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1.
PLoS Pathog ; 17(1): e1009226, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465137

RESUMO

Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.


Assuntos
/tratamento farmacológico , Infecções por Coronavirus/tratamento farmacológico , Exorribonucleases/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Infecções por Coronavirus/virologia , Exorribonucleases/genética , Humanos , Recombinação Genética/efeitos dos fármacos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética
2.
Int J Biol Macromol ; 168: 272-278, 2021 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-33309661

RESUMO

SARS-CoV-2is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. The nsp14 protein of SARS-CoV-2 houses a 3' to 5' exoribonuclease activity responsible for removing mismatches that arise during genome duplication. A homology model of nsp10-nsp14 complex was used to carry out in silico screening to identify molecules among natural products, or FDA approved drugs that can potentially inhibit the activity of nsp14. This exercise showed that ritonavir might bind to the exoribonuclease active site of the nsp14 protein. A model of the SARS-CoV-2-nsp10-nsp14 complex bound to substrate RNA showed that the ritonavir binding site overlaps with that of the 3' nucleotide of substrate RNA. A comparison of the calculated energies of binding for RNA and ritonavir suggested that the drug may bind to the active site of nsp14 with significant affinity. It is, therefore, possible that ritonavir may prevent association with substrate RNA and thus inhibit the exoribonuclease activity of nsp14. Overall, our computational studies suggest that ritonavir may serve as an effective inhibitor of the nsp14 protein. nsp14 is known to attenuate the inhibitory effect of drugs that function through premature termination of viral genome replication. Hence, ritonavir may potentiate the therapeutic properties of drugs such as remdesivir, favipiravir and ribavirin.


Assuntos
Antivirais/farmacologia , Exorribonucleases/antagonistas & inibidores , Ritonavir/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/administração & dosagem , Antivirais/química , Domínio Catalítico , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Exorribonucleases/química , Exorribonucleases/genética , Genoma Viral/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Pandemias , Ritonavir/administração & dosagem , Ritonavir/química , /fisiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos
3.
Nat Commun ; 11(1): 5362, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097710

RESUMO

Human C-terminal binding protein (CtBP)-interacting protein (CtIP) is a central regulator to initiate DNA end resection and homologous recombination (HR). Several studies have shown that post-translational modifications control the activity or expression of CtIP. However, it remains unclear whether and how cells restrain CtIP activity in unstressed cells and activate CtIP when needed. Here, we identify that USP52 directly interacts with and deubiquitinates CtIP, thereby promoting DNA end resection and HR. Mechanistically, USP52 removes the ubiquitination of CtIP to facilitate the phosphorylation and activation of CtIP at Thr-847. In addition, USP52 is phosphorylated by ATM at Ser-1003 after DNA damage, which enhances the catalytic activity of USP52. Furthermore, depletion of USP52 sensitizes cells to PARP inhibition in a CtIP-dependent manner in vitro and in vivo. Collectively, our findings reveal the key role of USP52 and the regulatory complexity of CtIP deubiquitination in DNA repair.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exorribonucleases/metabolismo , Ubiquitinação/fisiologia , Animais , Dano ao DNA , Exorribonucleases/genética , Feminino , Células HEK293 , Recombinação Homóloga , Humanos , Camundongos , Camundongos Nus , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Commun ; 11(1): 5496, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127896

RESUMO

Mechanical anisotropy is an essential property for many biomolecules to assume their structures, functions and applications, however, the mechanisms for their direction-dependent mechanical responses remain elusive. Herein, by using a single-molecule nanopore sensing technique, we explore the mechanisms of directional mechanical stability of the xrRNA1 RNA from ZIKA virus (ZIKV), which forms a complex ring-like architecture. We reveal extreme mechanical anisotropy in ZIKV xrRNA1 which highly depends on Mg2+ and the key tertiary interactions. The absence of Mg2+ and disruption of the key tertiary interactions strongly affect the structural integrity and attenuate mechanical anisotropy. The significance of ring structures in RNA mechanical anisotropy is further supported by steered molecular dynamics simulations in combination with force distribution analysis. We anticipate the ring structures can be used as key elements to build RNA-based nanostructures with controllable mechanical anisotropy for biomaterial and biomedical applications.


