RESUMO
The causes of Alzheimer's disease (AD) are poorly understood, although many genes are known to be involved in this pathology. To gain insights into the underlying molecular mechanisms, it is essential to identify the relationships between individual AD genes. Previous work has shown that the splice variant E of KLC1 (KLC1_vE) promotes AD, and that the CELF1 gene, which encodes an RNA-binding protein involved in splicing regulation, is at a risk locus for AD. Here, we identified a functional link between CELF1 and KLC1 in AD pathogenesis. Transcriptomic data from human samples from different ethnic groups revealed that CELF1 mRNA levels are low in AD brains, and the splicing pattern of KLC1 is strongly correlated with CELF1 expression levels. Specifically, KLC1_vE is negatively correlated with CELF1. Depletion and overexpression experiments in cultured cells demonstrated that the CELF1 protein down-regulates KLC1_vE. In a cross-linking and immunoprecipitation sequencing (CLIP-seq) database, CELF1 directly binds to KLC1 RNA, following which it likely modulates terminal exon usage, hence KLC1_vE formation. These findings reveal a new pathogenic pathway where a risk allele of CELF1 is associated with reduced CELF1 expression, which up-regulates KLC1_vE to promote AD.
Assuntos
Processamento Alternativo , Doença de Alzheimer , Proteínas CELF1 , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Proteínas CELF1/metabolismo , Proteínas CELF1/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Loss of von Hippel-Lindau protein pVHL function promotes VHL diseases, including sporadic and inherited clear cell Renal Cell Carcinoma (ccRCC). Mechanisms controlling pVHL function and regulation, including folding and stability, remain elusive. Here, we have identified the conserved cochaperone prefoldin complex in a screen for pVHL interactors. The prefoldin complex delivers non-native proteins to the chaperonin T-complex-protein-1-ring (TRiC) or Cytosolic Chaperonin containing TCP-1 (CCT) to assist folding of newly synthesized polypeptides. The pVHL-prefoldin interaction was confirmed in human cells and prefoldin knock-down reduced pVHL expression levels. Furthermore, when pVHL was expressed in Schizosaccharomyces pombe, all prefoldin mutants promoted its aggregation. We mapped the interaction of prefoldin with pVHL at the exon2-exon3 junction encoded region. Low levels of the PFDN3 prefoldin subunit were associated with poor survival in ccRCC patients harboring VHL mutations. Our results link the prefoldin complex with pVHL folding and this may impact VHL diseases progression.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Neoplasias Renais/genética , Chaperonas Moleculares/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Chaperonina com TCP-1 , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Chaperonas Moleculares/genética , Mutação , Ligação Proteica/genética , Dobramento de Proteína , Proteólise , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
CD44 mRNA contains nine consecutive cassette exons, v2 to v10. Upon alternative splicing, several isoforms are produced with different impacts on tumor biology. Here, we demonstrate the involvement of the RNA-binding proteins CELF1 and ELAVL1 in the control of CD44 splicing. We show by FRET-FLIM that these proteins directly interact in the nucleus. By combining RNAi-mediated depletion and exon array hybridization in HeLa cells, we observe that the exons v7 to v10 of CD44 are highly sensitive to CELF1 and ELAVL1 depletion. We confirm by RT-PCR that CELF1 and ELAVL1 together stimulate the inclusion of these exons in CD44 mRNA. Finally, we show in eight different tumor types that high expression of CELF1 and/or ELAVL1 is correlated with the inclusion of CD44 variable exons. These data point to functional interactions between CELF1 and ELAVL1 in the control of CD44 splicing in human cancers.
Assuntos
Processamento Alternativo , Receptores de Hialuronatos , Proteínas CELF1 , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Éxons/genética , Células HeLa , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Opacification of the ocular lens, termed cataract, is a common cause of blindness. To become transparent, lens fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1 post-transcriptionally controls key genes to regulate lens fiber cell differentiation. Celf1-targeted knockout mice and celf1-knockdown zebrafish and Xenopus morphants have severe eye defects/cataract. Celf1 spatiotemporally down-regulates the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 by interacting with its 5' UTR and mediating translation inhibition. Celf1 deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1 directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1 controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2 mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1-regulated molecular mechanisms in lens development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.
