RESUMO
BACKGROUND: Primary resistance to anti-EGFR therapies affects 40% of metastatic colorectal cancer patients harbouring wild-type RAS/RAF. YAP1 activation is associated with this resistance, prompting an investigation into AURKA's role in mediating YAP1 phosphorylation at Ser397, as observed in breast cancer. METHODS: We used transcriptomic analysis along with in vitro and in vivo models of RAS/RAF wild-type CRC to study YAP1 Ser397 phosphorylation as a potential biomarker for cetuximab resistance. We assessed cetuximab efficacy using CCK8 proliferation assays and cell cycle analysis. Additionally, we examined the effects of AURKA inhibition with alisertib and created a dominant-negative YAP1 Ser397 mutant to assess its impact on cancer stem cell features. RESULTS: The RAS/RAF wild-type CRC models exhibiting primary resistance to cetuximab prominently displayed elevated YAP1 phosphorylation at Ser397 primarily mediated by AURKA. AURKA-induced YAP1 phosphorylation was identified as a key trigger for cancer stem cell reprogramming. Consequently, we found that AURKA inhibition had the capacity to effectively restore cetuximab sensitivity and concurrently suppress the cancer stem cell phenotype. CONCLUSIONS: AURKA inhibition holds promise as a therapeutic approach to overcome cetuximab resistance in RAS/RAF wild-type colorectal cancer, offering a potential means to counter the development of cancer stem cell phenotypes associated with cetuximab resistance.
Assuntos
Aurora Quinase A , Neoplasias Colorretais , Humanos , Cetuximab/farmacologia , Cetuximab/metabolismo , Aurora Quinase A/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Hypoxia is a crucial factor contributing to maintenance of atherosclerotic lesions. The ability of ABCA1 to stimulate the efflux of cholesterol from cells in the periphery, particularly foam cells in atherosclerotic plaques, is an important anti-atherosclerotic mechanism. The posttranscriptional regulation by miRNAs represents a key regulatory mechanism of a number of signaling pathways involved in atherosclerosis. Previously, miR-199a-5p has been shown to be implicated in the endocytic and retrograde intracellular transport. Although the regulation of miR-199a-5p and ABCA1 by hypoxia has been already reported independently, the role of miR-199a-5p in macrophages and its possible role in atherogenic processes such us regulation of lipid homeostasis through ABCA1 has not been yet investigated. Here, we demonstrate that both ABCA1 and miR-199a-5p show an inverse regulation by hypoxia and Ac-LDL in primary macrophages. Moreover, we demonstrated that miR-199a-5p regulates ABCA1 mRNA and protein levels by directly binding to its 3'UTR. As a result, manipulation of cellular miR-199a-5p levels alters ABCA1 expression and cholesterol efflux in primary mouse macrophages. Taken together, these results indicate that the correlation between ABCA1-miR-199a-5p could be exploited to control macrophage cholesterol efflux during the onset of atherosclerosis, where cholesterol alterations and hypoxia play a pathogenic role.
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According to the stem cell theory for cancer, hepatocellular carcinomas are sustained by a group of cancer stem cells (CSCs) which are responsible for resistance to chemotherapy. In the present study we aimed to examine lipid metabolism in cancer stem cells induced by long-term treatment with sorafenib and its relationship with acquisition of a CSC-like phenotype. Two cell lines (HepG2SF1 and Huh7SF1) were generated by incubation with a step-wise increase of sorafenib concentrations for 10 months. These cell lines displayed stem-like characteristics like increase in the expression of ABCB1A, Nanog and Oct4 as well as an E-cadherin/N-cadherin switch. HepG2SF1 and Huh7SF1 cells showed intracellular accumulation of neutral lipids, assessed by flow cytometry and confocal microscopy. The exam of lipid metabolism revealed that HepG2SF1 and Huh7SF1 cells increased the expression of the enzymes involved in de novo lipid synthesis ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) and that of the fatty acid transporter CD36. In addition, these CSC-like cells had enhanced expression of the lipogenic transcription factor SREBP1c. Analysis of the key metabolic sensor AMP-activated kinase (AMPK) demonstrated that both AMPK phosphorylation and levels were decreased in the CSC-like cells compared to their parental cells. Interestingly, transfection of HepG2SF1 and Huh7SF1 cells with AMPK, restored the levels of the lipogenic enzymes and SREBP1c and decreased the intracellular lipid accumulation. Furthermore, AMPK transfection decreased the stemness markers and inhibited the E-cadherin/N-cadherin switch. Targeting AMPK and lipid metabolism of hepatocellular cancer stem cells is a promising strategy to face stemness and chemotherapy resistance.