Assuntos
Fenômenos Bioquímicos , Exorribonucleases/genética , Exorribonucleases/metabolismo , RNA Viral/química , Zika virus/genética , Anisotropia , Humanos , Magnésio/metabolismo , Fenômenos Mecânicos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Dobramento de RNA , RNA Viral/genética , Infecção por Zika virus/virologia
5.
Proc Natl Acad Sci U S A ; 117(38): 23982-23990, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32887800

RESUMO

MAC5 is a component of the conserved MOS4-associated complex. It plays critical roles in development and immunity. Here we report that MAC5 is required for microRNA (miRNA) biogenesis. MAC5 interacts with Serrate (SE), which is a core component of the microprocessor that processes primary miRNA transcripts (pri-miRNAs) into miRNAs and binds the stem-loop region of pri-miRNAs. MAC5 is essential for both the efficient processing and the stability of pri-miRNAs. Interestingly, the reduction of pri-miRNA levels in mac5 is partially caused by XRN2/XRN3, the nuclear-localized 5'-to-3' exoribonucleases, and depends on SE. These results reveal that MAC5 plays a dual role in promoting pri-miRNA processing and stability through its interaction with SE and/or pri-miRNAs. This study also uncovers that pri-miRNAs need to be protected from nuclear RNA decay machinery, which is connected to the microprocessor.


Assuntos
Proteínas de Arabidopsis , Exorribonucleases , MicroRNAs , Proteínas Nucleares , Proteínas de Ligação a RNA , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
6.
J Virol ; 94(23)2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32938769

RESUMO

Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations has now been found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless-as happened occasionally-reversion had first occurred. Only a single mutant was viable, likely because its E191D substitution is highly conservative. Remarkably, a SARS-CoV-2 ExoN knockout mutant was found to be unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity while not affecting N7-MTase activity. Our study strongly suggests that CoV nsp14 ExoN has an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that, based on previous MHV and SARS-CoV studies, was proposed to boost longer-term replication fidelity.IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.


Assuntos
Betacoronavirus/fisiologia , Exorribonucleases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Betacoronavirus/enzimologia , Betacoronavirus/genética , Domínio Catalítico , Linhagem Celular , Exorribonucleases/química , Exorribonucleases/genética , Fluoruracila/farmacologia , Técnicas de Inativação de Genes , Genoma Viral , Humanos , Metilação , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Mutação , RNA Viral/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Ensaio de Placa Viral , Dedos de Zinco
7.
Nucleic Acids Res ; 48(18): 10428-10440, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960265

RESUMO

Cellular exonucleases involved in the processes that regulate RNA stability and quality control have been shown to restrict or to promote the multiplication cycle of numerous RNA viruses. Influenza A viruses are major human pathogens that are responsible for seasonal epidemics, but the interplay between viral proteins and cellular exonucleases has never been specifically studied. Here, using a stringent interactomics screening strategy and an siRNA-silencing approach, we identified eight cellular factors among a set of 75 cellular proteins carrying exo(ribo)nuclease activities or involved in RNA decay processes that support influenza A virus multiplication. We show that the exoribonuclease ERI1 interacts with the PB2, PB1 and NP components of the viral ribonucleoproteins and is required for viral mRNA transcription. More specifically, we demonstrate that the protein-protein interaction is RNA dependent and that both the RNA binding and exonuclease activities of ERI1 are required to promote influenza A virus transcription. Finally, we provide evidence that during infection, the SLBP protein and histone mRNAs co-purify with vRNPs alongside ERI1, indicating that ERI1 is most probably recruited when it is present in the histone pre-mRNA processing complex in the nucleus.


