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1.
Plasmid ; 109: 102503, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32209400

RESUMO

Pseudomonas putida is a highly attractive production system for industrial needs. However, for its improvement as a biocatalyst at the industrial level, modulation of its gene expression is urgently needed. We report the construction of a plasmid expressing a small RNA-based system with the potential to be used for different purposes. Due to the small RNAs modular composition, the design facilities and ability to tune gene expression, they constitute a powerful tool in genetic and metabolic engineering. In the tool presented here, customized sRNAs are expressed from a plasmid and specifically directed to any region of a chosen target. Expression of these customized sRNAs is shown to differentially modulate the level of endogenous and heterologous reporter genes. The antisense interaction of the sRNA with the mRNA produces different outcomes. Depending on the particularity of each sRNA-target mRNA pair, we demonstrate the duality of this system, which is able either to decrease or increase the expression of the same given gene. This system combines high specificity with the potential to be widely applied, due to its predicted ability to modulate the expression of virtually any given gene. This plasmid can be used to redesign P. putida metabolism, fulfilling an important industrial gap.


Assuntos
Regulação Bacteriana da Expressão Gênica , Plasmídeos/genética , Pseudomonas putida/genética , RNA Bacteriano , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Engenharia Genética
2.
Biochem Soc Trans ; 47(5): 1279-1289, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31642890

RESUMO

The revolution of genomics and growth of systems biology urged the creation of synthetic biology, an engineering discipline aiming at recreating and reprogramming cellular functions for industrial needs. There has been a huge effort in synthetic biology to develop versatile and programmable genetic regulators that would enable the precise control of gene expression. Synthetic RNA components have emerged as a solution, offering a diverse range of programmable functions, including signal sensing, gene regulation and the modulation of molecular interactions. Owing to their compactness, structure and way of action, several types of RNA devices that act on DNA, RNA and protein have been characterized and applied in synthetic biology. RNA-based approaches are more 'economical' for the cell, since they are generally not translated. These RNA-based strategies act on a much shorter time scale than transcription-based ones and can be more efficient than protein-based mechanisms. In this review, we explore these RNA components as building blocks in the RNA synthetic biology field, first by explaining their natural mode of action and secondly discussing how these RNA components have been exploited to rewire bacterial regulatory circuitry.


Assuntos
Fenômenos Fisiológicos Bacterianos , Biologia Sintética , Regiões 5' não Traduzidas , Pareamento de Bases , Sistemas CRISPR-Cas , RNA Bacteriano/genética , Riboswitch
3.
RNA Biol ; 16(2): 211-219, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30608212

RESUMO

Ribosomes are macromolecular machines that carry out protein synthesis. After each round of translation, ribosome recycling is essential for reinitiating protein synthesis. Ribosome recycling factor (RRF), together with elongation factor G (EF-G), catalyse the transient split of the 70S ribosome into subunits. This splitting is then stabilized by initiation factor 3 (IF3), which functions as an anti-association factor. The correct amount of these factors ensures the precise level of 70S ribosomes in the cell. RNase R is a highly conserved exoribonuclease involved in the 3' to 5' degradation of RNAs. In this work we show that pneumococcal RNase R directly controls the expression levels of frr, fusA and infC mRNAs, the corresponding transcripts of RRF, EF-G and IF3, respectively. We present evidences showing that accumulation of these factors leads to a decreased amount of 70S active particles, as demonstrated by the altered sucrose gradient ribosomal pattern in the RNase R mutant strain. Furthermore, the single deletion of RNase R is shown to have a global impact on protein synthesis and cell viability, leading to a ~50% reduction in bacterial CFU/ml. We believe that the fine-tuned regulation of these transcripts by RNase R is essential for maintaining the precise amount of active ribosomal complexes required for proper mRNA translation and thus we propose RNase R as a new auxiliary factor in ribosome reassociation. Considering the overall impact of RNase R on protein synthesis, one of the main targets of antibiotics, this enzyme may be a promising target for antimicrobial treatment.


