RESUMO
Circular RNAs are garnering increasing interest as potential regulatory RNAs and a format for gene expression. The characterization of circular RNA using analytical techniques commonly employed in the literature, such as gel electrophoresis, can, under differing conditions, yield different results when attempting to distinguish circular RNA from linear RNA of similar molecular weights. Here, we describe circular RNA migration in different conditions, analyzed by gel electrophoresis and high-performance liquid chromatography (HPLC). We characterize key parameters that affect the migration pattern of circular RNA in gel electrophoresis systems, which include gel type, electrophoresis time, sample buffer composition, and voltage. Finally, we demonstrate the utility of orthogonal analytical tests for circular RNA that take advantage of its covalently closed structure to further distinguish circular RNA from linear RNA following in vitro synthesis.
Assuntos
RNA Circular , RNA , Eletroforese em Gel de Ágar/métodos , Peso Molecular , RNA/genética , RNA Circular/genéticaRESUMO
RNase P is an essential enzyme found across all domains of life that is responsible for the 5'-end maturation of precursor tRNAs. For decades, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations, especially in the model bacterium, Escherichia coli In E. coli, the temperature-sensitive (ts) rnpA49 mutation in the protein subunit of RNase P has arguably been one of the most well-studied mutations for examining the enzyme's activity in vivo. Here, we report for the first time naturally occurring temperature-resistant suppressor mutations of E. coli strains carrying the rnpA49 allele. We find that rnpA49 strains can partially compensate the ts defect via gene amplifications of either RNase P subunit (rnpA49 or rnpB) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies, and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insights into the behavior and complementation of the rnpA49 allele in vivo and provides direction for follow-up studies regarding RNase P regulation and turnover in E. coli.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Mutação , Fenótipo , Ribonuclease P , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ribonuclease P/genética , Ribonuclease P/metabolismo , Protease La/genética , Protease La/metabolismo , Supressão Genética , TemperaturaRESUMO
3' â 5' exoribonucleases play a critical role in many aspects of RNA metabolism. RNase R, PNPase, and RNase II are the major contributors to RNA processing, maturation, and quality control in bacteria. Bacteria don't seem to have dedicated RNA degradation machineries to process different classes of RNAs. Under different environmental and physiological conditions, their roles can be redundant and sometimes overlapping. Here, I discuss why PNPase and RNase R may have switched their physiological roles in some bacterial species to adapt to environmental conditions, despite being biochemically distinct exoribonucleases.
Assuntos
Adaptação Fisiológica , Exorribonucleases , Exorribonucleases/metabolismo , Exorribonucleases/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genéticaRESUMO
Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials.
Assuntos
Antibacterianos , Inibidores Enzimáticos , Escherichia coli , Exorribonucleases , Antibacterianos/farmacologia , Antibacterianos/química , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Domínio Catalítico , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Bactérias/efeitos dos fármacos , Bactérias/enzimologiaRESUMO
The alaW alaX operon encodes the Ala2 tRNAs, one of the two alanine tRNA isotypes in Escherichia coli. Our previous RNA-seq study showed that alaW alaX dicistronic RNA levels increased significantly in the absence of both RNase P and poly(A) polymerase I (PAP I), suggesting a role of polyadenylation in its stability. In this report, we show that RNase E initiates the processing of the primary alaW alaX precursor RNA by removing the Rho-independent transcription terminator, which appears to be the rate limiting step in the separation and maturation of the Ala2 pre-tRNAs by RNase P. Failure to separate the alaW and alaX pre-tRNAs by RNase P leads to poly(A)-mediated degradation of the dicistronic RNAs by polynucleotide phosphorylase (PNPase) and RNase R. Surprisingly, the thermosensitive RNase E encoded by the rne-1 allele is highly efficient in removing the terminator (>99%) at the nonpermissive temperature suggesting a significant caveat in experiments using this allele. Together, our data present a comprehensive picture of the Ala2 tRNA processing pathway and demonstrate that unprocessed RNase P substrates are degraded via a poly(A) mediated decay pathway.
