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Aging is associated with progressive phenotypic changes. Virtually all cellular phenotypes are produced by proteins, and their structural alterations can lead to age-related diseases. However, we still lack comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contributions to age-related phenotypes. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry (LiP-MS). The results, compiled in online ProtAge catalog, unraveled age-related functional changes in regulators of translation, protein folding, and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging, causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction, and led to lifespan extension. Altogether, this comprehensive map of protein structural changes enables identifying mechanisms of age-related phenotypes and offers opportunities for their reversal.
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Senescencia Celular , Longevidad , Longevidad/genética , Polimerizacion , AminoácidosRESUMEN
For the better part of the century, microbes have been a treasure trove for deciphering the inner workings of the cell, from early insights into DNA replication and restriction-enzyme-mediated antiviral responses, to unravelling the complexities of metabolic pathways and understanding gene expression and its regulatory mechanisms [...].
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Viruses are obligate intracellular parasites that, throughout evolution, have adapted numerous strategies to control the translation machinery, including the modulation of post-transcriptional modifications (PTMs) on transfer RNA (tRNA). PTMs are critical translation regulators used to further host immune responses as well as the expression of viral proteins. Yet, we lack critical insight into the temporal dynamics of infection-induced changes to the tRNA modification landscape (i.e., 'modificome'). In this study, we provide the first comprehensive quantitative characterization of the tRNA modificome in the marine bacterium Shewanella glacialimarina during Shewanella phage 1/4 infection. Specifically, we show that PTMs can be grouped into distinct categories based on modification level changes at various infection stages. Furthermore, we observe a preference for the UAC codon in viral transcripts expressed at the late stage of infection, which coincides with an increase in queuosine modification. Queuosine appears exclusively on tRNAs with GUN anticodons, suggesting a correlation between phage codon usage and PTM modification. Importantly, this work provides the basis for further studies into RNA-based regulatory mechanisms employed by bacteriophages to control the prokaryotic translation machinery.
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Viruses feature an evolutionary shaped minimal genome that is obligately dependent on the cellular transcription and translation machinery for propagation. To suppress host cell immune responses and ensure efficient replication, viruses employ numerous tactics to favor viral gene expression and protein synthesis. This necessitates a carefully balanced network of virus- and host-encoded components, of which the RNA-based regulatory mechanisms have emerged as particularly interesting albeit insufficiently studied, especially in unicellular organisms such as archaea, bacteria, and yeasts. Here, recent advances that further our understanding of RNA-based translation regulation, mainly through post-transcriptional chemical modification of ribonucleosides, codon usage, and (virus-encoded) transfer RNAs, will be discussed in the context of viral infection.
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Reverse transcription quantitative PCR (RT-qPCR) has emerged as the gold standard for virus detection and quantification, being utilized in numerous diagnostic and research applications. However, the direct detection of viruses has so far posed a challenge as the viral genome is often encapsidated by a proteinaceous layer surrounded by a lipid envelope. This necessitates an additional and undesired RNA extraction step prior to RT-qPCR amplification. To circumvent this limitation, we have developed a direct RT-qPCR method for the detection of RNA viruses. In our method, we provide a proof-of-concept using phage phi6, a safe-to-use proxy for pathogenic enveloped RNA viruses that is commonly utilized in e.g. aerosolization studies. First, the phage phi6 envelope is removed by 1% chloroform treatment and the virus is then directly quantified by RT-qPCR. To identify false negative results, firefly luciferase is included as a synthetic external control. Thanks to the duplex format, our direct RT-qPCR method reduces the reagents needed and provides an easy to implement and broadly applicable, fast, and cost-effective tool for the quantitative analysis of enveloped RNA viruses.â¢One-step direct RT-qPCR quantification of phage phi6 virus without prior RNA isolation.â¢Reduced reaction volume for sustainable and cost-effective analysis.
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Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography-mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae), and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.
