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1.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807682

RESUMO

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (-gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the -gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.


Assuntos
Quadruplex G , Proteínas de Ligação a RNA/metabolismo , /metabolismo , Dicroísmo Circular , Biologia Computacional/métodos , Bases de Dados Genéticas , Ensaio de Desvio de Mobilidade Eletroforética , Genoma Viral/fisiologia , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Replicação Viral/fisiologia
2.
Int J Nanomedicine ; 16: 2569-2584, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33833512

RESUMO

Background: Multidrug resistance (MDR) has emerged to be a major hindrance in cancer therapy, which contributes to the reduced sensitivity of cancer cells toward chemotherapeutic drugs mainly owing to the over-expression of drug efflux transporters. The combination of gene therapy and chemotherapy has been considered as a potential approach to improve the anti-cancer efficacy by reversing the MDR effect. Materials and Methods: The AS1411 aptamer-functionalized micelles were constructed through an emulsion/solvent evaporation strategy for the simultaneous co-delivery of doxorubicin and miR-519c. The therapeutic efficacy and related mechanism of micelles were explored based on the in vitro and in vivo active targeting ability and the suppression of MDR, using hepatocellular carcinoma cell line HepG2 as a model. Results: The micelle was demonstrated to possess favorable cellular uptake and tumor penetration ability by specifically recognizing the nucleolin in an AS1411 aptamer-dependent manner. Further, the intracellular accumulation of doxorubicin was significantly improved due to the suppression of ABCG2-mediated drug efflux by miR-519c, resulting in the efficient inhibition of tumor growth. Conclusion: The micelle-mediated co-delivery of doxorubicin and miR-519c provided a promising strategy to obtain ideal anti-cancer efficacy through the active targeting function and the reversion of MDR.


Assuntos
Aptâmeros de Nucleotídeos/administração & dosagem , Carcinoma Hepatocelular/terapia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Micelas , MicroRNAs/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Fosfoproteínas/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacologia , Apoptose , Aptâmeros de Nucleotídeos/química , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular , Movimento Celular , Proliferação de Células , Doxorrubicina/administração & dosagem , Resistência a Múltiplos Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oligodesoxirribonucleotídeos/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810326

RESUMO

Musashi-1 (MSI1) is an RNA-binding protein that regulates progenitor cells in adult and developing organisms to maintain self-renewal capacities. The role of musashi-1 in the bone healing environment and its relation with other osteogenic factors is unknown. In the current study, we analyze the expression of MSI1 in an experimental model of rat femoral bone fractures. We also analyze the relation between MSI1 expression and the expression of two osteogenic markers: periostin (POSTN) and runt-related transcription factor 2 (RUNX2). We use histological, immunohistochemical, and qPCR techniques to evaluate bone healing and the expression of MSI1, POSTN, and RUNX2 over time (4, 7, and 14 days). We compare our findings with non-fractured controls. We find that in bone calluses, the number of cells expressing MSI1 and RUNX2 increase over time and the intensity of POSTN expression decreases over time. Within bone calluses, we find the presence of MSI1 expression in mesenchymal stromal cells, osteoblasts, and osteocytes but not in hypertrophic chondrocytes. After 14 days, the expression of MSI1, POSTN, and RUNX2 was significantly correlated. Thus, we conclude that musashi-1 potentially serves in the osteogenic differentiation of mesenchymal stromal cells and bone healing. Therefore, further studies are needed to determine the possibility of musashi-1's role as a clinical biomarker of bone healing and therapeutic agent for bone regeneration.


