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1.
Nucleic Acids Res ; 51(2): 870-890, 2023 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-36620874

RESUMO

Hypoxia induces massive changes in alternative splicing (AS) to adapt cells to the lack of oxygen. Here, we identify the splicing factor SRSF6 as a key factor in the AS response to hypoxia. The SRSF6 level is strongly reduced in acute hypoxia, which serves a dual purpose: it allows for exon skipping and triggers the dispersal of nuclear speckles. Our data suggest that cells use dispersal of nuclear speckles to reprogram their gene expression during hypoxic adaptation and that SRSF6 plays an important role in cohesion of nuclear speckles. Down-regulation of SRSF6 is achieved through inclusion of a poison cassette exon (PCE) promoted by SRSF4. Removing the PCE 3' splice site using CRISPR/Cas9 abolishes SRSF6 reduction in hypoxia. Aberrantly high SRSF6 levels in hypoxia attenuate hypoxia-mediated AS and impair dispersal of nuclear speckles. As a consequence, proliferation and genomic instability are increased, while the stress response is suppressed. The SRSF4-PCE-SRSF6 hypoxia axis is active in different cancer types, and high SRSF6 expression in hypoxic tumors correlates with a poor prognosis. We propose that the ultra-conserved PCE of SRSF6 acts as a tumor suppressor and that its inclusion in hypoxia is crucial to reduce SRSF6 levels. This may prevent tumor cells from entering the metastatic route of hypoxia adaptation.


Assuntos
Hipóxia Celular , Salpicos Nucleares , Splicing de RNA , Fatores de Processamento de Serina-Arginina , Humanos , Processamento Alternativo , Éxons/genética , Fosfoproteínas/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Células HeLa
2.
Circ Res ; 130(1): 67-79, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34789007

RESUMO

BACKGROUND: Circular RNAs (circRNAs) are generated by back splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here, we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. METHODS AND RESULTS: Combining published endothelial RNA-sequencing data sets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localization and focal adhesion organization. Mechanistically, we identified the protein SDOS (syndesmos) to specifically interact with cZNF292 in endothelial cells by RNA-affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganization thereby recapitulating cZfp292 knockout phenotypes. CONCLUSIONS: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.


Assuntos
Proteínas de Ligação a DNA , Células Endoteliais , Endotélio Vascular , RNA Circular , Fatores de Transcrição , Animais , Humanos , Camundongos , Circulação Sanguínea , Proteínas de Ligação a DNA/genética , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Ligação Proteica , RNA Circular/genética , RNA Circular/metabolismo , Sindecana-4/metabolismo , Fatores de Transcrição/genética
3.
EMBO Rep ; 23(6): e54157, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35527520

RESUMO

Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA ) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.


Assuntos
RNA Longo não Codificante , Processamento Alternativo , Animais , Células Endoteliais/metabolismo , Camundongos , Permeabilidade , Isoformas de Proteínas/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Junções Íntimas/metabolismo
4.
Circ Res ; 126(9): 1127-1145, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32324505

RESUMO

The advent of deep sequencing technologies led to the identification of a considerable amount of noncoding RNA transcripts, which are increasingly recognized for their functions in controlling cardiovascular diseases. MicroRNAs have already been studied for a decade, leading to the identification of several vasculoprotective and detrimental species, which might be considered for therapeutic targeting. Other noncoding RNAs such as circular RNAs, YRNAs, or long noncoding RNAs are currently gaining increasing attention, and first studies provide insights into their functions as mediators or antagonists of vascular diseases in vivo. The present review article will provide an overview of the different types of noncoding RNAs controlling the vasculature and focus on the developing field of long noncoding RNAs.


Assuntos
MicroRNAs/genética , RNA Circular/genética , RNA Longo não Codificante/genética , Doenças Vasculares/genética , Animais , Regulação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , RNA Circular/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , Remodelação Vascular
5.
Am J Respir Crit Care Med ; 202(10): 1445-1457, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634060