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Methanosarcina/genética , Pseudomonas syringae/genética , ARN de Transferencia/química , Ribonucleósidos/análisis , Saccharomyces cerevisiae/genética , Carbono/química , Cromatografía Líquida de Alta Presión , Marcaje Isotópico , ARN de Archaea/genética , ARN Bacteriano/genética , ARN de Hongos/genética , Espectrometría de Masas en TándemRESUMEN
Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.
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Cromatografía Liquida , Espectrometría de Masas , Ribonucleósidos/análisis , Ribonucleósidos/química , Cromatografía Liquida/métodos , Grafito/química , Humanos , Espectrometría de Masas/métodos , ARN Bacteriano , ARN de Hongos , ARN de Transferencia/química , Ribonucleósidos/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Transfer RNA (tRNA) molecules are sumptuously decorated with evolutionary conserved post-transcriptional nucleoside modifications that are essential for structural stability and ensure efficient protein translation. The tRNA modification levels change significantly in response to physiological stresses, altering translation in a number of ways. For instance, tRNA hypomodification leads to translational slowdown, disrupting protein homeostasis and reducing cellular fitness. This highlights the importance of proper tRNA modification as a determinant for maintaining cellular function and viability during stress. Furthermore, the expression of several microbial virulence factors is induced by changes in environmental conditions; a process where tRNA 2-thiolation is unequivocal for pathogenicity. In this review, we discuss the multifaceted implications of tRNA modification for infection by examining the roles of nucleoside modification in tRNA biology. Future development of novel methods and combinatory utilization of existing technologies will bring tRNA modification-mediated regulation of cellular immunity and pathogenicity to the limelight.
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Interacciones Huésped-Patógeno/genética , Infecciones/genética , Procesamiento Postranscripcional del ARN , ARN de Transferencia/metabolismo , Virulencia/genética , Adaptación Fisiológica/genética , Animales , Anticodón/genética , Codón/genética , Código Genético , Humanos , Infecciones/fisiopatología , Redes y Vías Metabólicas/fisiología , Modelos Moleculares , Conformación de Ácido Nucleico , Estrés Oxidativo/genética , Biosíntesis de Proteínas , Estabilidad del ARN , ARN de Transferencia/química , ARN de Transferencia/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Ribosomas/metabolismo , Estrés Fisiológico/genética , Azufre/metabolismoRESUMEN
Quantitative mass spectrometry (MS) is a key technique in many research areas (1), including proteomics, metabolomics, glycomics, and lipidomics. Because all of the corresponding molecules can be described by chemical formulas, universal quantification tools are highly desirable. Here, we present pyQms, an open-source software for accurate quantification of all types of molecules measurable by MS. pyQms uses isotope pattern matching that offers an accurate quality assessment of all quantifications and the ability to directly incorporate mass spectrometer accuracy. pyQms is, due to its universal design, applicable to every research field, labeling strategy, and acquisition technique. This opens ultimate flexibility for researchers to design experiments employing innovative and hitherto unexplored labeling strategies. Importantly, pyQms performs very well to accurately quantify partially labeled proteomes in large scale and high throughput, the most challenging task for a quantification algorithm.
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Marcaje Isotópico/métodos , Proteoma/análisis , Proteómica/métodos , Programas Informáticos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Algoritmos , Cromatografía Liquida , Glicómica , MetabolómicaRESUMEN
Double-stranded RNA (dsRNA) is an inducer molecule of the RNA silencing (RNA interference, RNAi) pathway that is present in all higher eukaryotes and controls gene expression at the posttranscriptional level. This mechanism allows the cell to recognize aberrant genetic material in a highly sequence specific manner. This ultimately leads to degradation of the homologous target sequence, rendering the plant cell resistant to subcellular pathogens. Consequently, dsRNA-mediated resistance has been exploited in transgenic plants to convey resistance against viruses. In addition, it has been shown that enzymatically synthesized specific dsRNA molecules can be applied directly onto plant tissue to induce resistance against the cognate virus. This strongly implies that dsRNA molecules are applicable as efficacious agents in crop protection, which will fuel the demand for cost-effective dsRNA production methods. In this chapter, the different methods for dsRNA production-both in vitro and in vivo-are described in detail.