Assuntos
Exorribonucleases/genética , Vírus da Influenza A/genética , Influenza Humana/genética , Proteínas Nucleares/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Linhagem Celular , Histonas/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Interferente Pequeno , RNA Viral/genética , Ribonucleoproteínas/genética , Transcrição Genética/genética , Proteínas Virais/genética , Replicação Viral/genética
8.
Science ; 369(6503): 524-530, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732418

RESUMO

RNA molecules are frequently modified with a terminal 2',3'-cyclic phosphate group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2',3'-cyclic phosphates are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. We identified the predicted deadenylase angel homolog 2 (ANGEL2) as a human phosphatase that converts 2',3'-cyclic phosphates into 2',3'-OH nucleotides. We analyzed ANGEL2's substrate preference, structure, and reaction mechanism. Perturbing ANGEL2 expression affected the efficiency of pre-tRNA processing, X-box-binding protein 1 (XBP1) mRNA splicing during the unfolded protein response, and tRNA nucleotidyltransferase 1 (TRNT1)-mediated CCA addition onto tRNAs. Our results indicate that ANGEL2 is involved in RNA pathways that rely on the ligation or hydrolysis of 2',3'-cyclic phosphates.


Assuntos
Exorribonucleases/química , Nucleotidases/química , Ribonucleases/química , Cristalografia por Raios X , Exorribonucleases/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Nucleotidases/genética , Estrutura Secundária de Proteína , Precursores de RNA , Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/genética , Especificidade por Substrato , Proteína 1 de Ligação a X-Box/genética
9.
PLoS Genet ; 16(8): e1008893, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841241

RESUMO

All tRNAs are extensively modified, and modification deficiency often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of any of several tRNA body modifications results in rapid tRNA decay (RTD) of certain mature tRNAs by the 5'-3' exonucleases Rat1 and Xrn1. As tRNA quality control decay mechanisms are not extensively studied in other eukaryotes, we studied trm8Δ mutants in the evolutionarily distant fission yeast Schizosaccharomyces pombe, which lack 7-methylguanosine at G46 (m7G46) of their tRNAs. We report here that S. pombe trm8Δ mutants are temperature sensitive primarily due to decay of tRNATyr(GUA) and that spontaneous mutations in the RAT1 ortholog dhp1+ restored temperature resistance and prevented tRNA decay, demonstrating conservation of the RTD pathway. We also report for the first time evidence linking the RTD and the general amino acid control (GAAC) pathways, which we show in both S. pombe and S. cerevisiae. In S. pombe trm8Δ mutants, spontaneous GAAC mutations restored temperature resistance and tRNA levels, and the trm8Δ temperature sensitivity was precisely linked to GAAC activation due to tRNATyr(GUA) decay. Similarly, in the well-studied S. cerevisiae trm8Δ trm4Δ RTD mutant, temperature sensitivity was closely linked to GAAC activation due to tRNAVal(AAC) decay; however, in S. cerevisiae, GAAC mutations increased tRNA loss and exacerbated temperature sensitivity. A similar exacerbated growth defect occurred upon GAAC mutation in S. cerevisiae trm8Δ and other single modification mutants that triggered RTD. Thus, these results demonstrate a conserved GAAC activation coincident with RTD in S. pombe and S. cerevisiae, but an opposite impact of the GAAC response in the two organisms. We speculate that the RTD pathway and its regulation of the GAAC pathway is widely conserved in eukaryotes, extending to other mutants affecting tRNA body modifications.


Assuntos
Exorribonucleases/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA de Transferência/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , tRNA Metiltransferases/metabolismo , Aminoácidos/metabolismo , Evolução Molecular , Exorribonucleases/genética , RNA de Transferência/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , tRNA Metiltransferases/genética
10.
Nucleic Acids Res ; 48(13): 7404-7420, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32501509

RESUMO

RNA interference (RNAi) is a gene-silencing pathway that can play roles in viral defense, transposon silencing, heterochromatin formation and post-transcriptional gene silencing. Although absent from Saccharomyces cerevisiae, RNAi is present in other budding-yeast species, including Naumovozyma castellii, which have an unusual Dicer and a conventional Argonaute that are both required for gene silencing. To identify other factors that act in the budding-yeast pathway, we performed an unbiased genetic selection. This selection identified Xrn1p, the cytoplasmic 5'-to-3' exoribonuclease, as a cofactor of RNAi in budding yeast. Deletion of XRN1 impaired gene silencing in N. castellii, and this impaired silencing was attributable to multiple functions of Xrn1p, including affecting the composition of siRNA species in the cell, influencing the efficiency of siRNA loading into Argonaute, degradation of cleaved passenger strand and degradation of sliced target RNA.


