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Spermiogenesis is a highly orchestrated developmental process during which chromatin condensation decouples transcription from translation. Spermiogenic mRNAs are transcribed earlier and stored in a translationally inert state until needed for translation; however, it remains largely unclear how such repressed mRNAs become activated during spermiogenesis. We previously reported that the MIWI/piRNA machinery is responsible for mRNA elimination during late spermiogenesis in preparation for spermatozoa production. Here we unexpectedly discover that the same machinery is also responsible for activating translation of a subset of spermiogenic mRNAs to coordinate with morphological transformation into spermatozoa. Such action requires specific base-pairing interactions of piRNAs with target mRNAs in their 3' UTRs, which activates translation through coupling with cis-acting AU-rich elements to nucleate the formation of a MIWI/piRNA/eIF3f/HuR super-complex in a developmental stage-specific manner. These findings reveal a critical role of the piRNA system in translation activation, which we show is functionally required for spermatid development.
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Proteínas Argonautas/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN Interferente Pequeño/metabolismo , Espermatogénesis , Regiones no Traducidas 3' , Animales , Proteínas Argonautas/genética , Emparejamiento Base , Células Cultivadas , Proteína 1 Similar a ELAV/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
In a recent publication in Cell, Kowalski et al.1 developed an interdisciplinary and multiplexed approach to uncover regulatory modules of alternative polyadenylation, involving single-cell-based gene perturbation, isoform abundance analysis, machine learning of RNA motifs, and massively parallel reporter assays.
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Poliadenilación , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Aprendizaje Automático , Análisis de la Célula Individual , AnimalesRESUMEN
Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3' termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation.
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Regiones no Traducidas 3' , Poliadenilación , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Transporte Activo de Núcleo Celular , Exones , Regulación de la Expresión Génica , Humanos , Precursores del ARN/genética , Estabilidad del ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismoRESUMEN
Dysregulation of the DNA/RNA-binding protein FUS causes certain subtypes of ALS/FTD by largely unknown mechanisms. Recent evidence has shown that FUS toxic gain of function due either to mutations or to increased expression can disrupt critical cellular processes, including mitochondrial functions. Here, we demonstrate that in human cells overexpressing wild-type FUS or expressing mutant derivatives, the protein associates with multiple mRNAs, and these are enriched in mRNAs encoding mitochondrial respiratory chain components. Notably, this sequestration leads to reduced levels of the encoded proteins, which is sufficient to bring about disorganized mitochondrial networks, reduced aerobic respiration and increased reactive oxygen species. We further show that mutant FUS associates with mitochondria and with mRNAs encoded by the mitochondrial genome. Importantly, similar results were also observed in fibroblasts derived from ALS patients with FUS mutations. Finally, we demonstrate that FUS loss of function does not underlie the observed mitochondrial dysfunction, and also provides a mechanism for the preferential sequestration of the respiratory chain complex mRNAs by FUS that does not involve sequence-specific binding. Together, our data reveal that respiratory chain complex mRNA sequestration underlies the mitochondrial defects characteristic of ALS/FTD and contributes to the FUS toxic gain of function linked to this disease spectrum.
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Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Regulación de la Expresión Génica/genética , Mitocondrias/patología , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Línea Celular , Respiración de la Célula/genética , Células Cultivadas , Transporte de Electrón/genética , Genoma Mitocondrial , Humanos , Mitocondrias/genética , Mutación , Agregación Patológica de Proteínas/genética , Unión Proteica/genéticaRESUMEN
p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Ácido Mevalónico/metabolismo , Proteína p53 Supresora de Tumor/genética , Línea Celular Tumoral , Femenino , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Redes y Vías Metabólicas/efectos de los fármacos , Mutación , Prenilación , Regiones Promotoras Genéticas , Simvastatina/farmacología , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Alternative polyadenylation (APA) produces mRNA isoforms with different 3' UTR lengths. Previous studies indicated that 3' end processing and mRNA export are intertwined in gene regulation. Here, we show that mRNA export factors generally facilitate usage of distal cleavage and polyadenylation sites (PASs), leading to long 3' UTR isoform expression. By focusing on the export receptor NXF1, which exhibits the most potent effect on APA in this study, we reveal several gene features that impact NXF1-dependent APA, including 3' UTR size, gene size, and AT content. Surprisingly, NXF1 downregulation results in RNA polymerase II (Pol II) accumulation at the 3' end of genes, correlating with its role in APA regulation. Moreover, NXF1 cooperates with CFI-68 to facilitate nuclear export of long 3' UTR isoform with UGUA motifs. Together, our work reveals important roles of NXF1 in coordinating transcriptional dynamics, 3' end processing, and nuclear export of long 3' UTR transcripts, implicating NXF1 as a nexus of gene regulation.
