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1.
Protein Expr Purif ; 185: 105894, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33933612

RESUMO

The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19) has led to a world-wild pandemic. The replication of SARS-CoV-2 RNA genome involves the core replication-transcription complex (RTC, nsp12-nsp7-nsp8) and the proofreading complex (nsp14-nsp10) that can correct mismatched base pairs during replication. Structures and functions of SARS-CoV-2 RTC have been actively studied, yet little is known about SARS-CoV-2 nsp14-nsp10. Here, we purified, reconstituted, and characterized the SARS-CoV-2 nsp14-nsp10 proofreading nuclease in vitro. We show that SARS-CoV-2 nsp14 is activated by nsp10, functioning as a potent RNase that can hydrolyze RNAs in the context of single- and double-stranded RNA and RNA/DNA hybrid duplex. SARS-CoV-2 nsp14-nsp10 shows a metal-dependent nuclease activity but has different metal selectivity from RTC. While RTC is activated by Ca2+, nsp14-nsp10 is completely inhibited. Importantly, the reconstituted SARS-CoV-2 nsp14-nsp10 efficiently removed the A:A mismatch at the 3'-end of the primer, enabling the stalled RTC to restart RNA replication. Our collective results confirm that SARS-CoV-2 nsp14-nsp10 functions as the RNA proofreading complex in SARS-CoV-2 replication and provide a useful foundation to understand the structure and function of SARS-CoV-2 RNA metabolism.


Assuntos
COVID-19/virologia , Exorribonucleases/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Cálcio/metabolismo , Ativação Enzimática , Humanos , Hidrólise , Especificidade por Substrato
2.
Nucleic Acids Res ; 49(9): 5382-5392, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33956156

RESUMO

The emergence of SARS-CoV-2 infection has posed unprecedented threat to global public health. The virus-encoded non-structural protein 14 (nsp14) is a bi-functional enzyme consisting of an exoribonuclease (ExoN) domain and a methyltransferase (MTase) domain and plays a pivotal role in viral replication. Here, we report the structure of SARS-CoV-2 nsp14-ExoN domain bound to its co-factor nsp10 and show that, compared to the SARS-CoV nsp10/nsp14-full-length complex, SARS-CoV-2 nsp14-ExoN retains an integral exoribonuclease fold and preserves an active configuration in the catalytic center. Analysis of the nsp10/nsp14-ExoN interface reveals a footprint in nsp10 extensively overlapping with that observed in the nsp10/nsp16 structure. A marked difference in the co-factor when engaging nsp14 and nsp16 lies in helix-α1', which is further experimentally ascertained to be involved in nsp14-binding but not in nsp16-engagement. Finally, we also show that nsp10/nsp14-ExoN is enzymatically active despite the absence of nsp14-MTase domain. These data demonstrate that SARS-CoV-2 nsp10/nsp14-ExoN functions as an exoribonuclease with both structural and functional integrity.


Assuntos
Biocatálise , Exorribonucleases/química , Exorribonucleases/metabolismo , SARS-CoV-2/química , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Exorribonucleases/genética , Guanina , Metiltransferases/química , Metiltransferases/deficiência , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Domínios Proteicos/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/genética
3.
J Med Virol ; 93(7): 4258-4264, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33837972

RESUMO

The recent coronavirus disease 2019 (COVID-19), causing a global pandemic with devastating effects on healthcare and social-economic systems, has no special antiviral therapies available for human coronaviruses (CoVs). The severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) possesses a nonstructural protein (nsp14), with amino-terminal domain coding for proofreading exoribonuclease (ExoN) that is required for high-fidelity replication. The ability of CoVs during genome replication and transcription to proofread and exclude mismatched nucleotides has long hindered the development of anti-CoV drugs. The resistance of SARS-CoV-2 to antivirals, especially nucleoside analogs (NAs), shows the need to identify new CoV inhibition targets. Therefore, this review highlights the importance of nsp14-ExoN as a target for inhibition. Also, nucleoside analogs could be used in combination with existing anti-CoV therapeutics to target the proofreading mechanism.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Exorribonucleases/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos , Exorribonucleases/efeitos dos fármacos , Exorribonucleases/metabolismo , Genoma Viral/genética , Humanos , Metiltransferases/genética , Processamento Pós-Transcricional do RNA/fisiologia , RNA Viral/genética , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia
4.
Nucleic Acids Res ; 49(8): 4738-4749, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33788943

RESUMO

RNA 2'-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3'-5' exoribonuclease and is particularly sensitive to RNA 2'-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2'-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR's ribonuclease activity and significant sensitivity to RNA 2'-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2'-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2'-O-methylations research in biological and clinical samples.


