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1.
Biochemistry (Mosc) ; 86(9): 1053-1059, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34565311

RESUMO

"Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a longstanding collaboration between my research group and that of Alexander S. Spirin. Indeed, it was: we found that double-row polyribosomes can have both circular and linear arrangements of their mRNA [Afonina, Z. A., et al. (2013) Biochemistry (Moscow)], we figured out how eukaryotic ribosomes assemble on an mRNA to form supramolecular left-handed helices [Myasnikov, A. G., et al. (2014) Nat. Commun.], that the circularization of polyribosomes is poly-A and cap-independent [Afonina, Z. A., et al. (2014) Nucleic Acids Res.], and that intermediary polyribosomes with open structures exist after a transition from a juvenile phase to strongly translating polysomes of medium size [Afonina, Z. A., et al. (2015) Nucleic Acids Res.] until they form densely packed helical structures with reduced activity. Our joint fruitful exchanges, hence, led to major advances in the field, which are reviewed here from a personal and historical perspective in memory of Alexander S. Spirin.


Assuntos
Polirribossomos/química , Microscopia Crioeletrônica , Eucariotos/química , Eucariotos/genética , Eucariotos/metabolismo , Conformação de Ácido Nucleico , Poli A/química , Poli A/metabolismo , Polirribossomos/metabolismo , Capuzes de RNA/química , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo
2.
Viruses ; 13(9)2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34578302

RESUMO

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.


Assuntos
COVID-19/virologia , Metiltransferases/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Cromatografia Líquida , Regulação Viral da Expressão Gênica , Humanos , Espectrometria de Massas , Metilação , Metiltransferases/genética , Capuzes de RNA , RNA Viral/genética , SARS-CoV-2/genética , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/genética
3.
Methods Mol Biol ; 2351: 67-90, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382184

RESUMO

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation of the Mammalian Genome (FANTOM) consortia. These datasets constitute open resource for TSS annotations and gene expression analysis. Here, we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE "LQ-ssCAGE", which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50 ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.


Assuntos
Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Capuzes de RNA , Sítio de Iniciação de Transcrição , Bases de Dados Genéticas , Regulação da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Reprodutibilidade dos Testes , Fluxo de Trabalho
4.
Methods Mol Biol ; 2351: 201-210, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382191

RESUMO

Regulation of gene expression is a key feature for higher eukaryotes and how chromatin topology relates to gene activation is an intense area of research. Enhancer-promoter interactions are believed to mediate activation of target genes. Bidirectional transcription represents one hallmark of active enhancers that can be measured using transcriptome technologies such as Cap analysis of gene expression (CAGE). Recently, we have developed RNA and DNA interacting complexes ligated and sequenced (RADICL-Seq) a novel methodology to map genome-wide RNA-chromatin interactions in intact nuclei. Here, we describe how CAGE and RADICL-Seq data can be used to characterize enhancer elements and identify their target genes.


Assuntos
Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Capuzes de RNA , Algoritmos , Cromatina/genética , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala , Sítio de Iniciação de Transcrição , Transcrição Genética , Ativação Transcricional , Transcriptoma
5.
Viruses ; 13(8)2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34452352

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.


Assuntos
COVID-19/virologia , Metiltransferases/metabolismo , Capuzes de RNA/genética , RNA Viral/genética , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Anticorpos Monoclonais/análise , Humanos , Metiltransferases/análise , Metiltransferases/genética , Transporte Proteico , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/análise , Proteínas Virais Reguladoras e Acessórias/genética
6.
Antiviral Res ; 193: 105142, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34303749

RESUMO

SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 µM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Metiltransferases/antagonistas & inibidores , Nucleotidiltransferases/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , COVID-19/virologia , Linhagem Celular , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Metiltransferases/metabolismo , Nucleotidiltransferases/metabolismo , Capuzes de RNA , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
7.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34328830