Assuntos
Proteínas CELF1/fisiologia , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Endodesoxirribonucleases/genética , Cristalino/crescimento & desenvolvimento , Proteínas de Ligação a RNA/fisiologia , Proteínas de Xenopus/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Cristalino/citologia , Cristalino/metabolismo , Camundongos , Xenopus laevis , Peixe-ZebraRESUMO
BACKGROUND: Ocular lens clouding is termed as cataract, which depending on the onset, is classified as congenital or age-related. Developing new cataract treatments requires new models. Thus far, Xenopus embryos have not been evaluated as a system for studying cataract. RESULTS: We characterized the developmental process of lens formation in Xenopus laevis tailbuds and tadpoles, and we disrupted the orthologues of three mammalian cataract-linked genes in F0 by CRISPR/Cas9. We assessed the consequences of gene inactivation by combining external examination with histochemical analyses and functional vision assays. Inactivating the key metazoan eye development transcription factor gene pax6 produces a strong eye phenotype including an absence of eye tissue. Inactivating the genes for gap-junction protein and a nuclease, gja8 and dnase2b, produces lens defects that share several features of human cataracts, including impaired vision acuity, nuclei retention in lens fiber cells, and actin fibers disorganization. We tested the potential improvement of the visual acuity of gja8 crispant tadpoles upon treatment with the molecular chaperone 4-phenylbutyrate. CONCLUSION: Xenopus is a valuable model organism to understand the molecular pathology of congenital eye defects, including cataracts, and to screen molecules with a potential to prevent or reverse cataracts.
Assuntos
Xenopus laevis/fisiologia , Animais , Catarata/fisiopatologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Cristalino/fisiologiaRESUMO
The homeodomain transcription factors (TFs) Pax6 (OMIM: 607108) and Prox1 (OMIM: 601546) critically regulate gene expression in lens development. While PAX6 mutations in humans can cause cataract, aniridia, microphthalmia, and anophthalmia, among other defects, Prox1 deletion in mice causes severe lens abnormalities, in addition to other organ defects. Furthermore, the optimal dosage/spatiotemporal expression of these key TFs is essential for development. In lens development, Pax6 expression is elevated in cells of the anterior epithelium compared to fiber cells, while Prox1 exhibits the opposite pattern. Whether post-transcriptional regulatory mechanisms control these precise TF expression patterns is unknown. Here, we report the unprecedented finding that the cataract-linked RNA-binding protein (RBP), Celf1 (OMIM: 601074), post-transcriptionally regulates Pax6 and Prox1 protein expression in lens development. Immunostaining shows that Celf1 lens-specific conditional knockout (Celf1cKO) mice exhibit abnormal elevation of Pax6 protein in fiber cells and abnormal Prox1 protein levels in epithelial cells-directly opposite to their normal expression patterns in development. Furthermore, RT-qPCR shows no change in Pax6 and Prox1 transcript levels in Celf1cKO lenses, suggesting that Celf1 regulates these TFs on the translational level. Indeed, RNA-immunoprecipitation assays using Celf1 antibody indicate that Celf1 protein binds to Pax6 and Prox1 transcripts. Furthermore, reporter assays in Celf1 knockdown and Celf1-overexpression cells demonstrate that Celf1 negatively controls Pax6 and Prox1 translation via their 3' UTRs. These data define a new mechanism of RBP-based post-transcriptional regulation that enables precise control over spatiotemporal expression of Pax6 and Prox1 in lens development, thereby uncovering a new etiological mechanism for Celf1 deficiency-based cataract.
Assuntos
Proteínas CELF1/genética , Catarata/genética , Proteínas de Homeodomínio/genética , Cristalino/metabolismo , Fator de Transcrição PAX6/genética , Proteínas Supressoras de Tumor/genética , Animais , Proteínas CELF1/antagonistas & inibidores , Proteínas CELF1/deficiência , Catarata/patologia , Diferenciação Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Cristalino/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genéticaRESUMO
Prokaryotes and eukaryotes evolved relatively similar RNA-based molecular mechanisms to fight potentially deleterious nucleic acids coming from phages, transposons, or viruses. Short RNAs guide effector complexes toward their targets to be silenced or eliminated. These short immunity RNAs are transcribed from clustered loci. Unexpectedly and strikingly, bacterial and eukaryotic immunity RNA clusters share substantial functional and mechanistic resemblances in fighting nucleic acid intruders.
