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1.
Nat Commun ; 12(1): 5156, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526504

RESUMO

R-loops are by-products of transcription that must be tightly regulated to maintain genomic stability and gene expression. Here, we describe a mechanism for the regulation of the R-loop-specific helicase, senataxin (SETX), and identify the ubiquitin specific peptidase 11 (USP11) as an R-loop regulator. USP11 de-ubiquitinates SETX and its depletion increases SETX K48-ubiquitination and protein turnover. Loss of USP11 decreases SETX steady-state levels and reduces R-loop dissolution. Ageing of USP11 knockout cells restores SETX levels via compensatory transcriptional downregulation of the E3 ubiquitin ligase, KEAP1. Loss of USP11 reduces SETX enrichment at KEAP1 promoter, leading to R-loop accumulation, enrichment of the endonuclease XPF and formation of double-strand breaks. Overexpression of KEAP1 increases SETX K48-ubiquitination, promotes its degradation and R-loop accumulation. These data define a ubiquitination-dependent mechanism for SETX regulation, which is controlled by the opposing activities of USP11 and KEAP1 with broad applications for cancer and neurological disease.


Assuntos
DNA Helicases/genética , DNA/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Enzimas Multifuncionais/genética , Processamento de Proteína Pós-Traducional , Proteostase/genética , RNA Helicases/genética , Tioléster Hidrolases/genética , Linhagem Celular , Senescência Celular/genética , DNA/química , DNA/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/metabolismo , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/metabolismo , Ubiquitinação
2.
Nat Commun ; 12(1): 5512, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535666

RESUMO

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Hibridização de Ácido Nucleico , Edição de RNA , RNA/metabolismo , Adenosina Desaminase/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Deleção de Genes , Genes Reporter , Instabilidade Genômica , Proteínas de Fluorescência Verde/metabolismo , Recombinação Homóloga/genética , Humanos , Enzimas Multifuncionais/metabolismo , Estabilidade Proteica , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/genética , Proteína de Replicação A/metabolismo
3.
Int J Biol Macromol ; 190: 636-648, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517025

RESUMO

SARS-CoV-2 nucleocapsid (N) protein undergoes RNA-induced phase separation (LLPS) and sequesters the host key stress granule (SG) proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and 2 (G3BP1 and G3BP2) to inhibit SG formation. This will allow viral packaging and propagation in host cells. Based on a genomic-guided meta-analysis, here we identify upstream regulatory elements modulating the expression of G3BP1 and G3BP2 (collectively called G3BP1/2). Using this strategy, we have identified FOXA1, YY1, SYK, E2F-1, and TGFBR2 as activators and SIN3A, SRF, and AKT-1 as repressors of G3BP1/2 genes. Panels of the activators and repressors were then used to identify drugs that change their gene expression signatures. Two drugs, imatinib, and decitabine have been identified as putative modulators of G3BP1/2 genes and their regulators, suggesting their role as COVID-19 mitigation agents. Molecular docking analysis suggests that both drugs bind to G3BP1/2 with a much higher affinity than the SARS-CoV-2 N protein. This study reports imatinib and decitabine as candidate drugs against N protein and G3BP1/2 protein.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , COVID-19/tratamento farmacológico , Proteínas do Nucleocapsídeo de Coronavírus/química , DNA Helicases/química , Decitabina/química , Mesilato de Imatinib/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas de Ligação a RNA/química , SARS-CoV-2/química , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , COVID-19/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Decitabina/farmacologia , Sistemas de Liberação de Medicamentos , Genômica , Mesilato de Imatinib/farmacologia , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/metabolismo
4.
Nat Commun ; 12(1): 4848, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381037

RESUMO

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.


Assuntos
Metiltransferases/química , RNA Helicases/química , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
5.
J Mol Graph Model ; 108: 108001, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34388402

RESUMO

The 2015 Zika outbreak sparked major global concern and emphasized the reality and dangers still posed by mosquito borne pathogens. While efforts have been made to develop a vaccine and other therapeutics, there is still a great demand for antiviral drugs targeting Zika and other flaviviruses. The non-structural protein 3 (NS3) helicase is a vital component of the viral replication complex, tasked with unwinding the viral dsRNA molecule into single strands. Given this critical function, the Zika virus helicase is a potential therapeutic target and the focus of many ongoing research efforts. Using a combination of drug docking and molecular dynamics simulations, we have identified a list of competitive helicase inhibitors targeting the ATP hydrolysis site and have discovered a potential allosteric site capable of distorting both of the protein's active sites.


