RESUMO
Tardigrades are microscopic animals renowned for their ability to withstand extreme conditions, including high doses of ionizing radiation (IR). To better understand their radio-resistance, we first characterized induction and repair of DNA double- and single-strand breaks after exposure to IR in the model species Hypsibius exemplaris. Importantly, we found that the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in tardigrades' radio-resistance. To identify novel tardigrade-specific genes involved, we next conducted a comparative transcriptomics analysis across three different species. In all three species, many DNA repair genes were among the most strongly overexpressed genes alongside a novel tardigrade-specific gene, which we named Tardigrade DNA damage Response 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and preserve chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade-specific gene conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping cope with high levels of DNA damage inflicted by IR.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA , Radiação Ionizante , Tardígrados , Transcriptoma , Tardígrados/genética , Tardígrados/metabolismo , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Dano ao DNA , Tolerância a Radiação/genéticaRESUMO
OBJECTIVES: The mineralocorticoid receptor (MR), a hormone-activated transcription factor, besides its role in controlling hydroelectrolytic homeostasis, exerts pro-adipogenic and anti-thermogenic effects, inhibiting mitochondrial-uncoupling protein UCP1 expression in brown adipocytes. The aim of this study was to gain insight into the molecular mechanisms by which MR participates in such metabolic regulation. METHODS: We evaluated in vivo MR effects on cold-induced UCP1 expression in MR-overexpressing mice. Expression profiles of several transcriptional coregulators were analyzed during differentiation of the brown adipocyte T37i cell line. Given that UCP1 expression is inversely controlled by catecholamines/retinoic acid and corticosteroids, we investigated the mechanisms of MR's inhibitory effect on UCP1 transcription in T37i cells. Chromatin immunoprecipitation (ChIP) experiments enabled us to explore MR interaction with UCP1 promoter regions. RESULTS: Cold-induced UCP1 expression was blunted in the brown fat of MR-overexpressing mice. Along with induction of increasing mRNA levels for specific adipocyte markers during T37i differentiation, MR coactivator transcript levels significantly increased in intermediate states of differentiation, whereas expression of MR corepressors transiently increased approximately 2-fold. Such a simultaneous transient peak in coregulator expression is consistent with physiologically relevant cooperation occurring during brown adipogenesis. ChIP demonstrated that, after retinoic acid stimulation and aldosterone exposure, MR and PPARγ concomitantly bind to specific UCP1 promoter motifs. CONCLUSION: Our studies demonstrate that MR exerts a pivotal metabolic role by controlling energy expenditure, and provide novel information on how MR participates in the regulation of brown adipocyte function.
Assuntos
Tecido Adiposo Marrom/metabolismo , Receptores de Mineralocorticoides/fisiologia , Termogênese/fisiologia , Transcrição Gênica/fisiologia , Proteína Desacopladora 1/genética , Adipócitos/química , Adipócitos/fisiologia , Adipogenia/fisiologia , Aldosterona/farmacologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Temperatura Baixa , Metabolismo Energético/fisiologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/análise , Receptores de Mineralocorticoides/genética , Termogênese/genética , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
Circular RNAs constitute a unique class of RNAs whose precise functions remain to be elucidated. In particular, cancer-associated chromosomal translocations can give rise to fusion circular RNAs that play a role in leukemia progression. However, how and when fusion circular RNAs are formed and whether they are being selected in cancer cells remains unknown. Here, we used CRISPR/Cas9 to generate physiological translocation models of NPM1-ALK fusion gene. We showed that, in addition to generating fusion proteins and activating specific oncogenic pathways, chromosomal translocation induced by CRISPR/Cas9 led to the formation of de novo fusion circular RNAs. Specifically, we could recover different classes of circular RNAs composed of different circularization junctions, mainly back-spliced species. In addition, we identified fusion circular RNAs identical to those found in related patient tumor cells providing evidence that fusion circular RNAs arise early after chromosomal formation and are not just a consequence of the oncogenesis process.