Assuntos
Proteínas de Escherichia coli , Ribonuclease P , Ribonuclease P/genética , Ribonuclease P/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Poliadenilação , Óperon/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Bacteriano/metabolismoRESUMO
Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin-induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete thus permitting the re-initiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect. Analysis of temperature-sensitive mutant strains implicate RNase E, PNPase and RNase R in rifampicin-induced rRNA degradation. We cannot distinguish between a direct role for RNase E in rRNA degradation versus an indirect role involving a slowdown of mRNA degradation. Since mRNA and rRNA appear to be degraded by the same ribonucleases, competition by rRNA is likely to result in slower mRNA degradation rates in the presence of rifampicin than under normal growth conditions.
RESUMO
IMPORTANCE: Bacterial exoribonucleases play a crucial role in RNA maturation, degradation, quality control, and turnover. In this study, we have uncovered a previously unknown role of 3'-5' exoribonuclease RNase R of Pseudomonas syringae Lz4W in DNA damage and oxidative stress response. Here, we show that neither the exoribonuclease function of RNase R nor its association with the RNA degradosome complex is essential for this function. Interestingly, in P. syringae Lz4W, hydrolytic RNase R exhibits physiological roles similar to phosphorolytic 3'-5' exoribonuclease PNPase of E. coli. Our data suggest that during the course of evolution, mesophilic E. coli and psychrotrophic P. syringae have apparently swapped these exoribonucleases to adapt to their respective environmental growth conditions.
Assuntos
Escherichia coli , Exorribonucleases , Exorribonucleases/genética , Exorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Regiões Antárticas , Dano ao DNA , Estresse Oxidativo , RNA Bacteriano/genéticaRESUMO
BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor worldwide. Circular RNA (circRNA) is of great value in tumorigenesis progression. However, the mechanism of circFNDC3B in ESCC remains to be clarified. METHODS: Firstly, the circular characteristics of circFNDC3B were evaluated by Actinomycin D and RNase R measurements. The functions of circFNDC3B in ESCC cells were examined by CCK-8, EdU and flow cytometry. Subsequently, the molecular mechanism of circFNDC3B was explained using luciferase reporter gene detection. Finally, we constructed xenograft model to prove the role of circFNDC3B in vivo. RESULTS: Our study revealed that circFNDC3B was more stable than its linear RNA and prominently upregulated in ESCC. Functional findings suggested that silencing of circFNDC3B reduced the proliferation and enhanced apoptosis of ESCC cells in vitro. Meanwhile, knockdown of circFNDC3B attenuated tumor progression in vivo. Next, miR-370-3p/miR-136-5p was discovered to bind circFNDC3B. miR-370-3p/miR-136-5p reversed the promotive effect on cell proliferation and the inhibitory effect on cell apoptosis of circFNDC3B. MYO5A was a downstream target of miR-370-3p/miR-136-5p. CircFNDC3B served as a sponge for miR-370-3p/miR-136-5p and alleviated the prohibitory effect of miR-370-3p/miR-136-5p on MYO5A, which accelerated ESCC progression. CONCLUSION: circFNDC3B positively adjusted the MYO5A expression via spongy miR-370-3p/miR-136-5p, hence achieving the cancer-promoting effect on ESCC. circFNDC3B was a prospective diagnosis marker for ESCC.
Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , MicroRNAs , Miosina Tipo V , RNA Circular , Humanos , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Genes Reporter , MicroRNAs/genética , Cadeias Pesadas de Miosina , Estudos Prospectivos , RNA Circular/genéticaRESUMO
Pre-mRNAs from thousands of eukaryotic genes can be non-canonically spliced to generate circular RNAs (circRNAs) that have covalently linked ends. Most mature circular RNAs are expressed at low levels, but some have known physiological functions and/or accumulate to higher levels than their associated linear mRNAs. These observations have sparked great interest into this class of previously underappreciated RNAs and prompted the development of new experimental approaches to study them, especially methods to measure or modulate circular RNA expression levels. Nonetheless, each of these approaches has caveats and potential pitfalls that must be controlled for when designing experiments and interpreting results. Here, we provide practical advice, tips, and suggested guidelines for performing robust identification, validation, and functional characterization of circular RNAs. Beyond promoting rigor and reproducibility, these suggestions should help bring clarity to the field, especially how circular RNAs function and whether these transcripts may sponge microRNAs/proteins or serve as templates for translation.
