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1.
Methods Mol Biol ; 2203: 231-238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833216

RESUMO

Ribopuromycylation enables the visualization and quantitation of translation on a cellular level by immunofluorescence or in total using standard western blotting. This technique uses ribosome catalyzed puromycylation of nascent chains followed by immobilization on the ribosome by antibiotic chain elongation inhibitor emetine. Detection of puromycylated ribosome-bound nascent chains can then be achieved using a puromycin-specific antibody.


Assuntos
Coronavirus/genética , Puromicina/farmacologia , Infecções por Coronavirus , Imunofluorescência , Interações Hospedeiro-Patógeno , Humanos , Biossíntese de Proteínas , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
2.
PLoS Biol ; 18(8): e3000757, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32833957

RESUMO

In eukaryotes, conserved mechanisms ensure that cell growth is coordinated with nutrient availability. Overactive growth during nutrient limitation ("nutrient-growth dysregulation") can lead to rapid cell death. Here, we demonstrate that cells can adapt to nutrient-growth dysregulation by evolving major metabolic defects. Specifically, when yeast lysine-auxotrophic mutant lys- encountered lysine limitation, an evolutionarily novel stress, cells suffered nutrient-growth dysregulation. A subpopulation repeatedly evolved to lose the ability to synthesize organosulfurs (lys-orgS-). Organosulfurs, mainly reduced glutathione (GSH) and GSH conjugates, were released by lys- cells during lysine limitation when growth was dysregulated, but not during glucose limitation when growth was regulated. Limiting organosulfurs conferred a frequency-dependent fitness advantage to lys-orgS- by eliciting a proper slow growth program, including autophagy. Thus, nutrient-growth dysregulation is associated with rapid organosulfur release, which enables the selection of organosulfur auxotrophy to better tune cell growth to the metabolic environment. We speculate that evolutionarily novel stresses can trigger atypical release of certain metabolites, setting the stage for the evolution of new ecological interactions.


Assuntos
Adaptação Fisiológica/genética , Lisina/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Nutrientes/farmacologia , Saccharomyces cerevisiae/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Evolução Biológica , Glucose/metabolismo , Glucose/farmacologia , Lisina/deficiência , Redes e Vias Metabólicas/genética , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Nutrientes/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sirolimo/farmacologia , Estresse Fisiológico
3.
Nat Commun ; 11(1): 4013, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782250

RESUMO

Antibiotics that interfere with translation, when combined, interact in diverse and difficult-to-predict ways. Here, we explain these interactions by "translation bottlenecks": points in the translation cycle where antibiotics block ribosomal progression. To elucidate the underlying mechanisms of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using inducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks causes these interactions. We further show that growth laws, combined with drug uptake and binding kinetics, enable the direct prediction of a large fraction of observed interactions, yet fail to predict suppression. However, varying two translation bottlenecks simultaneously supports that dense traffic of ribosomes and competition for translation factors account for the previously unexplained suppression. These results highlight the importance of "continuous epistasis" in bacterial physiology.


Assuntos
Antibacterianos/farmacologia , Modelos Teóricos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Interações Medicamentosas , Epistasia Genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(34): 20530-20537, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32817463

RESUMO

Sarecycline is a new narrow-spectrum tetracycline-class antibiotic approved for the treatment of acne vulgaris. Tetracyclines share a common four-ring naphthacene core and inhibit protein synthesis by interacting with the 70S bacterial ribosome. Sarecycline is distinguished chemically from other tetracyclines because it has a 7-[[methoxy(methyl)amino]methyl] group attached at the C7 position of ring D. To investigate the functional role of this C7 moiety, we determined the X-ray crystal structure of sarecycline bound to the Thermus thermophilus 70S ribosome. Our 2.8-Å resolution structure revealed that sarecycline binds at the canonical tetracycline binding site located in the decoding center of the small ribosomal subunit. Importantly, unlike other tetracyclines, the unique C7 extension of sarecycline extends into the messenger RNA (mRNA) channel to form a direct interaction with the A-site codon to possibly interfere with mRNA movement through the channel and/or disrupt A-site codon-anticodon interaction. Based on our biochemical studies, sarecycline appears to be a more potent initiation inhibitor compared to other tetracyclines, possibly due to drug interactions with the mRNA, thereby blocking accommodation of the first aminoacyl transfer RNA (tRNA) into the A site. Overall, our structural and biochemical findings rationalize the role of the unique C7 moiety of sarecycline in antibiotic action.


