RESUMO
BACKGROUND: CUG-BP, Elav-like family member 1 (CELF1) is a multifunctional RNA binding protein found in a variety of adult and embryonic tissues. In the heart, CELF1 is found exclusively in the myocardium. However, the roles of CELF1 during cardiac development have not been completely elucidated. RESULTS: Myofibrillar organization is disrupted and proliferation is reduced following knockdown of CELF1 in cultured chicken primary embryonic cardiomyocytes. In vivo knockdown of Celf1 in developing Xenopus laevis embryos resulted in myofibrillar disorganization and a trend toward reduced proliferation in heart muscle, indicating conserved roles for CELF1 orthologs in embryonic cardiomyocytes. Loss of Celf1 also resulted in morphogenetic abnormalities in the developing heart and gut. Using optical coherence tomography, we showed that cardiac contraction was impaired following depletion of Celf1, while heart rhythm remained unperturbed. In contrast to cardiac muscle, loss of Celf1 did not disrupt myofibril organization in skeletal muscle cells, although it did lead to fragmentation of skeletal muscle bundles. CONCLUSIONS: CELF1 is required for normal myofibril organization, proliferation, morphogenesis, and contractile performance in the developing myocardium. Developmental Dynamics 245:854-873, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas CELF1/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Processamento Alternativo/genética , Animais , Western Blotting , Proteínas CELF1/genética , Células Cultivadas , Embrião de Galinha , Coração/embriologia , Imuno-Histoquímica , Morfogênese/genética , Morfogênese/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Xenopus laevisRESUMO
CUG-BP, Elav-like family member 1 (CELF1) is a highly conserved RNA binding protein that regulates pre-mRNA alternative splicing, polyadenylation, mRNA stability, and translation. In the heart, CELF1 is expressed in the myocardium, where its levels are tightly regulated during development. CELF1 levels peak in the heart during embryogenesis, and aberrant up-regulation of CELF1 in the adult heart has been implicated in cardiac pathogenesis in myotonic dystrophy type 1, as well as in diabetic cardiomyopathy. Either inhibition of CELF activity or over-expression of CELF1 in heart muscle causes cardiomyopathy in transgenic mice. Nonetheless, many of the cardiac targets of CELF1 regulation remain unknown. In this study, to identify cardiac targets of CELF1 we performed cross-linking immunoprecipitation (CLIP) for CELF1 from embryonic day 8 chicken hearts. We identified a previously unannotated exon in MYH7B as a novel target of CELF1-mediated regulation. We demonstrated that knockdown of CELF1 in primary chicken embryonic cardiomyocytes leads to increased inclusion of this exon and decreased MYH7B levels. We also investigated global changes in the transcriptome of primary embryonic cardiomyocytes following CELF1 knockdown in a published RNA-seq dataset. Pathway and network analyses identified strong associations between CELF1 and regulation of cell cycle and translation. Important regulatory proteins, including both RNA binding proteins and a cardiac transcription factor, were affected by loss of CELF1. Together, these data suggest that CELF1 is a key regulator of cardiomyocyte gene expression.
