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1.
Mol Cell ; 81(15): 3128-3144.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34216544

RESUMO

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.


Assuntos
Proteína BRCA1/genética , Replicação do DNA/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Linhagem Celular , Cisplatino/farmacologia , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos Endogâmicos NOD , RNA Helicases/genética , Rad51 Recombinase/genética , Proteína de Replicação A/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
2.
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
3.
BMJ Case Rep ; 14(6)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193451

RESUMO

Ataxia with oculomotor apraxia type 2 (AOA2), recently renamed as ATX-SETX, is an autosomal recessive, progressive neurodegenerative disorder belonging to inherited cerebellar ataxias. The pathogenic variants of the SETX gene have been implicated in ATX-SETX. We report the case of a 21-year-old woman presenting with ataxia, oculomotor apraxia and dystonia. She had elevated serum α-fetoprotein (AFP), follicle stimulating hormone (FSH) and luteinising hormone (LH) levels and moderate cerebellar atrophy. On further evaluation, she was found to have premature ovarian failure as well. Multiplex ligation-dependent probe amplification detected a heterozygous deletion in exon 6 of the SETX gene. A combination of cerebellar ataxia, oculomotor apraxia with elevated AFP and cerebellar atrophy are highly suggestive of ATX-SETX. In rare instances, it may be associated with premature ovarian failure with elevated FSH and LH levels, necessitating hormonal survey and fertility evaluation in all patients with ATX-SETX.


Assuntos
Apraxias , Ataxia Cerebelar , Adulto , Apraxias/genética , Ataxia , Ataxia Cerebelar/genética , DNA Helicases , Éxons/genética , Feminino , Humanos , Enzimas Multifuncionais , RNA Helicases/genética , Ataxias Espinocerebelares/congênito , Adulto Jovem
4.
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
5.
Arch Virol ; 166(9): 2529-2540, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34251549

RESUMO

RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as "non-positive" (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/genética , SARS-CoV-2/genética , Proteínas Virais/genética , COVID-19/epidemiologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/instrumentação , Cor , Proteínas do Envelope de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , França/epidemiologia , Expressão Gênica , Humanos , Limite de Detecção , Nasofaringe/virologia , Fosfoproteínas/genética , RNA Helicases/genética , RNA Polimerase Dependente de RNA/genética , Carga Viral
6.
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
7.
J Phys Chem B ; 125(31): 8787-8796, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34328740

RESUMO

The COVID-19 pandemic has demonstrated the need to develop potent and transferable therapeutics to treat coronavirus infections. Numerous antiviral targets are being investigated, but nonstructural protein 13 (nsp13) stands out as a highly conserved and yet understudied target. Nsp13 is a superfamily 1 (SF1) helicase that translocates along and unwinds viral RNA in an ATP-dependent manner. Currently, there are no available structures of nsp13 from SARS-CoV-1 or SARS-CoV-2 with either ATP or RNA bound, which presents a significant hurdle to the rational design of therapeutics. To address this knowledge gap, we have built models of SARS-CoV-2 nsp13 in Apo, ATP, ssRNA and ssRNA+ATP substrate states. Using 30 µs of a Gaussian-accelerated molecular dynamics simulation (at least 6 µs per substrate state), these models were confirmed to maintain substrate binding poses that are similar to other SF1 helicases. A Gaussian mixture model and linear discriminant analysis structural clustering protocol was used to identify key structural states of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inchworm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV, and V and suggest a power stroke-like motion of domain 2A relative to domain 1A. This structural and mechanistic insight of nsp13 RNA translocation presents novel targets for the further development of antivirals.


Assuntos
COVID-19 , SARS-CoV-2 , Trifosfato de Adenosina , Antivirais , Humanos , Pandemias , RNA Helicases/genética , RNA Viral/genética , Proteínas não Estruturais Virais/genética
8.
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
9.
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
10.
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
11.
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
12.
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
13.
J Immunol ; 206(10): 2453-2467, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33941659

RESUMO

The detection of intracellular nucleic acids is a fundamental mechanism of host defense against infections. The dysregulated nucleic acid sensing, however, is a major cause for a number of autoimmune diseases. In this study, we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for both intracellular DNA- and RNA-induced immune responses. We found that in both human and mouse cells, the deletion of G3BP1 led to the dampened cGAS activation by DNA and the insufficient binding of RNA by RIG-I. We further found that resveratrol (RSVL), a natural compound found in grape skin, suppressed both intracellular DNA- and RNA-induced type I IFN production through inhibiting G3BP1. Importantly, using experimental mouse models for Aicardi-Goutières syndrome, an autoimmune disorder found in humans, we demonstrated that RSVL effectively alleviated intracellular nucleic acid-stimulated autoimmune responses. Thus, our study demonstrated a broader role of G3BP1 in sensing different kinds of intracellular nucleic acids and presented RSVL as a potential treatment for autoimmune conditions caused by dysregulated nucleic acid sensing.