Assuntos
Antibacterianos/farmacologia , Ribossomos/efeitos dos fármacos , Tetraciclinas/farmacologia , Antibacterianos/química , RNA Ribossômico 16S/química , Tetraciclinas/química , Thermus thermophilus
5.
PLoS Genet ; 16(7): e1008917, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628663

RESUMO

Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite's AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology.


Assuntos
Quadruplex G , Malária/genética , Motivos de Nucleotídeos/genética , Plasmodium falciparum/genética , Aminoquinolinas/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma/efeitos dos fármacos , Humanos , Malária/tratamento farmacológico , Malária/parasitologia , Ácidos Picolínicos/farmacologia , Plasmodium falciparum/patogenicidade , Regiões Promotoras Genéticas/genética , Ribossomos/efeitos dos fármacos , Ribossomos/genética
6.
Nucleic Acids Res ; 48(10): 5201-5216, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32382758

RESUMO

High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.


Assuntos
Códon/metabolismo , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas/genética , Códon/genética , Haloferax volcanii/efeitos dos fármacos , Harringtoninas/farmacologia , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Elongação Traducional da Cadeia Peptídica/genética , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/genética , Biossíntese de Proteínas/efeitos dos fármacos , Pegadas de Proteínas , Fases de Leitura/genética , Ribossomos/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos
7.
Nat Commun ; 11(1): 1108, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111839

RESUMO

Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 107 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 103 rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.


Assuntos
Ribossomos/genética , Ribossomos/metabolismo , Antibacterianos/farmacologia , Clindamicina/farmacologia , Evolução Molecular Direcionada , Farmacorresistência Bacteriana/genética , Epistasia Genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Genótipo , Mutação , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Biologia Sintética
8.
PLoS Biol ; 18(1): e3000593, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31995552

RESUMO

During host colonization, bacteria use the alarmones (p)ppGpp to reshape their proteome by acting pleiotropically on DNA, RNA, and protein synthesis. Here, we elucidate how the initiating ribosome senses the cellular pool of guanosine nucleotides and regulates the progression towards protein synthesis. Our results show that the affinity of guanosine triphosphate (GTP) and the inhibitory concentration of ppGpp for the 30S-bound initiation factor IF2 vary depending on the programmed mRNA. The TufA mRNA enhanced GTP affinity for 30S complexes, resulting in improved ppGpp tolerance and allowing efficient protein synthesis. Conversely, the InfA mRNA allowed ppGpp to compete with GTP for IF2, thus stalling 30S complexes. Structural modeling and biochemical analysis of the TufA mRNA unveiled a structured enhancer of translation initiation (SETI) composed of two consecutive hairpins proximal to the translation initiation region (TIR) that largely account for ppGpp tolerance under physiological concentrations of guanosine nucleotides. Furthermore, our results show that the mechanism enhancing ppGpp tolerance is not restricted to the TufA mRNA, as similar ppGpp tolerance was found for the SETI-containing Rnr mRNA. Finally, we show that IF2 can use pppGpp to promote the formation of 30S initiation complexes (ICs), albeit requiring higher factor concentration and resulting in slower transitions to translation elongation. Altogether, our data unveil a novel regulatory mechanism at the onset of protein synthesis that tolerates physiological concentrations of ppGpp and that bacteria can exploit to modulate their proteome as a function of the nutritional shift happening during stringent response and infection.


Assuntos
Guanosina Tetrafosfato/farmacologia , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Ligação Competitiva , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Interações Hospedeiro-Patógeno/fisiologia , Cinética , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/fisiologia , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/química , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética
9.
OMICS ; 24(2): 96-109, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31895625

RESUMO

Ribosomopathies result in various cancers, neurodegenerative and viral diseases, and other pathologies such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Their pathophysiology at a proteome and functional level remains to be determined. Protein networks and highly connected hub proteins for ribosome biogenesis in Saccharomyces cerevisiae offer a potential as a model system to inform future therapeutic innovation in ribosomopathies. In this context, we report a ribosome biogenesis protein-protein interaction network in S. cerevisiae, created with 1772 proteins and 22,185 physical interactions connecting them. Moreover, by network decomposition analysis, we determined the linear pathways between the transcription factors and target proteins with a view to drug repurposing. While considering only the paths containing the three C/D box proteins (Nop56, Nop58, and Nop1), the most frequently encountered proteins were Aft1, Htz1, Ssa1, Ssb1, Ssb2, Gcn5, Cka1, Tef1, Nop1, Cdc28, Act1, Krr1, Rpl8B, and Tor1, which were then identified as potential drug targets. For drug repurposing, these candidate proteins were further searched in the DrugBank to find other diseases associated with them, as well as the drugs used to treat these diseases. To support the computational results, an experimental study was conducted using in-house manufactured microfluidic bioreactor platform, while the effect of the drug temsirolimus, Tor1 inhibitor, on yeast cells was investigated by following Nop56 protein expression. In conclusion, these results inform the ways in which ribosomopathies and associated common complex human diseases materialize and how drug repurposing might accelerate therapeutic innovation through bioinformatic studies of yeast.


