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1.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445336

RESUMO

Pseudomonas aeruginosa (Pae) is an opportunistic pathogen showing a high intrinsic resistance to a wide variety of antibiotics. It causes nosocomial infections that are particularly detrimental to immunocompromised individuals and to patients suffering from cystic fibrosis. We provide a snapshot on regulatory RNAs of Pae that impact on metabolism, pathogenicity and antibiotic susceptibility. Different experimental approaches such as in silico predictions, co-purification with the RNA chaperone Hfq as well as high-throughput RNA sequencing identified several hundreds of regulatory RNA candidates in Pae. Notwithstanding, using in vitro and in vivo assays, the function of only a few has been revealed. Here, we focus on well-characterized small base-pairing RNAs, regulating specific target genes as well as on larger protein-binding RNAs that sequester and thereby modulate the activity of translational repressors. As the latter impact large gene networks governing metabolism, acute or chronic infections, these protein-binding RNAs in conjunction with their cognate proteins are regarded as global post-transcriptional regulators.


Assuntos
Pseudomonas aeruginosa/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Interferência de RNA/fisiologia , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo
2.
Nat Commun ; 12(1): 5033, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413302

RESUMO

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.


Assuntos
Nucleotídeos de Adenina/química , COVID-19/diagnóstico , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Oligorribonucleotídeos/química , RNA Bacteriano/genética , Ribonucleases/metabolismo , SARS-CoV-2/genética , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Testes Diagnósticos de Rotina/métodos , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade
3.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
4.
Science ; 373(6556)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385369

RESUMO

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen Pseudomonas aeruginosa, analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identified numerous metabolic- and virulence-related transcriptional states that emerged dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm just several micrometers away, underscoring the importance of the microenvironment. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.


Assuntos
Pseudomonas aeruginosa/genética , Transcriptoma , Biofilmes/crescimento & desenvolvimento , Proteínas de Fímbrias/genética , Flagelina/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Hibridização in Situ Fluorescente , Fenótipo , Plâncton/genética , Plâncton/crescimento & desenvolvimento , Plâncton/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Piocinas/biossíntese , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , Análise Espaço-Temporal , Virulência/genética
5.
J Microbiol ; 59(9): 827-839, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34382149

RESUMO

Probiotics effectively prevent and improve metabolic diseases such as diabetes by regulating the intestinal microenvironment and gut microbiota. However, the effects of probiotics in gestational diabetes mellitus are not clear. Here, we showed that probiotic supplements significantly improved fasting blood glucose in a gestational diabetes mellitus rat model. To further understand the mechanisms of probiotics in gestational diabetes mellitus, the gut microbiota were analyzed via 16S rRNA sequencing. We found that compared with the normal pregnant group, the gestational diabetes mellitus rats had decreased diversity of gut microbiota. Moreover, probiotic supplementation restored the diversity of the gut microbiota in gestational diabetes mellitus rats, and the gut microbiota structure tended to be similar to that of normal pregnant rats. In particular, compared with gestational diabetes mellitus rats, the abundance of Firmicutes and Actinobacteria was higher after probiotic supplementation. Furthermore, activating carbohydrate metabolism and membrane transport pathways may be involved in the potential mechanisms by which probiotic supplements alleviate gestational diabetes mellitus. Overall, our results suggested that probiotic supplementation might be a novel approach to restore the gut microbiota of gestational diabetes mellitus rats and provided an experimental evidence for the use of probiotic supplements to treat gestational diabetes mellitus.


Assuntos
Diabetes Gestacional/tratamento farmacológico , Microbioma Gastrointestinal/efeitos dos fármacos , Probióticos/administração & dosagem , Animais , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Metabolismo dos Carboidratos , Carboidratos , Diabetes Gestacional/metabolismo , Diabetes Gestacional/microbiologia , Suplementos Nutricionais/análise , Feminino , Masculino , Gravidez , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Ratos , Ratos Sprague-Dawley
6.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
7.
Nat Microbiol ; 6(8): 1007-1020, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239075

