RESUMEN
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.
Asunto(s)
Antibacterianos , Inhibidores Enzimáticos , Escherichia coli , Exorribonucleasas , Antibacterianos/farmacología , Antibacterianos/química , Exorribonucleasas/antagonistas & inhibidores , Exorribonucleasas/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Dominio Catalítico , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Bacterias/efectos de los fármacos , Bacterias/enzimologíaRESUMEN
Mitochondrial RNA (mtRNA) in the cytosol can trigger the innate immune sensor MDA5, and autoinflammatory disease due to type I IFN. Here, we show that a dominant negative mutation in the gene encoding the mitochondrial exonuclease REXO2 may cause interferonopathy by triggering the MDA5 pathway. A patient characterized by this heterozygous de novo mutation (p.T132A) presented with persistent skin rash featuring hyperkeratosis, parakeratosis and acanthosis, with infiltration of lymphocytes and eosinophils around small blood vessels. In addition, circulating IgE levels and inflammatory cytokines, including IFNα, are found consistently elevated. Transcriptional analysis highlights a type I IFN gene signature in PBMC. Mechanistically, REXO2 (T132A) lacks the ability to cleave RNA and inhibits the activity of wild-type REXO2. This leads to an accumulation of mitochondrial dsRNA in the cytosol, which is recognized by MDA5, leading to the associated type I IFN gene signature. These results demonstrate that in the absence of appropriate regulation by REXO2, aberrant cellular nucleic acids may accumulate and continuously trigger innate sensors, resulting in an inborn error of immunity.
Asunto(s)
Heterocigoto , Interferón Tipo I , Helicasa Inducida por Interferón IFIH1 , Humanos , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferón Tipo I/metabolismo , Interferón Tipo I/genética , Mutación , Masculino , Mitocondrias/metabolismo , Mitocondrias/genética , Femenino , Inmunidad Innata/genética , Exonucleasas/metabolismo , Exonucleasas/genética , Células HEK293 , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Citosol/metabolismo , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Genes DominantesRESUMEN
The MUT-7 family of 3'-5' exoribonucleases is evolutionarily conserved across the animal kingdom and plays essential roles in small RNA production in the germline. Most MUT-7 homologues carry a C-terminal domain of unknown function named MUT7-C appended to the exoribonuclease domain. Our analysis shows that the MUT7-C is evolutionary ancient, as a minimal version of the domain exists as an individual protein in prokaryotes. In animals, MUT7-C has acquired an insertion that diverged during evolution, expanding its functions. Caenorhabditis elegans MUT-7 contains a specific insertion within MUT7-C, which allows binding to MUT-8 and, consequently, MUT-7 recruitment to germ granules. In addition, in C. elegans and human MUT-7, the MUT7-C domain contributes to RNA binding and is thereby crucial for ribonuclease activity. This RNA-binding function most likely represents the ancestral function of the MUT7-C domain. Overall, this study sheds light on MUT7-C and assigns two functions to this previously uncharacterized domain.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Exorribonucleasas , Dominios Proteicos , Animales , Exorribonucleasas/metabolismo , Exorribonucleasas/química , Exorribonucleasas/genética , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Humanos , Evolución Molecular , ARN/metabolismo , ARN/química , Secuencia de Aminoácidos , Unión ProteicaRESUMEN
The escape of mitochondrial double-stranded dsRNA (mt-dsRNA) into the cytosol has been recently linked to a number of inflammatory diseases. Here, we report that the release of mt-dsRNA into the cytosol is a general feature of senescent cells and a critical driver of their inflammatory secretome, known as senescence-associated secretory phenotype (SASP). Inhibition of the mitochondrial RNA polymerase, the dsRNA sensors RIGI and MDA5, or the master inflammatory signaling protein MAVS, all result in reduced expression of the SASP, while broadly preserving other hallmarks of senescence. Moreover, senescent cells are hypersensitized to mt-dsRNA-driven inflammation due to their reduced levels of PNPT1 and ADAR1, two proteins critical for mitigating the accumulation of mt-dsRNA and the inflammatory potency of dsRNA, respectively. We find that mitofusin MFN1, but not MFN2, is important for the activation of the mt-dsRNA/MAVS/SASP axis and, accordingly, genetic or pharmacologic MFN1 inhibition attenuates the SASP. Finally, we report that senescent cells within fibrotic and aged tissues present dsRNA foci, and inhibition of mitochondrial RNA polymerase reduces systemic inflammation associated to senescence. In conclusion, we uncover the mt-dsRNA/MAVS/MFN1 axis as a key driver of the SASP and we identify novel therapeutic strategies for senescence-associated diseases.