Assuntos
Autoimunidade/genética , DNA Helicases/deficiência , DNA Helicases/metabolismo , Espaço Intracelular/metabolismo , Ácidos Nucleicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/deficiência , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/deficiência , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Transdução de Sinais/genética , Células A549 , Animais , Autoimunidade/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Espaço Intracelular/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/genética , Resveratrol/administração & dosagem , Transdução de Sinais/imunologia , Transfecção
14.
Nat Commun ; 12(1): 3082, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035302

RESUMO

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5' splice-site (5'SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28's ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas Nucleares/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , RNA Helicases/genética , RNA Helicases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease H/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética
15.
J Proteome Res ; 20(6): 3165-3178, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33939924

RESUMO

Cytoplasmic stress granules (SGs) are dynamic foci containing translationally arrested mRNA and RNA-binding proteins (RBPs) that form in response to a variety of cellular stressors. It has been debated that SGs may evolve into cytoplasmic inclusions observed in many neurodegenerative diseases. Recent studies have examined the SG proteome by interrogating the interactome of G3BP1. However, it is widely accepted that multiple baits are required to capture the full SG proteome. To gain further insight into the SG proteome, we employed immunoprecipitation coupled with mass spectrometry of endogenous Caprin-1, an RBP implicated in mRNP granules. Overall, we identified 1543 proteins that interact with Caprin-1. Interactors under stressed conditions were primarily annotated to the ribosome, spliceosome, and RNA transport pathways. We validated four Caprin-1 interactors that localized to arsenite-induced SGs: ANKHD1, TALIN-1, GEMIN5, and SNRNP200. We also validated these stress-induced interactions in SH-SY5Y cells and further determined that SNRNP200 also associated with osmotic- and thermal-induced SGs. Finally, we identified SNRNP200 in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) spinal cord and motor cortex. Collectively, our findings provide the first description of the Caprin-1 protein interactome, identify novel cytoplasmic SG components, and reveal a SG protein in cytoplasmic aggregates in ALS patient neurons. Proteomic data collected in this study are available via ProteomeXchange with identifier PXD023271.


Assuntos
Grânulos Citoplasmáticos , DNA Helicases , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , Proteômica , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA , Proteínas de Ligação a RNA/genética
16.
PLoS Pathog ; 17(4): e1009530, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33909701

RESUMO

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3-METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.


Assuntos
Adenosina/análogos & derivados , RNA Helicases DEAD-box/fisiologia , Evasão da Resposta Imune/genética , Estabilidade de RNA/genética , Viroses , Adenosina/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Cricetinae , RNA Helicases DEAD-box/genética , Células HEK293 , Humanos , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Viroses/genética , Viroses/imunologia , Viroses/metabolismo , Replicação Viral/genética
17.
J Gen Virol ; 102(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33891535

RESUMO

RNA-remodelling proteins, including RNA helicases and chaperones, function to remodel structured RNAs and/or RNA-protein interactions and play indispensable roles in viral life cycles. Guaico Culex virus (GCXV) is the first uncovered animal-infected multicomponent virus with segmented positive-sense genomic RNAs. GCXV belongs to the Jingmenvirus group, a diverse clade of segmented viruses that are related to the prototypically unsegmented Flavivirus. However, little is known about the exact functions of the GCXV-encoded proteins. Here, we show that the putative non-structural protein (NSP) 2 on segment 2 of GCXV functions as an RNA helicase that unwinds RNA helix bidirectionally in an adenosine triphosphate (ATP)-dependent manner, and an RNA chaperone that remodels structured RNAs and facilitates RNA strand annealing independently of ATP. Together, our findings are the first demonstration of RNA-remodelling activity encoded by Jingmenvirus and highlight the functional significance of NSP2 in the GCXV life cycle.