Assuntos
Exorribonucleases/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Proteínas Argonauta/metabolismo , Clonagem Molecular , Exorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Saccharomyces/genética
11.
Am J Hum Genet ; 107(1): 24-33, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32502391

RESUMO

Zygotic cleavage failure (ZCF) is a unique early embryonic phenotype resulting in female infertility and recurrent failure of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). With this phenotype, morphologically normal oocytes can be retrieved and successfully fertilized, but they fail to undergo cleavage. Until now, whether this phenotype has a Mendelian inheritance pattern and which underlying genetic factors play a role in its development remained to be elucidated. B cell translocation gene 4 (BTG4) is a key adaptor of the CCR4-NOT deadenylase complex, which is involved in maternal mRNA decay in mice, but no human diseases caused by mutations in BTG4 have previously been reported. Here, we identified four homozygous mutations in BTG4 (GenBank: NM_017589.4) that are responsible for the phenotype of ZCF, and we found they followed a recessive inheritance pattern. Three of them-c.73C>T (p.Gln25Ter), c.1A>G (p.?), and c.475_478del (p.Ile159LeufsTer15)-resulted in complete loss of full-length BTG4 protein. For c.166G>A (p.Ala56Thr), although the protein level and distribution of mutant BTG4 was not altered in zygotes from affected individuals or in HeLa cells, the interaction between BTG4 and CNOT7 was abolished. In vivo studies further demonstrated that the process of maternal mRNA decay was disrupted in the zygotes of the affected individuals, which provides a mechanistic explanation for the phenotype of ZCF. Thus, we provide evidence that ZCF is a Mendelian phenotype resulting from mutations in BTG4. These findings contribute to our understanding of the role of BTG4 in human early embryonic development and provide a genetic marker for female infertility.


Assuntos
Proteínas de Ciclo Celular/genética , Infertilidade Feminina/genética , Mutação/genética , Zigoto/patologia , Animais , Linhagem Celular Tumoral , Desenvolvimento Embrionário/genética , Exorribonucleases/genética , Feminino , Células HeLa , Homozigoto , Humanos , Infertilidade Feminina/patologia , Camundongos , Fenótipo , Estabilidade de RNA/genética
12.
In Vivo ; 34(3 Suppl): 1629-1632, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32503821

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the coronavirus disease 2019 (COVID-19) pandemic. In the current analysis, we searched for SARS-CoV-2 sequences within the human genome. To compare the SARS-CoV-2 genome to the human genome, we used the blast-like alignment tool (BLAT) of the University of California, Santa Cruz Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. BLAT search results revealed a 117-base pair SARS-CoV-2 sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 orf1b (open reading frames) gene. The SARS-CoV-2 human sequence lies within non-structural proteins 14 and 15 (NSP14 and NSP15), and is quite close to the viral spike sequence, separated only by NSP16, a 904-base pair sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 and internalization of the complex by the host cell. It is probably no accident that a sequence from the SARS-CoV-2 orf1b gene is found in the human NTNG1 gene, implicated in schizophrenia, and that haloperidol, used to treat schizophrenia, may also be a treatment for COVID-19. We suggest, therefore, that it is important to investigate other haloperidol analogs. Among them are benperidol, bromperidol, bromperidol decanoate, droperidol, seperidol hydrochloride, and trifluperidol. These analogs might be valuable in the treatment of COVID-19 and other coronavirus infections.