Assuntos
Consolidação da Fratura , Proteínas do Tecido Nervoso/metabolismo , Osteogênese , Proteínas de Ligação a RNA/metabolismo , Animais , Calo Ósseo/citologia , Calo Ósseo/metabolismo , Calo Ósseo/fisiologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Proteínas do Tecido Nervoso/genética , Osteoblastos/metabolismo , Osteócitos/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Wistar
4.
Nat Commun ; 12(1): 2105, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33833232

RESUMO

Intestinal microbiota-derived metabolites have biological importance for the host. Polyamines, such as putrescine and spermidine, are produced by the intestinal microbiota and regulate multiple biological processes. Increased colonic luminal polyamines promote longevity in mice. However, no direct evidence has shown that microbial polyamines are incorporated into host cells to regulate cellular responses. Here, we show that microbial polyamines reinforce colonic epithelial proliferation and regulate macrophage differentiation. Colonisation by wild-type, but not polyamine biosynthesis-deficient, Escherichia coli in germ-free mice raises intracellular polyamine levels in colonocytes, accelerating epithelial renewal. Commensal bacterium-derived putrescine increases the abundance of anti-inflammatory macrophages in the colon. The bacterial polyamines ameliorate symptoms of dextran sulfate sodium-induced colitis in mice. These effects mainly result from enhanced hypusination of eukaryotic initiation translation factor. We conclude that bacterial putrescine functions as a substrate for symbiotic metabolism and is further absorbed and metabolised by the host, thus helping maintain mucosal homoeostasis in the intestine.


Assuntos
Colo/metabolismo , Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Putrescina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Colite/induzido quimicamente , Colite/patologia , Sulfato de Dextrana/toxicidade , Células Epiteliais/metabolismo , Feminino , Microbioma Gastrointestinal/fisiologia , Homeostase , Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
5.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802672

RESUMO

Raf kinase inhibitory protein (RKIP), also known as a phosphatidylethanolamine-binding protein 1 (PEBP1), functions as a tumor suppressor and regulates several signaling pathways, including ERK and NF-κκB. RKIP is severely downregulated in human malignant cancers, indicating a functional association with cancer metastasis and poor prognosis. The transcription regulation of RKIP gene in human cancers is not well understood. In this study, we suggested a possible transcription mechanism for the regulation of RKIP in human cancer cells. We found that Metadherin (MTDH) significantly repressed the transcriptional activity of RKIP gene. An analysis of publicly available datasets showed that the knockdown of MTDH in breast and endometrial cancer cell lines induced the expression RKIP. In addition, the results obtained from qRT-PCR and ChIP analyses showed that MTDH considerably inhibited RKIP expression. In addition, the RKIP transcript levels in MTDH-knockdown or MTDH-overexpressing MCF-7 cells were likely correlated to the protein levels, suggesting that MTDH regulates RKIP expression. In conclusion, we suggest that MTDH is a novel factor that controls the RKIP transcription, which is essential for cancer progression.


Assuntos
Progressão da Doença , Proteínas de Membrana/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Mutação/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Regulação para Cima/genética
6.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805769

RESUMO

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein-Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.


Assuntos
Linfócitos B/virologia , Loci Gênicos , Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Alelos , Linfócitos B/imunologia , Sequência de Bases , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , MicroRNAs/imunologia , Modelos Biológicos , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Esclerose Múltipla/virologia , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , RNA Mensageiro/imunologia , Proteínas de Ligação a RNA/imunologia , Transdução de Sinais
7.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805770

RESUMO

Pre-mRNA splicing plays an important role in muscle function and diseases. The RNA binding motif 20 (RBM20) is a splicing factor that is predominantly expressed in muscle tissues and primarily regulates pre-mRNA splicing of Ttn, encoding a giant muscle protein titin that is responsible for muscle function and diseases. RBM20-mediated Ttn splicing has been mostly studied in heart muscle, but not in skeletal muscle. In this study, we investigated splicing specificity in different muscle types in Rbm20 knockout rats and hormonal effects on RBM20-mediated splicing both in cellulo and in vivo studies. The results revealed that RBM20 is differentially expressed across muscles and RBM20-mediated splicing is muscle-type specific. In the presence of RBM20, Ttn splicing responds to hormones in a muscle-type dependent manner, while in the absence of RBM20, Ttn splicing is not affected by hormones. In differentiated and undifferentiated C2C12 cells, RBM20-mediated splicing in response to hormonal effects is mainly through genomic signaling pathway. The knowledge gained from this study may help further understand muscle-specific gene splicing in response to hormone stimuli in different muscle types.