Assuntos
Reposicionamento de Medicamentos , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Leveduras/efeitos dos fármacos , Leveduras/metabolismo , Biologia Computacional/métodos , Descoberta de Drogas , Ontologia Genética , Humanos , Modelos Teóricos , Anotação de Sequência Molecular , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo
10.
Nucleic Acids Res ; 48(4): e22, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31919515

RESUMO

In order to discover new antibiotics with improved activity and selectivity, we created a reliable in vitro reporter system to detect trans-translation activity, the main mechanism for recycling ribosomes stalled on problematic messenger RNA (mRNA) in bacteria. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. Our system is adapted for high-throughput screening of chemical compounds by fluorescence.


Assuntos
Bactérias/genética , Proteínas de Fluorescência Verde/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Antibacterianos/síntese química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Humanos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Ribossomos/efeitos dos fármacos , Ribossomos/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-31712205

RESUMO

Aminoglycoside resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant mechanism is overproduction of the SmeYZ efflux system. By studying laboratory-selected mutants and clinical isolates, we show here that damage to the 50S ribosomal protein L1 (RplA) activates SmeYZ production. We also show that gentamicin and minocycline, which target the ribosome, induce expression of smeYZ These findings explain the role of SmeYZ in both intrinsic and mutationally acquired aminoglycoside resistance.


Assuntos
Aminoglicosídeos/metabolismo , Antibacterianos/farmacologia , Proteínas Ribossômicas/genética , Ribossomos/efeitos dos fármacos , Stenotrophomonas maltophilia/genética , Farmacorresistência Bacteriana/genética , Gentamicinas/farmacologia , Testes de Sensibilidade Microbiana , Minociclina/farmacologia , Mutação , Stenotrophomonas maltophilia/efeitos dos fármacos , Stenotrophomonas maltophilia/metabolismo
12.
Brain ; 143(1): 94-111, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31855247

RESUMO

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance.


Assuntos
Antígenos CD/genética , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Adolescente , Ataxia/genética , Ataxia/fisiopatologia , Atrofia , Cerebelo/diagnóstico por imagem , Cerebelo/patologia , Colina/farmacologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/fisiopatologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/ultraestrutura , Transtornos de Deglutição/genética , Transtornos de Deglutição/fisiopatologia , Disartria/genética , Disartria/fisiopatologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Incontinência Fecal/genética , Incontinência Fecal/fisiopatologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Mutação da Fase de Leitura , Globo Pálido/diagnóstico por imagem , Transtornos Heredodegenerativos do Sistema Nervoso/diagnóstico por imagem , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Transtornos Heredodegenerativos do Sistema Nervoso/fisiopatologia , Homozigoto , Humanos , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/genética , Leucoencefalopatias/fisiopatologia , Imagem por Ressonância Magnética , Masculino , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Nootrópicos/farmacologia , Atrofia Óptica/genética , Atrofia Óptica/fisiopatologia , Linhagem , Ribossomos/efeitos dos fármacos , Ribossomos/ultraestrutura , Substância Negra/diagnóstico por imagem , Síndrome , Tremor/genética , Tremor/fisiopatologia , Incontinência Urinária/genética , Incontinência Urinária/fisiopatologia
13.
Am J Respir Cell Mol Biol ; 62(1): 74-86, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31264907