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Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Poliadenilación , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Regiones no Traducidas 3' , Transporte Activo de Núcleo Celular , Sitios de Unión , Núcleo Celular/genética , Células HEK293 , Células HeLa , Humanos , Cinética , Proteínas de Transporte Nucleocitoplasmático/genética , Unión Proteica , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.
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Factores de Transcripción , Zea mays , Zea mays/genética , Zea mays/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Bacterias/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Ceras/metabolismoRESUMEN
Long noncoding RNAs (lncRNAs) and promoter- or enhancer-associated unstable transcripts locate preferentially to chromatin, where some regulate chromatin structure, transcription and RNA processing1-13. Although several RNA sequences responsible for nuclear localization have been identified-such as repeats in the lncRNA Xist and Alu-like elements in long RNAs14-16-how lncRNAs as a class are enriched at chromatin remains unknown. Here we describe a random, mutagenesis-coupled, high-throughput method that we name 'RNA elements for subcellular localization by sequencing' (mutREL-seq). Using this method, we discovered an RNA motif that recognizes the U1 small nuclear ribonucleoprotein (snRNP) and is essential for the localization of reporter RNAs to chromatin. Across the genome, chromatin-bound lncRNAs are enriched with 5' splice sites and depleted of 3' splice sites, and exhibit high levels of U1 snRNA binding compared with cytoplasm-localized messenger RNAs. Acute depletion of U1 snRNA or of the U1 snRNP protein component SNRNP70 markedly reduces the chromatin association of hundreds of lncRNAs and unstable transcripts, without altering the overall transcription rate in cells. In addition, rapid degradation of SNRNP70 reduces the localization of both nascent and polyadenylated lncRNA transcripts to chromatin, and disrupts the nuclear and genome-wide localization of the lncRNA Malat1. Moreover, U1 snRNP interacts with transcriptionally engaged RNA polymerase II. These results show that U1 snRNP acts widely to tether and mobilize lncRNAs to chromatin in a transcription-dependent manner. Our findings have uncovered a previously unknown role of U1 snRNP beyond the processing of precursor mRNA, and provide molecular insight into how lncRNAs are recruited to regulatory sites to carry out chromatin-associated functions.
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Cromatina/genética , Cromatina/metabolismo , ARN Largo no Codificante/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Transcripción Genética , Animales , Línea Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Mutagénesis , Motivos de Nucleótidos , ARN Polimerasa II/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Sitios de Empalme de ARN , ARN Largo no Codificante/genética , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismoRESUMEN
Although it is widely accepted that herpesviruses utilize host RNA polymerase II (RNAPII) to transcribe viral genes, the mechanism of utilization varies significantly among herpesviruses. With the exception of herpes simplex virus 1 (HSV-1) in alpha-herpesviruses, the mechanism by which RNAPII transcribes viral genes in the remaining alpha-herpesviruses has not been reported. In this study, we investigated the transcriptional mechanism of an avian alpha-herpesvirus, Anatid herpesvirus 1 (AnHV-1). We discovered for the first time that hexamethylene-bis-acetamide-inducing protein 1 (HEXIM1), a major inhibitor of positive elongation factor B (P-TEFb), was significantly upregulated during AnHV-1 infection, and its expression was dynamically regulated throughout the progression of the disease. However, the expression level of HEXIM1 remained stable before and after HSV-1 infection. Excessive HEXIM1 assists AnHV-1 in progeny virus production, gene expression, and RNA polymerase II recruitment by promoting the formation of more inactive P-TEFb and the loss of RNAPII S2 phosphorylation. Conversely, the expression of some host survival-related genes, such as SOX8, CDK1, MYC, and ID2, was suppressed by HEXIM1 overexpression. Further investigation revealed that the C-terminus of the AnHV-1 US1 gene is responsible for the upregulation of HEXIM1 by activating its promoter but not by interacting with P-TEFb, which is the mechanism adopted by its homologs, HSV-1 ICP22. Additionally, the virus proliferation deficiency caused by US1 deletion during the early infection stage could be partially rescued by HEXIM1 overexpression, suggesting that HEXIM1 is responsible for AnHV-1 gaining transcription advantages when competing with cells. Taken together, this study revealed a novel HEXIM1-dependent AnHV-1 transcription mechanism, which has not been previously reported in herpesvirus or even DNA virus studies.IMPORTANCEHexamethylene-bis-acetamide-inducing protein 1 (HEXIM1) has been identified as an inhibitor of positive transcriptional elongation factor b associated with cancer, AIDS, myocardial hypertrophy, and inflammation. Surprisingly, no previous reports have explored the role of HEXIM1 in herpesvirus transcription. This study reveals a mechanism distinct from the currently known herpesvirus utilization of RNA polymerase II, highlighting the dependence on high HEXIM1 expression, which may be a previously unrecognized facet of the host shutoff manifested by many DNA viruses. Moreover, this discovery expands the significance of HEXIM1 in pathogen infection. It raises intriguing questions about whether other herpesviruses employ similar mechanisms to manipulate HEXIM1 and if this molecular target can be exploited to limit productive replication. Thus, this discovery not only contributes to our understanding of herpesvirus infection regulation but also holds implications for broader research on other herpesviruses, even DNA viruses.