Assuntos
Exorribonucleases/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Bactérias/genética , Bactérias/metabolismo , Sobrevivência Celular/genética , Mutação
4.
Biochem J ; 475(12): 2091-2105, 2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29802118

RESUMO

DIS3 (defective in sister chromatid joining) is the catalytic subunit of the exosome, a protein complex involved in the 3'-5' degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two protein-coding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN (PilT N-terminal) domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 and the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared with isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared with isoform 2, whereas acute myeloid leukaemia and chronic myelomonocytic leukaemia patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Taken together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers.


Assuntos
Processamento Alternativo , Complexo Multienzimático de Ribonucleases do Exossomo/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hematológicas/enzimologia , Proteínas de Neoplasias/biossíntese , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Células HEK293 , Células HeLa , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Proteínas de Neoplasias/genética , Células THP-1
5.
PLoS Genet ; 9(12): e1004001, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24367275

RESUMO

The presence of regulatory sequences in the 3' untranslated region (3'-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 3'-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 3'-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 3'-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 3'-UTRs may play in controlling mRNA expression. We showed that base pairing between the 3'-UTR and the Shine-Dalgarno (SD) region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG) within icaR 3'-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 3'-UTR with the 5'-UTR of the same mRNA.


Assuntos
Biossíntese de Proteínas , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Ribonucleico/genética , Staphylococcus aureus/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Pareamento de Bases , Biofilmes , Regulação Bacteriana da Expressão Gênica , Humanos , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/patologia , Staphylococcus aureus/patogenicidade
6.
Mol Biol Evol ; 31(8): 2170-80, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24855141

RESUMO

Retrogenes are functional processed copies of genes that originate via the retrotranscription of an mRNA intermediate and often exhibit testis-specific expression. Although this expression pattern appears to be favored by selection, the origin of such expression bias remains unexplained. Here, we study the regulation of two young testis-specific Drosophila retrogenes, Dntf-2r and Pros28.1A, using genetic transformation and the enhanced green fluorescent protein reporter gene in Drosophila melanogaster. We show that two different short (<24 bp) regions upstream of the transcription start sites (TSSs) act as testis-specific regulatory motifs in these genes. The Dntf-2r regulatory region is similar to the known ß2 tubulin 14-bp testis motif (ß2-tubulin gene upstream element 1 [ß2-UE1]). Comparative sequence analyses reveal that this motif was already present before the Dntf-2r insertion and was likely driving the transcription of a noncoding RNA. We also show that the ß2-UE1 occurs in the regulatory regions of other testis-specific retrogenes, and is functional in either orientation. In contrast, the Pros28.1A testes regulatory region in D. melanogaster appears to be novel. Only Pros28.1B, an older paralog of the Pros28.1 gene family, seems to carry a similar regulatory sequence. It is unclear how the Pros28.1A regulatory region was acquired in D. melanogaster, but it might have evolved de novo from within a region that may have been preprimed for testes expression. We conclude that relocation is critical for the evolutionary origin of male germline-specific cis-regulatory regions of retrogenes because expression depends on either the site of the retrogene insertion or the sequence changes close to the TSS thereafter. As a consequence we infer that positive selection will play a role in the evolution of these regulatory regions and can often act from the moment of the retrocopy insertion.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Proteínas de Transporte Nucleocitoplasmático/genética , Complexo de Endopeptidases do Proteassoma/genética , Testículo/crescimento & desenvolvimento , Animais , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Especificidade de Órgãos , Elementos Reguladores de Transcrição , Retroelementos , Seleção Genética
7.
Plasmid ; 78: 26-36, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25263573

RESUMO

In the last decade regulatory RNAs have emerged as powerful tools to regulate the expression of genes both in prokaryotes and in eukaryotes. RNases, by degrading these RNA molecules, control the right amount of regulatory RNAs, which is fundamental for an accurate regulation of gene expression in the cell. Remarkably the first antisense RNAs identified were plasmid-encoded and their detailed study was crucial for the understanding of prokaryotic antisense RNAs. In this review we highlight the role of RNases in the precise modulation of antisense RNAs that control plasmid replication, maintenance and transfer.