RESUMO

The 5' capped, message-sense RNA genome of Chikungunya virus (CHIKV) utilizes the host cell machinery for translation. Translation is regulated by eIF2 alpha at the initiation phase and by eIF4F at cap recognition. Translational suppression by eIF2 alpha phosphorylation occurs as an early event in many alphavirus infections. We observe that in CHIKV-infected HEK293 cells, this occurs as a late event, by which time the viral replication has reached an exponential phase, implying its minimal role in virus restriction. The regulation by eIF4F is mediated through the PI3K-Akt-mTOR, p38 MAPK and RAS-RAF-MEK-ERK pathways. A kinetic analysis revealed that CHIKV infection did not modulate AKT phosphorylation, but caused a significant reduction in p38 MAPK phosphorylation. It caused degradation of phospho-ERK 1/2 by increased autophagy, leaving the PI3K-Akt-mTOR and p38 MAPK pathways for pharmacological targeting. mTOR inhibition resulted in moderate reduction in viral titre, but had no effect on CHIKV E2 protein expression, indicating a minimal role of the mTOR complex in virus replication. Inhibition of p38 MAPK using SB202190 caused a significant reduction in viral titre and CHIKV E2 and nsP3 protein expression. Furthermore, inhibiting the two pathways together did not offer any synergism, indicating that inhibiting the p38 MAPK pathway alone is sufficient to cause restriction of CHIKV replication. Meanwhile, in uninfected cells the fully functional RAS-RAF-MEK-ERK pathway can circumvent the effect of p38 MAPK inhibition on cap-dependent translation. Thus, our results show that host-directed antiviral strategies targeting cellular p38 MAPK are worth exploring against Chikungunya as they could be selective against CHIKV-infected cells with minimal effects on uninfected host cells.


Assuntos
Autofagia , Vírus Chikungunya/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imidazóis/farmacologia , Biossíntese de Proteínas , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Apoptose , Linhagem Celular Tumoral , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Capuzes de RNA , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Replicação Viral/efeitos dos fármacos
8.
Biochem J ; 478(13): 2481-2497, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198328

RESUMO

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Capuzes de RNA/metabolismo , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Antivirais/química , Clorobenzenos/farmacologia , Chlorocebus aethiops , Ensaios Enzimáticos , Exorribonucleases/genética , Exorribonucleases/isolamento & purificação , Exorribonucleases/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Indazóis/farmacologia , Indenos/farmacologia , Indóis/farmacologia , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Nitrilas/farmacologia , Fenotiazinas/farmacologia , Purinas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato , Trifluperidol/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/isolamento & purificação , Proteínas Virais Reguladoras e Acessórias/metabolismo
9.
Elife ; 102021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34076576

RESUMO

During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5'UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a 'start-stop'. The 'start-stop' sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.


Assuntos
Proteínas de Homeodomínio/metabolismo , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Animais , Códon de Terminação , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Sítios Internos de Entrada Ribossomal , Conformação de Ácido Nucleico , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , Coelhos , Ribossomos/genética , Relação Estrutura-Atividade
10.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
11.
Nucleic Acids Res ; 49(12): 6722-6738, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34125914

RESUMO

The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation.


Assuntos
Ativação Linfocitária , Metiltransferases/fisiologia , Biossíntese de Proteínas , Ribossomos/metabolismo , Linfócitos T/enzimologia , Animais , Técnicas de Inativação de Genes , Guanosina/metabolismo , Ativação Linfocitária/genética , Metiltransferases/biossíntese , Metiltransferases/genética , Camundongos , Capuzes de RNA/química , Capuzes de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Regulação para Cima
12.
13.
Sci Signal ; 14(689)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34131072

RESUMO

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.


Assuntos
COVID-19/virologia , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Manganês/metabolismo , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Capuzes de RNA/química , Capuzes de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , Transdução de Sinais , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
14.
PLoS Pathog ; 17(5): e1009562, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33956914

RESUMO

Respiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the C-terminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7- and 2'-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5' end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at µM concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion.