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Anseriformes , Factor B de Elongación Transcripcional Positiva , Proteínas de Unión al ARN , Factores de Transcripción , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Transcripción Viral , AnimalesRESUMEN
The RNA polymerase II largest subunit C-terminal domain consists of repeated YSPTSPS heptapeptides. The role of tyrosine-1 (Tyr1) remains incompletely understood, as, for example, mutating all Tyr1 residues to Phe (Y1F) is lethal in vertebrates but a related mutant has only a mild phenotype in S. pombe. Here we show that Y1F substitution in budding yeast resulted in a strong slow-growth phenotype. The Y1F strain was also hypersensitive to several different cellular stresses that involve MAP kinase signaling. These phenotypes were all linked to transcriptional changes, and we also identified genetic and biochemical interactions between Tyr1 and both transcription initiation and termination factors. Further studies uncovered defects related to MAP kinase I (Slt2) pathways, and we provide evidence that Slt2 phosphorylates Tyr1 in vitro and in vivo. Our study has thus identified Slt2 as a Tyr1 kinase, and in doing so provided links between stress response activation and Tyr1 phosphorylation.
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Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Estrés Fisiológico , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Genotipo , Complejo Mediador/genética , Complejo Mediador/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Fenotipo , Fosforilación , Dominios Proteicos , ARN Polimerasa II/química , ARN Polimerasa II/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de Tiempo , Transducción Genética , TirosinaRESUMEN
Many long noncoding RNAs (lncRNAs) are unstable and rapidly degraded in the nucleus by the nuclear exosome. An exosome adaptor complex called NEXT (nuclear exosome targeting) functions to facilitate turnover of some of these lncRNAs. Here we show that knockdown of one NEXT subunit, Mtr4, but neither of the other two subunits, resulted in accumulation of two types of lncRNAs: prematurely terminated RNAs (ptRNAs) and upstream antisense RNAs (uaRNAs). This suggested a NEXT-independent Mtr4 function, and, consistent with this, we isolated a distinct complex containing Mtr4 and the zinc finger protein ZFC3H1. Strikingly, knockdown of either protein not only increased pt/uaRNA levels but also led to their accumulation in the cytoplasm. Furthermore, all pt/uaRNAs examined associated with active ribosomes, but, paradoxically, this correlated with a global reduction in heavy polysomes and overall repression of translation. Our findings highlight a critical role for Mtr4/ZFC3H1 in nuclear surveillance of naturally unstable lncRNAs to prevent their accumulation, transport to the cytoplasm, and resultant disruption of protein synthesis.
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Transporte Activo de Núcleo Celular/genética , Citoplasma/metabolismo , Regulación de la Expresión Génica/genética , ARN Helicasas/metabolismo , ARN Nuclear/metabolismo , Factores de Transcripción/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , ARN Helicasas/genética , Estabilidad del ARN , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The disease caused by Riemerella anatipestifer (R. anatipestifer, RA) results in large economic losses to the global duck industry every year. Serovar-related genomic variation, such as the O-antigen and capsular polysaccharide (CPS) gene clusters, has been widely used for serotyping in many gram-negative bacteria. RA has been classified into at least 21 serovars based on slide agglutination, but the molecular basis of serotyping is unknown. In this study, we performed a pan-genome-wide association study (Pan-GWAS) to identify the genetic loci associated with RA serovars. RESULTS: The results revealed a significant association between the putative CPS synthesis gene locus and the serological phenotype. Further characterization of the CPS gene clusters in 11 representative serovar strains indicated that they were highly diverse and serovar-specific. The CPS gene cluster contained the key genes wzx and wzy, which are involved in the Wzx/Wzy-dependent pathway of CPS synthesis. Similar CPS loci have been found in some other species within the family Weeksellaceae. We have also shown that deletion of the wzy gene in RA results in capsular defects and cross-agglutination. CONCLUSIONS: This study indicates that the CPS synthesis gene cluster of R. anatipestifer is a serotype-specific genetic locus. Importantly, our finding provides a new perspective for the systematic analysis of the genetic basis of the R anatipestifer serovars and a potential target for establishing a complete molecular serotyping scheme.