Assuntos
Betacoronavirus/genética , Cromossomos Humanos Par 1/genética , Exorribonucleases/genética , Genes Virais , Netrina-1/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Sequência de Bases , Infecções por Coronavirus/tratamento farmacológico , DNA Complementar/genética , Endorribonucleases/genética , Haloperidol/análogos & derivados , Haloperidol/farmacologia , Haloperidol/uso terapêutico , Humanos , Íntrons/genética , Pan troglodytes/genética , Pandemias , Pneumonia Viral/tratamento farmacológico , Poliproteínas , RNA Viral/genética , Esquizofrenia/tratamento farmacológico , Esquizofrenia/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie
13.
In Vivo ; 34(3 Suppl): 1633-1636, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32503822

RESUMO

In a previous study, we identified a 117 base severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 Orf1b gene in non-structural protein 14 (NSP14), which is an exonuclease and NSP15, an endoribonuclease. In the current study we compared the human genome with other viral genomes to determine some of the characteristics of human sequences found in the latter. Most of the viruses had human sequences, but they were short. Hepatitis A and St Louis encephalitis had human sequences that were longer than the 117 base SARS-Cov-2 sequence, but they were in non-coding regions of the human genome. The SARS-Cov-2 sequence was the only long sequence found in a human gene (NTNG1). The related coronaviruses SARS-Cov had a 41 BP human sequence on chromosome 3 that was not part of a human gene, and MERS had no human sequence. The 117 base SARS-CoV-2 human sequence is relatively close to the viral spike sequence, separated only by NSP16, a 904 base sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 (ACE2) and internalization of the complex by the host cell. We have no explanation for the NSP14 and NSP15 SARS-Cov-2 sequences we observed here or how they might relate to infectiousness. Further studies are warranted.


Assuntos
Betacoronavirus/genética , Exorribonucleases/genética , Genoma Viral , Vírus da SARS/genética , Proteínas não Estruturais Virais/genética , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 3/genética , Vírus de DNA/genética , Endorribonucleases , Proteínas Ligadas por GPI/genética , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Netrinas/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Proteínas Virais/genética
14.
Nat Commun ; 11(1): 2765, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488030

RESUMO

MicroRNAs (miRNAs) associated with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3' ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability. However, the underlying mechanism is not well understood. Here, by genetic and biochemical studies as well as deep sequencing analyses, we find that AGO mutations disrupting miRNA 3' binding are sufficient to trigger extensive miRNA 3' modifications in HEK293T cells and in cancer patients. Comparing these modifications in TUT4, TUT7 and DIS3L2 knockout cells, we find that TUT7 is more robust than TUT4 in oligouridylating mature miRNAs, which in turn leads to their degradation by the DIS3L2 exonuclease. Our findings indicate a decay machinery removing AGO-associated miRNAs with an exposed 3' end. A set of endogenous miRNAs including miR-7, miR-222 and miR-769 are targeted by this machinery presumably due to target-directed miRNA degradation.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , MicroRNAs/metabolismo , RNA Nucleotidiltransferases/metabolismo , Proteínas Argonauta/genética , Proteínas de Ligação a DNA/genética , Exorribonucleases/genética , Técnicas de Inativação de Genes , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/genética , RNA Nucleotidiltransferases/genética
15.
Emerg Microbes Infect ; 9(1): 1418-1428, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32529952

RESUMO

The Coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2 virus, is now causing a tremendous global health concern. Since its first appearance in December 2019, the outbreak has already caused over 5.8 million infections worldwide (till 29 May 2020), with more than 0.35 million deaths. Early virus-mediated immune suppression is believed to be one of the unique characteristics of SARS-CoV-2 infection and contributes at least partially to the viral pathogenesis. In this study, we identified the key viral interferon antagonists of SARS-CoV-2 and compared them with two well-characterized SARS-CoV interferon antagonists, PLpro and orf6. Here we demonstrated that the SARS-CoV-2 nsp13, nsp14, nsp15 and orf6, but not the unique orf8, could potently suppress primary interferon production and interferon signalling. Although SARS-CoV PLpro has been well-characterized for its potent interferon-antagonizing, deubiquitinase and protease activities, SARS-CoV-2 PLpro, despite sharing high amino acid sequence similarity with SARS-CoV, loses both interferon-antagonising and deubiquitinase activities. Among the 27 viral proteins, SARS-CoV-2 orf6 demonstrated the strongest suppression on both primary interferon production and interferon signalling. Orf6-deleted SARS-CoV-2 may be considered for the development of intranasal live-but-attenuated vaccine against COVID-19.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Interferons/antagonistas & inibidores , Interferons/metabolismo , Metiltransferases/metabolismo , Pneumonia Viral/metabolismo , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Endorribonucleases/genética , Exorribonucleases/genética , Interações Hospedeiro-Patógeno , Humanos , Interferons/genética , Metiltransferases/genética , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Helicases/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
16.
Nucleic Acids Res ; 48(11): 6136-6148, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374864