Assuntos
Proteínas CELF1/genética , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Miocárdio/metabolismo , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Embrião de Galinha , Reagentes de Ligações Cruzadas , Éxons , Humanos , Imunoprecipitação , Íntrons , Camundongos , Miócitos Cardíacos/citologia , Cadeias Pesadas de Miosina/genética , Precursores de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNARESUMO
BACKGROUND: Development of the valves and septa of the heart depends on the formation and remodeling of the endocardial cushions in the atrioventricular canal and outflow tract. These cushions are populated by mesenchyme produced from the endocardium by epithelial-mesenchymal transition (EMT). The endocardial cushions are remodeled into the valves at post-EMT stages via differentiation of the mesenchyme and changes in the extracellular matrix (ECM). Transforming growth factor ß (TGFß) signaling has been implicated in both the induction of EMT in the endocardial cushions and the remodeling of the valves at post-EMT stages. We previously identified the RNA binding protein muscleblind-like 1 (MBNL1) as a negative regulator of TGFß signaling and EMT in chicken endocardial cushions ex vivo. Here, we investigate the role of MBNL1 in endocardial cushion development and valvulogenesis in Mbnl1(∆E3/∆E3) mice, which are null for MBNL1 protein. METHODS: Collagen gel invasion assays, histology, immunohistochemistry, real-time RT-PCR, optical coherence tomography, and echocardiography were used to evaluate EMT and TGFß signaling in the endocardial cushions, and morphogenesis, ECM composition, and function of the heart valves. RESULTS: As in chicken, the loss of MBNL1 promotes precocious TGFß signaling and EMT in the endocardial cushions. Surprisingly, this does not lead to the production of excess mesenchyme, but later valve morphogenesis is aberrant. Adult Mbnl1(∆E3/∆E3) mice exhibit valve dysmorphia with elevated TGFß signaling, changes in ECM composition, and increased pigmentation. This is accompanied by a high incidence of regurgitation across both inflow and outflow valves. Mbnl1(∆E3/∆E3) mice also have a high incidence of ostium secundum septal defects accompanied by atrial communication, but do not develop overt cardiomyopathy. CONCLUSIONS: Together, these data indicate that MBNL1 plays a conserved role in negatively regulating TGFß signaling, and is required for normal valve morphogenesis and homeostasis in vivo.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Coxins Endocárdicos/embriologia , Valvas Cardíacas/embriologia , Organogênese , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Animais , Proteínas de Ligação a DNA/genética , Coxins Endocárdicos/metabolismo , Transição Epitelial-Mesenquimal , Coração/embriologia , Valvas Cardíacas/citologia , Valvas Cardíacas/metabolismo , Camundongos , Proteínas de Ligação a RNA/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
CUG-BP, Elav-like family member 1 (CELF1) is a multi-functional RNA binding protein that regulates pre-mRNA alternative splicing in the nucleus, as well as polyadenylation status, mRNA stability, and translation in the cytoplasm [1]. Dysregulation of CELF1 has been implicated in cardiomyopathies in myotonic dystrophy type 1 and diabetes [2-5], but the targets of CELF1 regulation in the heart have not been systematically investigated. We previously demonstrated that in the developing heart CELF1 expression is restricted to the myocardium and peaks during embryogenesis [6-8]. To identify transcripts regulated by CELF1 in the embryonic myocardium, RNA-seq was used to compare the transcriptome of primary embryonic cardiomyocytes following siRNA-mediated knockdown of CELF1 to that of controls. Raw data files of the RNA-seq reads have been deposited in NCBI's Gene Expression Omnibus [9] under the GEO Series accession number GSE67360. These data can be used to identify transcripts whose levels or alternative processing (i.e., alternative splicing or polyadenylation site usage) are regulated by CELF1, and should provide insight into the pathways and processes modulated by this important RNA binding protein during normal heart development and during cardiac pathogenesis.
RESUMO
CUG-BP, Elav-like family (CELF) proteins regulate cell type- and developmental stage-specific alternative splicing in the heart. Repression of CELF-mediated splicing activity via expression of a nuclear dominant negative CELF protein in heart muscle was previously shown to induce dysregulation of alternative splicing, cardiac dysfunction, cardiac hypertrophy, and dilated cardiomyopathy in MHC-CELFΔ transgenic mice. A "mild" line of MHC-CELFΔ mice that expresses a lower level of the dominant negative protein exhibits cardiac dysfunction and myopathy at a young age, but spontaneously recovers normal cardiac function and heart size with age despite the persistence of splicing defects. To the best of our knowledge, this was the first example of a genetically induced cardiomyopathy that spontaneously recovers without intervention. In this study, we explored the basis for this recovery. We examined whether a transcriptional program regulated by serum response factor (SRF) that is dysregulated in juvenile MHC-CELFΔ mice is restored in the mild line with age, and evaluated global changes in gene expression by microarray analyses. We found that differences in gene expression between the mild line and wild type hearts are greatly reduced in older animals, including a partial recovery of SRF target gene expression. We did not find evidence of a new compensatory pathway being activated in the mild line with age, and propose that recovery may occur due to developmental stage-specific compatibility of CELF-dependent splice variants with the cellular environment of the cardiomyocyte.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Cardiomiopatias/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Miocárdio/patologia , Processamento Alternativo , Animais , Proteína delta de Ligação ao Facilitador CCAAT/genética , Cálcio/metabolismo , Cardiomiopatias/patologia , Feminino , Coração/fisiologia , Hemizigoto , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Resposta Sérica/metabolismo , Transcrição GênicaRESUMO
In vivo, RNA molecules are constantly accompanied by RNA binding proteins (RBPs), which are intimately involved in every step of RNA biology, including transcription, editing, splicing, transport and localization, stability, and translation. RBPs therefore have opportunities to shape gene expression at multiple levels. This capacity is particularly important during development, when dynamic chemical and physical changes give rise to complex organs and tissues. This review discusses RBPs in the context of heart development. Since the targets and functions of most RBPs--in the heart and at large--are not fully understood, this review focuses on the expression and roles of RBPs that have been implicated in specific stages of heart development or developmental pathology. RBPs are involved in nearly every stage of cardiogenesis, including the formation, morphogenesis, and maturation of the heart. A fuller understanding of the roles and substrates of these proteins could ultimately provide attractive targets for the design of therapies for congenital heart defects, cardiovascular disease, or cardiac tissue repair.