Asunto(s)
Senescencia Celular , Citosol , Inflamación , Mitocondrias , ARN Bicatenario , ARN Bicatenario/metabolismo , Humanos , Citosol/metabolismo , Mitocondrias/metabolismo , Inflamación/metabolismo , Inflamación/patología , Inflamación/genética , Animales , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Fenotipo Secretor Asociado a la Senescencia , Helicasa Inducida por Interferón IFIH1/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ratones , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Transducción de SeñalRESUMEN
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.
Asunto(s)
Adaptación Fisiológica , Exorribonucleasas , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
The hibernation-promoting factor (Hpf) in Staphylococcus aureus binds to 70S ribosomes and induces the formation of the 100S complex (70S dimer), leading to translational avoidance and occlusion of ribosomes from RNase R-mediated degradation. Here, we show that the 3'-5' exoribonuclease YhaM plays a previously unrecognized role in modulating ribosome stability. Unlike RNase R, which directly degrades the 16S rRNA of ribosomes in S. aureus cells lacking Hpf, YhaM destabilizes ribosomes by indirectly degrading the 3'-hpf mRNA that carries an intrinsic terminator. YhaM adopts an active hexameric assembly and robustly cleaves ssRNA in a manganese-dependent manner. In vivo, YhaM appears to be a low-processive enzyme, trimming the hpf mRNA by only 1 nucleotide. Deletion of yhaM delays cell growth. These findings substantiate the physiological significance of this cryptic enzyme and the protective role of Hpf in ribosome integrity, providing a mechanistic understanding of bacterial ribosome turnover.
Asunto(s)
Proteínas Bacterianas , Exorribonucleasas , ARN Mensajero , Proteínas Ribosómicas , Ribosomas , Staphylococcus aureus , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Ribosomas/metabolismo , Ribosomas/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Staphylococcus aureus/genética , Staphylococcus aureus/enzimología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Estabilidad del ARN/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismoRESUMEN
Many viruses have evolved structured RNA elements that can influence transcript abundance and translational efficiency, and help evade host immune factors by hijacking cellular machinery during replication. Here, we evaluated the functional impact of sub-genomic flaviviral RNAs (sfRNAs) known to stall exoribonuclease activity, by incorporating these elements into recombinant adeno-associated viral (AAV) genome cassettes. Specifically, sfRNAs from Dengue, Zika, Japanese Encephalitis, Yellow Fever, Murray Valley Encephalitis, and West Nile viruses increased transcript stability and transgene expression compared to a conventional woodchuck hepatitis virus element (WPRE). Further dissection of engineered transcripts revealed that sfRNA elements (i) require incorporation in cis within the 3' untranslated region (UTR) of AAV genomes, (ii) require minimal dumbbell structures to exert the observed effects, and (iii) can stabilize AAV transcripts independent of 5'-3' exoribonuclease 1 (XRN1)-mediated decay. Additionally, preliminary in vivo assessment of AAV vectors bearing sfRNA elements in mice achieved increased transcript abundance and expression in cardiac tissue. Leveraging the functional versatility of engineered viral RNA elements may help improve the potency of AAV vector-based gene therapies. IMPORTANCE: Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer.
Asunto(s)
Dependovirus , Vectores Genéticos , ARN Viral , Dependovirus/genética , Animales , ARN Viral/genética , ARN Viral/metabolismo , Vectores Genéticos/genética , Ratones , Humanos , Estabilidad del ARN , Flaviviridae/genética , Transgenes , Células HEK293 , Genoma Viral , Regiones no Traducidas 3'/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genéticaRESUMEN
In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.