Assuntos
Exorribonucleases/química , Mycoplasma genitalium/química , RNA/química , Ribose/metabolismo , Catálise , Domínio Catalítico/genética , Cromatografia Líquida , Cristalografia por Raios X , Escherichia coli/metabolismo , Exorribonucleases/metabolismo , Metilação , Mutagênese , Mutação , Mycoplasma genitalium/enzimologia , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Estabilidade de RNA , Proteínas Recombinantes , Especificidade por Substrato , Espectrometria de Massas em Tandem
5.
Mol Cell ; 81(9): 1935-1950.e6, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33735606

RESUMO

Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly because of technical limitations in purifying authentic nascent transcripts. We present a new approach to characterize nascent RNA, called polymerase intact nascent transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5' ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano), and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5' P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that co-transcriptional splicing either occurs immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.


Assuntos
Nanotecnologia , RNA Polimerase II/metabolismo , Precursores de RNA/biossíntese , Splicing de RNA , RNA Mensageiro/biossíntese , RNA-Seq , Transcrição Genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Células HCT116 , Células HeLa , Humanos , Cinética , Poliadenilação , Capuzes de RNA , RNA Polimerase II/genética , Precursores de RNA/genética , RNA Mensageiro/genética
6.
Life Sci ; 268: 118996, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33412213

RESUMO

AIMS: CNOT7 plays an important role in many biological processes, providing attractive opportunities for the treatment of malignant tumors. However, the functions and mechanism of CNOT7 in ovarian cancer (OC) have not been elucidated. The purpose of this study was to assess the role of CNOT7 in OC. MATERIALS AND METHODS: SKOV3 and A2780 cells were chosen as the cell lines for the experiments of this manuscript via the analysis of the expression of CNOT7 protein and the mRNA level in ovarian surface epithelium (OSE) cells, SKOV3, HO8910 and A2780 cells. The expression of CNOT7 was detected by western blot assays and RT-PCR in A2780 and SKOV3 cells. The MTT assays, colony formation assays and EdU assays were used to measure cell proliferation when CNOT7 was knocked down or overexpressed in A2780 and SKOV3 cells. Furthermore, cell migration and invasion ability were achieved from transwell assays. Cell cycle and apoptosis rate after small interference RNA-CNOT7 (siRNA-CNOT7) were detected by flow cytometry assays. Finally, the cell proliferation, migration and invasion ability were detected when A2780 and SKOV3 cells with CNOT7 overexpression were treated with LY294002. KEY FINDINGS: The expression of CNOT7 protein in OC cells, including SKOV3, HO8910 and A2780 cells were significantly higher than that in OSE cells (P < 0.05). The mRNA level of CNOT7 in HO8910 and A2780 cells were significantly higher than that in OSE cells (P < 0.01). However, the mRNA level of CNOT7 in SKOV3 cells was no significant difference compared with OSE cells (P > 0.05). The results suggested that knockdown of CNOT7 could inhibit the cell proliferation, migration and invasion ability in A2780 and SKOV3 cells, and increase cell apoptosis and autophagy. The expression of apoptosis-related molecules (PARP, Caspase3 and Caspase9) and autophagy-related protein (LC3B) were up-regulated after CNOT7 knockdown, while the expression of cycle-related protein (CDK6) and the anti-apoptotic gene (Bcl2) were downregulated. Meanwhile, the opposite results were observed when CNOT7 was overexpressed in A2780 and SKOV3 cells. It is worth noting that the effect of CNOT7 overexpression in A2780 and SKOV3 cells could be partially or completely eliminated by treatment with AKT inhibitor LY294002. SIGNIFICANCE: CNOT7 has a carcinogenic effect in OC, and the carcinogenic effect may be achieved via the AKT signaling pathway.


Assuntos
Exorribonucleases/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cromonas/farmacologia , Exorribonucleases/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Morfolinas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Proteínas Repressoras/genética , Ensaio Tumoral de Célula-Tronco
7.
Nucleic Acids Res ; 49(7): e41, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33511416

RESUMO

Modifications in RNA are numerous (∼170) and in higher numbers compared to DNA (∼5) making the ability to sequence an RNA molecule to identify these modifications highly tenuous using next generation sequencing (NGS). The ability to immobilize an exoribonuclease enzyme, such as XRN1, to a solid support while maintaining its activity and capability to cleave both the canonical and modified ribonucleotides from an intact RNA molecule can be a viable approach for single-molecule RNA sequencing. In this study, we report an enzymatic reactor consisting of covalently attached XRN1 to a solid support as the groundwork for a novel RNA exosequencing technique. The covalent attachment of XRN1 to a plastic solid support was achieved using EDC/NHS coupling chemistry. Studies showed that the solid-phase digestion efficiency of model RNAs was 87.6 ± 2.8%, while the XRN1 solution-phase digestion for the same model was 78.3 ± 4.4%. The ability of immobilized XRN1 to digest methylated RNA containing m6A and m5C ribonucleotides was also demonstrated. The processivity and clipping rate of immobilized XRN1 secured using single-molecule fluorescence measurements of a single RNA transcript demonstrated a clipping rate of 26 ± 5 nt s-1 and a processivity of >10.5 kb at 25°C.