Assuntos
Proteínas Argonautas/imunologia , Sistemas CRISPR-Cas/imunologia , Eucariotos/imunologia , Células Procarióticas/imunologia , RNA Interferente Pequeno/imunologia , Proteínas Argonautas/genética , Bacteriófagos/genética , Bacteriófagos/patogenicidade , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Elementos de DNA Transponíveis/imunologia , Eucariotos/genética , Eucariotos/virologia , Plasmídeos/química , Plasmídeos/imunologia , Células Procarióticas/virologia , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/imunologia , RNA Interferente Pequeno/genéticaRESUMO
Regulation of alternative splicing is an important process for cell differentiation and development. Down-regulation of Ptbp1, a regulatory RNA-binding protein, leads to developmental skin defects in Xenopus laevis. To identify Ptbp1-dependent splicing events potentially related to the phenotype, we conducted RNAseq experiments following Ptbp1 depletion. We systematically compared exon-centric and junction-centric approaches to detect differential splicing events. We showed that the junction-centric approach performs far better than the exon-centric approach in Xenopus laevis. We carried out the same comparisons using simulated data in human, which led us to propose that the better performances of the junction-centric approach in Xenopus laevis essentially relies on an incomplete exonic annotation associated with a correct transcription unit annotation. We assessed the capacity of the exon-centric and junction-centric approaches to retrieve known and to discover new Ptbp1-dependent splicing events. Notably, the junction-centric approach identified Ptbp1-controlled exons in agfg1, itga6, actn4, and tpm4 mRNAs, which were independently confirmed. We conclude that the junction-centric approach allows for a more complete and informative description of splicing events, and we propose that this finding might hold true for other species with incomplete annotations.
Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/genética , Animais , Simulação por Computador , Embrião não Mamífero/metabolismo , Éxons/genética , Biblioteca Gênica , Modelos Genéticos , Anotação de Sequência Molecular , Morfolinos/farmacologia , RNA Mensageiro/genética , Alinhamento de Sequência , Análise de Sequência de RNA , Xenopus laevis/embriologiaRESUMO
In humans, genetic diseases affecting skin integrity (genodermatoses) are generally caused by mutations in a small number of genes that encode structural components of the dermal-epidermal junctions. In this article, we first show that inactivation of both exosc9, which encodes a component of the RNA exosome, and ptbp1, which encodes an RNA-binding protein abundant in Xenopus embryonic skin, impairs embryonic Xenopus skin development, with the appearance of dorsal blisters along the anterior part of the fin. However, histological and electron microscopy analyses revealed that the two phenotypes are distinct. Exosc9 morphants are characterized by an increase in the apical surface of the goblet cells, loss of adhesion between the sensorial and peridermal layers, and a decrease in the number of ciliated cells within the blisters. Ptbp1 morphants are characterized by an altered goblet cell morphology. Gene expression profiling by deep RNA sequencing showed that the expression of epidermal and genodermatosis-related genes is also differentially affected in the two morphants, indicating that alterations in post-transcriptional regulations can lead to skin developmental defects through different routes. Therefore, the developing larval epidermis of Xenopus will prove to be a useful model for dissecting the post-transcriptional regulatory network involved in skin development and stability with significant implications for human diseases.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/genética , Técnicas de Silenciamento de Genes , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Pele/embriologia , Pele/patologia , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Nadadeiras de Animais/embriologia , Animais , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/patologia , Embrião não Mamífero/ultraestrutura , Epiderme/efeitos dos fármacos , Epiderme/patologia , Epiderme/ultraestrutura , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Hibridização In Situ , Morfolinos/farmacologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas de Xenopus/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by an expanded (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The toxic RNA transcripts produced from the mutant allele alter the function of RNA-binding proteins leading to the functional depletion of muscleblind-like (MBNL) proteins and an increase in steady state levels of CUG-BP1 (CUGBP-ETR-3 like factor 1, CELF1). The role of increased CELF1 in DM1 pathogenesis is well studied using genetically engineered mouse models. Also, as a potential therapeutic strategy, the benefits of increasing MBNL1 expression have recently been reported. However, the effect of reduction of CELF1 is not yet clear. In this study, we generated CELF1 knockout mice, which also carry an inducible toxic RNA transgene to test the effects of CELF1 reduction in RNA toxicity. We found that the absence of CELF1 did not correct splicing defects. It did however mitigate the increase in translational targets of CELF1 (MEF2A and C/EBPß). Notably, we found that loss of CELF1 prevented deterioration of muscle function by the toxic RNA, and resulted in better muscle histopathology. These data suggest that while reduction of CELF1 may be of limited benefit with respect to DM1-associated spliceopathy, it may be beneficial to the muscular dystrophy associated with RNA toxicity.