Asunto(s)
Citosol , Mitocondrias , Prohibitinas , ARN Bicatenario , ARN Mitocondrial , Humanos , Citosol/metabolismo , Mitocondrias/metabolismo , ARN Bicatenario/metabolismo , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , Línea Celular Tumoral , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Transporte de ARN , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas MitocondrialesRESUMEN
Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the mechanism determining the switch between continuous transcription and premature termination to generate RNA primers for mitochondrial DNA (mtDNA) replication remains unclear. The pentatricopeptide repeat domain of mtRNAP exhibits exoribonuclease activity, which is required for the initiation of mtDNA replication in Drosophila. In this review, we explain how this exonuclease activity contributes to primer synthesis in strand-coupled mtDNA replication, and discuss how its regulation might co-ordinate mtDNA replication and transcription in both Drosophila and mammals.
Asunto(s)
Replicación del ADN , ADN Mitocondrial , Mitocondrias , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Animales , Mitocondrias/metabolismo , Mitocondrias/genética , Humanos , ARN Polimerasas Dirigidas por ADN/metabolismo , Transcripción Genética , Drosophila/genética , Drosophila/metabolismo , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMEN
To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of 14-3-3σ (Stratifin) was analyzed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of PTEN-mediated AKT regulation (K14.creP/Δ5PTENflx/flx). Consistent with 14-3-3σ roles in epidermal differentiation, HK1.ras hyperplasia and papillomas displayed elevated 14-3-3σ expression in supra-basal keratinocytes, paralleled by supra-basal p-MDM2166 activation and sporadic p-AKT473 expression. In bi-genic HK1.fos/Δ5PTENflx/flx hyperplasia, basal-layer 14-3-3σ expression appeared, and alongside p53/p21, was associated with keratinocyte differentiation and keratoacanthoma etiology. Tri-genic HK1.ras/fos-Δ5PTENflx/flx hyperplasia/papillomas initially displayed increased basal-layer 14-3-3σ, suggesting attempts to maintain supra-basal p-MDM2166 and protect basal-layer p53. However, HK1.ras/fos-Δ5PTENflx/flx papillomas exhibited increasing basal-layer p-MDM2166 activation that reduced p53, which coincided with malignant conversion. Despite p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCCs) and alongside elevated p21, limited malignant progression via inhibiting p-AKT1473 expression; until 14-3-3σ/p21 loss facilitated progression to aggressive SCC exhibiting uniform p-AKT1473. Analysis of TPA-promoted HK1.ras-Δ5PTENflx/flx mouse skin, demonstrated early loss of 14-3-3σ/p53/p21 in hyperplasia and papillomas, with increased p-MDM2166/p-AKT1473 that resulted in rapid malignant conversion and progression to poorly differentiated SCC. In 2D/3D cultures, membranous 14-3-3σ expression observed in normal HaCaT and SP1ras61 papilloma keratinocytes was unexpectedly detected in malignant T52ras61/v-fos SCC cells cultured in monolayers, but not invasive 3D-cells. Collectively, these data suggest 14-3-3σ/Stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms; while persistent p53-independent expression in early wdSCC may involve p21-mediated AKT1 inhibition to limit malignant progression.
Asunto(s)
Proteínas 14-3-3 , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-mdm2 , Neoplasias Cutáneas , Proteína p53 Supresora de Tumor , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Ratones , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Carcinogénesis/metabolismo , Carcinogénesis/genética , Carcinogénesis/patología , Progresión de la Enfermedad , Humanos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Queratinocitos/metabolismo , Queratinocitos/patología , Regulación Neoplásica de la Expresión GénicaRESUMEN
This study explored the role of 14-3-3σ in carbon ion-irradiated pancreatic adenocarcinoma (PAAD) cells and xenografts and clarified the underlying mechanism. The clinical significance of 14-3-3σ in patients with PAAD was explored using publicly available databases. 14-3-3σ was silenced or overexpressed and combined with carbon ions to measure cell proliferation, cell cycle, and DNA damage repair. Immunoblotting and immunofluorescence (IF) assays were used to determine the underlying mechanisms of 14-3-3σ toward carbon ion radioresistance. We used the BALB/c mice to evaluate the biological behavior of 14-3-3σ in combination with carbon ions. Bioinformatic analysis revealed that PAAD expressed higher 14-3-3σ than normal pancreatic tissues; its overexpression was related to invasive clinicopathological features and a worse prognosis. Knockdown or overexpression of 14-3-3σ demonstrated that 14-3-3σ promoted the survival of PAAD cells after carbon ion irradiation. And 14-3-3σ was upregulated in PAAD cells during DNA damage (carbon ion irradiation, DNA damaging agent) and promotes cell recovery. We found that 14-3-3σ resulted in carbon ion radioresistance by promoting RPA2 and RAD51 accumulation in the nucleus in PAAD cells, thereby increasing homologous recombination repair (HRR) efficiency. Blocking the HR pathway consistently reduced 14-3-3σ overexpression-induced carbon ion radioresistance in PAAD cells. The enhanced radiosensitivity of 14-3-3σ depletion on carbon ion irradiation was also demonstrated in vivo. Altogether, 14-3-3σ functions in tumor progression and can be a potential target for developing biomarkers and treatment strategies for PAAD along with incorporating carbon ion irradiation.
