RESUMO
Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and in vitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA in vitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.
Assuntos
DNA Topoisomerases Tipo I , RNA Polimerase II , Proteínas de Ligação a RNA , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/genética , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Ligação Proteica , DNA/metabolismo , DNA/genética , Transcrição Gênica , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA/metabolismo , RNA/genética , Linhagem Celular Tumoral , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/genética , Células HCT116 , Conformação de Ácido NucleicoRESUMO
Neurofibromatosis type 1 is a rare neurogenetic syndrome, characterized by pigmentary abnormalities, learning and social deficits, and a predisposition for benign and malignant tumor formation caused by germline mutations in the NF1 gene. With the cloning of the NF1 gene and the recognition that the encoded protein, neurofibromin, largely functions as a negative regulator of RAS activity, attention has mainly focused on RAS and canonical RAS effector pathway signaling relevant to disease pathogenesis and treatment. However, as neurofibromin is a large cytoplasmic protein the RAS regulatory domain of which occupies only 10% of its entire coding sequence, both canonical and non-canonical RAS pathway modulation, as well as the existence of potential non-RAS functions, are becoming apparent. In this Special article, we discuss our current understanding of neurofibromin function.
Assuntos
Neurofibromatose 1 , Neurofibromina 1 , Genes da Neurofibromatose 1 , Humanos , Neurofibromatose 1/genética , Neurofibromina 1/genética , Proteínas , Transdução de Sinais/genéticaRESUMO
Since the discovery that enhancers can support transcription, the roles of enhancer RNAs have remained largely elusive. We identified that enhancer RNAs interact with and augment bromodomain engagement with acetylated chromatin. Here, we discuss our recent findings and the potential mechanisms underlying the regulation and functions of enhancer RNA-bromodomain associations.
Assuntos
Elementos Facilitadores Genéticos/genética , Epigênese Genética/genética , RNA/genética , Animais , Regulação da Expressão Gênica/genética , HumanosRESUMO
Cardiac functions can be altered by changes in the microstructure of the heart, i.e., remodeling of the cardiac tissue, which may activate pathologies such as hypertrophy, dilation, or cardiac fibrosis. Cardiac fibrosis can develop due to an excessive deposition of extracellular matrix proteins, which are products of the activation of fibroblasts. In this context, the anatomical-histological change may interfere with the functioning of the cardiac tissue, which requires specialized cells for its operation. The purpose of the present study was to determine the cellular interactions and morphological changes in cocultures of 3T3 fibroblasts and RL-14 cardiomyocytes via the generation of a platform an in vitro model. For this purpose, a platform emulating the biological characteristics of endomyocardial fibrosis was generated using a cell patterning technique to study morphological cellular changes in compact and irregular patterns of fibrosis. It was found that cellular patterns emulating the geometrical distributions of endomyocardial fibrosis generated morphological changes after interaction of the RL-14 cardiomyocytes with the 3T3 fibroblasts. Through this study, it was possible to evaluate biological characteristics such as cell proliferation, adhesion, and spatial distribution, which are directly related to the type of emulated endomyocardial fibrosis. This research concluded that fibroblasts inhibited the proliferation of cardiomyocytes via their interaction with specific microarchitectures. This behavior is consistent with the histopathological distribution of cardiac fibrosis; therefore, the platform developed in this research could be useful for the in vitro assessment of cellular microdomains. This would allow for the experimental determination of interactions with drugs, substrates, or biomaterials within the engineering of cardiac tissues.
Assuntos
Comunicação Celular , Técnicas de Cocultura/métodos , Fibrose Endomiocárdica/etiologia , Fibroblastos/patologia , Miócitos Cardíacos/patologia , Células 3T3 , Animais , Adesão Celular , Linhagem Celular , Proliferação de Células , Fibrose Endomiocárdica/patologia , Humanos , Camundongos , Modelos BiológicosRESUMO
La colangitis biliar primaria es una enfermedad hepática autoinmune que conduce a la destrucción progresiva de los conductos biliares intrahepáticos, lo que aumenta el riesgo de desarrollar cirrosis e hipertensión portal. Actualmente, el ácido ursodesoxicólico es el medicamento de primera línea para el tratamiento de esta entidad. Este medicamento desplaza los ácidos biliares hidrofóbicos y aumenta las concentraciones de ácidos biliares hidrofílicos en la bilis, lo cual favorece la integridad de los conductos biliares, adicionalmente, tiene efectos antiinflamatorios y propiedades inmunomo-duladoras y antiapoptóticas. En los últimos 40 años, numerosos ensayos clínicos han respaldado la eficacia clínica del ácido ursodesoxicólico y su seguridad cuando se utiliza en pacientes con colan-gitis biliar primaria. Se realiza una revisión del ácido ursodesoxicólico en el contexto de colangitis biliar primaria, se describe su historia, mecanismos de acción, efectos secundarios y dosificación. Finalmente, se menciona su uso en situaciones especiales como son el embarazo y la lactancia
Primary biliary cholangitis is an autoimmune liver disease that leads to progressive destruction of intrahepatic bile ducts, increasing the risk of developing cirrhosis and portal hypertension. Currently, ursodeoxycholic acid is the first-line drug for the treatment of this condition. This drug displaces hy-drophobic bile acids and increases concentrations of hydrophilic bile acids in the bile, which favors the integrity of the bile ducts, additionally, it has anti-inflammatory effects and immunoprotective and antiapoptotic properties. Over the past 40 years numerous clinical trials have supported the clinical efficacy of ursodeoxycholic acid and its safety when used in patients with primary biliary cholangitis. A review of ursodeoxycholic acid in the context of primary biliary cholangitis is carried out, and its history, mechanisms of action, side effects and dosage are described. Finally, its use in special situations such as pregnancy and lactation are discussed.