Assuntos
Coração/embriologia , Proteínas de Ligação a RNA/metabolismo , Animais , Doenças Cardiovasculares/embriologia , Doenças Cardiovasculares/genética , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Humanos , MorfogêneseRESUMO
Members of the CUG-BP, Elav-like family (CELF) regulate alternative splicing in the heart. In MHC-CELFΔ transgenic mice, CELF splicing activity is inhibited postnatally in heart muscle via expression of a nuclear dominant negative CELF protein under an α-myosin heavy chain promoter. MHC-CELFΔ mice develop dilated cardiomyopathy characterized by alternative splicing defects, enlarged hearts, and severe contractile dysfunction. In this study, gene expression profiles in the hearts of wild type, high- and low-expressing lines of MHC-CELFΔ mice were compared using microarrays. Gene ontology and pathway analyses identified contraction and calcium signaling as the most affected processes. Network analysis revealed that the serum response factor (SRF) network is highly affected. Downstream targets of SRF were up-regulated in MHC-CELFΔ mice compared to the wild type, suggesting an increase in SRF activity. Although SRF levels remained unchanged, known inhibitors of SRF activity were down-regulated. Conversely, we found that these inhibitors are up-regulated and downstream SRF targets are down-regulated in the hearts of MCKCUG-BP1 mice, which mildly over-express CELF1 in heart and skeletal muscle. This suggests that changes in SRF activity are a consequence of changes in CELF-mediated regulation rather than a secondary result of compensatory pathways in heart failure. In MHC-CELFΔ males, where the phenotype is only partially penetrant, both alternative splicing changes and down-regulation of inhibitors of SRF correlate with the development of cardiomyopathy. Together, these results strongly support a role for CELF-mediated alternative splicing in the regulation of contractile gene expression, achieved in part through modulating the activity of SRF, a key cardiac transcription factor.
Assuntos
Processamento Alternativo/genética , Proteína delta de Ligação ao Facilitador CCAAT/genética , Contração Miocárdica/genética , Fator de Resposta Sérica/genética , Animais , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Regulação da Expressão Gênica , Complexo Principal de Histocompatibilidade/genética , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Miosinas VentricularesRESUMO
Alternative splicing is an important mechanism for generating transcript and protein diversity. In the brain, alternative splicing is particularly prevalent, and alternative splicing factors are highly enriched. These include the six members of the CUG-BP, Elav-like family (CELF). This review summarizes what is known about the expression of different CELF proteins in the nervous system and the evidence that they are important in neural development and function. The involvement of CELF proteins in the pathogenesis of a number of neurodegenerative disorders, including myotonic dystrophy, spinocerebellar ataxia, fragile X syndrome, spinal muscular atrophy, and spinal and bulbar muscular atrophy is discussed. Finally, the known targets of CELF-mediated alternative splicing regulation in the nervous system and the functional consequences of these splicing events are reviewed. This article is part of a Special Issue entitled "RNA and splicing regulation in neurodegeneration."