Asunto(s)
Proteínas 14-3-3 , Ratones Endogámicos BALB C , Neoplasias Pancreáticas , Reparación del ADN por Recombinación , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/radioterapia , Animales , Humanos , Ratones , Línea Celular Tumoral , Regulación hacia Abajo , Tolerancia a Radiación/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Radioterapia de Iones Pesados , Carbono , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Masculino , Daño del ADN , FemeninoRESUMEN
The yeast Saccharomyces cerevisiae and most eukaryotes carry two 5' â 3' exoribonuclease paralogs. In yeast, they are called Xrn1, which shuttles between the nucleus and the cytoplasm, and executes major cytoplasmic messenger RNA (mRNA) decay, and Rat1, which carries a strong nuclear localization sequence (NLS) and localizes to the nucleus. Xrn1 is 30% identical to Rat1 but has an extra ~500 amino acids C-terminal extension. In the cytoplasm, Xrn1 can degrade decapped mRNAs during the last round of translation by ribosomes, a process referred to as "cotranslational mRNA decay." The division of labor between the two enzymes is still enigmatic and serves as a paradigm for the subfunctionalization of many other paralogs. Here we show that Rat1 is capable of functioning in cytoplasmic mRNA decay, provided that Rat1 remains cytoplasmic due to its NLS disruption (cRat1). This indicates that the physical segregation of the two paralogs plays roles in their specific functions. However, reversing segregation is not sufficient to fully complement the Xrn1 function. Specifically, cRat1 can partially restore the cell volume, mRNA stability, the proliferation rate, and 5' â 3' decay alterations that characterize xrn1Δ cells. Nevertheless, cotranslational decay is only slightly complemented by cRat1. The use of the AlphaFold prediction for cRat1 and its subsequent docking with the ribosome complex and the sequence conservation between cRat1 and Xrn1 suggest that the tight interaction with the ribosome observed for Xrn1 is not maintained in cRat1. Adding the Xrn1 C-terminal domain to Rat1 does not improve phenotypes, which indicates that lack of the C-terminal is not responsible for partial complementation. Overall, during evolution, it appears that the two paralogs have acquired specific characteristics to make functional partitioning beneficial.
Asunto(s)
Exorribonucleasas , Estabilidad del ARN , ARN Mensajero , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Citoplasma/metabolismo , Biosíntesis de ProteínasRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome encodes 29 proteins including four structural, 16 nonstructural (nsps), and nine accessory proteins (https://epimedlab.org/sars-cov-2-proteome/). Many of these proteins contain potential targetable sites for the development of antivirals. Despite the widespread use of vaccinations, the emergence of variants necessitates the investigation of new therapeutics and antivirals. Here, the EpiMed Coronabank Chemical Collection (https://epimedlab.org/crl/) was utilized to investigate potential antivirals against the nsp14 exoribonuclease (ExoN) domain. Molecular docking was performed to evaluate the binding characteristics of our chemical library against the nsp14 ExoN site. Based on the initial screen, trisjuglone, ararobinol, corilagin, and naphthofluorescein were identified as potential lead compounds. Molecular dynamics (MD) simulations were subsequently performed, with the results highlighting the stability of the lead compounds in the nsp14 ExoN site. Protein-RNA docking revealed the potential for the lead compounds to disrupt the interaction with RNA when bound to the ExoN site. Moreover, hypericin, cyanidin-3-O-glucoside, and rutin were previously identified as lead compounds targeting the papain-like protease (PLpro) naphthalene binding site. Through performing MD simulations, the stability and interactions of lead compounds with PLpro were further examined. Overall, given the critical role of the exonuclease activity of nsp14 in ensuring viral fidelity and the multifunctional role of PLpro in viral pathobiology and replication, these nsps represent important targets for antiviral drug development. Our databases can be utilized for in silico studies, such as the ones performed here, and this approach can be applied to other potentially druggable SARS-CoV-2 protein targets.