Assuntos
Infecção por Zika virus , Zika virus , Animais , Simulação de Dinâmica Molecular , RNA Helicases/metabolismo , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais
6.
Molecules ; 26(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206406

RESUMO

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.


Assuntos
Antivirais/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteases/farmacologia , Vírus de RNA/enzimologia , COVID-19/tratamento farmacológico , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Avaliação Pré-Clínica de Medicamentos , Vírus da Encefalite Transmitidos por Carrapatos/enzimologia , Corantes Fluorescentes/química , Humanos , RNA Helicases/antagonistas & inibidores , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , SARS-CoV-2/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
7.
Biochem J ; 478(13): 2405-2423, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198322

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Chlorocebus aethiops , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , RNA Helicases/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Suramina/farmacologia , Células Vero , Proteínas não Estruturais Virais/metabolismo
8.
Biomolecules ; 11(7)2021 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206878

RESUMO

Helicase proteins are known to use the energy of ATP to unwind nucleic acids and to remodel protein-nucleic acid complexes. They are involved in almost every aspect of DNA and RNA metabolisms and participate in numerous repair mechanisms that maintain cellular integrity. The archaeal Lhr-type proteins are SF2 helicases that are mostly uncharacterized. They have been proposed to be DNA helicases that act in DNA recombination and repair processes in Sulfolobales and Methanothermobacter. In Thermococcales, a protein annotated as an Lhr2 protein was found in the network of proteins involved in RNA metabolism. To investigate this, we performed in-depth phylogenomic analyses to report the classification and taxonomic distribution of Lhr-type proteins in Archaea, and to better understand their relationship with bacterial Lhr. Furthermore, with the goal of envisioning the role(s) of aLhr2 in Thermococcales cells, we deciphered the enzymatic activities of aLhr2 from Thermococcus barophilus (Tbar). We showed that Tbar-aLhr2 is a DNA/RNA helicase with a significant annealing activity that is involved in processes dependent on DNA and RNA transactions.


Assuntos
DNA Helicases/genética , RNA Helicases/genética , Thermococcales/enzimologia , Adenosina Trifosfatases/genética , Proteínas Arqueais/química , DNA/química , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Filogenia , RNA/química , RNA Helicases/isolamento & purificação , RNA Helicases/metabolismo , Homologia de Sequência de Aminoácidos , Thermococcales/genética , Thermococcales/metabolismo
9.
J Virol ; 95(19): e0015321, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34287041

RESUMO

Orf virus (ORFV) is a highly epitheliotropic parapoxvirus with zoonotic significance that induces proliferative lesions in the skin of sheep, goats, and humans. Several viral proteins carried by ORFV, including nuclear factor-κB (NF-κB) inhibitors, play important roles in hijacking host-associated proteins for viral evasion of the host innate immune response. However, the roles of proteins with unknown functions in viral replication and latent infection remain to be explored. Here, we present data demonstrating that the ORF120, an early-late ORFV-encoded protein, activates the NF-κB pathway in the early phase of infection, which implies that ORFV may regulate NF-κB through a biphasic mechanism. A DUAL membrane yeast two-hybrid system and coimmunoprecipitation experiments revealed that the ORF120 protein interacts with Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1). The overexpression of the ORF120 protein can efficiently increase the expression of G3BP1 and nuclear translocation of NF-κB-p65 in primary ovine fetal turbinate (OFTu) and HeLa cells. The knockdown of G3BP1 significantly decreased ORF120-induced NF-κB activation, indicating that G3BP1 is involved in ORF120-induced NF-κB pathway activation. A dual-luciferase reporter assay revealed that ORF120 could positively regulate the NF-κB pathway through the full-length G3BP1 or the domain of G3BP1RRM+RGG. In conclusion, we demonstrate, for the first time, that the ORF120 protein is capable of positively regulating NF-κB signaling by interacting with G3BP1, providing new insights into ORFV pathogenesis and a theoretical basis for antiviral drug design. IMPORTANCE As part of the host innate response, the nuclear factor-κB (NF-κB) pathway plays a partial antiviral role in nature by regulating the innate immune response. Thus, the NF-κB pathway is probably the most frequently targeted intracellular pathway for subversion by anti-immune modulators that are carried by a wide range of pathogens. Various viruses, including poxviruses, carry several proteins that prepare the host cell for viral replication by inhibiting cytoplasmic events, leading to the initiation of NF-κB transcriptional activity. However, NF-κB activity is hypothesized to facilitate viral replication to a great extent. The significance of our research is in the exploration of the activation mechanism of NF-κB induced by the Orf virus (ORFV) ORF120 protein interacting with G3BP1, which helps not only to explain the ability of ORFV to modulate the immune response through the positive regulation of NF-κB but also to show the mechanism by which the virus evades the host innate immune response.