Assuntos
Fatores de Transcrição MEF2/metabolismo , Músculo Esquelético/patologia , Distrofia Miotônica/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas CELF1 , Modelos Animais de Doenças , Feminino , Humanos , Fatores de Transcrição MEF2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Distrofia Miotônica/patologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , TransgenesRESUMO
Defects in the development of the ocular lens can cause congenital cataracts. To understand the various etiologies of congenital cataracts, it is important to characterize the genes linked to this developmental defect and to define their downstream pathways that are relevant to lens biology and pathology. Deficiency or alteration of several RNA-binding proteins, including the conserved RBP Celf1 (CUGBP Elav-like family member 1), has been described to cause lens defects and early onset cataracts in animal models and/or humans. Celf1 is involved in various aspects of post-transcriptional gene expression control, including regulation of mRNA stability/decay, alternative splicing and translation. Celf1 germline knockout mice and lens conditional knockout (Celf1cKO) mice develop fully penetrant cataracts in early postnatal stages. To define the genome-level changes in RNA transcripts that result from Celf1 deficiency, we performed high-throughput RNA-sequencing of Celf1cKO mouse lenses at postnatal day (P) 0. Celf1cKO lenses exhibit 987 differentially expressed genes (DEGs) at cut-offs of >1.0 log2 counts per million (CPM), ≥±0.58 log2 fold-change and <0.05 false discovery rate (FDR). Of these, 327 RNAs were reduced while 660 were elevated in Celf1cKO lenses. The DEGs were subjected to various downstream analyses including iSyTE lens enriched-expression, presence in Cat-map, and gene ontology (GO) and representation of regulatory pathways. Further, a comparative analysis was done with previously generated microarray datasets on Celf1cKO lenses P0 and P6. Together, these analyses validated and prioritized several key genes mis-expressed in Celf1cKO lenses that are relevant to lens biology, including known cataract-linked genes (e.g., Cryab, Cryba2, Cryba4, Crybb1, Crybb2, Cryga, Crygb, Crygc, Crygd, Cryge, Crygf, Dnase2b, Bfsp1, Gja3, Pxdn, Sparc, Tdrd7, etc.) as well as novel candidates (e.g., Ell2 and Prdm16). Together, these data have defined the alterations in lens transcriptome caused by Celf1 deficiency, in turn uncovering downstream genes and pathways (e.g., structural constituents of eye lenses, lens fiber cell differentiation, etc.) associated with lens development and early-onset cataracts.
Assuntos
Proteínas CELF1 , Catarata , Cristalino , Animais , Humanos , Camundongos , Catarata/metabolismo , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Cristalino/metabolismo , Camundongos Knockout , RNA/metabolismo , Transcriptoma/genéticaRESUMO
Cataract, the opacification of the lens, is the leading cause of blindness worldwide. Although effective, cataract surgery is costly and can lead to complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined three-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization and biology. These organoids can be rapidly produced in large amounts. High-throughput RNA sequencing (RNA-seq) on specific organoid regions isolated via laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display a spatiotemporal expression of key lens genes, e.g., Jag1, Pax6, Prox1, Hsf4 and Cryab. Further, these lens organoids are amenable to the induction of opacities. Finally, the knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1, induces opacities in these organoids, indicating their use in rapidly screening for genes that are functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataracts.