Assuntos
Conectina/genética , Músculo Esquelético/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Precursores de RNA/genética , Processamento de RNA , Proteínas de Ligação a RNA/genética , Animais , Antitireóideos/farmacologia , Linhagem Celular , Conectina/metabolismo , Cruzamentos Genéticos , Feminino , Humanos , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Especificidade de Órgãos , Propiltiouracila/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Estreptozocina/farmacologia , Tri-Iodotironina/farmacologia
8.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807542

RESUMO

Genetic analyses of patients with amyotrophic lateral sclerosis (ALS) have identified disease-causing mutations and accelerated the unveiling of complex molecular pathogenic mechanisms, which may be important for understanding the disease and developing therapeutic strategies. Many disease-related genes encode RNA-binding proteins, and most of the disease-causing RNA or proteins encoded by these genes form aggregates and disrupt cellular function related to RNA metabolism. Disease-related RNA or proteins interact or sequester other RNA-binding proteins. Eventually, many disease-causing mutations lead to the dysregulation of nucleocytoplasmic shuttling, the dysfunction of stress granules, and the altered dynamic function of the nucleolus as well as other membrane-less organelles. As RNA-binding proteins are usually components of several RNA-binding protein complexes that have other roles, the dysregulation of RNA-binding proteins tends to cause diverse forms of cellular dysfunction. Therefore, understanding the role of RNA-binding proteins will help elucidate the complex pathophysiology of ALS. Here, we summarize the current knowledge regarding the function of disease-associated RNA-binding proteins and their role in the dysfunction of membrane-less organelles.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Humanos , RNA/metabolismo
9.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799602

RESUMO

RNAs transmit information from DNA to encode proteins that perform all cellular processes and regulate gene expression in multiple ways. From the time of synthesis to degradation, RNA molecules are associated with proteins called RNA-binding proteins (RBPs). The RBPs play diverse roles in many aspects of gene expression including pre-mRNA processing and post-transcriptional and translational regulation. In the last decade, the application of modern techniques to identify RNA-protein interactions with individual proteins, RNAs, and the whole transcriptome has led to the discovery of a hidden landscape of these interactions in plants. Global approaches such as RNA interactome capture (RIC) to identify proteins that bind protein-coding transcripts have led to the identification of close to 2000 putative RBPs in plants. Interestingly, many of these were found to be metabolic enzymes with no known canonical RNA-binding domains. Here, we review the methods used to analyze RNA-protein interactions in plants thus far and highlight the understanding of plant RNA-protein interactions these techniques have provided us. We also review some recent protein-centric, RNA-centric, and global approaches developed with non-plant systems and discuss their potential application to plants. We also provide an overview of results from classical studies of RNA-protein interaction in plants and discuss the significance of the increasingly evident ubiquity of RNA-protein interactions for the study of gene regulation and RNA biology in plants.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Proteínas de Plantas/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética , Tabaco/genética , Arabidopsis/metabolismo , Sequência de Bases , Oryza/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Ligação Proteica , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Tabaco/metabolismo , Transcriptoma
10.
Science ; 372(6537): 91-94, 2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33795458

RESUMO

Neurons are the longest-lived cells in our bodies and lack DNA replication, which makes them reliant on a limited repertoire of DNA repair mechanisms to maintain genome fidelity. These repair mechanisms decline with age, but we have limited knowledge of how genome instability emerges and what strategies neurons and other long-lived cells may have evolved to protect their genomes over the human life span. A targeted sequencing approach in human embryonic stem cell-induced neurons shows that, in neurons, DNA repair is enriched at well-defined hotspots that protect essential genes. These hotspots are enriched with histone H2A isoforms and RNA binding proteins and are associated with evolutionarily conserved elements of the human genome. These findings provide a basis for understanding genome integrity as it relates to aging and disease in the nervous system.