Assuntos
Regulação Bacteriana da Expressão Gênica , Plasmídeos/genética , RNA Antissenso , Ribonucleases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Conjugação Genética , Replicação do DNA , Proteínas de Escherichia coli/genética , Fator F/genética , Feromônios/metabolismo , RNA Antissenso/genética , RNA Bacteriano/genética , Ribonucleases/genética
8.
J Mol Biol ; 436(18): 168711, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-39019106

RESUMO

Previous studies on RNase R have highlighted significant effects of this ribonuclease in several processes of Streptococcus pneumoniae biology. In this work we show that elimination of RNase R results in overexpression of most of genes encoding the components of type II fatty acid biosynthesis (FASII) cluster. We demonstrate that RNase R is implicated in the turnover of most of transcripts from this pathway, affecting the outcome of the whole FASII cluster, and ultimately leading to changes in the membrane fatty acid composition. Our results show that the membrane of the deleted strain contains higher proportion of unsaturated and long-chained fatty acids than the membrane of the wild type strain. These alterations render the RNase R mutant more prone to membrane lipid peroxidation and are likely the reason for the increased sensitivity of this strain to detergent lysis and to the action of the bacteriocin nisin. Reprogramming of membrane fluidity is an adaptative cell response crucial for bacterial survival in constantly changing environmental conditions. The data presented here is suggestive of a role for RNase R in the composition of S. pneumoniae membrane, with strong impact on pneumococci adaptation to different stress situations.


Assuntos
Membrana Celular , Ácidos Graxos , Fluidez de Membrana , Streptococcus pneumoniae , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos/biossíntese , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Endorribonucleases/metabolismo , Endorribonucleases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Peroxidação de Lipídeos
9.
Plasmid ; 70(2): 247-53, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23707902

RESUMO

A new replicon suitable for cloning and gene expression was successfully introduced into Streptococcus pneumoniae. The non-integrative lactococcal vectors pIL253 (higher-copy) and pIL252 (lower-copy), which are based on the promiscuous theta-replicating plasmid pAMß1, were established in pneumococcus. The stability and the small size of these plasmids, together with the presence of a helpful multi-cloning site make them a useful genetic tool for gene expression in this bacterium. The functionality of the system was tested by cloning and expressing the pneumococcal RNase R gene in pIL253. Full constitutive expression of the cloned gene was observed, clearly demonstrating that this plasmid can be used as an expression vector in S. pneumoniae. Moreover, gene expression can be regulated by the use of the lower- or higher-copy number vector versions. The existence of other replicative plasmids based on this family, which are also probably functional in pneumococcus, further broadens the cloning possibilities. We also show that S. pneumoniae cells can accommodate simultaneously pIL252 or pIL253 together with pLS1, a pMV158 derivative, which replicates via a rolling circle mechanism. This fact greatly increases the ability to manipulate this bacterium. The availability of a new family of replicative vectors for genetic manipulation in S. pneumoniae is an important contribution to the study of this pathogenic microorganism.


Assuntos
Clonagem Molecular/métodos , Expressão Gênica , Plasmídeos/genética , Replicon/genética , Streptococcus pneumoniae/genética , Western Blotting , Variações do Número de Cópias de DNA/genética , Primers do DNA/genética , Vetores Genéticos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Nucleic Acids Res ; 39(7): 2918-30, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21138960

RESUMO

MicA is a trans-encoded small non-coding RNA, which downregulates porin-expression in stationary-phase. In this work, we focus on the role of endoribonucleases III and E on Salmonella typhimurium sRNA MicA regulation. RNase III is shown to regulate MicA in a target-coupled way, while RNase E is responsible for the control of free MicA levels in the cell. We purified both Salmonella enzymes and demonstrated that in vitro RNase III is only active over MicA when in complex with its targets (whether ompA or lamB mRNAs). In vivo, MicA is demonstrated to be cleaved by RNase III in a coupled way with ompA mRNA. On the other hand, RNase E is able to cleave unpaired MicA and does not show a marked dependence on its 5' phosphorylation state. The main conclusion of this work is the existence of two independent pathways for MicA turnover. Each pathway involves a distinct endoribonuclease, having a different role in the context of the fine-tuned regulation of porin levels. Cleavage of MicA by RNase III in a target-dependent fashion, with the concomitant decay of the mRNA target, strongly resembles the eukaryotic RNAi system, where RNase III-like enzymes play a pivotal role.