RESUMO

Fusobacterium nucleatum, long known as a constituent of the oral microflora, has recently garnered renewed attention for its association with several different human cancers. The growing interest in this emerging cancer-associated bacterium contrasts with a paucity of knowledge about its basic gene expression features and physiological responses. As fusobacteria lack all established small RNA-associated proteins, post-transcriptional networks in these bacteria are also unknown. In the present study, using differential RNA-sequencing, we generate high-resolution global RNA maps for five clinically relevant fusobacterial strains-F. nucleatum subspecies nucleatum, animalis, polymorphum and vincentii, as well as F. periodonticum-for early, mid-exponential growth and early stationary phase. These data are made available in an online browser, and we use these to uncover fundamental aspects of fusobacterial gene expression architecture and a suite of non-coding RNAs. Developing a vector for functional analysis of fusobacterial genes, we discover a conserved fusobacterial oxygen-induced small RNA, FoxI, which serves as a post-transcriptional repressor of the major outer membrane porin FomA. Our findings provide a crucial step towards delineating the regulatory networks enabling F. nucleatum adaptation to different environments, which may elucidate how these bacteria colonize different compartments of the human body.


Assuntos
Infecções por Fusobacterium/microbiologia , Fusobacterium nucleatum/genética , Neoplasias/microbiologia , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fusobacterium nucleatum/classificação , Fusobacterium nucleatum/crescimento & desenvolvimento , Fusobacterium nucleatum/fisiologia , Humanos , Porinas/genética , Porinas/metabolismo , RNA Bacteriano/metabolismo
8.
Chem Commun (Camb) ; 57(61): 7517-7520, 2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34235521

RESUMO

We demonstrate that a recombinase ribozyme achieves multiple functions in the same reaction network: self-reproduction, iterative elongation and circularization of other RNAs, leading to synthesis of diverse products predicted by a kinetic model. This shows that key mechanisms can be integrated and controlled toward Darwinian evolution in RNA reaction networks.


Assuntos
RNA Bacteriano/genética , RNA Catalítico/genética , RNA/genética , Azoarcus/enzimologia , Biocatálise , Fenômenos Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Sequências Repetidas Invertidas , Cinética , RNA/química , RNA Bacteriano/química , RNA Catalítico/química , Recombinases/química , Recombinases/genética
9.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299177

RESUMO

Adaptation of bacteria to a changing environment is often accompanied by remodeling of the transcriptome. In the facultative phototroph Rhodobacter sphaeroides the alternative sigma factors RpoE, RpoHI and RpoHII play an important role in a variety of stress responses, including heat, oxidative stress and nutrient limitation. Photooxidative stress caused by the simultaneous presence of chlorophylls, light and oxygen is a special challenge for phototrophic organisms. Like alternative sigma factors, several non-coding sRNAs have important roles in the defense against photooxidative stress. RNAseq-based transcriptome data pointed to an influence of the stationary phase-induced StsR sRNA on levels of mRNAs and sRNAs with a role in the photooxidative stress response. Furthermore, StsR also affects expression of photosynthesis genes and of genes for regulators of photosynthesis genes. In vivo and in vitro interaction studies revealed that StsR, that is under control of the RpoHI and RpoHII sigma factors, targets rpoE mRNA and affects its abundance by altering its stability. RpoE regulates expression of the rpoHII gene and, consequently, expression of stsR. These data provide new insights into a complex regulatory network of protein regulators and sRNAs involved in defense against photooxidative stress and the regulation of photosynthesis genes.


Assuntos
Proteínas de Bactérias/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , RNA Bacteriano/genética , Rhodobacter sphaeroides/crescimento & desenvolvimento , Fator sigma/metabolismo , Transcriptoma , Proteínas de Bactérias/genética , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Fator sigma/genética
10.
Int J Biol Macromol ; 185: 582-591, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34216660

RESUMO

The effects of a novel Flammulina velutipes polysaccharide (FVP) on intestinal microbiota, immune repertoire and heart transcriptome were investigated in this study. The results showed that FVP treatment could effectively regulate the abundance of colonic microbiota. And FVP exhibited obvious immunoregulatory effect by influencing V gene and J gene fragments usage on TCRα chain. The usage frequency of TRBV1, TRBJ1-6 and TRBJ1-5 were significantly altered, and 41 V-J pairs were identified with obvious difference after FVP treatment. Furthermore, the mRNA of mice heart was analyzed by transcriptome assay. Total 525 genes and 1587 mRNA were significantly changed after FVP treatment. KEGG annotation indicated that the up-regulated mRNA was enriched in 17 pathways including adherens junction, mTOR signaling pathway, insulin signaling pathway, mitophagy, tight junction, PPAR signaling pathway and TNF signaling pathway, etc. Meanwhile, the down-regulated mRNA was gathered in AMPK signaling pathway, metabolism of xenobiotics by cytochrome P450, apelin signaling pathway, PPAR signaling pathway, PI3K-Akt signaling pathway, insulin signaling pathway, cardiac muscle contraction, adrenergic signaling in cardiomyocytes, Fc gamma R-mediated phagocytosis, etc. The great potential exhibited by FVP could make it an ideal candidate as complementary medicine or functional food for promotion of health.