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Técnicas Genéticas , Virus de Plantas/genética , ARN Bicatenario/biosíntesis , Escherichia coli/genética , Silenciador del Gen , Reacción en Cadena de la Polimerasa/métodos , Pseudomonas syringae/genética , Nicotiana/genéticaRESUMEN
Chemical modifications of transfer RNA (tRNA) molecules are evolutionarily well conserved and critical for translation and tRNA structure. Little is known how these nucleoside modifications respond to physiological stress. Using mass spectrometry and complementary methods, we defined tRNA modification levels in six yeast species in response to elevated temperatures. We show that 2-thiolation of uridine at position 34 (s(2)U34) is impaired at temperatures exceeding 30°C in the commonly used Saccharomyces cerevisiae laboratory strains S288C and W303, and in Saccharomyces bayanus. Upon stress relief, thiolation levels recover and we find no evidence that modified tRNA or s(2)U34 nucleosides are actively removed. Our results suggest that loss of 2-thiolation follows accumulation of newly synthesized tRNA that lack s(2)U34 modification due to temperature sensitivity of the URM1 pathway in S. cerevisiae and S. bayanus. Furthermore, our analysis of the tRNA modification pattern in selected yeast species revealed two alternative phenotypes. Most strains moderately increase their tRNA modification levels in response to heat, possibly constituting a common adaptation to high temperatures. However, an overall reduction of nucleoside modifications was observed exclusively in S288C. This surprising finding emphasizes the importance of studies that utilize the power of evolutionary biology, and highlights the need for future systematic studies on tRNA modifications in additional model organisms.
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Procesamiento Postranscripcional del ARN , ARN de Hongos/genética , ARN de Transferencia/genética , Saccharomyces cerevisiae/genética , Evolución Molecular , Filogenia , ARN de Hongos/biosíntesis , ARN de Transferencia/biosíntesis , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae , Compuestos de Sulfhidrilo/metabolismo , Temperatura , Transcripción GenéticaRESUMEN
Chemical RNA modifications are present in all kingdoms of life and many of these post-transcriptional modifications are conserved throughout evolution. However, most of the research has been performed on single cell organisms, whereas little is known about how RNA modifications contribute to the development of metazoans. In recent years, the identification of RNA modification genes in genome wide association studies (GWAS) has sparked new interest in previously neglected genes. In this review, we summarize recent findings that connect RNA modification defects and phenotypes in higher eukaryotes. Furthermore, we discuss the implications of aberrant tRNA modification in various human diseases including metabolic defects, mitochondrial dysfunctions, neurological disorders, and cancer. As the molecular mechanisms of these diseases are being elucidated, we will gain first insights into the functions of RNA modifications in higher eukaryotes and finally understand their roles during development.
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Procesamiento Postranscripcional del ARN , ARN de Transferencia/metabolismo , ARN/genética , ARN/metabolismo , ARNt Metiltransferasas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Disautonomía Familiar/genética , Disautonomía Familiar/metabolismo , Disautonomía Familiar/patología , Epilepsia Rolándica/genética , Epilepsia Rolándica/metabolismo , Epilepsia Rolándica/patología , Estudio de Asociación del Genoma Completo , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Conformación de Ácido Nucleico , Fenotipo , ARN Mitocondrial , ARN de Transferencia/genética , ARNt Metiltransferasas/genéticaRESUMEN
Recent advances in the field of RNA interference and new cost-effective approaches for large-scale double-stranded RNA (dsRNA) synthesis have fuelled the demand for robust high-performance purification techniques suitable for dsRNA molecules of various lengths. To address this issue, we developed an improved dsRNA purification method based on anion exchange chromatography utilizing convective interaction media (CIM) monolithic columns. To evaluate column performance we synthesized a selection of dsRNA molecules (58-1810 bp) in a one-step enzymatic reaction involving bacteriophage T7 DNA-dependent RNA polymerase and phi6 RNA-dependent RNA polymerase. In addition, small interfering RNAs (siRNAs) of 25-27 bp were generated by Dicer digestion of the genomic dsRNA of bacteriophage phi6. We demonstrated that linearly scalable CIM monolithic quaternary amine (QA) columns can be used as a fast and superior alternative to standard purification methods (e.g. LiCl precipitation) to obtain highly pure dsRNA preparations. The impurities following Dicer treatment were quickly and efficiently removed with the QA CIM monolithic column, yielding siRNA molecules of high purity suitable for potential therapeutic applications. Moreover, baseline separation of dsRNA molecules up to 1 kb in non-denaturing conditions was achieved.