Assuntos
Precursores de RNA , RNA Circular , RNA/genética , RNA/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , Reprodutibilidade dos TestesRESUMO
Circular RNAs (circRNAs) are non-coding RNAs (ncRNAs) originating from a post-transcriptional modification process called back-splicing. Despite circRNAs being traditionally considered by-products rather than independently functional, circRNAs play many vital roles, such as in host immunity during viral infection. However, in shrimp, these remain largely unexplored. Therefore, this study aims to identify circRNAs in Litopenaeus vannamei in the context of WSSV infection, one of the most eradicative pathogens threatening shrimp populations worldwide. We identified 290 differentially expressed circRNAs (DECs) in L. vannamei upon WSSV infection. Eight DECs were expressed from their parental genes, including alpha-1-inhibitor-3, calpain-B, integrin-V, hemicentin-2, hemocytin, mucin-17, proPO2, and rab11-FIP4. These were examined quantitatively by qRT-PCR, which revealed the relevant expression profiles to those obtained from circRNA-Seq. Furthermore, the structural and chemical validation of the DECs conformed to the characteristics of circRNAs. One of the functional properties of circRNAs as a miRNA sponge was examined via the interaction network between DECs and WSSV-responsive miRNAs, which highlighted the targets of miRNA sponges. Our discovery could provide insight into the participation of these ncRNAs in shrimp antiviral responses.
Assuntos
MicroRNAs , Viroses , Animais , Transcriptoma , RNA Circular/genética , MicroRNAs/metabolismoRESUMO
Circular RNAs (circRNAs) have gained recent attraction due to their functional versatility and particular structure connected to human diseases. Current investigations are focused on the interplay between their ability to sponge smaller species of RNAs, such as microRNAs (miRNAs), thus influencing their regulatory activity on gene expression and protein templates. Therefore, their reported implication in various biological processes axis has resulted in an accumulating number of studies. While the testing and annotation methods of novel circular transcripts are still under development, there is still a plethora of transcript candidates suitable for investigation in human disease. The discordance in the literature regarding the approaches used in circRNAs quantification and validation methods, especially regarding qRT-PCR, the current golden standard procedure, leads to high result variability and undermines the replicability of the studies. Therefore, our study will offer several valuable insights into bioinformatic data for experimental design for circRNA investigation and in vitro aspects. Specifically, we will highlight key aspects such as circRNA database annotation divergent primer design and several processing steps, such as RNAse R treatment optimization and circRNA enrichment assessment. Additionally, we will provide insights into the exploration of circRNA-miRNA interactions, a prerequisite for further functional investigations. With this, we aim to contribute to the methodological consensus in a currently expanding field with possible implications for assessing therapeutic targets and biomarker discovery.
Assuntos
MicroRNAs , RNA Circular , Humanos , RNA Circular/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase , Biologia Computacional/métodos , Redes Reguladoras de Genes , Perfilação da Expressão Gênica/métodosRESUMO
Stable Intronic Sequence RNA (sisRNA) is a relatively new class of non-coding RNA. Found in many organisms, these sisRNA produced from their host genes are generally involved in regulatory roles, controlling gene expression at multiple levels through active involvement in regulatory feedback loops. Large scale identification of sisRNA via genome-wide RNA sequencing has been difficult, largely in part due to its low abundance. Done on its own, RNA sequencing often yields a large mass of information that is ironically uninformative; the potential sisRNA reads being masked by other highly abundant RNA species like ribosomal RNA and messenger RNA. In this review, we present a practical workflow for the enrichment of circular sisRNA through the use of transcriptionally quiescent systems, rRNA-depletion, and RNase R treatment prior to deep sequencing. This workflow allows circular sisRNA to be reliably detected. We also present various methods to experimentally validate the circularity and stability of the circular sisRNA identified, as well as a few methods for further functional characterisation.