Assuntos
Distrofina/genética , Enzimas Imobilizadas/metabolismo , Exorribonucleases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , RNA/metabolismo , Análise de Sequência de RNA/métodos , Humanos , Clivagem do RNA
8.
J Struct Biol ; 213(1): 107690, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33383190

RESUMO

COVID-19 pandemic, caused by SARS-CoV-2, has drastically affected human health all over the world. After the emergence of the pandemic the major focus of efforts to attenuate the infection has been on repurposing the already approved drugs to treat COVID-19 adopting a fast-track strategy. However, to date a specific regimen to treat COVID-19 is not available. Over the last few months a substantial amount of data about the structures of various key proteins and their recognition partners involved in the SARS-CoV-2 pathogenesis has emerged. These studies have not only provided the molecular level descriptions ofthe viral pathogenesis but also laid the foundation for rational drug design and discovery. In this review, we have recapitulated the structural details of four key viral enzymes, RNA-dependent RNA polymerase, 3-chymotrypsin like protease, papain-like protease and helicase, and two host factors including angiotensin-converting enzyme 2 and transmembrane serine protease involved in the SARS-CoV-2 pathogenesis, and described the potential hotspots present on these structures which could be explored for therapeutic intervention. We have also discussed the significance of endoplasmic reticulum α-glucosidases as potential targets for anti-SARS-CoV-2 drug discovery.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/virologia , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Exorribonucleases/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/metabolismo , RNA Helicases/metabolismo , SARS-CoV-2/fisiologia , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo
9.
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
10.
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
11.
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
12.
J Proteome Res ; 19(11): 4670-4677, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32907334

RESUMO

The global pandemic of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to the death of more than 675,000 worldwide and over 150,000 in the United States alone. However, there are currently no approved effective pharmacotherapies for COVID-19. Here, we combine homology modeling, molecular docking, molecular dynamics simulation, and binding affinity calculations to determine potential targets for toremifene, a selective estrogen receptor modulator which we have previously identified as a SARS-CoV-2 inhibitor. Our results indicate the possibility of inhibition of the spike glycoprotein by toremifene, responsible for aiding in fusion of the viral membrane with the cell membrane, via a perturbation to the fusion core. An interaction between the dimethylamine end of toremifene and residues Q954 and N955 in heptad repeat 1 (HR1) perturbs the structure, causing a shift from what is normally a long, helical region to short helices connected by unstructured regions. Additionally, we found a strong interaction between toremifene and the methyltransferase nonstructural protein (NSP) 14, which could be inhibitory to viral replication via its active site. These results suggest potential structural mechanisms for toremifene by blocking the spike protein and NSP14 of SARS-CoV-2, offering a drug candidate for COVID-19.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/virologia , Exorribonucleases , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus , Toremifeno , Proteínas não Estruturais Virais , Antivirais/química , Antivirais/metabolismo , Reposicionamento de Medicamentos , Exorribonucleases/química , Exorribonucleases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Pandemias , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Toremifeno/química , Toremifeno/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
13.
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
14.
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
15.
Int J Mol Sci ; 21(16)2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32824072

RESUMO

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a severe global health crisis. In this paper, we used docking and simulation methods to identify potential targets and the mechanism of action of chloroquine (CQ) and hydroxychloroquine (HCQ) against SARS-CoV-2. Our results showed that both CQ and HCQ influenced the functionality of the envelope (E) protein, necessary in the maturation processes of the virus, due to interactions that modify the flexibility of the protein structure. Furthermore, CQ and HCQ also influenced the proofreading and capping of viral RNA in SARS-CoV-2, performed by nsp10/nsp14 and nsp10/nsp16. In particular, HCQ demonstrated a better energy binding with the examined targets compared to CQ, probably due to the hydrogen bonding of the hydroxyl group of HCQ with polar amino acid residues.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Cloroquina/farmacologia , Exorribonucleases/metabolismo , Hidroxicloroquina/farmacologia , Metiltransferases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Humanos , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Proteínas do Envelope Viral/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos
16.
Nucleic Acids Res ; 48(14): 7653-7664, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32667657