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Proteínas ELAV/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Proteínas ELAV/genética , Humanos , Doenças Neurodegenerativas/genética , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/metabolismoRESUMO
BACKGROUND: Alternative splicing is often subjected to complex regulatory control that involves many protein factors and cis-acting RNA sequence elements. One major challenge is to identify all of the protein players and define how they control alternative expression of a particular exon in a combinatorial manner. The Muscleblind-like (MBNL) and CUG-BP and ELAV-Like family (CELF) proteins are splicing regulatory proteins, which function as antagonists in the regulation of several alternative exons. Currently only a limited number of common targets of MBNL and CELF are known that are antagonistically regulated by these two groups of proteins. RESULTS: Recently, we identified neurofibromatosis type 1 (NF1) exon 23a as a novel target of negative regulation by CELF proteins. Here we report that MBNL family members are positive regulators of this exon. Overexpression of MBNL proteins promote exon 23a inclusion in a low MBNL-expressing cell line, and simultaneous siRNA-mediated knockdown of MBNL1 and MBNL2 family members in a high MBNL-expressing cell line promotes exon 23a skipping. Importantly, these two groups of proteins antagonize each other in regulating inclusion of exon 23a. Furthermore, we analyzed the binding sites of these proteins in the intronic sequences upstream of exon 23a by UV cross-linking assays. We show that in vitro, in addition to the previously identified preferred binding sequence UGCUGU, the MBNL proteins need the neighboring sequences for optimal binding. CONCLUSION: This study along with our previous work that demonstrated roles for Hu, CELF, and TIA-1 and TIAR proteins in the regulation of NF1 exon 23a establish that this exon is under tight, complex control.
Assuntos
Processamento Alternativo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Neurofibromatose 1/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteínas CELF1 , Linhagem Celular , Éxons , Células HeLa , Humanos , Neurofibromatose 1/metabolismo , Interferência de RNA , Precursores de RNA/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , RatosRESUMO
BACKGROUND: Valvulogenesis and septation in the developing heart depend on the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). These cushions are invaded by a subpopulation of endocardial cells that undergo an epithelial-mesenchymal transition in response to paracrine and autocrine transforming growth factor ß (TGFß) signals. We previously demonstrated that the RNA binding protein muscleblind-like 1 (MBNL1) is expressed specifically in the cushion endocardium, and knockdown of MBNL1 in stage 14 embryonic chicken AVC explants enhances TGFß-dependent endocardial cell invasion. RESULTS: In this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGFß3 after it is no longer required to induce basal levels of invasion. TGFß3, but not TGFß2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGFß3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGFß3 production in the endocardium. More TGFß3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGFß3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGFß3 secretion, and early exposure to excess TGFß3 induces precocious invasion. MBNL1 expression precedes TGFß3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGFß3 signaling. The stimulatory effects of MBNL1 knockdown on invasion are lost in stage 16 AVC explants. Knockdown of MBNL1 in OFT explants similarly enhances cell invasion, but not activation. TGFß is necessary and sufficient to mediate this effect. CONCLUSIONS: Taken together, these data support a model in which MBNL1 negatively regulates cell invasion in the endocardial cushions by restricting the magnitude and timing of endocardial-derived TGFß3 production.
Assuntos
Proteínas Aviárias/genética , Coxins Endocárdicos/embriologia , Coração/embriologia , Proteínas de Ligação a RNA/genética , Fator de Crescimento Transformador beta3/metabolismo , Animais , Comunicação Autócrina , Proteínas Aviárias/metabolismo , Movimento Celular , Embrião de Galinha , Coxins Endocárdicos/citologia , Coxins Endocárdicos/metabolismo , Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Mesoderma/citologia , Mesoderma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Técnicas de Cultura de Tecidos , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/fisiologiaRESUMO
Myotonic dystrophy type 1 (DM1) is caused by the expansion of CUG repeats in the 3' UTR of DMPK transcripts. DM1 pathogenesis has been attributed in part to alternative splicing dysregulation via elevation of CUG-BP, Elav-like family member 1 (CELF1). Several therapeutic approaches have been tested in cells and mice, but no previous studies had specifically targeted CELF1. Here, we show that repressing CELF activity rescues CELF-dependent alternative splicing in cell culture and transgenic mouse models of DM1. CELF-independent splicing, however, remained dysregulated. These data highlight both the potential and limitations of targeting CELF1 for the treatment of DM1.