Assuntos
Bactérias/isolamento & purificação , Flammulina/química , Perfilação da Expressão Gênica/métodos , Miocárdio/química , Polissacarídeos/administração & dosagem , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Animais , Bactérias/efeitos dos fármacos , Bactérias/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Masculino , Camundongos , Anotação de Sequência Molecular , Filogenia , Polissacarídeos/farmacologia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de RNA , Xenobióticos
11.
Nat Commun ; 12(1): 4433, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290242

RESUMO

The small, regulatory RNA RepG (Regulator of polymeric G-repeats) regulates the expression of the chemotaxis receptor TlpB in Helicobacter pylori by targeting a variable G-repeat in the tlpB mRNA leader. Here, we show that RepG additionally controls lipopolysaccharide (LPS) phase variation by also modulating the expression of a gene (hp0102) that is co-transcribed with tlpB. The hp0102 gene encodes a glycosyltransferase required for LPS O-chain biosynthesis and in vivo colonization of the mouse stomach. The G-repeat length defines a gradual (rather than ON/OFF) control of LPS biosynthesis by RepG, and leads to gradual resistance to a membrane-targeting antibiotic. Thus, RepG-mediated modulation of LPS structure might impact host immune recognition and antibiotic sensitivity, thereby helping H. pylori to adapt and persist in the host.


Assuntos
Farmacorresistência Bacteriana , Helicobacter pylori/fisiologia , Lipopolissacarídeos/biossíntese , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Regiões 5' não Traduzidas , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Lipopolissacarídeos/química , Camundongos , Antígenos O/biossíntese , Antígenos O/química , Polimixina B/farmacologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Sequências Repetitivas de Ácido Nucleico , Estresse Salino , Estômago/microbiologia
12.
Methods Mol Biol ; 2314: 481-512, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235667

RESUMO

RNA sequencing (RNAseq) in bacteria has become a transformative tool for many applications, including the identification of mechanisms that contribute to pathogenesis, environmental adaptation, and drug response. The kinds of analysis outputs achievable from RNA-seq depend heavily on several key technical parameters during the sample preparation, sequencing, and data processing steps. In this chapter, we will describe the process of preparing Mycobacterium tuberculosis samples into sequencing libraries, selecting the appropriate sequencing platform, and performing data processing compatible with gene expression quantification. We will also discuss how each parameter could affect outcomes. The protocols described below produce consistently high yields. This chapter should inform on the technical considerations that impact sequencing output and enable the reader to decide on the best parameters to implement based on their own experimental goals.


Assuntos
Proteínas de Bactérias/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Mycobacterium tuberculosis/genética , RNA Bacteriano/genética , Análise de Sequência de RNA/métodos , Humanos , RNA Bacteriano/análise , Fluxo de Trabalho
13.
Methods Mol Biol ; 2314: 513-531, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235668

RESUMO

Next-generation sequencing technologies facilitate the analysis of multiple important properties of transcriptomes in addition to gene expression levels. Here, we describe a method for mapping RNA 5' ends in Mycobacterium tuberculosis and Mycobacterium smegmatis, which allows the determination of transcription start sites (TSSs), comparative analysis of promoter usage under different conditions, and mapping of endoribonucleolytic cleavage sites. We describe in detail the procedures for constructing RNA sequencing libraries appropriate for RNA 5' end mapping using an Illumina sequencing platform, as well as bioinformatic procedures for data analysis.