RESUMO

High CO2 retention, or hypercapnia, is associated with worse outcomes in patients with chronic pulmonary diseases. Skeletal muscle wasting is also an independent predictor of poor outcomes in patients with acute and chronic pulmonary diseases. Although previous evidence indicates that high CO2 accelerates skeletal muscle catabolism via AMPK (AMP-activated protein kinase)-FoxO3a-MuRF1 (E3-ubiquitin ligase muscle RING finger protein 1), little is known about the role of high CO2 in regulating skeletal muscle anabolism. In the present study, we investigated the potential role of high CO2 in attenuating skeletal muscle protein synthesis. We found that locomotor muscles from patients with chronic CO2 retention demonstrated depressed ribosomal gene expression in comparison with locomotor muscles from non-CO2-retaining individuals, and analysis of the muscle proteome of normo- and hypercapnic mice indicates reduction of important components of ribosomal structure and function. Indeed, mice chronically kept under a high-CO2 environment show evidence of skeletal muscle downregulation of ribosomal biogenesis and decreased protein synthesis as measured by the incorporation of puromycin into skeletal muscle. Hypercapnia did not regulate the mTOR pathway, and rapamycin-induced deactivation of mTOR did not cause a decrease in ribosomal gene expression. Loss-of-function studies in cultured myotubes showed that AMPKα2 regulates CO2-mediated reductions in ribosomal gene expression and protein synthesis. Although previous evidence has implicated TIF1A (transcription initiation factor-1α) and KDM2A (lysine-specific demethylase 2A) in AMPK-driven regulation of ribosomal gene expression, we found that these mediators were not required in the high CO2-induced depressed protein anabolism. Our research supports future studies targeting ribosomal biogenesis and protein synthesis to alleviate the effects of high CO2 on skeletal muscle turnover.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Dióxido de Carbono/efeitos adversos , Regulação para Baixo/efeitos dos fármacos , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Adolescente , Animais , Proteínas F-Box/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Pneumopatias/etiologia , Pneumopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Ribossomos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(1): 761-770, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871206

RESUMO

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga Chlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show that Chlamydomonas VIG1, an ortholog of the Drosophila melanogaster Vasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. A VIG1-deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavage-mediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts.


Assuntos
Chlamydomonas reinhardtii/fisiologia , Proteínas de Plantas/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas Argonauta/metabolismo , Cicloeximida/farmacologia , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas , Íntrons/genética , Mutação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Polirribossomos/genética , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
15.
Nat Commun ; 10(1): 5611, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31819057

RESUMO

Oxidation and alkylation of nucleobases are known to disrupt their base-pairing properties within RNA. It is, however, unclear whether organisms have evolved general mechanism(s) to deal with this damage. Here we show that the mRNA-surveillance pathway of no-go decay and the associated ribosome-quality control are activated in response to nucleobase alkylation and oxidation. Our findings reveal that these processes are important for clearing chemically modified mRNA and the resulting aberrant-protein products. In the absence of Xrn1, the level of damaged mRNA significantly increases. Furthermore, deletion of LTN1 results in the accumulation of protein aggregates in the presence of oxidizing and alkylating agents. This accumulation is accompanied by Hel2-dependent regulatory ubiquitylation of ribosomal proteins. Collectively, our data highlight the burden of chemically damaged mRNA on cellular homeostasis and suggest that organisms evolved mechanisms to counter their accumulation.


Assuntos
Estresse Oxidativo , Ribossomos/metabolismo , 4-Nitroquinolina-1-Óxido/metabolismo , Alquilação , Adutos de DNA/metabolismo , Dano ao DNA , Células HEK293 , Humanos , Metanossulfonato de Metila/farmacologia , Mutação/genética , Oxirredução , Peptídeos/metabolismo , Polirribossomos/metabolismo , Agregados Proteicos , Quinolonas/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
16.
Sci Rep ; 9(1): 18397, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804585

RESUMO

In recent years, a number of small RNA molecules derived from snoRNAs have been observed. Findings concerning the functions of snoRNA-derived small RNAs (sdRNAs) in cells are limited primarily to their involvement in microRNA pathways. However, similar molecules have been observed in Saccharomyces cerevisiae, which is an organism lacking miRNA machinery. Here we examined the subcellular localization of sdRNAs in yeast. Our findings reveal that both sdRNAs and their precursors, snoRNAs, are present in the cytoplasm at levels dependent upon stress conditions. Moreover, both sdRNAs and snoRNAs may interact with translating ribosomes in a stress-dependent manner. Likely consequential to their ribosome association and protein synthesis suppression features, yeast sdRNAs may exert inhibitory activity on translation. Observed levels of sdRNAs and snoRNAs in the cytoplasm and their apparent presence in the ribosomal fractions suggest independent regulation of these molecules by yet unknown factors.


Assuntos
Regulação Fúngica da Expressão Gênica , Biossíntese de Proteínas , RNA Nucleolar Pequeno/genética , Ribossomos/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Resposta ao Choque Frio , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Resposta ao Choque Térmico , Conformação de Ácido Nucleico , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Nucleolar Pequeno/classificação , RNA Nucleolar Pequeno/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Salinidade , Cloreto de Sódio/farmacologia , Sorbitol/farmacologia , Estresse Fisiológico/genética , Raios Ultravioleta
17.
Nat Commun ; 10(1): 4563, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594941

RESUMO

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.