Assuntos
Reparo do DNA , Genoma Humano , Instabilidade Genômica , Neurônios/metabolismo , Envelhecimento/genética , Dano ao DNA , DNA Intergênico , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Células-Tronco Embrionárias , Histonas/metabolismo , Humanos , Mitose , Mutação , Doenças do Sistema Nervoso/genética , Neurônios/citologia , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de DNA , Transcrição Genética
11.
World J Surg Oncol ; 19(1): 131, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33882945

RESUMO

BACKGROUND: Radiotherapy is a main therapeutic method for cancers, including colon cancer. In the current study, we aim to explore the effects of circular RNA (circRNA) circ_0055625 in the progression and radiosensitivity of colon cancer and the underlying mechanism. METHODS: The expression of circ_0055625 and musashi homolog 1 (MSI1) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). MSI1 protein expression was determined by Western blot. Cell proliferation was assessed by cell counting kit-8 (CCK-8) and colony formation assays. Cell survival fraction, apoptosis, and invasion were investigated by colony formation assay, flow cytometry analysis, and transwell invasion assay, respectively. Cell migration was detected by wound-healing and transwell migration assays. The binding relationship between microRNA-338-3p (miR-338-3p) and circ_0055625 or MSI1 was predicted by online databases and identified by Dual-Luciferase Reporter Assay. The effects of circ_0055625 silencing on the tumor formation and radiosensitivity of colon cancer in vivo were explored by in vivo tumor formation assay. RESULTS: Circ_0055625 and MSI1 were upregulated in colon cancer tissues and cells relative to control groups. Radiation treatment apparently increased the expression of circ_0055625 and MSI1 in colon cancer cells. Circ_0055625 knockdown or MSI1 silencing repressed cell proliferation, migration, and invasion and promoted cell apoptosis and radiosensitivity in colon cancer. Also, circ_0055625 silencing-mediated effects were attenuated by MSI1 overexpression. Additionally, circ_0055625 silencing reduced MSI1 expression, which could be attenuated by miR-338-3p inhibitor. Mechanically, circ_0055625 acted as a sponge for miR-338-3p to regulate MSI1. Furthermore, circ_0055625 knockdown hindered tumor growth and improved radiosensitivity in vivo. CONCLUSION: Circ_0055625 repression inhibited the progression and radioresistance of colon cancer by downregulating MSI1 through sponging miR-338-3p. This result might provide a theoretical basis for improving the therapy of colon cancer with radiation.


Assuntos
Neoplasias do Colo , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , RNA Circular/genética , Proteínas de Ligação a RNA/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/radioterapia , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Inativação de Genes , Inativação Gênica , Humanos , Proteínas do Tecido Nervoso/biossíntese , Prognóstico , Proteínas de Ligação a RNA/biossíntese , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos da radiação , Transfecção
12.
Cytokine ; 142: 155492, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33711707

RESUMO

BACKGROUND AND AIMS: The interferon-induced transmembrane protein 3 (IFITM3) plays an important role in the adaptive and innate immune response by inhibiting viral membrane hemifusion between the host and viral cell cytoplasm. Single nucleotide polymorphisms (SNPs) in the gene IFITM3 have been associated with susceptibility and severity of influenza or other viral infections. We aimed to analyze the role of SNPs in the gene IFITM3 in SARS-CoV-2 infection. METHODS: We performed genotyping of the SNPs rs12252 and rs34481144 in the gene IFITM3 in 239 SARS-CoV-2-positive and 253 SARS-CoV-2-negative patients. We analyzed the association of the SNPs with susceptibility to SARS-CoV-2 infection and severity of COVID-19. RESULTS: SARS-CoV-2-positive and SARS-CoV-2-negative patients did not differ regarding demographics. Neither IFITM3 rs12252 nor rs34481144 polymorphisms were related to SARS-CoV-2 infection risk or severity of COVID-19. Interestingly, we observed the putative deleterious rs12252 CC genotype only in SARS-CoV-2-positive patients (N = 2). Also, we found a non-significant higher frequency of rs34481144 A-allele carriers in the patients with 'serious' COVID-19. CONCLUSIONS: In summary, we could not confirm the recently reported influence of polymorphisms in the gene IFITM3 on SARS-CoV-2 infection risk or severity of COVID-19 in a German cohort. Additional studies are needed to clarify the influence of the rs12252 CC genotype on SARS-CoV-2 infection risk and the rs34481144 A-allele on course of COVID-19.