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Resinas de Intercambio Aniónico , Cromatografía por Intercambio Iónico/métodos , ARN Bicatenario/aislamiento & purificaciónRESUMEN
Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae, infects young salmon, with a severe impact on the commercial sea farming industry. Of the five mature proteins encoded by the IPNV genome, the multifunctional VP3 has an essential role in morphogenesis; interacting with the capsid protein VP2, the viral double-stranded RNA (dsRNA) genome and the RNA-dependent RNA polymerase VP1. Here we investigate one of these VP3 functions and present the crystal structure of the C-terminal 12 residues of VP3 bound to the VP1 polymerase. This interaction, visualized for the first time, reveals the precise molecular determinants used by VP3 to bind the polymerase. Competition binding studies confirm that this region of VP3 is necessary and sufficient for VP1 binding, while biochemical experiments show that VP3 attachment has no effect on polymerase activity. These results indicate how VP3 recruits the polymerase into birnavirus capsids during morphogenesis.
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Virus de la Necrosis Pancreática Infecciosa/química , ARN Polimerasa Dependiente del ARN/química , Proteínas Estructurales Virales/química , Cristalografía por Rayos X , Unión Proteica , Conformación Proteica , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Estructurales Virales/metabolismoRESUMEN
The activation of the interferon (IFN) system, which is triggered largely by the recognition of viral nucleic acids, is one of the most important host defense reactions against viral infections. Although influenza A and B viruses, which both have segmented negative-strand RNA genomes, share major structural similarities, they have evolutionarily diverged, with total genetic incompatibility. Here we compare antiviral-inducing mechanisms during infections with type A and B influenza viruses in human dendritic cells. We observed that IFN responses are induced significantly faster in cells infected with influenza B virus than in cells infected with type A influenza virus and that the early induction of antiviral gene expression is mediated by the activation of the transcription factor IFN regulatory factor 3 (IRF3). We further demonstrate that influenza A virus infection activates IFN responses only after viral RNA (vRNA) synthesis, whereas influenza B virus induces IFN responses even if its infectivity is destroyed by UV treatment. Thus, initial viral transcription, replication, and viral protein synthesis are dispensable for influenza B virus-induced antiviral responses. Moreover, vRNA molecules from both type A and B viruses are equally potent activators of IFN induction, but incoming influenza B virus structures are recognized directly in the cytosol, while influenza A virus is able to evade early recognition. Collectively, our data provide new evidence of a novel antiviral evasion strategy for influenza A virus without a contribution of the viral NS1 protein, and this opens up new insights into different influenza virus pathogenicities.
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Células Dendríticas/virología , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Virus de la Influenza B/inmunología , Virus de la Influenza B/patogenicidad , Factor 3 Regulador del Interferón/metabolismo , Interferones/biosíntesis , Animales , Línea Celular , Células Dendríticas/inmunología , Perros , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Evasión Inmune , Gripe Humana/inmunología , Interferones/inmunología , Células de Riñón Canino Madin Darby , ARN Viral/biosíntesis , Internalización del VirusRESUMEN
Double-stranded RNA viruses encode a single protein species containing RNA-dependent RNA polymerase (RdRP) motifs. This protein is responsible for RNA transcription and replication. The architecture of viral RdRPs resembles that of a cupped right hand with fingers, palm and thumb domains. Those using de novo initiation have a flexible structural elaboration that constitutes the priming platform. Here we investigate the properties of the C-terminal priming domain of bacteriophage Ï6 to get insights into the role of an extended loop connecting this domain to the main body of the polymerase. Proteolyzed Ï6 RdRP that possesses a nick in the hinge region of this loop was better suited for de novo initiation. The clipped C-terminus remained associated with the main body of the polymerase via the anchor helix. The structurally flexible hinge region appeared to be involved in the control of priming platform movement. Moreover, we detected abortive initiation products for a bacteriophage RdRP.
