validation rate of 88.5% (153 validated out of 173 tested). AVAILABILITY: MADS+ scripts, documentations and annotation files are available at http://www.medicine.uiowa.edu/Labs/Xing/MADSplus/.