RESUMO

In eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. Here, we report that these enzymes can also remove FAD and dephospho-CoA (dpCoA) non-canonical caps from RNA, and we have named these activities deFADding and deCoAping. The crystal structures of mammalian DXO with 3'-FADP or CoA and fission yeast Rai1 with 3'-FADP provide elegant insight to these activities. FAD and CoA are accommodated in the DXO/Rai1 active site by adopting folded conformations. The flavin of FAD and the pantetheine group of CoA contact the same region at the bottom of the active site tunnel, which undergoes conformational changes to accommodate the different cap moieties. We have developed FAD-capQ to detect and quantify FAD-capped RNAs and determined that FAD caps are present on short RNAs (with less than ∼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO.


Assuntos
Coenzima A/metabolismo , Exorribonucleases/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Kluyveromyces/enzimologia , Proteínas Nucleares/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Animais , Exorribonucleases/química , Exorribonucleases/genética , Flavina-Adenina Dinucleotídeo/análise , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , NAD/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Capuzes de RNA/análise , Especificidade por Substrato , Transcrição Genética
17.
J Virol ; 94(16)2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32461321

RESUMO

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.


Assuntos
Exorribonucleases/metabolismo , Interferon Tipo I/biossíntese , Interferons/biossíntese , Vírus da Diarreia Epidêmica Suína/fisiologia , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Exorribonucleases/genética , Expressão Gênica , Guanina/metabolismo , Imunidade Inata , Metilação , Mutação , Vírus da Diarreia Epidêmica Suína/enzimologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Suínos , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral
18.
Nat Commun ; 11(1): 2619, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457326

RESUMO

DIS3L2-mediated decay (DMD) is a surveillance pathway for certain non-coding RNAs (ncRNAs) including ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and RMRP. While mutations in DIS3L2 are associated with Perlman syndrome, the biological significance of impaired DMD is obscure and pathological RNAs have not been identified. Here, by ribosome profiling (Ribo-seq) we find specific dysregulation of endoplasmic reticulum (ER)-targeted mRNA translation in DIS3L2-deficient cells. Mechanistically, DMD functions in the quality control of the 7SL ncRNA component of the signal recognition particle (SRP) required for ER-targeted translation. Upon DIS3L2 loss, sustained 3'-end uridylation of aberrant 7SL RNA impacts ER-targeted translation and causes ER calcium leakage. Consequently, elevated intracellular calcium in DIS3L2-deficient cells activates calcium signaling response genes and perturbs ESC differentiation. Thus, DMD is required to safeguard ER-targeted mRNA translation, intracellular calcium homeostasis, and stem cell differentiation.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Exorribonucleases/metabolismo , Macrossomia Fetal/microbiologia , RNA Mensageiro/metabolismo , Tumor de Wilms/microbiologia , Animais , Sinalização do Cálcio/genética , Diferenciação Celular , Células-Tronco Embrionárias , Exorribonucleases/deficiência , Exorribonucleases/genética , Macrossomia Fetal/enzimologia , Macrossomia Fetal/genética , Regulação da Expressão Gênica , Humanos , Insulina/metabolismo , Camundongos , Biossíntese de Proteínas , RNA Citoplasmático Pequeno/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Uridina Monofosfato/metabolismo , Tumor de Wilms/enzimologia , Tumor de Wilms/genética
19.
Antiviral Res ; 178: 104793, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32283108