Assuntos
/genética , Predisposição Genética para Doença , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Ligação a RNA/genética , Índice de Gravidade de Doença , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
13.
Nat Commun ; 12(1): 1569, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33692367

RESUMO

Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.


Assuntos
Proteínas de Ligação a RNA/química , RNA/química , Animais , Sítios de Ligação , Humanos , Imunoprecipitação , RNA/metabolismo , RNA/efeitos da radiação , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/efeitos da radiação , Raios Ultravioleta
14.
Chem Biol Interact ; 339: 109432, 2021 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-33684387

RESUMO

Mitochondrial dependent oxidative stress (OS) and subsequent cell death are considered as the major cytotoxicity caused by Triethylene glycol dimethacrylate (TEGDMA), a commonly monomer of many resin-based dental composites. Under OS microenvironment, autophagy serves as a cell homeostatic mechanism and maintains redox balance through degradation or turnover of cellular components in order to promote cell survival. However, whether autophagy is involved in the mitochondrial oxidative damage and apoptosis induced by TEGDMA, and the cellular signaling pathways underlying this process remain unclear. In the present study, we demonstrated that TEGDMA induced mouse preodontoblast cell line (mDPC6T) dysfunctional mitochondrial oxidative response. In further exploring the underlying mechanisms, we found that TEGDMA impaired autophagic flux, as evidenced by increased LC3-II expression and hindered p62 degradation, thereby causing both mitochondrial oxidative damage and cell apoptosis. These results were further verified by treatment with chloroquine (autophagy inhibitor) and rapamycin (autophagy promotor). More importantly, we found that the JNK/MAPK pathway was the key upstream regulator of above injury process. Collectively, our finding firstly demonstrated that TEGDMA induced JNK-dependent autophagy, thereby promoting mitochondrial dysfunction-associated oxidative damage and apoptosis in preodontoblast.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Ácidos Polimetacrílicos/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Oxirredução/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia
15.
Medicine (Baltimore) ; 100(10): e25031, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33725886

RESUMO

ABSTRACT: Adrenocortical carcinoma (ACC) is considered a rare cancer with poor prognosis. We used public datasets from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases to assess the relationships between N6-methyladenosine (m6A)-related genes and ACC.We used the Wilcoxon signed-rank test to compare m6A-related gene expression in ACC tissues with that in normal tissues. Then, ACC patients were grouped based on a cluster analysis of m6A-related gene expression. m6A-related genes that were significantly associated with survival were incorporated into a risk signature, and 2 groups were divided according to median risk score. Fisher exact tests were utilized to analyze differences in clinical variables between groups. We compared the overall survival (OS) rates of the groups by means of Kaplan-Meier curves and Cox regression analyses.We found that RBM15, ZC3H3, YTDHF1, YTDHF2, and ALBH5 were overexpressed in ACC and that KIAA1429, YTHDC1, HNRNPC, WTAP, METTL3, and FTO were down regulated in ACC. In addition, membership in cluster 2 or the high-risk group was associated with advanced clinical factors and poor prognosis. The univariable and multivariable Cox regression analyses showed that risk score can be considered an independent prognostic factor for ACC.We found that the expression of m6A-related genes could be used as an independent prognostic factor in ACC. However, the current study has some limitations, and further studies of m6A-related genes in ACC are needed.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Biomarcadores Tumorais/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Adenosina/análogos & derivados , Adenosina/metabolismo , Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/mortalidade , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/mortalidade , Carcinoma Adrenocortical/patologia , Biomarcadores Tumorais/metabolismo , Conjuntos de Dados como Assunto , Regulação para Baixo , Feminino , Humanos , Masculino , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Pessoa de Meia-Idade , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Prognóstico , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA-Seq , Taxa de Sobrevida , Regulação para Cima
16.
Int J Nanomedicine ; 16: 1805-1817, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33692623