Assuntos
Catarata , Cristalino , Animais , Camundongos , Cristalino/metabolismo , Catarata/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a RNA/metabolismo , Organoides/metabolismoRESUMO
The ocular lens, along with the cornea, focuses light on the retina to generate sharp images. Opacification of the lens, or cataract, is the leading cause of blindness worldwide. Presently, the best approach for cataract treatment is to surgically remove the diseased lens and replace it with an artificial implant. Although effective, this is costly and can have post-surgical complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and anti-cataract drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined 3-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization at the tissue, cellular and transcriptomic levels. These 3D cultured lens organoids can be rapidly produced in large amounts. High-throughput RNA-sequencing (RNA-seq) on specific organoid regions isolated by laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display spatiotemporal expression of key lens genes, e.g. , Jag1 , Pax6 , Prox1 , Hsf4 and Cryab . Further, these lens organoids are amenable to induction of opacities. Finally, knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1 , induces opacities in these organoids, indicating their use in rapidly screening for genes functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology, and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataract.
RESUMO
Targeted inactivations of RNA-binding proteins (RNA-BPs) can lead to huge phenotypical defects. These defects are due to the deregulation of certain mRNAs. However, we generally do not know, among the hundreds of mRNAs that are normally controlled by one RNA-BP, which are responsible for the observed phenotypes. Here, we designed an antisense oligonucleotide ("target protector") that masks the binding site of the RNA-BP CUG-binding protein 1 (CUGBP1) on the mRNA Suppressor of Hairless [Su(H)] that encodes a key player of Notch signaling. We showed that injecting this oligonucleotide into Xenopus embryos specifically inhibited the binding of CUGBP1 to the mRNA. This caused the derepression of Su(H) mRNA, the overexpression of Su(H) protein, and a phenotypic defect, loss of somitic segmentation, similar to that caused by a knockdown of CUGBP1. To demonstrate a causal relationship between Su(H) derepression and the segmentation defects, a rescue experiment was designed. Embryonic development was restored when the translation of Su(H) mRNA was re-repressed and the level of Su(H) protein was reduced to a normal level. This "target protector and rescue assay" demonstrates that the phenotypic defects associated with CUGBP1 inactivation in Xenopus are essentially due to the deregulation of Su(H) mRNA. Similar approaches may be largely used to uncover the links between the phenotype caused by the inactivation of an RNA-BP and the identity of the RNAs associated with that protein.
Assuntos
RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Proteínas CELF1 , Embrião não Mamífero , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Marcação de Genes/métodos , Modelos Biológicos , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , Ligação Proteica/fisiologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
The TP63 gene encodes the p63 transcription factor. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, ß, γ, and δ. The regulatory functions of p63 are isoform specific. The α isoform inhibits the epithelial-to-mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Using The Cancer Genome Atlas data, we observed that a higher proportion of the TP63γ isoform is a detrimental factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC) and is accompanied by the downregulation of desmosomal genes. By a correlation-based approach, we investigated the regulation of the production of the TP63γ isoform. According to our analysis of GTEx data, the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ in several tissues. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines, keratinocyte or Xenopus embryos leads to an increase in TP63γ isoform abundance. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63γ-specific exon. Intronic regions around the TP63γ-specific exon were sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Together, these results identify TP63γ as an unfavorable prognostic marker in HNSCC, and identify PTBP1 as the first direct splicing regulator of TP63γ production and a potential route toward TP63 isoform control. Significance: Quantifying TP63γ isoforms in patients' tumors could allow for the early detection of patients with HNSCC with an early loss in desmosomal gene expression and poor prognostic. The identification of PTBP1 as a transacting factor controlling TP63γ production may allow to control TP63γ expression.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Humanos , Fatores de Processamento de RNA/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Isoformas de Proteínas/genética , Processamento Alternativo/genética , Fatores de Transcrição/genética , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Proteínas Supressoras de Tumor/genética , Ribonucleoproteínas Nucleares Heterogêneas/genéticaRESUMO
Reverse genetics consists in the modification of the activity of a target gene to analyse the phenotypic consequences. Four main approaches are used towards this goal and will be explained in this review. Two of them are centred on genome alterations. Mutations produced by random chemical or insertional mutagenesis can be screened to recover only mutants in a specific gene of interest. Alternatively, these alterations may be specifically targeted on a gene of interest by HR (homologous recombination). The other two approaches are centred on mRNA. RNA interference is a powerful method to reduce the level of gene products, while MO (morpholino) antisense oligonucleotides alter mRNA metabolism or translation. Some model species, such as Drosophila, are amenable to most of these approaches, whereas other model species are restricted to one of them. For example, in mice and yeasts, gene targeting by HR is prevalent, whereas in Xenopus and zebrafish MO oligonucleotides are mainly used. Genome-wide collections of mutants or inactivated models obtained in several species by these approaches have been made and will help decipher gene functions in the post-genomic era.