RESUMO
RNA processing is important for generating protein diversity and modulating levels of protein expression. The CUG-BP, Elav-like family (CELF) of RNA-binding proteins regulate several steps of RNA processing in the nucleus and cytoplasm, including pre-mRNA alternative splicing, C to U RNA editing, deadenylation, mRNA decay, and translation. In vivo, CELF proteins have been shown to play roles in gametogenesis and early embryonic development, heart and skeletal muscle function, and neurosynaptic transmission. Dysregulation of CELF-mediated programs has been implicated in the pathogenesis of human diseases affecting the heart, skeletal muscles, and nervous system.
Assuntos
Proteínas de Ligação a RNA/genética , Animais , Displasia Arritmogênica Ventricular Direita/metabolismo , Humanos , Doenças Musculares/metabolismo , Doenças do Sistema Nervoso/metabolismo , Fenótipo , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND: CUG-BP and ETR-3-like factor (CELF) proteins regulate tissue- and developmental stage-specific alternative splicing in striated muscle. We previously demonstrated that heart muscle-specific expression of a nuclear dominant negative CELF protein in transgenic mice (MHC-CELFΔ) effectively disrupts endogenous CELF activity in the heart in vivo, resulting in impaired cardiac function. In this study, transgenic mice that express the dominant negative protein under a skeletal muscle-specific promoter (Myo-CELFΔ) were generated to investigate the role of CELF-mediated alternative splicing programs in normal skeletal muscle. METHODOLOGY/PRINCIPAL FINDINGS: Myo-CELFΔ mice exhibit modest changes in CELF-mediated alternative splicing in skeletal muscle, accompanied by a reduction of endomysial and perimysial spaces, an increase in fiber size variability, and an increase in slow twitch muscle fibers. Weight gain and mean body weight, total number of muscle fibers, and overall muscle strength were not affected. CONCLUSIONS/SIGNIFICANCE: Although these findings demonstrate that CELF activity contributes to the normal alternative splicing of a subset of muscle transcripts in vivo, the mildness of the effects in Myo-CELFΔ muscles compared to those in MHC-CELFΔ hearts suggests CELF activity may be less determinative for alternative splicing in skeletal muscle than in heart muscle. Nonetheless, even these small changes in CELF-mediated splicing regulation were sufficient to alter muscle organization and muscle fiber properties affected in myotonic dystrophy. This lends further evidence to the hypothesis that dysregulation of CELF-mediated alternative splicing programs may be responsible for the disruption of these properties during muscle pathogenesis.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Genes Dominantes/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Processamento Alternativo/genética , Animais , Fenômenos Biomecânicos/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , Camundongos , Camundongos Transgênicos , Força Muscular/fisiologia , Mutação/genética , Miogenina/metabolismo , Tamanho do ÓrgãoRESUMO
INTRODUCTION: Myotonic dystrophy, or dystrophia myotonica (DM), is characterized by prominent muscle wasting and weakness as well as delayed muscle relaxation resulting from persistent electrical discharges. METHODS: We hypothesized heterogeneity among muscles in degree of weakness and myotonia in an expanded [(CUG)(250)] repeats transgenic (HSA(LR)) mouse DM model. Muscle contraction was compared among diaphragm, extensor digitorum longus (EDL), and soleus muscles. RESULTS: Myotonia was found only in EDL, as manifested by longer late-relaxation time and elevated myotonic index. EDL, but not the other two muscles, had impaired force over a wide range of stimulation frequencies. During fatigue-inducing stimulation, DM EDL muscle force per cross-sectional area was significantly impaired during 25-Hz stimulation, whereas there were no differences in fatigue response for DM diaphragm or soleus. CONCLUSION: In an expanded repeats model of DM the EDL is more susceptible to myotonia and force impairment than muscles with lower proportions of fast-twitch fibers.