RESUMO
Mineralocorticoid receptor (MR) mediates the sodium-retaining action of aldosterone in the distal nephron. Herein, we decipher mechanisms by which hypotonicity increases MR expression in renal principal cells. We identify HuR (human antigen R), an mRNA-stabilizing protein, as an important posttranscriptional regulator of MR expression. Hypotonicity triggers a rapid and reversible nuclear export of HuR in renal KC3AC1 cells, as quantified by high-throughput microscopy. We also identify a key hairpin motif in the 3'-untranslated region of MR transcript, pivotal for the interaction with HuR and its stabilizing function. Next, we show that hypotonicity increases MR recruitment onto Sgk1 promoter, a well-known MR target gene, thereby enhancing aldosterone responsiveness. Our data shed new light on the crucial role of HuR as a stabilizing factor for the MR transcript and provide evidence for a short autoregulatory loop in which expression of a nuclear receptor transcriptionally regulating water and sodium balance is controlled by osmotic tone.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Rim/metabolismo , Mineralocorticoides/metabolismo , Pressão Osmótica/fisiologia , Proteínas de Ligação a RNA/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transdução de Sinais/fisiologia , Regiões 3' não Traduzidas/genética , Transporte Ativo do Núcleo Celular/genética , Aldosterona/metabolismo , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/metabolismo , Rim/fisiologia , Osmose/fisiologia , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/genéticaRESUMO
Aldosterone and the Mineralocorticoid Receptor (MR) control hydroelectrolytic homeostasis and alterations of mineralocorticoid signaling pathway are involved in the pathogenesis of numerous human diseases, justifying the need to decipher molecular events controlling MR expression level. Here, we show in renal cells that the RNA-Binding Protein, Human antigen R (HuR), plays a central role in the editing of MR transcript as revealed by a RNA interference strategy. We identify a novel Δ6 MR splice variant, which lacks the entire exon 6, following a HuR-dependent exon skipping event. Using isoform-specific TaqMan probes, we show that Δ6 MR variant is expressed in all MR-expressing tissues and cells and demonstrate that extracelullar tonicity regulates its renal expression. More importantly, this splice variant exerts dominant-negative effects on transcriptional activity of the full-length MR protein. Collectively, our data highlight a crucial role of HuR as a master posttranscriptional regulator of MR expression in response to osmotic stress. We demonstrate that hypotonicity, not only enhances MR mRNA stability, but also decreases expression of the Δ6 MR variant, thus potentiating renal MR signaling. These findings provide compelling evidence for an autoregulatory feedback loop for the control of sodium homeostasis through posttranscriptional events, likely relevant in renal pathophysiological situations.
Assuntos
Processamento Alternativo , Proteína Semelhante a ELAV 1/genética , Rim/metabolismo , Osmorregulação/genética , Receptores de Mineralocorticoides/genética , Sódio na Dieta/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Proteína Semelhante a ELAV 1/metabolismo , Éxons , Retroalimentação Fisiológica , Furosemida/farmacologia , Homeostase/genética , Humanos , Íntrons , Rim/efeitos dos fármacos , Camundongos , Modelos Moleculares , Concentração Osmolar , Pressão Osmótica , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores de Mineralocorticoides/metabolismo , Sódio na Dieta/administração & dosagem , Homologia Estrutural de Proteína , Privação de Água , Intoxicação por Água/genética , Intoxicação por Água/metabolismo , Intoxicação por Água/fisiopatologiaRESUMO
Gene editing techniques have been extensively used to attempt to model recurrent genomic rearrangements found in tumor cells. These methods involve the induction of double-strand breaks at endogenous loci followed by the identification of breakpoint junctions within a population, which typically arise by nonhomologous end joining. The low frequency of these events, however, has hindered the cloning of cells with the desired rearrangement before oncogenic transformation. Here we present a strategy combining CRISPR-Cas9 technology and homology-directed repair to allow for the selection of human mesenchymal stem cells harboring the oncogenic translocation EWSR1-WT1 found in the aggressive desmoplastic small round cell tumor. The expression of the fusion transcript is under the control of the endogenous EWSR1 promoter and, importantly, can be conditionally expressed using Cre recombinase. This method is easily adapted to generate any cancer-relevant rearrangement.