Assuntos
Antibacterianos/farmacologia , Azóis/farmacologia , Agentes de Controle Biológico/farmacologia , Peptídeos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/metabolismo , Azóis/química , Azóis/metabolismo , Agentes de Controle Biológico/química , Agentes de Controle Biológico/metabolismo , Microscopia Crioeletrônica , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Testes de Sensibilidade Microbiana , Família Multigênica , Biossíntese Peptídica/genética , Peptídeos/química , Peptídeos/metabolismo , Phaseolus/microbiologia , RNA Ribossômico 23S/metabolismo , RNA Ribossômico 23S/ultraestrutura , Rhizobium/genética , Rhizobium/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Especificidade da Espécie , Simbiose
18.
Appl Environ Microbiol ; 86(1)2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31604775

RESUMO

Vegetative cells of Bacillus subtilis can recover from injury after high-hydrostatic-pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that substantial numbers of ribosomal genes and translation-related genes (e.g., translation initiation factors) were upregulated during the growth arrest phase after HHP treatment. The transcript levels of cold shock-responsive genes, whose products play key roles in efficient translation, and heat shock-responsive genes, whose products mediate correct protein folding or degrade misfolded proteins, were also upregulated. In contrast, the transcript level of hpf, whose product (Hpf) is involved in ribosome inactivation through the dimerization of 70S ribosomes, was downregulated during the growth arrest phase. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. We also found that cell growth after HHP-induced injury was apparently inhibited by the addition of Mn2+ or Zn2+ to the recovery medium. Ribosome reconstruction in the HHP-injured cells was also significantly delayed in the presence of Mn2+ or Zn2+ Moreover, Zn2+, but not Mn2+, promoted dimer formation of 70S ribosomes in the HHP-injured cells. Disruption of the hpf gene suppressed the Zn2+-dependent accumulation of ribosome dimers, partially relieving the inhibitory effect of Zn2+ on the growth recovery of HHP-treated cells. In contrast, it was likely that Mn2+ prevented ribosome reconstruction without stimulating ribosome dimerization. Our results suggested that both Mn2+ and Zn2+ can prevent ribosome reconstruction, thereby delaying the growth recovery of HHP-injured B. subtilis cells.IMPORTANCE HHP treatment is used as a nonthermal processing technology in the food industry to inactivate bacteria while retaining high quality of foods under suppressed chemical reactions. However, some populations of bacterial cells may survive the inactivation. Although the survivors are in a transient nongrowing state due to HHP-induced injury, they can recover from the injury and then start growing, depending on the postprocessing conditions. The recovery process in terms of cellular components after the injury remains unclear. Transcriptome analysis using vegetative cells of Bacillus subtilis revealed that the translational machinery can preferentially be reconstructed after HHP treatment. We found that both Mn2+ and Zn2+ prolonged the growth-arrested stage of HHP-injured cells by delaying ribosome reconstruction. It is likely that ribosome reconstruction is crucial for the recovery of growth ability in HHP-injured cells. This study provides further understanding of the recovery process in HHP-injured B. subtilis cells.


Assuntos
Bacillus subtilis , Pressão Hidrostática/efeitos adversos , Viabilidade Microbiana , Ribossomos , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Manganês/farmacologia , Compostos de Manganês/farmacologia , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Ribossomos/metabolismo , Sais/farmacologia , Transcriptoma , Compostos de Zinco/farmacologia
20.
Res Microbiol ; 170(8): 435-447, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31563533

RESUMO

The ATP binding cassette protein superfamily comprises ATPase enzymes which are, for the most part, involved in transmembrane transport. Within this superfamily however, some protein families have other functions unrelated to transport. One example is the ABC-F family, which comprises an extremely diverse set of cytoplasmic proteins. All of the proteins in the ABC-F family characterized to date act on the ribosome and are translation factors. Their common function is ATP-dependent modulation of the stereochemistry of the peptidyl transferase center (PTC) in the ribosome coupled to changes in its global conformation and P-site tRNA binding geometry. In this review, we give an overview of the function, structure, and theories for the mechanisms-of-action of microbial proteins in the ABC-F family, including those involved in mediating resistance to ribosome-binding antibiotics.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Farmacorresistência Bacteriana Múltipla/fisiologia , Escherichia coli/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Transporte Biológico/fisiologia , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla/genética , Biossíntese de Proteínas/genética , Conformação Proteica , Domínios Proteicos , Ribossomos/metabolismo
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