Assuntos
Splicing de RNA , RNA , Sequência de Bases , Íntrons/genética , RNA/genética , Estabilidade de RNA , Análise de Sequência de RNARESUMO
Exoribonuclease R (RNase R) is a 3' hydrolytic exoribonuclease that can degrade structured RNA. Mutation in RNase R affects virulence of certain human pathogenic bacteria. The aim of this study was to determine whether RNase R is necessary for virulence of the phytopathogen that causes bacterial blight in rice, Xanthomonas oryzae pv. oryzae (Xoo). In silico analysis has indicated that RNase R is highly conserved among various xanthomonads. Amino acid sequence alignment of Xoo RNase R with RNase R from various taxa indicated that Xoo RNase R clustered with RNase R of order Xanthomonadales. To study its role in virulence, we generated a gene disruption mutant of Xoo RNase R. The Xoo rnr- mutant is moderately virulence deficient, and the complementing strain (rnr-/pHM1::rnr) rescued the virulence deficiency of the mutant. We investigated swimming and swarming motilities in both nutrient-deficient minimal media and nutrient-optimal media. We observed that RNase R mutation has adversely affected the swimming and swarming motilities of Xoo in optimal media. However, in nutrient-deficient media only swimming motility was noticeably affected. Growth curves in optimal media at suboptimal temperature (15°C cold stress) indicate that the Xoo rnr- mutant grows more slowly than the Xoo wild type and complementing strain (rnr-/pHM1::rnr). Given these findings, we report for the first time that RNase R function is necessary for complete virulence of Xoo in rice. It is also important for motility of Xoo in media and for growth of Xoo at suboptimal temperature.
Assuntos
Oryza , Xanthomonas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Exorribonucleases/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , VirulênciaRESUMO
tRNA maturation and quality control are crucial for proper functioning of these transcripts in translation. In several organisms, defective tRNAs were shown to be tagged by poly(A) or CCACCA tails and subsequently degraded by 3'-exonucleases. In a deep-sequencing analysis of tRNA 3'-ends, we detected the CCACCA tag also in Escherichia coli However, this tag closely resembles several 3'-trailers of tRNA precursors targeted for maturation and not for degradation. Here, we investigate the ability of two important exonucleases, RNase R and RNase T, to distinguish tRNA precursors with a native 3'-trailer from tRNAs with a CCACCA tag. Our results show that the degrading enzyme RNase R breaks down both tRNAs primed for degradation as well as precursor transcripts, indicating that it is a rather nonspecific RNase. RNase T, a main processing exonuclease involved in trimming of 3'-trailers, is very inefficient in converting the CCACCA-tagged tRNA into a mature transcript. Hence, while both RNases compete for trailer-containing tRNA precursors, the inability of RNase T to process CCACCA tails ensures that defective tRNAs cannot reenter the functional tRNA pool, representing a safeguard to avoid detrimental effects of tRNAs with erroneous integrity on protein synthesis. Furthermore, these data indicate that the RNase T-mediated end turnover of the CCA sequence represents a means to deliver a tRNA to a repeated quality control performed by the CCA-adding enzyme. Hence, originally described as a futile side reaction, the tRNA end turnover seems to fulfill an important function in the maintenance of the tRNA pool in the cell.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Exorribonucleases/metabolismo , RNA Nucleotidiltransferases/metabolismo , RNA de Transferência/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Exorribonucleases/genética , Conformação de Ácido Nucleico , Processamento de Terminações 3' de RNA , RNA Nucleotidiltransferases/genética , Estabilidade de RNA , RNA de Transferência/químicaRESUMO
Recent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins (RBPs). The biochemical enrichment of circRNAs by exoribonuclease treatment or by depletion of polyadenylated RNAs coupled with deep-sequencing is widely used for the systematic identification of circRNAs. Although these methods enrich circRNAs substantially, they do not eliminate efficiently non-polyadenylated and highly-structured RNAs. Here, we describe a method we termed RPAD, based on initial RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion. These joint interventions drastically depleted linear RNAs leading to isolation of highly pure circRNAs from total RNA pools. By facilitating the isolation of highly pure circRNAs, RPAD enables the elucidation of circRNA biogenesis, sequence, and function.
Assuntos
Biologia Computacional/métodos , Poli A/genética , RNA Mensageiro/genética , RNA/isolamento & purificação , Análise de Sequência de RNA/métodos , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto , Exorribonucleases/genética , Exorribonucleases/metabolismo , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli A/metabolismo , Poliadenilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA/genética , RNA/metabolismo , RNA Circular , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3' and 5' ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.