RESUMO

Rationale: Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Their role in pulmonary arterial hypertension (PAH) remains to be explored.Objectives: To elucidate the role of TYKRIL (tyrosine kinase receptor-inducing lncRNA) as a regulator of p53/ PDGFRß (platelet-derived growth factor receptor ß) signaling pathway and to investigate its role in PAH.Methods: Pericytes and pulmonary arterial smooth muscle cells exposed to hypoxia and derived from patients with idiopathic PAH were analyzed with RNA sequencing. TYKRIL knockdown was performed in above-mentioned human primary cells and in precision-cut lung slices derived from patients with PAH.Measurements and Main Results: Using RNA sequencing data, TYKRIL was identified to be consistently upregulated in pericytes and pulmonary arterial smooth muscles cells exposed to hypoxia and derived from patients with idiopathic PAH. TYKRIL knockdown reversed the proproliferative (n = 3) and antiapoptotic (n = 3) phenotype induced under hypoxic and idiopathic PAH conditions. Owing to the poor species conservation of TYKRIL, ex vivo studies were performed in precision-cut lung slices from patients with PAH. Knockdown of TYKRIL in precision-cut lung slices decreased the vascular remodeling (n = 5). The number of proliferating cell nuclear antigen-positive cells in the vessels was decreased and the number of terminal deoxynucleotide transferase-mediated dUTP nick end label-positive cells in the vessels was increased in the LNA (locked nucleic acid)-treated group compared with control. Expression of PDGFRß, a key player in PAH, was found to strongly correlate with TYKRIL expression in the patient samples (n = 12), and TYKRIL knockdown decreased PDGFRß expression (n = 3). From the transcription factor-screening array, it was observed that TYKRIL knockdown increased the p53 activity, a known repressor of PDGFRß. RNA immunoprecipitation using various p53 mutants demonstrated that TYKRIL binds to the N-terminal of p53 (an important region for p300 interaction with p53). The proximity ligation assay revealed that TYKRIL interferes with the p53-p300 interaction (n = 3) and regulates p53 nuclear translocation.Conclusions: TYKRIL plays an important role in PAH by regulating the p53/PDGFRß axis.


Assuntos
Expressão Gênica , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Proteínas Tirosina Quinases/genética , RNA Longo não Codificante , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
6.
Circulation ; 139(10): 1320-1334, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30586743

RESUMO

BACKGROUND: The majority of the human genome comprises noncoding sequences, which are in part transcribed as long noncoding RNAs (lncRNAs). lncRNAs exhibit multiple functions, including the epigenetic control of gene expression. In this study, the effect of the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) on atherosclerosis was examined. METHODS: The effect of MALAT1 on atherosclerosis was determined in apolipoprotein E-deficient (Apoe-/-) MALAT1-deficient (Malat1-/-) mice that were fed with a high-fat diet and by studying the regulation of MALAT1 in human plaques. RESULTS: Apoe-/- Malat1-/- mice that were fed a high-fat diet showed increased plaque size and infiltration of inflammatory CD45+ cells compared with Apoe-/- Malat1+/+ control mice. Bone marrow transplantation of Apoe-/- Malat1-/- bone marrow cells in Apoe-/- Malat1+/+ mice enhanced atherosclerotic lesion formation, which suggests that hematopoietic cells mediate the proatherosclerotic phenotype. Indeed, bone marrow cells isolated from Malat1-/- mice showed increased adhesion to endothelial cells and elevated levels of proinflammatory mediators. Moreover, myeloid cells of Malat1-/- mice displayed enhanced adhesion to atherosclerotic arteries in vivo. The anti-inflammatory effects of MALAT1 were attributed in part to reduction of the microRNA miR-503. MALAT1 expression was further significantly decreased in human plaques compared with normal arteries and was lower in symptomatic versus asymptomatic patients. Lower levels of MALAT1 in human plaques were associated with a worse prognosis. CONCLUSIONS: Reduced levels of MALAT1 augment atherosclerotic lesion formation in mice and are associated with human atherosclerotic disease. The proatherosclerotic effects observed in Malat1-/- mice were mainly caused by enhanced accumulation of hematopoietic cells.


Assuntos
Aorta/metabolismo , Aortite/metabolismo , Aterosclerose/metabolismo , Células da Medula Óssea/metabolismo , Hematopoese , Placa Aterosclerótica , RNA Longo não Codificante/metabolismo , Animais , Aorta/patologia , Aortite/genética , Aortite/patologia , Aterosclerose/genética , Aterosclerose/patologia , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Estudos de Casos e Controles , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Transdução de Sinais
7.
Circ Res ; 117(10): 884-90, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26377962