Asunto(s)
Antivirales , Simulación del Acoplamiento Molecular , SARS-CoV-2 , Antivirales/química , Antivirales/farmacología , Sitios de Unión , SARS-CoV-2/efectos de los fármacos , Humanos , Exorribonucleasas/metabolismo , Exorribonucleasas/química , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Naftalenos/química , Naftalenos/farmacología , Unión Proteica , Tratamiento Farmacológico de COVID-19 , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Simulación de Dinámica Molecular , Dominios ProteicosRESUMEN
We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC50 values between 100 nM and 1 µM in cells, which align with the EC50 values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.
Asunto(s)
Proteínas 14-3-3 , Unión Proteica , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Humanos , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genéticaRESUMEN
Until recently, the general 5'-3' mRNA decay was placed in the cytosol after the mRNA was released from ribosomes. However, the discovery of an additional 5' to 3' pathway, the Co-Translational mRNA Decay (CTRD), changed this paradigm. Up to date, defining the real contribution of CTRD in the general mRNA turnover has been hardly possible as the enzyme involved in this pathway is also involved in cytosolic decay. Here we overcame this obstacle and created an Arabidopsis line specifically impaired for CTRD called XRN4ΔCTRD. Through a genome-wide analysis of mRNA decay rate in shoot and root, we tested the importance of CTRD in mRNA turnover. First, we observed that mRNAs tend to be more stable in root than in shoot. Next, using XRN4ΔCTRD line, we demonstrated that CTRD is a major determinant in mRNA turnover. In shoot, the absence of CTRD leads to the stabilization of thousands of transcripts while in root its absence is highly compensated resulting in faster decay rates. We demonstrated that this faster decay rate is partially due to the XRN4-dependent cytosolic decay. Finally, we correlated this organ-specific effect with XRN4ΔCTRD line phenotypes revealing a crucial role of CTRD in mRNA homeostasis and proper organ development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas , Brotes de la Planta , Estabilidad del ARN , ARN Mensajero , Arabidopsis/genética , Arabidopsis/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/genética , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brotes de la Planta/metabolismo , Brotes de la Planta/genética , Genoma de Planta , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Biosíntesis de Proteínas , ARN de Planta/metabolismo , ARN de Planta/genética , Citosol/metabolismoRESUMEN
RNA-binding proteins (RBPs) regulate key aspects of RNA processing including alternative splicing, mRNA degradation and localization by physically binding RNA molecules. Current methods to map these interactions, such as CLIP, rely on purifying single proteins at a time. Our new method, ePRINT, maps RBP-RNA interaction networks on a global scale without purifying individual RBPs. ePRINT uses exoribonuclease XRN1 to precisely map the 5' end of the RBP binding site and uncovers direct and indirect targets of an RBP of interest. Importantly, ePRINT can also uncover RBPs that are differentially activated between cell fate transitions, including neural progenitor differentiation into neurons.
Asunto(s)
Proteínas de Unión al ARN , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , Exorribonucleasas/metabolismo , Humanos , ARN/metabolismo , Animales , Unión ProteicaRESUMEN
Therapeutic fluoropyrimidines 5-fluorouracil (5-FU) and 5-fluorocytosine (5-FC) are in long use for treatment of human cancers and severe invasive fungal infections, respectively. 5-Fluorouridine triphosphate represents a bioactive metabolite of both drugs and is incorporated into target cells' RNA. Here we use the model fungus Saccharomyces cerevisiae to define fluorinated tRNA as a key mediator of 5-FU and 5-FC cytotoxicity when specific tRNA methylations are absent. tRNA methylation deficiency caused by loss of Trm4 and Trm8 was previously shown to trigger an RNA quality control mechanism resulting in partial destabilization of hypomodified tRNAValAAC. We demonstrate that, following incorporation into tRNA, fluoropyrimidines strongly enhance degradation of yeast tRNAValAAC lacking Trm4 and Trm8 dependent methylations. At elevated temperature, such effect occurs already in absence of Trm8 alone. Genetic approaches and quantification of tRNA modification levels reveal that enhanced fluoropyrimidine cytotoxicity results from additional, drug induced uridine modification loss and activation of tRNAValAAC decay involving the exonuclease Xrn1. These results suggest that inhibition of tRNA methylation may be exploited to boost therapeutic efficiency of 5-FU and 5-FC.