Assuntos
Regiões 5' não Traduzidas/genética , Mycobacterium tuberculosis/genética , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , Análise de Sequência de RNA/métodos , Sítio de Iniciação de Transcrição , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Bacteriano/análise , Transcriptoma
14.
Front Cell Infect Microbiol ; 11: 604511, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34322396

RESUMO

Bacterial small RNAs (sRNAs) are critical post-transcriptional regulators that exert broad effects on cell physiology. One class of sRNAs, referred to as trans-acting sRNAs, base-pairs with mRNAs to cause changes in their stability or translation. Another class of sRNAs sequesters RNA-binding proteins that in turn modulate mRNA expression. RNA chaperones play key roles in these regulatory events by promoting base-pairing of sRNAs to mRNAs, increasing the stability of sRNAs, inducing conformational changes on mRNA targets upon binding, or by titrating sRNAs away from their primary targets. In pathogenic bacteria, sRNAs and their chaperones exert broad impacts on both cell physiology and virulence, highlighting the central role of these systems in pathogenesis. This review provides an overview of the growing number and roles of these chaperone proteins in sRNA regulation, highlighting how these proteins contribute to bacterial pathogenesis.


Assuntos
Pequeno RNA não Traduzido , Bactérias/genética , Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Virulência
15.
Front Cell Infect Microbiol ; 11: 696533, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34327153

RESUMO

Bacterial small RNAs (sRNAs) play a vital role in pathogenesis by enabling rapid, efficient networks of gene attenuation during infection. In recent decades, there has been a surge in the number of proposed and biochemically-confirmed sRNAs in both Gram-positive and Gram-negative pathogens. However, limited homology, network complexity, and condition specificity of sRNA has stunted complete characterization of the activity and regulation of these RNA regulators. To streamline the discovery of the expression of sRNAs, and their post-transcriptional activities, we propose an integrative in vivo data-mining approach that couples DNA protein occupancy, RNA-seq, and RNA accessibility data with motif identification and target prediction algorithms. We benchmark the approach against a subset of well-characterized E. coli sRNAs for which a degree of in vivo transcriptional regulation and post-transcriptional activity has been previously reported, finding support for known regulation in a large proportion of this sRNA set. We showcase the abilities of our method to expand understanding of sRNA RseX, a known envelope stress-linked sRNA for which a cellular role has been elusive due to a lack of native expression detection. Using the presented approach, we identify a small set of putative RseX regulators and targets for experimental investigation. These findings have allowed us to confirm native RseX expression under conditions that eliminate H-NS repression as well as uncover a post-transcriptional role of RseX in fimbrial regulation. Beyond RseX, we uncover 163 putative regulatory DNA-binding protein sites, corresponding to regulation of 62 sRNAs, that could lead to new understanding of sRNA transcription regulation. For 32 sRNAs, we also propose a subset of top targets filtered by engagement of regions that exhibit binding site accessibility behavior in vivo. We broadly anticipate that the proposed approach will be useful for sRNA-reliant network characterization in bacteria. Such investigations under pathogenesis-relevant environmental conditions will enable us to deduce complex rapid-regulation schemes that support infection.


Assuntos
Pequeno RNA não Traduzido , Mineração de Dados , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética
16.
Science ; 372(6546): 1057-1062, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34083482

RESUMO

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.


Assuntos
Códon , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/virologia , Compostos Macrocíclicos/metabolismo , Polímeros/metabolismo , Biossíntese de Proteínas , Fagos T/crescimento & desenvolvimento , Aminoácidos/metabolismo , Bacteriólise , Uso do Códon , Códon de Terminação , Evolução Molecular Direcionada , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Deleção de Genes , Código Genético , Genoma Bacteriano , Compostos Macrocíclicos/química , Mutagênese , Fatores de Terminação de Peptídeos/genética , Polímeros/química , RNA Bacteriano/genética , RNA de Transferência/genética , RNA de Transferência de Serina/genética , Ubiquitina/biossíntese , Ubiquitina/genética
17.
Mol Cell ; 81(14): 2901-2913.e5, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34157309

RESUMO

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Bacteriano/genética , Domínio Catalítico/genética , Endorribonucleases/genética , Exorribonucleases/genética , Regulação Bacteriana da Expressão Gênica/genética , Chaperonas Moleculares/genética , Estabilidade de RNA/genética , Pequeno RNA não Traduzido/genética
18.
Methods Mol Biol ; 2323: 249-265, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086286

RESUMO

Research on RNA function and therapeutic potential is dominated by the use of chemoengineered RNA mimics. Recent efforts have led to the establishment of novel technologies for the production of recombinant or bioengineered RNA molecules, which should better recapitulate the structures, functions and safety profiles of natural RNAs because both are produced and folded in living cells. Herein, we describe a robust approach for reproducible fermentation production of bioengineered RNA agents (BERAs) carrying warhead miRNAs, siRNAs, aptamers, or other forms of small RNAs, based upon an optimal hybrid tRNA/pre-miRNA carrier. Target BERA/sRNAs are readily purified by fast protein liquid chromatography (FPLC) to a high degree of homogeneity (>97%). This approach offers a consistent high-level expression (>30% of total bacterial RNAs) and large-scale production of ready-to-use BERAs (multiple to tens milligrams from 1 L bacterial culture).