Assuntos
Carcinoma Hepatocelular/metabolismo , Metabolismo dos Lipídeos/fisiologia , Células-Tronco Neoplásicas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Acetil-CoA Carboxilase/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/genética , Lipogênese , Neoplasias Hepáticas/metabolismo , Metformina/farmacologia , Fosforilação , Sorafenibe/farmacologiaRESUMO
Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. HCC treatment is hindered by the frequent emergence of chemoresistance to the multikinase inhibitor sorafenib, which has been related to the presence of cancer stem cells (CSCs) that self-renew and often escape therapy. The key metabolic sensor AMP-activated kinase (AMPK) has recently been recognized as a tumour growth regulator. In this study, we aimed to elucidate the role of AMPK in the development of a stem cell phenotype in HCC cells. To this end, we enriched the CSC population in HCC cell lines that showed increased expression of drug resistance (ALDH1A1, ABCB1A) and stem cell (CD133, Nanog, Oct4, alpha fetoprotein) markers and demonstrated their stemness phenotype. These cells were refractory to sorafenib-induced cell death. We report that sorafenib-resistant cells had lower levels of total and phosphorylated AMPK as well as its downstream substrate, ACC, compared with the parental cells. Interestingly, AMPK knockdown with siRNA or inhibition with dorsomorphin increased the expression of stem cell markers in parental cells and blocked sorafenib-induced cell death. Conversely, the upregulation of AMPK, either by transfection or by pharmacological activation with A-769662, decreased the expression of ALDH1A1, ABCB1A, CD133, Nanog, Oct4, and alpha fetoprotein, and restored sensitivity to sorafenib. Analysis of the underlying mechanism points to hypoxia-inducible factor HIF-1α as a regulator of stemness. In vivo studies in a xenograft mouse model demonstrated that stem-like cells have greater tumourigenic capacity. AMPK activation reduced xenograft tumour growth and decreased the expression of stem cell markers. Taken together, these results indicate that AMPK may serve as a novel target to overcome chemoresistance in HCC.
Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Biomarcadores Tumorais , Carcinoma Hepatocelular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Hepáticas , Proteínas de Neoplasias , Células-Tronco Neoplásicas , Pironas/farmacologia , Sorafenibe/farmacologia , Tiofenos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Compostos de Bifenilo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Obesity, a major risk factor for chronic diseases such as type 2 diabetes (T2D), represents a serious primary health problem worldwide. Dietary habits are of special interest to prevent and counteract the obesity and its associated metabolic disorders, including lipid steatosis. Capsaicin, a pungent compound of chili peppers, has been found to ameliorate diet-induced obesity in rodents and humans. The purpose of this study was to examine the effect of capsaicin on hepatic lipogenesis and to delineate the underlying signaling pathways involved, using HepG2 cells as an experimental model. Cellular neutral lipids, stained with BODIPY493/503, were quantified by flow cytometry, and the protein expression and activity were determined by immunoblotting. Capsaicin reduced basal neutral lipid content in HepG2 cells, as well that induced by troglitazone or by oleic acid. This effect of capsaicin was prevented by dorsomorphin and GW9662, pharmacological inhibitors of AMPK and PPARγ, respectively. In addition, capsaicin activated AMPK and inhibited the AKT/mTOR pathway, major regulators of hepatic lipogenesis. Furthermore, capsaicin blocked autophagy and increased PGC-1α protein. These results suggest that capsaicin behaves as an anti-lipogenic compound in HepG2 cells.