RESUMO

The rapid global emergence of SARS-CoV-2 has been the cause of significant health concern, highlighting the immediate need for antivirals. Viral RNA-dependent RNA polymerases (RdRp) play essential roles in viral RNA synthesis, and thus remains the target of choice for the prophylactic or curative treatment of several viral diseases, due to high sequence and structural conservation. To date, the most promising broad-spectrum class of viral RdRp inhibitors are nucleoside analogues (NAs), with over 25 approved for the treatment of several medically important viral diseases. However, Coronaviruses stand out as a particularly challenging case for NA drug design due to the presence of an exonuclease (ExoN) domain capable of excising incorporated NAs and thus providing resistance to many of these available antivirals. Here we use the available structures of the SARS-CoV RdRp and ExoN proteins, as well as Lassa virus N exonuclease to derive models of catalytically competent SARS-CoV-2 enzymes. We then map a promising NA candidate, GS-441524 (the active metabolite of Remdesivir) to the nucleoside active site of both proteins, identifying the residues important for nucleotide recognition, discrimination, and excision. Interestingly, GS-441524 addresses both enzyme active sites in a manner consistent with significant incorporation, delayed chain termination, and altered excision due to the ribose 1'-CN group, which may account for the increased antiviral effect compared to other available analogues. Additionally, we propose structural and function implications of two previously identified RdRp resistance mutations in relation to resistance against Remdesivir. This study highlights the importance of considering the balance between incorporation and excision properties of NAs between the RdRp and ExoN.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antimetabólitos/farmacologia , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Exorribonucleases/química , Proteínas não Estruturais Virais/química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/química , Alanina/farmacologia , Antimetabólitos/química , Antivirais/química , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , Domínio Catalítico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Farmacorresistência Viral , Exorribonucleases/genética , Exorribonucleases/metabolismo , Humanos , Modelos Moleculares , Mutação , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Conformação Proteica , RNA Viral/química , RNA Viral/genética , /metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
20.
Nucleic Acids Res ; 48(10): 5349-5365, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32313933

RESUMO

Growing mammalian oocytes accumulate substantial amounts of RNA, most of which is degraded during subsequent meiotic maturation. The growth-to-maturation transition begins with germinal vesicle or nuclear envelope breakdown (GVBD) and is critical for oocyte quality and early development. The molecular machinery responsible for the oocyte transcriptome transition remains unclear. Here, we report that an exosome-associated RNase, EXOSC10, sculpts the transcriptome to facilitate the growth-to-maturation transition of mouse oocytes. We establish an oocyte-specific conditional knockout of Exosc10 in mice using CRISPR/Cas9 which results in female subfertility due to delayed GVBD. By performing multiple single oocyte RNA-seq, we document dysregulation of several types of RNA, and the mRNAs that encode proteins important for endomembrane trafficking and meiotic cell cycle. As expected, EXOSC10-depleted oocytes have impaired endomembrane components including endosomes, lysosomes, endoplasmic reticulum and Golgi. In addition, CDK1 fails to activate, possibly due to persistent WEE1 activity, which blocks lamina phosphorylation and disassembly. Moreover, we identified rRNA processing defects that cause higher percentage of developmentally incompetent oocytes after EXOSC10 depletion. Collectively, we propose that EXOSC10 promotes normal growth-to-maturation transition in mouse oocytes by sculpting the transcriptome to degrade RNAs encoding growth-phase factors and, thus, support the maturation phase of oogenesis.


Assuntos
Exorribonucleases/fisiologia , Complexo Multienzimático de Ribonucleases do Exossomo/fisiologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oogênese , Transcriptoma , Animais , Proteína Quinase CDC2/metabolismo , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Feminino , Infertilidade Feminina/genética , Membranas Intracelulares/metabolismo , Camundongos , Lâmina Nuclear/metabolismo , Poli A , RNA/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , RNA-Seq
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