Assuntos
Metilação de DNA , Metiltransferases/metabolismo , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas não Estruturais Virais/metabolismo , Humanos , Imunidade Inata , Metiltransferases/genética , Capuzes de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/metabolismo , Proteínas não Estruturais Virais/genética , Replicação Viral
15.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-33972410

RESUMO

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.


Assuntos
Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/química , Cristalografia , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Capuzes de RNA/química , RNA Mensageiro/química , RNA Viral/química , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Síncrotrons , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo
16.
Biochem Biophys Res Commun ; 558: 189-195, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33940551

RESUMO

In RNA field, the demarcation between coding and non-coding has been negotiated by the recent discovery of occasionally translated circular RNAs (circRNAs). Although absent of 5' cap structure, circRNAs can be translated cap-independently. Complementary intron-mediated overexpression is one of the most utilized methodologies for circRNA research but not without bearing echoing skepticism for its poorly defined mechanism and latent coexistent side products. In this study, leveraging such circRNA overexpression system, we have interrogated the protein-coding potential of 30 human circRNAs containing infinite open reading frames in HEK293T cells. Surprisingly, pervasive translation signals are detected by immunoblotting. However, intensive mutagenesis reveals that numerous translation signals are generated independently of circRNA synthesis. We have developed a dual tag strategy to isolate translation noise and directly demonstrate that the spurious translation signals originate from cryptically spliced linear transcripts. The concomitant linear RNA byproducts, presumably concatemers, can be translated to allow pseudo rolling circle translation signals, and can involve backsplicing junction (BSJ) to disqualify the BSJ-based evidence for circRNA translation. We also find non-AUG start codons may engage in the translation initiation of circRNAs. Taken together, our systematic evaluation sheds light on heterogeneous translational outputs from circRNA overexpression vector and comes with a caveat that ectopic overexpression technique necessitates extremely rigorous control setup in circRNA translation and functional investigation.


Assuntos
RNA Circular/genética , RNA Circular/metabolismo , Códon de Iniciação , Células HEK293 , Humanos , Íntrons , Modelos Genéticos , Mutagênese , Fases de Leitura Aberta , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Regulação para Cima
17.
J Virol ; 95(15): e0077721, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011549

RESUMO

Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5' end. The capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5'-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.


Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Capuzes de RNA/genética , RNA Viral/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genética , Animais , Encefalomielite Equina Venezuelana/patologia , Encefalomielite Equina Venezuelana/virologia , Cavalos , Humanos , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , Nucleotidiltransferases/metabolismo , Proteínas não Estruturais Virais/genética
18.
PLoS One ; 16(4): e0249928, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33836032

RESUMO

Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Nepovirus/genética , Capuzes de RNA/fisiologia , RNA Viral/biossíntese , Sequência de Bases , Códon de Iniciação , Genes Reporter , Lycopersicon esculentum/virologia , Mutagênese , RNA Viral/genética , Alinhamento de Sequência
19.
SLAS Discov ; 26(6): 757-765, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33874769

RESUMO

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.


Assuntos
Adenosina/análogos & derivados , Ensaios de Triagem em Larga Escala , Capuzes de RNA/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Adenosina/química , Adenosina/farmacologia , COVID-19/virologia , Clonagem Molecular , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Metilação , Modelos Moleculares , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Trítio , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
20.
Nucleic Acids Res ; 49(9): 5159-5176, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33893802

RESUMO

The eIF4E are a family of initiation factors that bind the mRNA 5' cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active during stress and that it selects specific mRNA populations based on 5' TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have 'moon-lighting' functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote 'stress resistance' and adaptation.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/metabolismo , Biossíntese de Proteínas , Estresse Fisiológico/genética , Animais , Células Cultivadas , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Camundongos , Naftiridinas/farmacologia , Capuzes de RNA/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores
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