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Capsaicina/farmacologia , Lipogênese/efeitos dos fármacos , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Autofagia/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Hep G2 , Humanos , Lipídeos/análise , Modelos Biológicos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Current chemotherapy for castration-resistant prostate cancer is established on taxane-based compounds like docetaxel. However, eventually, the development of toxic side effects and resistance limits the therapeutic benefit being the major concern in the treatment of prostate cancer. Combination therapies in many cases, enhance drug efficacy and delay the appearance of undesired effects, representing an important option for the treatment of castration-resistant prostate cancer. In this study, we tested the efficacy of the combination of docetaxel and capsaicin, the pungent ingredient of hot chili peppers, on prostate cancer cells proliferation. METHODS: Prostate cancer LNCaP and PC3 cell lines were used in this study. Levels of total and phosphorylated forms of Akt, mTOR, S6, LKB1, AMPK and ACC were determined by Western blot. AMPK, LKB1 and Akt knock down was performed by siRNA. PTEN was overexpressed by transient transfection with plasmids. Xenograft prostate tumors were induced in nude mice and treatments (docetaxel and capsaicin) were administered intraperitoneally. Statistical analyses were performed with GraphPad software. Combination index was calculated with Compusyn software. RESULTS: Docetaxel and capsaicin synergistically inhibited the growth of LNCaP and PC3 cells, with a combination index lower than 1 for most of the combinations tested. Co-treatment with docetaxel and capsaicin notably decreased Akt and its downstream targets mTOR and S6 phosphorylation. Overexpression of PTEN phosphatase abrogated the synergistic antiproliferative effect of docetaxel and capsaicin. The combined treatment also increased the phosphorylation of AMP-activated kinase (AMPK) and the phosphorylation of its substrate ACC. In addition, pharmacological inhibition of AMPK with dorsomorphin (compound C) as well as knock down by siRNA of AMPK or its upstream kinase LKB1, abolished the synergy of docetaxel and capsaicin. Mechanistically, we showed that the synergistic anti-proliferative effect may be attributed to two independent effects: Inhibition of the PI3K/Akt/mTOR signaling pathway by one side, and AMPK activation by the other. In vivo experiments confirmed the synergistic effects of docetaxel and capsaicin in reducing the tumor growth of PC3 cells. CONCLUSION: Combination of docetaxel and capsaicin represents a therapeutically relevant approach for the treatment of Prostate Cancer.
RESUMO
Capsaicin is a natural compound present in chili and red peppers and the responsible of their spicy flavor. It has recently provoked interest because of its antitumoral effects in many cell types although its action mechanism is not clearly understood. As metabolic dysregulation is one of the hallmarks of cancer cells and the key metabolic sensor in the AMP-activated kinase (AMPK), in this study we explored the ability of capsaicin to modulate AMPK activity. We found that capsaicin activated AMPK in HepG2 cells by increasing AMPK phosphorylation and its downstream target ACC. Mechanistically, we determined that capsaicin activated AMPK through the calcium/calmodulin-dependent protein kinase kinase ß, CaMKKß as either the CaMKK inhibitor STO-609 or CaMKK knock down with siRNA abrogated the activation of AMPK. Moreover, capsaicin decreased cell viability, inhibited Akt/mTOR pathway and increased reactive oxygen species (ROS) in HepG2 cells. AMPK activation was involved in the underpinning mechanism of capsaicin-induced cell death.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Capsaicina/farmacologia , Capsicum/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fármacos do Sistema Sensorial/farmacologia , Benzimidazóis/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Sobrevivência Celular , Ativação Enzimática , Células Hep G2 , Humanos , Naftalimidas/farmacologia , Fosforilação , Transdução de SinaisRESUMO
Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Reparo de DNA por Recombinação/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.