Assuntos
Marcação de Genes , Animais , Animais Geneticamente Modificados , Genoma , Camundongos , Modelos Genéticos , Oligonucleotídeos Antissenso/genética , Interferência de RNA , Recombinação Genética , Peixe-ZebraRESUMO
Several different mRNAs can be produced from a given pre-mRNA by regulated alternative splicing, or as the result of deregulations that may lead to pathological states. Analysing splicing patterns is therefore of importance to describe and understand developmental programs, cellular responses to internal or external cues, or human diseases. We describe here a method, Pyrosequencing Analysis of Splicing Patterns (PASP), that combines RT-PCR and pyrosequencing of PCR products. We demonstrated that: (i) Ratios of two pure RNAs mixed in various proportions were accurately measured by PASP; (ii) PASP can be adapted to virtually any splicing event, including mutually exclusive exons, complex patterns of exon skipping or inclusion, and alternative 3' terminal exons; (iii) In extracts from different organs, the proportions of RNA isoforms measured by PASP reflected those measured by other methods. The PASP method is therefore reliable for analysing splicing patterns. All steps are done in 96-wells microplates, without gel electrophoresis, opening the way to high-throughput comparisons of RNA from several sources.
Assuntos
Processamento Alternativo , Análise de Sequência de RNA , Animais , Éxons , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , XenopusRESUMO
CUGBP1 is an RNA-binding protein controlling alternative splicing, mRNA translation and stability. In this work we used a motif scoring approach to identify putative CUGBP1 binding sites for genes located on the human chromosome 12. This allowed us to identify the gene CD9 as a presumptive target for CUGBP1-mediated regulation. In a number of cancers, the tetraspanin CD9 is down-regulated, an event correlated with a bad prognostic. Using a combination of biochemical approaches and CUGBP1 knockdown, we showed that CUGBP1 directly controls CD9 expression.
Assuntos
Antígenos CD/genética , Cromossomos Humanos Par 12/genética , Glicoproteínas de Membrana/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Sítios de Ligação , Proteínas CELF1 , Células Cultivadas , Biologia Computacional/métodos , Regulação para Baixo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Análise de Sequência de DNA/métodos , Tetraspanina 29RESUMO
CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1(-/-) mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1(-/-) males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1(-/-) males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.
Assuntos
Transtornos do Crescimento/congênito , Proteínas Mutantes/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espermatogênese/fisiologia , Animais , Apoptose , Biomarcadores , Proteínas CELF1 , Sobrevivência Celular , Cruzamentos Genéticos , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/citologia , Epididimo/anormalidades , Epididimo/citologia , Epididimo/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genótipo , Células Germinativas/citologia , Infertilidade Masculina , Masculino , Camundongos , Proteínas Mutantes/genética , Fenótipo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Testículo/anormalidades , Testículo/citologia , Testículo/embriologiaRESUMO
Neurodevelopment requires precise regulation of gene expression, including post-transcriptional regulatory events such as alternative splicing and mRNA translation. However, translational regulation of specific isoforms during neurodevelopment and the mechanisms behind it remain unknown. Using RNA-seq analysis of mouse neocortical polysomes, here we report translationally repressed and derepressed mRNA isoforms during neocortical neurogenesis whose orthologs include risk genes for neurodevelopmental disorders. We demonstrate that the translation of distinct mRNA isoforms of the RNA binding protein (RBP), Elavl4, in radial glia progenitors and early neurons depends on its alternative 5' UTRs. Furthermore, 5' UTR-driven Elavl4 isoform-specific translation depends on upstream control by another RBP, Celf1. Celf1 regulation of Elavl4 translation dictates development of glutamatergic neurons. Our findings reveal a dynamic interplay between distinct RBPs and alternative 5' UTRs in neuronal development and underscore the risk of post-transcriptional dysregulation in co-occurring neurodevelopmental disorders.