Assuntos
Edição de Genes/métodos , Proteína EWS de Ligação a RNA/genética , Translocação Genética , Proteínas WT1/genética , Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras GenéticasRESUMO
TPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3'-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein-protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function.
Assuntos
Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Regiões 3' não Traduzidas , Animais , Células COS , Técnicas de Cultura de Células , Chlorocebus aethiops , AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endorribonucleases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Tristetraprolina/metabolismoRESUMO
Aldosterone regulates sodium homeostasis by activating the mineralocorticoid receptor (MR), a member of the nuclear receptor superfamily. Hyperaldosteronism leads todeleterious effects on the kidney, blood vessels, and heart. Although steroidal antagonists such as spironolactone and eplerenone are clinically useful for the treatment of cardiovascular diseases, they are associated with several side effects. Finerenone, a novel nonsteroidal MR antagonist, is presently being evaluated in two clinical phase IIb trials. Here, we characterized the molecular mechanisms of action of finerenone and spironolactone at several key steps of the MR signaling pathway. Molecular modeling and mutagenesis approaches allowed identification of Ser-810 and Ala-773 as key residues for the high MR selectivity of finerenone. Moreover, we showed that, in contrast to spironolactone, which activates the S810L mutant MR responsible for a severe form of early onset hypertension, finerenone displays strict antagonistic properties. Aldosterone-dependent phosphorylation and degradation of MR are inhibited by both finerenone and spironolactone. However, automated quantification of MR subcellular distribution demonstrated that finerenone delays aldosterone-induced nuclear accumulation of MR more efficiently than spironolactone. Finally, chromatin immunoprecipitation assays revealed that, as opposed to spironolactone, finerenone inhibits MR, steroid receptor coactivator-1, and RNA polymerase II binding at the regulatory sequence of the SCNN1A gene and also remarkably reduces basal MR and steroid receptor coactivator-1 recruitment, unraveling a specific and unrecognized inactivating mechanism on MR signaling. Overall, our data demonstrate that the highly potent and selective MR antagonist finerenone specifically impairs several critical steps of the MR signaling pathway and therefore represents a promising new generation MR antagonist.
Assuntos
Aldosterona/farmacologia , Naftiridinas/farmacologia , Coativador 1 de Receptor Nuclear/metabolismo , Receptores de Mineralocorticoides/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Western Blotting , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Células HEK293 , Humanos , Cinética , Microscopia de Fluorescência , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Receptores de Mineralocorticoides/genética , Transdução de Sinais/efeitos dos fármacos , Espironolactona/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
Aldosterone exerts its effects mainly by activating the mineralocorticoid receptor (MR), a transcription factor that regulates gene expression through complex and dynamic interactions with coregulators and transcriptional machinery, leading to fine-tuned control of vectorial ionic transport in the distal nephron. To identify genome-wide aldosterone-regulated MR targets in human renal cells, we set up a chromatin immunoprecipitation (ChIP) assay by using a specific anti-MR antibody in a differentiated human renal cell line expressing green fluorescent protein (GFP)-MR. This approach, coupled with high-throughput sequencing, allowed identification of 974 genomic MR targets. Computational analysis identified an MR response element (MRE) including single or multiple half-sites and palindromic motifs in which the AGtACAgxatGTtCt sequence was the most prevalent motif. Most genomic MR-binding sites (MBSs) are located >10 kb from the transcriptional start sites of target genes (84%). Specific aldosterone-induced recruitment of MR on the first most relevant genomic sequences was further validated by ChIP-quantitative (q)PCR and correlated with concomitant and positive aldosterone-activated transcriptional regulation of the corresponding gene, as assayed by RT-qPCR. It was notable that most MBSs lacked MREs but harbored DNA recognition motifs for other transcription factors (FOX, EGR1, AP1, PAX5) suggesting functional interaction. This work provides new insights into aldosterone MR-mediated renal signaling and opens relevant perspectives for mineralocorticoid-related pathophysiology.