Assuntos
Predisposição Genética para Doença/genética , Força Muscular , Debilidade Muscular/genética , Debilidade Muscular/fisiopatologia , Músculo Esquelético/fisiopatologia , Distrofia Miotônica/genética , Distrofia Miotônica/fisiopatologia , Animais , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Eletromiografia/métodos , Camundongos , Camundongos Transgênicos , Contração Muscular/genética , Força Muscular/genética , Debilidade Muscular/diagnóstico , Distrofia Miotônica/diagnóstico , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Muscleblind-like (MBNL) proteins have been shown to regulate pre-mRNA alternative splicing, and MBNL1 has been implicated in regulating fetal-to-adult transitions in alternative splicing in the heart. MBNL1 is highly conserved, exhibiting more than 95% identity at the amino acid level between birds and mammals. To investigate MBNL1 expression during embryonic heart development, we examined MBNL1 transcript and protein expression in the embryonic chicken heart from the formation of the primitive heart tube through cardiac morphogenesis (embryonic days 1.5 through 8). MBNL1 transcript levels remained steady throughout these stages, whereas MBNL1 protein levels increased and exhibited a shift in isoforms. MBNL1 has several alternatively spliced exons. Using RT-PCR, we determined that the inclusion of one of these, exon 5, decreases dramatically during cardiac morphogenesis. This developmental transition is conserved in mice. Functional analyses of MBNL1 isoforms containing or lacking exon 5-encoded sequences revealed that exon 5 is important for the regulation of the subcellular localization, RNA binding affinity, and alternative splicing activity of MBNL1 proteins. A second MBNL protein, MBNL2, is also expressed in the embryonic heart. We found that MBNL2 exon 5, which is paralogous to MBNL1 exon 5, is similarly regulated during embryonic heart development. Analysis of MBNL1 and MBNL2 transcripts in several embryonic tissues in chicken and mouse indicate that exon 5 alternative splicing is highly conserved and tissue-specific. Thus, we propose that conserved developmental stage- and tissue-specific alternative splicing of MBNL transcripts is an important mechanism by which MBNL activity is regulated during embryonic development.
Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Embrião de Galinha , Evolução Molecular , Éxons/genética , Coração/embriologia , Humanos , Camundongos , Especificidade de Órgãos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismoRESUMO
The CUG-BP and ETR-3 like factors (CELF) are a family of six highly conserved RNA-binding proteins that preferentially bind to UG-rich sequences. One of the key functions of these proteins is to mediate alternative splicing in a number of tissues, including brain, heart and muscle. To fully understand the function of CELF proteins, it is important to identify downstream targets of CELF proteins. In this communication, we report that neurofibromatosis type I (NF1) exon 23a is a novel target of CELF protein-mediated splicing regulation in neuron-like cells. NF1 regulates Ras signaling, and the isoform that excludes exon 23a shows 10 times greater ability to down-regulate Ras signaling than the isoform that includes exon 23a. Five of the six CELF proteins strongly suppress the inclusion of NF1 exon 23a. Over-expression or siRNA knockdown of these proteins in cell transfection experiments altered the levels of NF1 exon 23a inclusion. In vitro binding and splicing analyses demonstrate that CELF proteins block splicing through interfering with binding of U2AF(65). These studies, combined with our previous investigations demonstrating a role for Hu proteins and TIA-1/TIAR in controlling NF1 exon 23a inclusion, highlight the complex nature of regulation of this important alternative splicing event.
Assuntos
Processamento Alternativo , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Neurofibromina 1/genética , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Éxons , Células HeLa , Humanos , Dados de Sequência Molecular , Neurofibromina 1/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/química , Ratos , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AFRESUMO
The development of the valves and septa of the heart depends on the formation and remodeling of endocardial cushions. Here, we report that the alternative splicing regulator muscleblind-like 1 (MBNL1) exhibits a regionally restricted pattern of expression in canal region endocardium and ventricular myocardium during endocardial cushion development in chicken. Knockdown of MBNL1 in atrioventricular explants leads to a transforming growth factor beta-dependent increase in epithelial-mesenchymal transition (EMT) of endocardial cells. This reveals a novel role for MBNL1 during embryonic development, and represents the first evidence that an alternative splicing regulator is a key player in endocardial cushion development.