Assuntos
Bioengenharia/métodos , MicroRNAs/isolamento & purificação , RNA Bacteriano/isolamento & purificação , RNA de Transferência/isolamento & purificação , RNA não Traduzido/isolamento & purificação , RNA/isolamento & purificação , Sequência de Bases , Cromatografia por Troca Iônica/métodos , Clonagem Molecular/métodos , Contaminação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Endotoxinas/análise , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fermentação , MicroRNAs/biossíntese , MicroRNAs/genética , Desnaturação de Ácido Nucleico , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , RNA/biossíntese , RNA/genética , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA de Transferência/biossíntese , RNA de Transferência/genética , RNA não Traduzido/genética
19.
Biomed Pharmacother ; 140: 111778, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34062416

RESUMO

Liraglutide has been demonstrated to alleviate hepatic steatosis in clinical practice, but the underlying mechanism remains unclear. Our previous study indicated that the HIF-2α/PPARα pathway was involved in hepatic lipid accumulation induced by hypoxia.We aimed to investigate whether liraglutide could alleviate lipid-induced hepatic steatosis via the HIF-2α/PPARα pathway. Whole-body HIF-2α heterozygous knockout (HIF-2α+/-) mice and littermate wild-type (WT) mice were successfully established. Male mice challenged with a high-fat diet were treated with liraglutide (0.6 mg/kg/d) or normal saline by intraperitoneal injection for 4 weeks. We observed that, compared with WT mice, many indicators of HIF-2α+/- mice improved, including GTT, ITT, fasting blood glucose, body weight, liver weight, and lipid profile in serum or liver lipid deposition, and the expression level of PPARα, mitochondrial function genes, and fatty acid oxidation genes were upregulated, while those of HIF-2α and lipogenesis genes were downregulated significantly. After liraglutide treatment in WT mice, we found that significant improvements were observed in the fat mass, GTT, ITT, fasting blood glucose, body weight, liver weight, lipid profile in serum or liver lipid deposition; the ß-oxidation genes were upregulated and the lipogenesis genes were downregulated; and the abundance of intestinal Akkermansia muciniphila increased significantly. However, the effects of liraglutide on WT mice were not observed in HIF-2α+/- mice. In addition, in the HepG2 steatotic hepatocyte model, liraglutide alleviated lipid deposits by repressing lipid synthesis and enhancing fatty acid ß-oxidation, which were substantially suppressed by the HIF-2α modulators. Therefore, the HIF-2α/PPARα pathway is essential for liraglutide-alleviated lipid-induced hepatic steatosis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipoglicemiantes/uso terapêutico , Metabolismo dos Lipídeos/efeitos dos fármacos , Liraglutida/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , PPAR alfa/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Dieta Hiperlipídica , Microbioma Gastrointestinal/genética , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Liraglutida/farmacologia , Masculino , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/microbiologia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética
20.
Nat Commun ; 12(1): 3282, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078900

RESUMO

Bacterial processes necessary for adaption to stressful host environments are potential targets for new antimicrobials. Here, we report large-scale transcriptomic analyses of 32 human bacterial pathogens grown under 11 stress conditions mimicking human host environments. The potential relevance of the in vitro stress conditions and responses is supported by comparisons with available in vivo transcriptomes of clinically important pathogens. Calculation of a probability score enables comparative cross-microbial analyses of the stress responses, revealing common and unique regulatory responses to different stresses, as well as overlapping processes participating in different stress responses. We identify conserved and species-specific 'universal stress responders', that is, genes showing altered expression in multiple stress conditions. Non-coding RNAs are involved in a substantial proportion of the responses. The data are collected in a freely available, interactive online resource (PATHOgenex).


Assuntos
Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , RNA Bacteriano/genética , Estresse Fisiológico/genética , Transcriptoma , Adaptação Fisiológica/genética , Atlas como Assunto , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Genes Bacterianos , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Positivas/patogenicidade , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Internet , Microbiota/genética , Filogenia , RNA Bacteriano/metabolismo
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