Assuntos
Sequência Conservada , Evolução Molecular , RNA de Transferência/genética , Elementos de Resposta , Ribossomos/metabolismo , Anticódon/genética , Epistasia Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Sequência Rica em GC , Mutação , Fatores de Iniciação de Peptídeos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA de Transferência/químicaRESUMO
High-throughput RNA sequencing and novel bioinformatic pipelines have identified thousands of circular (circ)RNAs containing backsplice junction sequences. However, circRNAs generated from multiple exons may contain different combinations of exons and/or introns arising from alternative splicing, while the backsplice junction sequence is the same. To be able to identify circRNA splice variants, we developed a method termed circRNA-Rolling Circle Amplification (circRNA-RCA). This method detects full-length circRNA sequences by performing reverse transcription (RT) in the absence of RNase H activity, followed by polymerase chain reaction (PCR) amplification of full-length circRNAs using a forward primer spanning the backsplice junction sequence and a reverse primer exactly upstream of the forward primer. By sequencing the PCR products, circRNA splice variants bearing the same backsplice junctions, which were otherwise only predicted computationally, could be experimentally validated. The splice variants were further predicted to associate with different subsets of target RNA-binding proteins and microRNAs, supporting the notion that different circRNA splice variants can have different biological impacts. In sum, the circRNA-RCA method allows the accurate identification of full-length circRNA sequences, offering unique insight into their individual function.
Assuntos
DNA Complementar/genética , Splicing de RNA , RNA Circular/genética , Processamento Alternativo , Sequência de Bases , Células HeLa , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de RNA/métodosRESUMO
A novel RNase R, psrnr, was cloned from the Antarctic bacterium Psychrobacter sp. ANT206 and expressed in Escherichia coli (E. coli). A bioinformatics analysis of the psrnr gene revealed that it contained an open reading frame of 2313 bp and encoded a protein (PsRNR) of 770 amino acids. Homology modeling indicated that PsRNR had reduced hydrogen bonds and salt bridges, which might be the main reason for the catalytic efficiency at low temperatures. A site directed mutation exhibited that His 667 in the active site was absolutely crucial for the enzyme catalysis. The recombinant PsRNR (rPsRNR) showed maximum activity at 30 °C and had thermal instability, suggesting that rPsRNR was a cold-adapted enzyme. Interestingly, rPsRNR displayed remarkable salt tolerance, remaining stable at 0.5-3.0 M NaCl. Furthermore, rPsRNR had a higher kcat value, contributing to its efficient catalytic activity at a low temperature. Overall, cold-adapted RNase R in this study was an excellent candidate for antimicrobial treatment.
Assuntos
Adaptação Biológica , Temperatura Baixa , Microbiologia Ambiental , Camada de Gelo/microbiologia , Psychrobacter/fisiologia , Ribonucleases/metabolismo , Tolerância ao Sal , Sequência de Aminoácidos , Regiões Antárticas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ativação Enzimática , Cinética , Modelos Biológicos , Conformação Molecular , Estrutura Molecular , Psychrobacter/isolamento & purificação , Ribonucleases/genéticaRESUMO
BACKGROUND: Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives in the stationary phase. The genome-wide mRNA half-lives were determined by a dynamic analysis of transcriptomes after transcription arrest. We have combined the analysis of mRNA half-lives with the steady-state concentrations (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. RESULTS: The values of mRNA half-lives demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. CONCLUSIONS: This work had identified PNPase as a central player associated with mRNA degradation in stationary phase.
Assuntos
Escherichia coli/citologia , Escherichia coli/enzimologia , Exorribonucleases/metabolismo , Estabilidade de RNA , Genoma Bacteriano , Meia-Vida , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Transcriptoma/genéticaRESUMO
This review describes how, using the power of genetic suppressor analysis, mRNA turnover in bacteria was shown to be an essential function of RNase E. RNase E is an essential multifunctional enzyme in bacteria, involved in the processing of stable RNAs to their mature forms (rRNAs and tRNAs) and in the turnover of most mRNAs. Genetic suppressor analysis was successfully used to address whether mRNA turnover is one of the essential functions of RNase E. Conditional lethal mutations in rne were shown to be suppressible by three different classes of extragenic suppressors, including a class that caused overexpression of RelE. The only known function of RelE is the cleavage of mRNA in the ribosomal A-site. Suppression of the conditional lethal defect in rne by RelE overexpression provides strong genetic evidence that mRNA turnover is one of the essential functions of RNase E. Several hypotheses that could explain why mRNA turnover is essential are discussed. Suppressor analysis is an old-fashioned but very powerful approach that can be usefully applied to address a wide variety of important questions in biology and genetics. In this work suppressor analysis has revealed that mRNA turnover is an essential function of RNase E, a conclusion that raises a host of interesting questions for future research.