Assuntos
Domínio Catalítico/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Polimerase Dirigida por DNA/genética , Células-Tronco Embrionárias/citologia , Proteína de Replicação A/antagonistas & inibidores , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Replicação A/genética , Relação Estrutura-Atividade , Translocação Genética/genética , DNA Polimerase tetaRESUMO
DNA polymerase θ (Polθ) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Polθ transfers nucleotides to the 3' terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Polθ template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Polθ terminal transferase activity is most efficient on DNA containing 3' overhangs, is facilitated by an insertion loop and conserved residues that hold the 3' primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Polθ to generate genetic diversity during alt-EJ and characterizes Polθ as among the most proficient terminal transferases known.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Nucleotidilexotransferase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Animais , Coenzimas/metabolismo , Células-Tronco Embrionárias , Manganês/metabolismo , Camundongos , DNA Polimerase tetaRESUMO
The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions. Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.
Assuntos
Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Genética , Telômero/genética , Telômero/metabolismo , Animais , Sequência de Bases , Morte Celular/genética , Linhagem Celular , Aberrações Cromossômicas , Cromossomos de Mamíferos/genética , DNA Polimerase Dirigida por DNA/deficiência , Genes BRCA1 , Genes BRCA2 , Células HeLa , Humanos , Camundongos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Rad51 Recombinase/metabolismo , Recombinação Genética/genética , Reparo de DNA por Recombinação/genética , Translocação Genética/genética , DNA Polimerase tetaRESUMO
Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5' end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3' end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies.
Assuntos
Coronavirus/fisiologia , RNA Viral/metabolismo , Montagem de Vírus , Animais , Linhagem Celular , Coronavirus/genética , Análise Mutacional de DNA , Mutação Puntual , RNA Viral/genética , Deleção de Sequência , Vírion/metabolismo , Replicação ViralRESUMO
The mammalian telomere-binding protein Rap1 was recently found to have additional nontelomeric functions, acting as a transcriptional cofactor and a regulator of the NF-κB pathway. Here, we assess the effect of disrupting mouse Rap1 in vivo and report on its unanticipated role in metabolic regulation and body-weight homeostasis. Rap1 inhibition causes dysregulation in hepatic as well as adipose function, leading to glucose intolerance, insulin resistance, liver steatosis, and excess fat accumulation. Furthermore, Rap1 appears to play a pivotal role in the transcriptional cascade that controls adipocyte differentiation in vitro. Using a separation-of-function allele, we show that the metabolic function of Rap1 is independent of its recruitment to TTAGGG binding elements found at telomeres and at other interstitial loci. In conclusion, our study underscores an additional function for the most conserved telomere-binding protein, forging a link between telomere biology and metabolic signaling.
Assuntos
Peso Corporal/genética , Obesidade/genética , Telômero/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Obesidade/metabolismo , Homologia de Sequência de Aminoácidos , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Coronavirus (CoV) transcription requires a high-frequency recombination process that links newly synthesized minus-strand subgenomic RNA copies to the leader region, which is present only once, at the 5' end of the genome. This discontinuous RNA synthesis step is based on the complementarity between the transcription-regulating sequences (TRSs) at the leader region and those preceding each gene in the nascent minus-strand RNA. Furthermore, the template switch requires the physical proximity of RNA genome domains located between 20,000 and 30,000 nucleotides apart. In this report, it is shown that the efficacy of this recombination step is promoted by novel additional long-distance RNA-RNA interactions between RNA motifs located close to the TRSs controlling the expression of each gene and their complementary sequences mapping close to the 5' end of the genome. These interactions would bring together the motifs involved in the recombination process. This finding indicates that the formation of high-order RNA structures in the CoV genome is necessary to control the expression of at least the viral N gene. The requirement of these long-distance interactions for transcription was shown by the engineering of CoV replicons in which the complementarity between the newly identified sequences was disrupted. Furthermore, disruption of complementarity in mutant viruses led to mutations that restored complementarity, wild-type transcription levels, and viral titers by passage in cell cultures. The relevance of these high-order structures for virus transcription is reinforced by the phylogenetic conservation of the involved RNA motifs in CoVs.