RESUMO

Introduction: RNA interference is a promising therapy in glioma treatment. However, the application of RNA interference has been limited in glioma therapy by RNA instability and the lack of tumor targeting. Here, we report a novel DNA tetrahedron, which can effectively deliver small interfering RNA to glioma cells and induce apoptosis. Methods: siRNA, a small interfering RNA that can suppress the expression of survivin in glioma, was loaded into the DNA tetrahedron (TDN). To enhance the ability of active targeting of this nanoparticle, we modified one side of the DNA nanostructure with aptamer as1411 (As-TDN-R), which can selectively recognize the nucleolin in the cytomembrane of tumor cells. The modified nanoparticles were characterized by agarose gel electrophoresis, dynamic light scattering, and transmission electron microscopy. The serum stability was evaluated by agarose gel electrophoresis. Nucleolin was detected by Western blot and immunofluorescence, and targeted cellular uptake was examined by flow cytometry. The TUNEL assay, flow cytometry, and Western Blot were used to detect apoptosis in U87 cells. The gene silencing of survivin was examined by qPCR, Western Blot, and immunofluorescence. Results: As-TDN-R alone showed better stability towards siRNA, indicating that TDN was a good siRNA protector. Compared with TDN alone, there was increased intercellular uptake of As-TDN-R by U87 cells, evidenced by overexpressed nucleolin in glioma cell lines. TUNEL assay, flow cytometry, and Western Blot revealed increased apoptosis in the As-TDN-R group. The downregulation of survivin protein and mRNA expression levels indicated that As-TDN-R effectively silenced the target gene. Conclusion: The novel nanoparticle can serve as a good carrier for targeting siRNA delivery in glioma. Further exploration of the DNA nanostructure can greatly promote the application of DNA-based drug systems in glioma.


Assuntos
DNA/química , Técnicas de Transferência de Genes , Glioma/terapia , Nanoestruturas/química , RNA Interferente Pequeno/administração & dosagem , Apoptose/efeitos dos fármacos , Aptâmeros de Nucleotídeos/química , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação para Baixo , Endocitose , Inativação Gênica , Glioma/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Nanoestruturas/ultraestrutura , Oligodesoxirribonucleotídeos/química , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Survivina/metabolismo
17.
Nat Commun ; 12(1): 1394, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654093

RESUMO

N6-methyladenosine (m6A) is a reversible mRNA modification that has been shown to play important roles in various biological processes. However, the roles of m6A modification in macrophages are still unknown. Here, we discover that ablation of Mettl3 in myeloid cells promotes tumour growth and metastasis in vivo. In contrast to wild-type mice, Mettl3-deficient mice show increased M1/M2-like tumour-associated macrophage and regulatory T cell infiltration into tumours. m6A sequencing reveals that loss of METTL3 impairs the YTHDF1-mediated translation of SPRED2, which enhances the activation of NF-kB and STAT3 through the ERK pathway, leading to increased tumour growth and metastasis. Furthermore, the therapeutic efficacy of PD-1 checkpoint blockade is attenuated in Mettl3-deficient mice, identifying METTL3 as a potential therapeutic target for tumour immunotherapy.


Assuntos
Adenosina/análogos & derivados , Reprogramação Celular , Macrófagos/metabolismo , Macrófagos/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , RNA Neoplásico/metabolismo , Adenosina/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Polaridade Celular , Proliferação de Células , Citocinas/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Neoplasias Pulmonares/secundário , Metilação , Metiltransferases/metabolismo , Camundongos Knockout , Células Mieloides/metabolismo , NF-kappa B/metabolismo , Metástase Neoplásica , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras , Fator de Transcrição STAT3/metabolismo , Linfócitos T Reguladores/imunologia , Microambiente Tumoral
18.
Int J Mol Sci ; 22(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669383