Assuntos
Pequeno RNA não Traduzido/metabolismo , Ribonuclease III/metabolismo , Salmonella typhimurium/enzimologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Pareamento de Bases , Mutação , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/análise , Pequeno RNA não Traduzido/química , Ribonuclease III/genética , Salmonella typhimurium/genética
11.
BMC Microbiol ; 12: 268, 2012 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-23167513

RESUMO

BACKGROUND: Ribonuclease R (RNase R) is an exoribonuclease that recognizes and degrades a wide range of RNA molecules. It is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. RNase R has also been implicated in the trans-translation process. Transfer-messenger RNA (tmRNA/SsrA RNA) and SmpB are the main effectors of trans-translation, an RNA and protein quality control system that resolves challenges associated with stalled ribosomes on non-stop mRNAs. Trans-translation has also been associated with deficiencies in stress-response mechanisms and pathogenicity. RESULTS: In this work we study the expression of RNase R in the human pathogen Streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery (SmpB and tmRNA/SsrA). We show that RNase R is induced after a 37°C to 15°C temperature downshift and that its levels are dependent on SmpB. On the other hand, our results revealed a strong accumulation of the smpB transcript in the absence of RNase R at 15°C. Transcriptional analysis of the S. pneumoniae rnr gene demonstrated that it is co-transcribed with the flanking genes, secG and smpB. Transcription of these genes is driven from a promoter upstream of secG and the transcript is processed to yield mature independent mRNAs. This genetic organization seems to be a common feature of Gram positive bacteria, and the biological significance of this gene cluster is further discussed. CONCLUSIONS: This study unravels an additional contribution of RNase R to the trans-translation system by demonstrating that smpB is regulated by this exoribonuclease. RNase R in turn, is shown to be under the control of SmpB. These proteins are therefore mutually dependent and cross-regulated. The data presented here shed light on the interactions between RNase R, trans-translation and cold-shock response in an important human pathogen.


Assuntos
Exorribonucleases/biossíntese , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Estabilidade de RNA , Proteínas de Ligação a RNA/biossíntese , Streptococcus pneumoniae/genética , Transcrição Gênica , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/metabolismo , Temperatura
12.
Microorganisms ; 10(2)2022 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-35208772

RESUMO

Pneumococcal infections have increasingly high mortality rates despite the availability of vaccines and antibiotics. Therefore, the identification of new virulence determinants and the understanding of the molecular mechanisms behind pathogenesis have become of paramount importance in the search of new targets for drug development. The exoribonuclease RNase R has been involved in virulence in a growing number of pathogens. In this work, we used Galleria mellonella as an infection model to demonstrate that the presence of RNase R increases the pneumococcus virulence. Larvae infected with the RNase R mutant show an increased expression level of antimicrobial peptides. Furthermore, they have a lower bacterial load in the hemolymph in the later stages of infection, leading to a higher survival rate of the larvae. Interestingly, pneumococci expressing RNase R show a sudden drop in bacterial numbers immediately after infection, resembling the eclipse phase observed after intravenous inoculation in mice. Concomitantly, we observed a lower number of mutant bacteria inside larval hemocytes and a higher susceptibility to oxidative stress when compared to the wild type. Together, our results indicate that RNase R is involved in the ability of pneumococci to evade the host immune response, probably by interfering with internalization and/or replication inside the larval hemocytes.

13.
Microorganisms ; 10(2)2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-35208797

RESUMO

The pandemic caused by SARS-CoV-2 is not over yet, despite all the efforts from the scientific community. Vaccination is a crucial weapon to fight this virus; however, we still urge the development of antivirals to reduce the severity and progression of the COVID-19 disease. For that, a deep understanding of the mechanisms involved in viral replication is necessary. nsp15 is an endoribonuclease critical for the degradation of viral polyuridine sequences that activate host immune sensors. This enzyme is known as one of the major interferon antagonists from SARS-CoV-2. In this work, a biochemical characterization of SARS-CoV-2 nsp15 was performed. We saw that nsp15 is active as a hexamer, and zinc can block its activity. The role of conserved residues from SARS-CoV-2 nsp15 was investigated, and N164 was found to be important for protein hexamerization and to contribute to the specificity to degrade uridines. Several chemical groups that impact the activity of this ribonuclease were also identified. Additionally, FDA-approved drugs with the capacity to inhibit the in vitro activity of nsp15 are reported in this work. This study is of utmost importance by adding highly valuable information that can be used for the development and rational design of therapeutic strategies.