Assuntos
Coronavirus/fisiologia , Regulação Viral da Expressão Gênica , RNA Viral/biossíntese , Recombinação Genética , Transcrição Gênica , Animais , Linhagem Celular , Cricetinae , Humanos , Conformação de Ácido Nucleico , Hibridização de Ácido NucleicoRESUMO
Coronavirus subgenomic mRNA (sgmRNA) transcription requires a discontinuous RNA synthesis mechanism driven by the transcription-regulating sequences (TRSs), located at the 3' end of the genomic leader (TRS-L) and also preceding each gene (TRS-B). In transmissible gastroenteritis virus (TGEV), the free energy of TRS-L and cTRS-B (complement of TRS-B) duplex formation is one of the factors regulating the transcription of sgmRNAs. In addition, N gene sgmRNA transcription is controlled by a transcription-regulating motif, including a long-distance RNA-RNA interaction between complementary proximal and distal elements. The extension of complementarity between these two sequences increased N gene transcription. An active domain, a novel essential component of the transcription-regulating motif, has been identified. The active domain primary sequence was necessary for its activity. Relocation of the active domain upstream of the N gene TRS core sequence in the absence of the proximal and distal elements also enhanced sgmRNA N transcription. According to the proposed working model for N gene transcriptional activation, the long-distance RNA-RNA interaction relocates the distant active domain in close proximity with the N gene TRS, which probably increases the frequency of template switching during the synthesis of negative RNA. The transcription-regulating motif has been optimized to a minimal sequence showing a 4-fold activity increase in relation to the native RNA motif. Full-length TGEV infectious viruses were generated with the optimized transcription-regulating motif, which enhanced by 5-fold the transcription of the 3a gene and can be used in expression vectors based in coronavirus genomes.
Assuntos
Proteínas do Nucleocapsídeo/biossíntese , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , RNA Viral/genética , Transcrição Gênica , Vírus da Gastroenterite Transmissível/fisiologia , Pareamento de Bases , Proteínas do Nucleocapsídeo de Coronavírus , RNA Complementar/genética , RNA Complementar/metabolismo , Vírus da Gastroenterite Transmissível/genéticaRESUMO
The coronavirus (CoV) discontinuous transcription mechanism is driven by long-distance RNA-RNA interactions between transcription-regulating sequences (TRSs) located at the 5' terminal leader (TRS-L) and also preceding each mRNA-coding sequence (TRS-B). The contribution of host cell proteins to CoV transcription needs additional information. Polypyrimidine tract-binding protein (PTB) was reproducibly identified in association with positive-sense RNAs of transmissible gastroenteritis coronavirus (TGEV) TRS-L and TRS-B by affinity chromatography and mass spectrometry. A temporal regulation of PTB cytoplasmic levels was observed during infection, with a significant increase from 7 to 16 h postinfection being inversely associated with a decrease in viral replication and transcription. Silencing the expression of PTB with small interfering RNA in two cell lines (Huh7 and HEK 293T) led to a significant increase of up to 4-fold in mRNA levels and virus titer, indicating a negative effect of PTB on CoV RNA accumulation. During CoV infection, PTB relocalized from the nucleus to novel cytoplasmic structures different from replication-transcription sites in which stress granule markers T-cell intracellular antigen-1 (TIA-1) and TIA-1-related protein (TIAR) colocalized. PTB was detected in these modified stress granules in TGEV-infected swine testis cells but not in stress granules induced by oxidative stress. Furthermore, viral genomic and subgenomic RNAs were detected in association with PTB and TIAR. These cytoplasmic ribonucleoprotein complexes might be involved in posttranscriptional regulation of virus gene expression.