RESUMO

RATIONALE: Circular RNAs (circRNAs) are noncoding RNAs generated by back splicing. Back splicing has been considered a rare event, but recent studies suggest that circRNAs are widely expressed. However, the expression, regulation, and function of circRNAs in vascular cells is still unknown. OBJECTIVE: Here, we characterize the expression, regulation, and function of circRNAs in endothelial cells. METHODS AND RESULTS: Endothelial circRNAs were identified by computational analysis of ribo-minus RNA generated from human umbilical venous endothelial cells cultured under normoxic or hypoxic conditions. Selected circRNAs were biochemically characterized, and we found that the majority of them lacks polyadenylation, is resistant to RNase R digestion and localized to the cytoplasm. We further validated the hypoxia-induced circRNAs cZNF292, cAFF1, and cDENND4C, as well as the downregulated cTHSD1 by reverse transcription polymerase chain reaction in cultured endothelial cells. Cloning of cZNF292 validated the predicted back splicing of exon 4 to a new alternative exon 1A. Silencing of cZNF292 inhibited cZNF292 expression and reduced tube formation and spheroid sprouting of endothelial cells in vitro. The expression of pre-mRNA or mRNA of the host gene was not affected by silencing of cZNF292. No validated microRNA-binding sites for cZNF292 were detected in Argonaute high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation data sets, suggesting that cZNF292 does not act as a microRNA sponge. CONCLUSIONS: We show that the majority of the selected endothelial circRNAs fulfill all criteria of bona fide circRNAs. The circRNA cZNF292 exhibits proangiogenic activities in vitro. These data suggest that endothelial circRNAs are regulated by hypoxia and have biological functions.


Assuntos
Proteínas de Transporte/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas do Tecido Nervoso/genética , RNA/genética , Proteínas de Transporte/metabolismo , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Análise por Conglomerados , Simulação por Computador , Éxons , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , RNA/metabolismo , Interferência de RNA , Splicing de RNA , RNA Circular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
8.
Genes Dev ; 23(14): 1650-64, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19605687

RESUMO

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity.


Assuntos
Proteínas do Complexo SMN/metabolismo , Trypanosoma brucei brucei/metabolismo , Proteínas Centrais de snRNP/metabolismo , Sequência de Aminoácidos , Animais , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN/química , Alinhamento de Sequência , Spliceossomos/metabolismo , Proteínas Centrais de snRNP/química
10.
JCI Insight ; 8(5)2023 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-36883566

RESUMO

The adult mammalian heart has limited regenerative capacity, while the neonatal heart fully regenerates during the first week of life. Postnatal regeneration is mainly driven by proliferation of preexisting cardiomyocytes and supported by proregenerative macrophages and angiogenesis. Although the process of regeneration has been well studied in the neonatal mouse, the molecular mechanisms that define the switch between regenerative and nonregenerative cardiomyocytes are not well understood. Here, using in vivo and in vitro approaches, we identified the lncRNA Malat1 as a key player in postnatal cardiac regeneration. Malat1 deletion prevented heart regeneration in mice after myocardial infarction on postnatal day 3 associated with a decline in cardiomyocyte proliferation and reparative angiogenesis. Interestingly, Malat1 deficiency increased cardiomyocyte binucleation even in the absence of cardiac injury. Cardiomyocyte-specific deletion of Malat1 was sufficient to block regeneration, supporting a critical role of Malat1 in regulating cardiomyocyte proliferation and binucleation, a landmark of mature nonregenerative cardiomyocytes. In vitro, Malat1 deficiency induced binucleation and the expression of a maturation gene program. Finally, the loss of hnRNP U, an interaction partner of Malat1, induced similar features in vitro, suggesting that Malat1 regulates cardiomyocyte proliferation and binucleation by hnRNP U to control the regenerative window in the heart.


Assuntos
Coração , Ribonucleoproteínas Nucleares Heterogêneas Grupo U , Infarto do Miocárdio , Miócitos Cardíacos , RNA Longo não Codificante , Regeneração , Animais , Camundongos , Coração/fisiologia , Coração/fisiopatologia , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/fisiopatologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiologia , Mamíferos , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Regeneração/genética , Regeneração/fisiologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
11.
Int J Med Microbiol ; 302(4-5): 221-4, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22964417

RESUMO

The parasitic unicellular trypanosomatids are responsible for several fatal diseases in humans and livestock. Regarding their biochemistry and molecular biology, they possess a multitude of special features such as polycistronic transcription of protein-coding genes. The resulting long primary transcripts need to be processed by coupled trans-splicing and polyadenylation reactions, thereby generating mature mRNAs. Catalyzed by a large ribonucleoprotein complex termed the spliceosome, trans-splicing attaches a 39-nucleotide leader sequence, which is derived from the Spliced Leader (SL) RNA, to each protein-coding gene. Recent genome-wide studies demonstrated that alternative trans-splicing increases mRNA and protein diversity in these organisms. In this mini-review we give an overview of the current state of research on trans-splicing.