Asunto(s)
Flucitosina , Fluorouracilo , ARN de Transferencia , Saccharomyces cerevisiae , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Flucitosina/farmacología , Fluorouracilo/farmacología , Metilación , Estabilidad del ARN/efectos de los fármacos , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , ARNt Metiltransferasas/metabolismo , ARNt Metiltransferasas/genética , Uridina/metabolismoRESUMEN
Recent progress in the investigation of microRNA (miRNA) biogenesis and the miRNA processing machinery has revealed previously unknown roles of posttranscriptional regulation in gene expression. The molecular mechanistic interplay between miRNAs and their regulatory factors, RNA-binding proteins (RBPs) and exoribonucleases, has been revealed to play a critical role in tumorigenesis. Moreover, recent studies have shown that the proliferation of hepatocellular carcinoma (HCC)-causing hepatitis C virus (HCV) is also characterized by close crosstalk of a multitude of host RBPs and exoribonucleases with miR-122 and its RNA genome, suggesting the importance of the mechanistic interplay among these factors during the proliferation of HCV. This review primarily aims to comprehensively describe the well-established roles and discuss the recently discovered understanding of miRNA regulators, RBPs and exoribonucleases, in relation to various cancers and the proliferation of a representative cancer-causing RNA virus, HCV. These have also opened the door to the emerging potential for treating cancers as well as HCV infection by targeting miRNAs or their respective cellular modulators.
Asunto(s)
Exorribonucleasas , Regulación Neoplásica de la Expresión Génica , Hepacivirus , MicroARNs , Neoplasias , Proteínas de Unión al ARN , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Neoplasias/genética , Neoplasias/metabolismo , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Animales , Hepacivirus/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Hepatitis C/metabolismo , Hepatitis C/genética , Hepatitis C/virologíaRESUMEN
N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.
Asunto(s)
Adenosina , Adenosina/análogos & derivados , ARN Helicasas DEAD-box , Exorribonucleasas , Inestabilidad Genómica , Metiltransferasas , Estructuras R-Loop , ARN Polimerasa II , Terminación de la Transcripción Genética , Humanos , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Adenosina/metabolismo , Adenosina/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Células HEK293 , Cromatina/metabolismo , Cromatina/genética , Daño del ADN , Células HeLa , ARN/metabolismo , ARN/genética , Transcripción Genética , Metilación de ARNRESUMEN
BACKGROUND: Xrn1 exoribonuclease is the major mRNA degradation enzyme in Saccharomyces cerevisiae. In exponentially growing cells, Xrn1 is localised in the yeast cells and directs the degradation of mRNA molecules. Xrn1 is gradually deposited and presumably inactivated in the processing bodies (P-bodies) as the yeast population ages. Xrn1 can also localise to the membrane compartment of the arginine permease Can1/eisosome compartment at the yeast plasma membrane. This localisation correlates with the metabolic (diauxic) shift from glucose fermentation to respiration, although the relevance of this Xrn1 localisation remains unknown. METHODS: We monitored the growth rates and morphology of Xrn1-green fluorescent protein (GFP) cells compared to wild-type and Δxrn1 cells and observed the Xrn1-GFP localisation pattern in different media types for up to 72 hours using fluorescence microscopy. RESULTS: We present the dynamic changes in the localisation of Xrn1 as a versatile tool for monitoring the growth of yeast populations at the single-cell level using fluorescence microscopy. CONCLUSIONS: The dynamic changes in the localisation of Xrn1 can be a versatile tool for monitoring the growth of yeast populations at the single-cell level. Simultaneously, Xrn1 localisation outside of P-bodies in post-diauxic cells supports its storage and cytoprotective function, yet the role of P-bodies in cell metabolism has still not yet been entirely elucidated.