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Bacteriófago phi 6/química , Bacteriófago phi 6/enzimología , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Transcripción Genética , Replicación ViralRESUMEN
Enveloped double-stranded RNA (dsRNA) bacterial virus Pseudomonas phage Ï6 has been developed into an advanced assembly system where purified virion proteins and genome segments self-assemble into infectious viral particles, inferring the assembly pathway. The most intriguing step is the membrane assembly occurring inside the bacterial cell. Here, we demonstrate that the middle virion shell, made of protein 8, associates with the expanded viral core particle and the virus-specific membrane vesicle.
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Bacteriófago phi 6/fisiología , Proteínas de la Cápside/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Metabolismo de los Lípidos , Unión Proteica , Ensamble de VirusRESUMEN
The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV) is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its apo form and at 2.2 Å resolution bound to the catalytically-activating metal magnesium. We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxy)nucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase) and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA:polymerase complexes, providing a mechanism for the genesis of viral genome:polymerase complexes observed in vivo.
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ARN Polimerasas Dirigidas por ADN/química , Genoma Viral , Virus de la Necrosis Pancreática Infecciosa/enzimología , Dominio Catalítico , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/metabolismo , Magnesio , Unión Proteica , Conformación Proteica , ARN Viral/biosíntesis , Transcripción GenéticaRESUMEN
Recognition of viral genetic material takes place via several different receptor systems, such as retinoic acid-inducible gene I-like receptors and TLRs 3, 7, 8, and 9. At present, systematic comparison of the ability of different types of RNAs to induce innate immune responses in human immune cells has been limited. In this study, we generated bacteriophage 6 and influenza A virus-specific ssRNA and dsRNA molecules ranging from 58 to 2956 nt. In human monocyte-derived dendritic cells (moDCs), short dsRNAs efficiently upregulated the expression of IFN (IFN-α, IFN-ß, and IFN-λ1) and proinflammatory (TNF-α, IL-6, IL-12, and CXCL10) cytokine genes. These genes were also induced by ssRNA molecules, but size-specific differences were not as pronounced as with dsRNA molecules. Dephosphorylation of short ssRNA and dsRNA molecules led to a dramatic reduction in their ability to stimulate innate immune responses. Such a difference was not detected for long ssRNAs. RNA-induced cytokine responses correlated well with IFN regulatory factor 3 phosphorylation, suggesting that IFN regulatory factor 3 plays a major role in both ssRNA- and dsRNA-activated responses in human moDCs. We also found that IFN gene expression was efficiently stimulated following recognition of short dsRNAs by retinoic acid-inducible gene I and TLR3 in human embryonic kidney 293 cells, whereas ssRNA-induced responses were less dependent on the size of the RNA molecule. Our data suggest that human moDCs are extremely sensitive in recognizing foreign RNA, and the responses depend on RNA size, form (ssRNA versus dsRNA), and the level of 5' phosphorylation.
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Bacteriófago phi 6/inmunología , Células Dendríticas/inmunología , Inmunidad Innata/inmunología , Virus de la Influenza A/inmunología , Monocitos/inmunología , ARN Bicatenario/inmunología , ARN Viral/inmunología , Citocinas/inmunología , Células HEK293 , Humanos , Inmunidad Innata/efectos de los fármacos , Factor 3 Regulador del Interferón/inmunología , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , ARN Bicatenario/farmacología , ARN Viral/farmacología , Receptores Toll-Like/inmunología , Transactivadores , Factores de Transcripción/inmunología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3' terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage phi 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi 6 RdRP can be greatly enhanced.