Assuntos
Culex/virologia , RNA Helicases/genética , Proteínas não Estruturais Virais/genética , Vírus não Classificados/genética , Animais , Chaperonas Moleculares/genética , Dobramento de Proteína , RNA Viral/metabolismo , Replicação Viral
18.
Viruses ; 13(3)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799742

RESUMO

The genus Flavivirus includes related, unclassified segmented flavi-like viruses, two segments of which have homology with flavivirus RNA-dependent RNA polymerase NS5 and RNA helicase-protease NS3. This group includes such viruses as Jingmen tick virus, Alongshan virus, Yanggou tick virus and others. We detected the Yanggou tick virus in Dermacentor nuttalli and Dermacentor marginatus ticks in two neighbouring regions of Russia. The virus prevalence ranged from 0.5% to 8.0%. We detected RNA of the Alongshan virus in 44 individuals or pools of various tick species in eight regions of Russia. The virus prevalence ranged from 0.6% to 7.8%. We demonstrated the successful replication of the Yanggou tick virus and Alongshan virus in IRE/CTVM19 and HAE/CTVM8 tick cell lines without a cytopathic effect. According to the phylogenetic analysis, we divided the Alongshan virus into two groups: an Ixodes persulcatus group and an Ixodes ricinus group. In addition, the I. persulcatus group can be divided into European and Asian subgroups. We found amino acid signatures specific to the I. ricinus and I. persulcatus groups and also distinguished between the European and Asian subgroups of the I. persulcatus group.


Assuntos
Dermacentor/virologia , Infecções por Flaviviridae/epidemiologia , Flaviviridae/genética , Ixodes/virologia , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos/genética , Animais , Vetores Aracnídeos/virologia , Linhagem Celular , Culicidae/virologia , Flaviviridae/isolamento & purificação , Filogenia , RNA Helicases/genética , RNA Viral/genética , Federação Russa/epidemiologia , Serina Endopeptidases/genética
19.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804185

RESUMO

The progress of the cell cycle is directly regulated by modulation of cyclins and cyclin-dependent kinases. However, many proteins that control DNA replication, RNA transcription and the synthesis and degradation of proteins can manage the activity or levels of master cell cycle regulators. Among them, RNA helicases are key participants in RNA metabolism involved in the global or specific tuning of cell cycle regulators at the level of transcription and translation. Several RNA helicases have been recently evaluated as promising therapeutic targets, including eIF4A, DDX3 and DDX5. However, targeting RNA helicases can result in side effects due to the influence on the cell cycle. In this review, we discuss direct and indirect participation of RNA helicases in the regulation of the cell cycle in order to draw attention to downstream events that may occur after suppression or inhibition of RNA helicases.


Assuntos
Ciclo Celular/genética , Replicação do DNA/genética , RNA Helicases/genética , Divisão Celular/genética , Quinases Ciclina-Dependentes/genética , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/genética , Humanos
20.
PLoS Pathog ; 17(4): e1009496, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33872335

RESUMO

LINE-1 (L1) retrotransposons are autonomous transposable elements that can affect gene expression and genome integrity. Potential consequences of exogenous viral infections for L1 activity have not been studied to date. Here, we report that hepatitis C virus (HCV) infection causes a significant increase of endogenous L1-encoded ORF1 protein (L1ORF1p) levels and translocation of L1ORF1p to HCV assembly sites at lipid droplets. HCV replication interferes with retrotransposition of engineered L1 reporter elements, which correlates with HCV RNA-induced formation of stress granules and can be partially rescued by knockdown of the stress granule protein G3BP1. Upon HCV infection, L1ORF1p localizes to stress granules, associates with HCV core in an RNA-dependent manner and translocates to lipid droplets. While HCV infection has a negative effect on L1 mobilization, L1ORF1p neither restricts nor promotes HCV infection. In summary, our data demonstrate that HCV infection causes an increase of endogenous L1 protein levels and that the observed restriction of retrotransposition of engineered L1 reporter elements is caused by sequestration of L1ORF1p in HCV-induced stress granules.


Assuntos
Carcinoma Hepatocelular/virologia , DNA Helicases/metabolismo , Hepacivirus/fisiologia , Hepatite C/virologia , Neoplasias Hepáticas/virologia , Elementos Nucleotídeos Longos e Dispersos/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular Tumoral , Grânulos Citoplasmáticos/virologia , DNA Helicases/genética , Humanos , Gotículas Lipídicas/virologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Ribonucleoproteínas/genética
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