RESUMO

Recurrent protein folding motifs include various types of helical bundles formed by α-helices that supercoil around each other. While specific patterns of amino acid residues (heptad repeats) characterize the highly versatile folding motif of four-α-helical bundles, the significance of the polypeptide chain directionality is not sufficiently understood, although it determines sequence patterns, helical dipoles, and other parameters for the folding and oligomerization processes of bundles. To investigate directionality aspects in sequence-structure relationships, we reversed the amino acid sequences of two well-characterized, highly regular four-α-helical bundle proteins and studied the folding, oligomerization, and structural properties of the retro-proteins, using Circular Dichroism Spectroscopy (CD), Size Exclusion Chromatography combined with Multi-Angle Laser Light Scattering (SEC-MALS), and Small Angle X-ray Scattering (SAXS). The comparison of the parent proteins with their retro-counterparts reveals that while the α-helical character of the parents is affected to varying degrees by sequence reversal, the folding states, oligomerization propensities, structural stabilities, and shapes of the new molecules strongly depend on the characteristics of the heptad repeat patterns. The highest similarities between parent and retro-proteins are associated with the presence of uninterrupted heptad patterns in helical bundles sequences.


Assuntos
Proteínas de Bactérias/química , Dobramento de Proteína , Proteínas de Ligação a RNA/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cromatografia em Gel , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Peptídeos , Conformação Proteica em alfa-Hélice , Proteínas de Ligação a RNA/genética , Espalhamento a Baixo Ângulo , Difração de Raios X
19.
Mol Cell ; 81(7): 1439-1452.e9, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33705709

RESUMO

The ATPase Prp16 governs equilibrium between the branching (B∗/C) and exon ligation (C∗/P) conformations of the spliceosome. Here, we present the electron cryomicroscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 Å resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. The exon-ligation factors Prp18 and Slu7 bind to Ci before ATP hydrolysis by Prp16 can destabilize the branching conformation. Biochemical assays suggest that these pre-bound factors prime the C complex for conversion to C∗ by Prp16. A complete model of the Prp19 complex (NTC) reveals how the branching factors Yju2 and Isy1 are recruited by the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain tethered to the NTC in the C∗ complex to promote exon ligation. Our results explain how Prp16 action modulates the dynamic binding of step-specific factors to alternatively stabilize the C or C∗ conformation and establish equilibrium of the catalytic spliceosome.


Assuntos
Modelos Químicos , Processamento de RNA , RNA Fúngico/química , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Spliceossomos/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo
20.
Life Sci ; 274: 119366, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33741419

RESUMO

AIMS: N6-methyladenosine (m6A) is the most prevalent internal chemical RNA modification in mammal mRNAs. Accumulating evidence has shown the critical role of m6A in cardiovascular diseases including cardiac hypertrophy, heart failure, ischemic heart disease, vascular calcification, restenosis, and aortic aneurysm. However, whether m6A participates in the occurrence and development of hypoxic pulmonary hypertension (HPH) remains largely unknown. The present study aims to explore the role of key transferase METTL3, in the development of HPH. MATERIALS AND METHODS: Pulmonary artery smooth muscle cells (PASMCs) and hypoxic rat models were used to research the METTL3-mediated m6A in HPH. EdU, transwell and TUNEL were performed to evaluate the proliferation, migration and apoptosis rates. m6A RNA Methylation Quantification Kit and m6A-qPCR were utilized to measure the total m6A level and m6A level of PTEN mRNA. RNA immunoprecipitation was used to detect the interaction between METTL3 and PTEN mRNA. KEY FINDINGS: Both METTL3 mRNA and protein were found abnormally upregulated in vivo and in vitro. Furthermore, downregulation of METTL3 attenuated PASMCs proliferation and migration. In addition, m6A binding protein YTHDF2 was found significantly increased in PASMCs under hypoxia. YTHDF2 recognized METTL3 mediated m6A modified PTEN mRNA and promoted the degradation of PTEN. Decreased PTEN led to over-proliferation of PASMCs through activation of PI3K/Akt signaling pathway. SIGNIFICANCE: METTL3/YTHDF2/PTEN axis exerts a significant role in hypoxia induced PASMCs proliferation, providing a novel therapeutic target for HPH.


Assuntos
Adenosina/análogos & derivados , Hipóxia/fisiopatologia , Metiltransferases/metabolismo , Miócitos de Músculo Liso/patologia , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/patologia , RNA Mensageiro/metabolismo , Adenosina/química , Animais , Apoptose , Proliferação de Células , Masculino , Metilação , Metiltransferases/genética , Miócitos de Músculo Liso/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
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