Assuntos
RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Trans-Splicing , Trypanosoma/genética , Processamento Alternativo , Poliadenilação , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA de Protozoário/genética , RNA Líder para Processamento/genética , RNA Líder para Processamento/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Transcrição Gênica , Trypanosoma/metabolismo
13.
Noncoding RNA ; 8(4)2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35893232

RESUMO

Besides transcription, RNA decay accounts for a large proportion of regulated gene expression and is paramount for cellular functions. Classical RNA surveillance pathways, like nonsense-mediated decay (NMD), are also implicated in the turnover of non-mutant transcripts. Whereas numerous protein factors have been assigned to distinct RNA decay pathways, the contribution of long non-coding RNAs (lncRNAs) to RNA turnover remains unknown. Here we identify the lncRNA CALA as a potent regulator of RNA turnover in endothelial cells. We demonstrate that CALA forms cytoplasmic ribonucleoprotein complexes with G3BP1 and regulates endothelial cell functions. A detailed characterization of these G3BP1-positive complexes by mass spectrometry identifies UPF1 and numerous other NMD factors having cytoplasmic G3BP1-association that is CALA-dependent. Importantly, CALA silencing impairs degradation of NMD target transcripts, establishing CALA as a non-coding regulator of RNA steady-state levels in the endothelium.

14.
RNA Biol ; 8(1): 90-100, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21282982

RESUMO

Pre-mRNA splicing in trypanosomes requires the SMN-mediated assembly of small nuclear ribonucleoproteins (snRNPs). In contrast to higher eukaryotes, the cellular localization of snRNP biogenesis and the involvement of nuclear-cytoplasmic trafficking in trypanosomes are controversial. By using RNAi knockdown of SMN in T. brucei to investigate its functional role in snRNP assembly, we found dramatic changes in the steady-state levels of snRNAs and snRNPs: The SL RNA accumulates, whereas U1, U4, and U5 snRNA levels decrease, and Sm core assembly in particular of the SL RNA is strongly reduced. In addition, SMN depletion blocks U4/U6 di-snRNP formation; the variant Sm core of the U2 snRNP, however, still forms efficiently after SMN knockdown. Concerning the longstanding question, whether nuclear-cytoplasmic trafficking is involved in trypanosomal snRNP biogenesis, fluorescence in situ hybridization (FISH) and immunofluorescence assays revealed that the SL RNA genes and transcripts colocalize with SMN. Remarkably, SMN silencing leads to a nucleoplasmic accumulation of both SL RNA and the Sm proteins. In sum, our data demonstrate an essential and snRNA-selective role of SMN in snRNP biogenesis in vivo and strongly argue for a nucleoplasmic Sm core assembly of the SL RNP.


Assuntos
RNA de Protozoário/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN/metabolismo , Trypanosoma brucei brucei/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Imunofluorescência , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Loci Gênicos , Hibridização in Situ Fluorescente , Interferência de RNA , Splicing de RNA , RNA de Protozoário/genética , RNA Nuclear Pequeno/análise , RNA Líder para Processamento/metabolismo , Proteínas do Complexo SMN/genética , Trypanosoma brucei brucei/metabolismo
15.
Eukaryot Cell ; 9(3): 379-86, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20081062

RESUMO

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.


Assuntos
Multimerização Proteica/fisiologia , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/biossíntese , Trypanosoma brucei brucei/metabolismo , Proteínas Centrais de snRNP/metabolismo , Proliferação de Células , Centrifugação com Gradiente de Concentração , Expressão Gênica/genética , Espaço Intranuclear/metabolismo , Espectrometria de Massas , Ligação Proteica/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Nuclear Pequeno/genética , Ribonuclease H/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Trypanosoma brucei brucei/genética , Proteínas Centrais de snRNP/genética
16.
Nat Commun ; 11(1): 2039, 2020 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-32341350

RESUMO

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.


Assuntos
Apoptose , Infarto do Miocárdio/genética , Miócitos Cardíacos/citologia , RNA Longo não Codificante/genética , Envelhecimento , Animais , Proteínas de Transporte/genética , Sobrevivência Celular , Coenzima A-Transferases/genética , Modelos Animais de Doenças , Inativação Gênica , Humanos , Proteínas com Domínio LIM/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/genética , RNA Antissenso/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição de p300-CBP/genética
17.
Vascul Pharmacol ; 114: 13-22, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30910127

RESUMO

The advent of deep sequencing technologies recently unraveled the complexity of the human genome: Although almost entirely transcribed, only a very minor part of our genome actually accounts for protein coding exons and most is considered non-coding. Among the non-coding transcripts, long non-coding RNAs (lncRNAs) constitute a rather heterogeneous group of linear as well as circular RNAs (circRNAs). LncRNAs act via multiple mechanisms and several lncRNAs were shown to be involved in vascular development, growth and remodeling. For example, the lncRNAs PUNISHER, MALAT1, MEG3, and GATA6-AS regulate vessel formation in vivo, whereas lincRNA-p21 controls smooth muscle cell function and neointima formation. For several other lncRNAs (e.g. SENCR, SMILR, and HypERlnc) functional roles in smooth muscle cells/pericytes have been described in vitro. Less information is available with respect to the function of circRNAs. Here most studies report on expression profiles but some circRNAs (e.g. cANRIL or cZNF292) may also play critical roles in smooth muscle or endothelial cells in vitro. This review summarizes the current knowledge of lncRNA and circRNA functions in vascular biology and disease and discusses their potential use as biomarkers.