Assuntos
DNA Helicases/metabolismo , Ectima Contagioso/virologia , NF-kappa B/metabolismo , Vírus do Orf/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , DNA Helicases/química , Células HeLa , Humanos , Vírus do Orf/genética , Vírus do Orf/crescimento & desenvolvimento , Vírus do Orf/patogenicidade , Proteínas de Ligação a Poli-ADP-Ribose/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ovinos , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Transcrição Genética , Ativação Transcricional , Proteínas Virais/genética , Virulência
10.
Nat Commun ; 12(1): 4268, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257295

RESUMO

Drosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct-processive or distributive-modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2's cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated.


Assuntos
Proteínas de Drosophila/metabolismo , RNA Helicases/metabolismo , RNA de Cadeia Dupla/genética , Ribonuclease III/metabolismo , Imagem Individual de Molécula/métodos , Animais , Drosophila , Proteínas de Drosophila/genética , Modelos Teóricos , RNA Helicases/genética , Interferência de RNA/fisiologia , Ribonuclease III/genética
11.
Nat Commun ; 12(1): 4451, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294712

RESUMO

Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.


Assuntos
DNA Fúngico/química , Estruturas R-Loop , RNA Fúngico/química , Ciclo Celular/genética , Ciclo Celular/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos , Instabilidade Genômica , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estruturas R-Loop/genética , Estruturas R-Loop/fisiologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Cancer Sci ; 112(9): 3884-3894, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34077586

RESUMO

Gene alterations are recognized as important events in acute myeloid leukemia (AML) progression. Studies on hematopoiesis of altered genes contribute to a better understanding on their roles in AML progression. Our previous work reported a DEAH box helicase 15 (DHX15) R222G mutation in AML patients, and we showed DHX15 overexpression is associated with poor prognosis in AML patients. In this work, we further study the role of dhx15 in zebrafish developmental hematopoiesis by generating dhx15-/- zebrafish using transcription activator-like effector nuclease technology. Whole-mount in situ hybridization (WISH) analysis showed hematopoietic stem/progenitor cells were dramatically perturbed when dhx15 was deleted. Immunofluorescence staining indicated inhibited hematopoietic stem/progenitor cell (HSPC) proliferation instead of accelerated apoptosis were detected in dhx15-/- zebrafish. Furthermore, our data showed that HSPC defect is mediated through the unfolded protein response (UPR) pathway. DHX15 R222G mutation, a recurrent mutation identified in AML patients, displayed a compromised function in restoring HSPC failure in dhx15-/- ; Tg (hsp: DHX15 R222G) zebrafish. Collectively, this work revealed a vital role of dhx15 in the maintenance of definitive hematopoiesis in zebrafish through the unfolded protein respone pathway. The study of DHX15 and DHX15 R222G mutation could hold clinical significance for evaluating prognosis of AML patients with aberrant DHX15 expression.


Assuntos
RNA Helicases DEAD-box/metabolismo , Hematopoese/genética , Leucemia Mieloide Aguda/genética , Resposta a Proteínas não Dobradas/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Apoptose/genética , Proliferação de Células/genética , RNA Helicases DEAD-box/genética , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/metabolismo , Humanos , Hibridização In Situ , Leucemia Mieloide Aguda/metabolismo , Mutação , RNA Helicases/genética , RNA Helicases/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
13.
Nat Commun ; 12(1): 3686, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140498

RESUMO

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.


Assuntos
Hipóxia Celular , Dano ao DNA/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica/genética , Enzimas Multifuncionais/metabolismo , Estruturas R-Loop/genética , RNA Helicases/metabolismo , Resposta a Proteínas não Dobradas/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , DNA Helicases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Enzimas Multifuncionais/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oxigênio/farmacologia , Estruturas R-Loop/efeitos dos fármacos , RNA Helicases/genética , RNA-Seq , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima , Zinostatina/farmacologia , eIF-2 Quinase/metabolismo
14.
Science ; 373(6551): 236-241, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34083449

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.