14.
Biochem Biophys Res Commun ; 411(1): 50-5, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21708124

RESUMO

The Escherichia coli bolA morphogene is very important in adaptation to stationary phase and stress response mechanisms. Genes of this family are widespread in gram negative bacteria and in eukaryotes. The expression of this gene is tightly regulated at transcriptional and post-transcriptional levels and its overexpression is known to induce round cellular morphology. The results presented in this report demonstrate that the H-NS protein, a pleiotropic regulator of gene expression, is a new transcriptional modulator of the bolA gene. In this work we show that and in vivo the levels of bolA are down-regulated by H-NS and in vitro this global regulator interacts directly with the bolA promoter region. Moreover, DNaseI foot-printing experiments mapped the interaction regions of H-NS and bolA and revealed that this global regulator binds not only one but both bolA promoters. We provide a new insight into the bolA regulation network demonstrating that H-NS represses the transcription of this important gene.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Transcrição Gênica
15.
FEBS J ; 288(17): 5130-5147, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33705595

RESUMO

SARS-CoV-2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS-CoV-2. Nsp14 is a multifunctional protein with two distinct activities, an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14-nsp10 complex from SARS-CoV-2. We confirm the 3'-5' exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS-CoV-2 nsp14-nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14-mediated ExoN activity of SARS-CoV-2. We studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function, contrasting to the functionality of these residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS-CoV-2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication.


Assuntos
COVID-19/genética , Exorribonucleases/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/genética , Antivirais/química , Antivirais/uso terapêutico , COVID-19/virologia , Desenho de Fármacos , Exorribonucleases/antagonistas & inibidores , Humanos , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/genética , Mapas de Interação de Proteínas/genética , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Replicação Viral/genética , Tratamento Farmacológico da COVID-19
16.
Biochemistry ; 48(50): 11848-57, 2009 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-19863111

RESUMO

Maturation, turnover, and quality control of RNA are performed by many different classes of ribonucleases. Escherichia coli RNase II is the prototype of the RNase II family of ribonucleases, a ubiquitous family of hydrolytic, processive 3' --> 5' exonucleases crucial in RNA metabolism. RNase R is a member of this family that is modulated in response to stress and has been implicated in virulence. In this work, RNase II-like proteins were characterized in the human pathogens Salmonella typhimurium and Streptococcus pneumoniae. By sequence analysis, only one member of the RNase II family was identified in S. pneumoniae, while both RNase II and RNase R were found in Sa. typhimurium. These enzymes were cloned, expressed, purified, and characterized with regard to their biochemical features and modular architecture. The specificity of substrates and the final products generated by the enzymes were clearly demonstrated. Sa. typhimurium RNase II and RNase R behaved essentially as their respective E. coli counterparts. We have shown that the only hydrolytic RNase found in S. pneumoniae was able to degrade structured RNAs as is the case with E. coli RNase R. Our results further showed that there are differences with regard to the activity and ability to bind RNA from enzymes belonging to two distinct pneumococcal strains, and this may be related to a single amino acid substitution in the catalytic domain. Since ribonucleases have not been previously characterized in S. pneumoniae or Sa. typhimurium, this work provides an important first step in the understanding of post-transcriptional control in these pathogens.


Assuntos
Proteínas de Bactérias/química , Exorribonucleases/química , Família Multigênica , Salmonella typhimurium/enzimologia , Streptococcus pneumoniae/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Simulação por Computador , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Exorribonucleases/genética , Exorribonucleases/isolamento & purificação , Exorribonucleases/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Salmonella typhimurium/patogenicidade , Homologia de Sequência de Aminoácidos , Streptococcus pneumoniae/patogenicidade , Especificidade por Substrato , Virulência
17.
Gene Expr Patterns ; 8(3): 141-7, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18178530