RESUMO

Small molecule-based modulation of a triple helix in the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been proposed as an attractive avenue for cancer treatment and a model system for understanding small molecule:RNA recognition. To elucidate fundamental recognition principles and structure-function relationships, we designed and synthesized nine novel analogs of a diphenylfuran-based small molecule DPFp8, a previously identified lead binder of MALAT1. We investigated the role of recognition modalities in binding and in silico studies along with the relationship between affinity, stability and in vitro enzymatic degradation of the triple helix. Specifically, molecular docking studies identified patterns driving affinity and selectivity, including limited ligand flexibility, as observed by ligand preorganization and 3D shape complementarity for the binding pocket. The use of differential scanning fluorimetry allowed rapid evaluation of ligand-induced thermal stabilization of the triple helix, which correlated with decreased in vitro degradation of this structure by the RNase R exonuclease. The magnitude of stabilization was related to binding mode and selectivity between the triple helix and its precursor stem loop structure. Together, this work demonstrates the value of scaffold-based libraries in revealing recognition principles and of raising broadly applicable strategies, including functional assays, for small molecule-RNA targeting.


Assuntos
Furanos/química , RNA Longo não Codificante/química , Exorribonucleases/metabolismo , Furanos/síntese química , Ligantes , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Longo não Codificante/metabolismo
17.
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
18.
Eur J Med Chem ; 201: 112557, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32563813

RESUMO

The spreading of new viruses is known to provoke global human health threat. The current COVID-19 pandemic caused by the recently emerged coronavirus SARS-CoV-2 is one significant and unfortunate example of what the world will have to face in the future with emerging viruses in absence of appropriate treatment. The discovery of potent and specific antiviral inhibitors and/or vaccines to fight these massive outbreaks is an urgent research priority. Enzymes involved in the capping pathway of viruses and more specifically RNA N7- or 2'O-methyltransferases (MTases) are now admitted as potential targets for antiviral chemotherapy. We designed bisubstrate inhibitors by mimicking the transition state of the 2'-O-methylation of the cap RNA in order to block viral 2'-O MTases. This work resulted in the synthesis of 16 adenine dinucleosides with both adenosines connected by various nitrogen-containing linkers. Unexpectedly, all the bisubstrate compounds were barely active against 2'-O MTases of several flaviviruses or SARS-CoV but surprisingly, seven of them showed efficient and specific inhibition against SARS-CoV N7-MTase (nsp14) in the micromolar to submicromolar range. The most active nsp14 inhibitor identified is as potent as but particularly more specific than the broad-spectrum MTase inhibitor, sinefungin. Molecular docking suggests that the inhibitor binds to a pocket formed by the S-adenosyl methionine (SAM) and cap RNA binding sites, conserved among SARS-CoV nsp14. These dinucleoside SAM analogs will serve as starting points for the development of next inhibitors for SARS-CoV-2 nsp14 N7-MTase.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Nucleosídeos/química , Pneumonia Viral/tratamento farmacológico , Capuzes de RNA/metabolismo , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Adenina/química , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Exorribonucleases/metabolismo , Humanos , Metilação , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo
19.
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
20.
Nucleic Acids Res ; 48(13): 7027-7040, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542340

RESUMO

Methylation of miRNAs at the 2'-hydroxyl group on the ribose at 3'-end (2'-O-methylation, 2'Ome) is critical for miRNA function in plants and Drosophila. Whether this methylation phenomenon exists for mammalian miRNA remains unknown. Through LC-MS/MS analysis, we discover that majority of miR-21-5p isolated from human non-small cell lung cancer (NSCLC) tissue possesses 3'-terminal 2'Ome. Predominant 3'-terminal 2'Ome of miR-21-5p in cancer tissue is confirmed by qRT-PCR and northern blot after oxidation/ß-elimination procedure. Cancerous and the paired non-cancerous lung tissue miRNAs display different pattern of 3'-terminal 2'Ome. We further identify HENMT1 as the methyltransferase responsible for 3'-terminal 2'Ome of mammalian miRNAs. Compared to non-methylated miR-21-5p, methylated miR-21-5p is more resistant to digestion by 3'→5' exoribonuclease polyribonucleotide nucleotidyltransferase 1 (PNPT1) and has higher affinity to Argonaute-2, which may contribute to its higher stability and stronger inhibition on programmed cell death protein 4 (PDCD4) translation, respectively. Our findings reveal HENMT1-mediated 3'-terminal 2'Ome of mammalian miRNAs and highlight its role in enhancing miRNA's stability and function.


Assuntos
Proteínas Argonauta/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Exorribonucleases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Metilação , Proteínas de Ligação a RNA/metabolismo
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