Assuntos
Aldosterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Rim/metabolismo , Receptores de Mineralocorticoides/biossíntese , Elementos de Resposta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Aldosterona/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Humanos , Rim/citologia , Receptores de Mineralocorticoides/genética , Transdução de Sinais/fisiologiaRESUMO
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant inheritable vascular dysplasia caused by mutations in genes encoding either endoglin or activin receptor-like kinase-1 (ALK1). Functional significance of endoglin missense mutations remains largely unknown leading to a difficult discrimination between polymorphisms and pathogenic mutations. In order to study the functional significance of endoglin mutations and to help HHT1 diagnosis, we developed a cellular assay based on the ability of endoglin to enhance ALK1 response to bone morphogenetic protein 9 (BMP9). We generated and characterized 31 distinct ENG mutants reproducing human HHT1 missense mutations identified in patients of the Molecular Genetics Department in Lyon. We found that 16 mutants behaved like wild-type (WT) endoglin, and thus corresponded to benign rare variants. The 15 other variants showed defects in BMP9 response and were identified as pathogenic mutations. Interestingly, two mutants (S278P and F282V) had lost their ability to bind BMP9, identifying two crucial amino acids for BMP9 binding to endoglin. For all the others, the functional defect was correlated with a defective trafficking to the cell surface associated with retention in the endoplasmic reticulum. Further, we demonstrated that some intracellular mutants dimerized with WT endoglin and impaired its cell-surface expression thus acting as dominant-negatives. Taken together, we show that endoglin loss-of-function can result from different mechanisms in HHT1 patients. We also provide a diagnostic tool helping geneticists in screening for novel or conflicting ENG mutations.
Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Mutação , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Telangiectasia Hemorrágica Hereditária/genética , Telangiectasia Hemorrágica Hereditária/metabolismo , Animais , Antígenos CD/química , Linhagem Celular , Membrana Celular/metabolismo , Endoglina , Expressão Gênica , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/metabolismo , Humanos , Camundongos , Fenótipo , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Receptores de Superfície Celular/química , Telangiectasia Hemorrágica Hereditária/diagnósticoRESUMO
The mineralocorticoid receptor (MR) mediates the Na(+)-retaining action of aldosterone. MR is highly expressed in the distal nephron, which is submitted to intense variations in extracellular fluid tonicity generated by the corticopapillary gradient. We previously showed that post-transcriptional events control renal MR abundance. Here, we report that hypertonicity increases expression of the mRNA-destabilizing protein Tis11b, a member of the tristetraprolin/ZFP36 family, and thereby, decreases MR expression in renal KC3AC1 cells. The 3'-untranslated regions (3'-UTRs) of human and mouse MR mRNA, containing several highly conserved adenylate/uridylate-rich elements (AREs), were cloned downstream of a reporter gene. Luciferase activities of full-length or truncated MR Luc-3'-UTR mutants decreased drastically when cotransfected with Tis11b plasmid, correlating with an approximately 50% shorter half-life of ARE-containing transcripts. Using site-directed mutagenesis and RNA immunoprecipitation, we identified a crucial ARE motif within the MR 3'-UTR, to which Tis11b must bind for destabilizing activity. Coimmunoprecipitation experiments suggested that endogenous Tis11b physically interacts with MR mRNA in KC3AC1 cells, and Tis11b knockdown prevented hypertonicity-elicited repression of MR. Moreover, hypertonicity blunted aldosterone-stimulated expression of glucocorticoid-induced leucine-zipper protein and the α-subunit of the epithelial Na(+) channel, supporting impaired MR signaling. Challenging the renal osmotic gradient by submitting mice to water deprivation, diuretic administration, or high-Na(+) diet increased renal Tis11b and decreased MR expression, particularly in the cortex, thus establishing a mechanistic pathway for osmotic regulation of MR expression in vivo. Altogether, we uncovered a mechanism by which renal MR expression is regulated through mRNA turnover, a post-transcriptional control that seems physiologically relevant.