Assuntos
Interações Hospedeiro-Patógeno , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Viral/metabolismo , Vírus da Gastroenterite Transmissível/patogenicidade , Replicação Viral , Animais , Humanos , Suínos , Transcrição GênicaRESUMO
Coronavirus (CoV) RNA synthesis includes the replication of the viral genome, and the transcription of sgRNAs by a discontinuous mechanism. Both processes are regulated by RNA sequences such as the 5' and 3' untranslated regions (UTRs), and the transcription regulating sequences (TRSs) of the leader (TRS-L) and those preceding each gene (TRS-Bs). These distant RNA regulatory sequences interact with each other directly and probably through protein-RNA and protein-protein interactions involving viral and cellular proteins. By analogy to other plus-stranded RNA viruses, such as polioviruses, in which translation and replication switch involves a cellular factor (PCBP) and a viral protein (3CD) it is conceivable that in CoVs the switch between replication and transcription is also associated with the binding of proteins that are specifically recruited by the replication or transcription complexes. Complexes between RNA motifs such as TRS-L and the TRS-Bs located along the CoV genome are probably formed previously to the transcription start, and most likely promote template-switch of the nascent minus RNA to the TRS-L region. Many cellular proteins interacting with regulatory CoV RNA sequences are members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA-binding proteins, involved in mRNA processing and transport, which shuttle between the nucleus and the cytoplasm. In the context of CoV RNA synthesis, these cellular ribonucleoproteins might also participate in RNA-protein complexes to bring into physical proximity TRS-L and distant TRS-B, as proposed for CoV discontinuous transcription. In this review, we summarize RNA-RNA and RNA-protein interactions that represent modest examples of complex quaternary RNA-protein structures required for the fine-tuning of virus replication. Design of chemically defined replication and transcription systems will help to clarify the nature and activity of these structures.
Assuntos
Coronavirus/genética , RNA Viral/genética , RNA Viral/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo , Replicação Viral , Animais , Coronavirus/fisiologia , Genoma Viral/genética , Humanos , Proteínas Virais/genéticaRESUMO
Transmissible gastroenteritis coronavirus (TGEV) genomic RNA transcription generates 5'- and 3'-coterminal subgenomic mRNAs. This process involves a discontinuous step during the synthesis of minus-sense RNA that is modulated by transcription-regulating sequences located at the 3' end of the leader (TRS-L) and also preceding each viral gene (TRS-Bs). TRSs include a highly conserved core sequence (CS) (5'-CUAAAC-3') and variable flanking sequences. It has been previously proposed that TRS-Bs act as attenuation or stop signals during the synthesis of minus-sense RNAs. The nascent minus-stranded RNA would then be transferred by a template switch process to the TRS-L, which acts as the acceptor RNA. To study whether the TRS-L is structured and to determine whether this structure has a functional impact on genomic and subgenomic viral RNA synthesis, we have used a combination of nuclear magnetic resonance (NMR) spectroscopy and UV thermal denaturation approaches together with site-directed mutagenesis and in vivo transcriptional analyses. The results indicated that a 36-nucleotide oligomer encompassing the wild-type TRS-L forms a structured hairpin closed by an apical AACUAAA heptaloop. This loop contains most of the CS and is isolated from a nearby internal loop by a short Watson-Crick base-paired stem. TRS-L mutations altering the structure and the stability of the TRS-L hairpin affected replication and transcription, indicating the requirement of a functional RNA hairpin structure in these processes.
Assuntos
RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Elementos Reguladores de Transcrição , Transcrição Gênica , Vírus da Gastroenterite Transmissível/genética , Pareamento de Bases , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Espectrofotometria Ultravioleta , Vírus da Gastroenterite Transmissível/fisiologiaRESUMO
Purified nucleocapsid protein (N protein) from transmissible gastroenteritis virus (TGEV) enhanced hammerhead ribozyme self-cleavage and favored nucleic acid annealing, properties that define RNA chaperones, as previously reported. Several TGEV N-protein deletion mutants were expressed in Escherichia coli and purified, and their RNA binding ability and RNA chaperone activity were evaluated. The smallest N-protein domain analyzed with RNA chaperone activity, facilitating DNA and RNA annealing, contained the central unstructured region (amino acids 117 to 268). Interestingly, N protein and its deletion mutants with RNA chaperone activity enhanced template switching in a retrovirus-derived heterologous system, reinforcing the concept that TGEV N protein is an RNA chaperone that could be involved in template switching. This result is in agreement with the observation that in vivo, N protein is not necessary for TGEV replication, but it is required for efficient transcription.