RESUMO

The vitamin A-derived morphogen retinoic acid (RA) plays important roles during the development of chordate animals. The Aldh1a-family of RA-synthesizing enzymes consists of three members, Aldh1a1-3 (Raldh1-3), that are dynamically expressed throughout development. We have searched the known teleost genomes for the presence of Raldh family members and have found that teleost fish possess orthologs of Aldh1a2 and Aldh1a3 only. Here we describe the expression of aldh1a3 in the zebrafish, Danio rerio. Whole mount in situ hybridization shows that aldh1a3 is expressed during eye development in the retina flanking the optic stalks and later is expressed ventrally, opposite the expression domain of aldh1a2. During inner ear morphogenesis, aldh1a3 is expressed in developing sensory epithelia of the cristae and utricular macula and is specifically up-regulated in epithelial projections throughout the formation of the walls of the semicircular canals and endolymphatic duct. In contrast to the mouse inner ear, which expresses all three Raldhs, we find that only aldh1a3 is expressed in the zebrafish otocyst, while aldh1a2 is present in the periotic mesenchyme. During larval stages, additional expression domains of aldh1a3 appear in the anterior pituitary and the swim bladder. Our analyses provide a starting point for genetic studies to examine the role of RA in these organs and emphasize the suitability of the zebrafish inner ear in dissecting the contribution of RA signaling to the development of the vestibular system.


Assuntos
Aldeído Desidrogenase/genética , Isoenzimas/genética , Retinal Desidrogenase/genética , Peixe-Zebra/genética , Aldeído Desidrogenase/biossíntese , Aldeído Desidrogenase/deficiência , Família Aldeído Desidrogenase 1 , Animais , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Orelha Interna/embriologia , Orelha Interna/enzimologia , Evolução Molecular , Perfilação da Expressão Gênica , Isoenzimas/biossíntese , Isoenzimas/deficiência , Mesoderma/embriologia , Mesoderma/enzimologia , Retinal Desidrogenase/biossíntese , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
18.
Biochimie ; 114: 113-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25542646

RESUMO

Gene expression not only depends on the rate of transcription but is also largely controlled at the post-transcriptional level. Translation rate and mRNA decay greatly influence the final protein levels. Surveillance mechanisms are essential to ensure the quality of the RNA and proteins produced. Trans-translation is one of the most important systems in the quality control of bacterial translation. This process guarantees the destruction of abnormal proteins and also leads to degradation of the respective defective RNAs through the action of Ribonuclease R (RNase R). This exoribonuclease hydrolyzes RNAs starting from their 3' end. Besides its involvement in trans-translation, RNase R also participates in the quality control of rRNA molecules involved in ribosomal biogenesis. RNase R is thus emerging as a key factor in ensuring translation accuracy. This review focuses on issues related to the quality control of translation, with special emphasis on the role of RNase R.


Assuntos
Proteínas de Escherichia coli/fisiologia , Exorribonucleases/fisiologia , Biossíntese de Proteínas , Ribossomos/fisiologia , Bactérias/genética , Bactérias/metabolismo , RNA Bacteriano/fisiologia
19.
J Microbiol Biotechnol ; 24(1): 8-12, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24150497

RESUMO

Biofilm formation and antibiotic resistance are important determinants for bacterial pathogenicity. Ribonucleases control RNA degradation and there is increasing evidence that they have an important role in virulence mechanisms. In this report, we show that ribonucleases affect susceptibility against ribosome-targeting antibiotics and biofilm formation in Salmonella.


Assuntos
Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana , Ribonucleases/metabolismo , Salmonella typhimurium/enzimologia , Salmonella typhimurium/fisiologia , Biofilmes/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacos
20.
Curr Opin Microbiol ; 18: 105-15, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24704578

RESUMO

Ribonucleases (RNases) are key factors in the control of biological processes, since they modulate the processing, degradation and quality control of RNAs. This review gives many illustrative examples of the role of RNases in the regulation of small RNAs (sRNAs). RNase E and PNPase have been shown to degrade the free pool of sRNAs. RNase E can also be recruited to cleave mRNAs when they are interacting with sRNAs. RNase III cleaves double-stranded structures, and can cut both the sRNA and its RNA target when they are hybridized. Overall, ribonucleases act as conductors in the control of sRNAs. Therefore, it is very important to further understand their role in the post-transcriptional control of gene expression.


Assuntos
Regulação da Expressão Gênica , Pequeno RNA não Traduzido/metabolismo , Ribonucleases/metabolismo , Biossíntese de Proteínas
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