Assuntos
Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/química , Óxidos N-Cíclicos/farmacologia , Ferro/metabolismo , SARS-CoV-2/efeitos dos fármacos , Enxofre/metabolismo , Motivos de Aminoácidos , Animais , Antivirais/farmacologia , Sítios de Ligação , Domínio Catalítico , Chlorocebus aethiops , Coenzimas/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/farmacologia , Ferro/química , Domínios Proteicos , RNA Helicases/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Marcadores de Spin , Enxofre/química , Células Vero , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Zinco/metabolismo
15.
Nat Commun ; 12(1): 3965, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172724

RESUMO

Eukaryotic gene expression is constantly controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation, resulting in phosphorylation of the central NMD factor UPF1 and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional connection between the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm exhaustive NMD inhibition resulting in massive transcriptomic alterations. Intriguingly, we find that the functionally underestimated SMG5 can substitute the role of SMG7 and individually activate NMD. Furthermore, the presence of either SMG5 or SMG7 is sufficient to support SMG6-mediated endonucleolysis of NMD targets. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.


Assuntos
Proteínas de Transporte/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Feminino , Técnicas de Inativação de Genes , Humanos , Fosforilação , RNA Helicases/genética , RNA Helicases/metabolismo , Telomerase/metabolismo , Transativadores/genética , Transativadores/metabolismo
16.
Nat Commun ; 12(1): 3849, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158508

RESUMO

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Estruturas R-Loop , RNA Helicases/metabolismo , Transativadores/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Células HCT116 , Humanos , Hibridização de Ácido Nucleico , RNA/genética , RNA/metabolismo , RNA Helicases/genética , Interferência de RNA , Telômero/genética , Telômero/metabolismo , Transativadores/genética
17.
Nat Commun ; 12(1): 3542, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112789

RESUMO

R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA/genética , Estruturas R-Loop , RNA Longo não Codificante/metabolismo , Telômero/metabolismo , Proteína BRCA1/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromatografia Líquida , Ilhas de CpG , DNA Helicases/metabolismo , Exorribonucleases/metabolismo , Humanos , Hibridização in Situ Fluorescente , Espectrometria de Massas , Enzimas Multifuncionais/metabolismo , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Estruturas R-Loop/genética , RNA Helicases/metabolismo , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Telômero/genética
18.
Front Immunol ; 12: 609543, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093517

RESUMO

The RLRs play critical roles in sensing and fighting viral infections especially RNA virus infections. Despite the extensive studies on RLRs in humans and mice, there is a lack of systemic investigation of livestock animal RLRs. In this study, we characterized the porcine RLR members RIG-I, MDA5 and LGP2. Compared with their human counterparts, porcine RIG-I and MDA5 exhibited similar signaling activity to distinct dsRNA and viruses, via similar and cooperative recognitions. Porcine LGP2, without signaling activity, was found to positively regulate porcine RIG-I and MDA5 in transfected porcine alveolar macrophages (PAMs), gene knockout PAMs and PK-15 cells. Mechanistically, LGP2 interacts with RIG-I and MDA5 upon cell activation, and promotes the binding of dsRNA ligand by MDA5 as well as RIG-I. Accordingly, porcine LGP2 exerted broad antiviral functions. Intriguingly, we found that porcine LGP2 mutants with defects in ATPase and/or dsRNA binding present constitutive activity which are likely through RIG-I and MDA5. Our work provided significant insights into porcine innate immunity, species specificity and immune biology.


Assuntos
Proteína DEAD-box 58/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , RNA Helicases/metabolismo , Animais , Linhagem Celular , Proteína DEAD-box 58/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Helicase IFIH1 Induzida por Interferon/genética , Mutação , Ligação Proteica , RNA Helicases/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Suínos
19.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065512

RESUMO

Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an "ancient" RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.


Assuntos
Antígenos de Neoplasias/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células T Matadoras Naturais/metabolismo , RNA Helicases/metabolismo , Espermatogênese/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Ativação Linfocitária/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo
20.
J Biol Chem ; 297(1): 100829, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34048711

RESUMO

Brr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar but enzymatically inactive C-terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide-binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide-binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active N-terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate these long-range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicing.


Assuntos
Nucleotídeos de Adenina/metabolismo , RNA Helicases/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Aminoácidos/metabolismo , Sítios de Ligação , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutação/genética , Domínios Proteicos , Ribonucleoproteínas Nucleares Pequenas/química , Especificidade por Substrato
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