Assuntos
Coxins Endocárdicos/embriologia , Endocárdio/embriologia , Células Epiteliais/fisiologia , Mesoderma/embriologia , Proteínas de Ligação a RNA/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Processamento Alternativo/genética , Animais , Animais Geneticamente Modificados , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Embrião de Galinha , Regulação para Baixo/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Coxins Endocárdicos/efeitos dos fármacos , Coxins Endocárdicos/metabolismo , Endocárdio/efeitos dos fármacos , Endocárdio/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Mesoderma/fisiologia , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
During the transition from juvenile to adult life, the heart undergoes programmed remodeling at the levels of transcription and alternative splicing. Members of the CUG-BP and ETR-3-like factor (CELF) family have been implicated in driving developmental transitions in alternative splicing of cardiac transcripts during maturation of the heart. Here, we investigated the timing of the requirement for CELF activity in the postnatal heart using a previously described transgenic mouse model (MHC-CELFDelta). In MHC-CELFDelta mice, nuclear CELF activity has been disrupted specifically in the heart by cardiac-specific expression of a dominant negative CELF protein. Longitudinal analyses of two lines of MHC-CELFDelta mice with differing levels of dominant negative protein expression demonstrate that CELF splicing activity is required for healthy cardiac function during juvenile, but not adult, life. Cardiac function, chamber dilation, and heart size all recover with age in the mild line of MHC-CELFDelta mice without a loss of dominant negative protein expression or change in expression of endogenous CELF proteins or known CELF antagonists. This is the first example of a mouse model with genetically induced cardiomyopathy that spontaneously recovers without intervention. Our results suggest that CELF proteins are key players in the integrated gene expression program involved in postnatal cardiac remodeling and maturation.
Assuntos
Envelhecimento/metabolismo , Processamento Alternativo/fisiologia , Proteína delta de Ligação ao Facilitador CCAAT/biossíntese , Coração/crescimento & desenvolvimento , Proteínas Musculares/biossíntese , Miocárdio/metabolismo , Envelhecimento/genética , Animais , Proteína delta de Ligação ao Facilitador CCAAT/genética , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Tamanho do Órgão/fisiologia , Especificidade de Órgãos/fisiologiaRESUMO
The human selenoproteome is composed of approximately 25 selenoproteins, which cotranslationally incorporate selenocysteine, the 21st amino acid. Selenoprotein expression requires an unusual translation mechanism, as selenocysteine is encoded by the UGA stop codon. SECIS-binding protein 2 (SBP2) is an essential component of the selenocysteine insertion machinery. SBP2 is also the only factor known to differentiate among selenoprotein mRNAs, thereby modulating the relative expression of the individual selenoproteins. Here, we show that expression of SBP2 protein varies widely across tissues and cell types examined, despite previous observations of only modest variation in SBP2 mRNA levels. This discrepancy between SBP2 mRNA and protein levels implies translational regulation, which is often mediated via untranslated regions (UTRs) in regulated transcripts. We have identified multiple sequences in the SBP2 3' UTR that are highly conserved. The proximal short conserved region is GU rich and was subsequently shown to be a binding site for CUG-BP1. The distal half of the 3' UTR is largely conserved, and multiple proteins interact with this region. One of these proteins was identified as HuR. Both CUG-BP1 and HuR are members of the Turnover and Translation Regulatory RNA-Binding Protein family (TTR-RBP). Members of this protein family are linked by the common ability to rapidly effect gene expression through alterations in the stability and translatability of target mRNAs. The identification of CUG-BP1 and HuR as factors that bind to the SBP2 3' UTR suggests that TTR-RBPs play a role in the regulation of SBP2, which then dictates the expression of the selenoproteome.
Assuntos
Regulação da Expressão Gênica , Proteínas de Ligação a RNA/química , Regiões 3' não Traduzidas , Motivos de Aminoácidos , Animais , Núcleo Celular/metabolismo , Códon de Terminação , Citoplasma/metabolismo , Cavalos , Humanos , Mutação , Proteoma , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , RatosRESUMO
The CUG-BP and ETR-3-like factor (CELF) protein family has been implicated in the regulation of pre-mRNA alternative splicing, mRNA stability, and translation. Here we discuss the evolution and radiation of the CELF protein subfamilies, and report the cloning of the chicken CELF family members. In this study, we examined the embryonic expression patterns of the CELF family in the chick by in situ hybridization. We found that the tissue specificity reported for CELF proteins in the adult is established early during embryogenesis. Members of one subfamily, CUG-BP1 and ETR-3, are broadly expressed in the early embryo, while members of the second subfamily, CELF4-6, are restricted primarily to the nervous system. Expression patterns of individual CELF genes in several tissues, including the heart, liver, eye, and neural tube, exhibit distinct, yet overlapping, expression patterns. This suggests that different members of the CELF family play distinct functional roles during embryogenesis.