Assuntos
Aterosclerose/metabolismo , Vasos Sanguíneos/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Vasos Sanguíneos/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Marcadores Genéticos , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , RNA Longo não Codificante/genética , Transdução de Sinais
18.
Nat Commun ; 9(1): 237, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29339785

RESUMO

Impaired or excessive growth of endothelial cells contributes to several diseases. However, the functional involvement of regulatory long non-coding RNAs in these processes is not well defined. Here, we show that the long non-coding antisense transcript of GATA6 (GATA6-AS) interacts with the epigenetic regulator LOXL2 to regulate endothelial gene expression via changes in histone methylation. Using RNA deep sequencing, we find that GATA6-AS is upregulated in endothelial cells during hypoxia. Silencing of GATA6-AS diminishes TGF-ß2-induced endothelial-mesenchymal transition in vitro and promotes formation of blood vessels in mice. We identify LOXL2, known to remove activating H3K4me3 chromatin marks, as a GATA6-AS-associated protein, and reveal a set of angiogenesis-related genes that are inversely regulated by LOXL2 and GATA6-AS silencing. As GATA6-AS silencing reduces H3K4me3 methylation of two of these genes, periostin and cyclooxygenase-2, we conclude that GATA6-AS acts as negative regulator of nuclear LOXL2 function.


Assuntos
Aminoácido Oxirredutases/metabolismo , Células Endoteliais/metabolismo , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica/genética , Hipóxia/genética , Neovascularização Fisiológica/genética , RNA Antissenso/genética , RNA Longo não Codificante/genética , Animais , Moléculas de Adesão Celular/genética , Ciclo-Oxigenase 2/genética , Epigênese Genética , Transição Epitelial-Mesenquimal , Inativação Gênica , Código das Histonas/genética , Histonas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Metilação , Camundongos
19.
J Am Coll Cardiol ; 67(10): 1214-1226, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26965544

RESUMO

Recent studies suggest that the majority of the human genome is transcribed, but only about 2% accounts for protein-coding exons. Long noncoding RNAs (lncRNAs) constitute a heterogenic class of RNAs that includes, for example, intergenic lncRNAs, antisense transcripts, and enhancer RNAs. Moreover, alternative splicing can lead to the formation of circular RNAs. In support of putative functions, GWAS for cardiovascular diseases have shown predictive single-nucleotide polymorphisms in lncRNAs, such as the 9p21 susceptibility locus that encodes the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL). Many lncRNAs are regulated during disease. For example, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and myocardial infarction-associated transcript (MIAT) were shown to affect endothelial cell functions and diabetic retinopathy, whereas lincRNA-p21 controls neointima formation. In the heart, several lncRNAs were shown to act as microRNA sponges and to control ischemia-reperfusion injury or act as epigenetic regulators. In this review, the authors summarize the current understanding of lncRNA functions and their role as biomarkers in cardiovascular diseases.


Assuntos
Regulação da Expressão Gênica , Infarto do Miocárdio/genética , RNA Longo não Codificante/genética , RNA/genética , Biomarcadores/sangue , Predisposição Genética para Doença , Humanos , Infarto do Miocárdio/sangue , RNA Longo não Codificante/sangue
20.
Nat Med ; 22(10): 1140-1150, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27595325

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA (CTSS), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3' untranslated region (3' UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx+ regions, which form a long stem-loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1) to the 3' UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-γ and tumor-necrosis-factor-α induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases.


Assuntos
Adenosina Desaminase/genética , Aterosclerose/genética , Catepsinas/genética , Proteína Semelhante a ELAV 1/genética , Edição de RNA/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas , Adenosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aneurisma Aórtico/genética , Doenças das Artérias Carótidas/genética , Doença da Artéria Coronariana/genética , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia/genética , Immunoblotting , Inosina/metabolismo , Interferon gama/farmacologia , Masculino , Pessoa de Meia-Idade , Edição de RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Fator de Necrose Tumoral alfa/farmacologia
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