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Enfermedades de las Aves de Corral , Riemerella , Animales , Serogrupo , Estudio de Asociación del Genoma Completo , Riemerella/genética , Patos/genética , Patos/microbiología , Enfermedades de las Aves de Corral/microbiologíaRESUMEN
BACKGROUND & AIMS: The changes of HBV-specific B-cells in chronic hepatitis B (CHB) patients underwent pegylated interferon-alfa (PEG-IFNα) treatment and achieved functional cure remain unclear. We aimed to evaluate the alterations in HBV-specific B-cells during treatment and therefore explored the mechanism of functional recovery of HBsAg-specific B-cells. METHODS: We included 39 nucleos(t)ide analogues-treated CHB patients who received sequential combination therapy with PEG-IFNα and 8 treatment-naive CHB patients. HBV-specific B-cells were characterized ex vivo using fluorescent labeled HBsAg and HBcAg. The frequency, phenotype, and subsets of HBV-specific B-cells and follicular helper T cells (Tfh-cells) were detected using flow cytometry. The functionality of HBV-specific B-cells was quantified through ELISpot assays. RESULTS: During treatment, the fraction of activated memory B-cells (MBCs) among HBsAg-specific B-cells and the expression of IgG, CXCR3, and CD38 increased. Antibody-secretion capacity of HBsAg-specific B-cell was restored after treatment only in patients with a functional cure and it showed a positive correlation with serum hepatitis B surface antibody levels. The phenotype and function of HBsAg-specific B-cells differed between patients with and without functional cure. Patients with functional cure exhibited IgG+ classical MBCs and plasmablasts in HBsAg-specific B-cells. HBcAg-specific B-cells displayed both attenuated antibody secretion with reduced IgG expression and an IgM+ atypical type of MBCs after treatment, irrespective of with and without functional cure. The number of CD40L+ Tfh-cells increased after PEG-IFNα treatment and positively correlated with HBsAg-specific B-cell activation. CONCLUSIONS: After PEG-IFNα treatment, HBsAg- and HBcAg-specific B-cells exhibit various changes in antibody secretion. Their functional differences are reflected in the alterations in phenotypes and subtypes. The presence of CD40L+ Tfh-cells is associated with the active recovery of HBsAg-specific B-cells. IMPACT AND IMPLICATIONS: HBV-related complications and hepatocellular carcinoma remain the leading causes of mortality from chronic liver disease worldwide, and a cure is rarely achieved with antiviral therapies. Elucidating the immunological mechanisms underlying the functional cure of CHB patients offers a promising therapeutic strategy for viral clearance, such as therapeutic vaccine. We analyzed the alterations in HBV-specific B-cells in patients treated with PEG-IFNα and identified novel pathways for immunotherapeutic boosting of B cell immunity.
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Integrating self-healing capabilities into printed stretchable electronic devices is important for improving performance and extending device life. However, achieving printed self-healing stretchable electronic devices with excellent device-level healing ability and stretchability while maintaining outstanding electrical performance remains challenging. Herein, a series of printed device-level self-healing stretchable electronic devices is achieved by depositing liquid metal/silver fractal dendrites/polystyrene-block-polyisoprene-block-polystyrene (LM/Ag FDs/SIS) conductive inks onto a self-healing thermoplastic polyurethane (TPU) film via screen printing method. Owing to the fluidic properties of the LM and the interfacial hydrogen bonding and disulfide bonds of TPU, the as-obtained stretchable electronic devices maintain good electronic properties under strain and exhibit device-level self-healing properties without external stimulation. Printed self-healing stretchable electrodes possess high electrical conductivity (1.6 × 105 S m-1), excellent electromechanical properties, and dynamic stability, with only a 2.5-fold increase in resistance at 200% strain, even after a complete cut and re-healing treatment. The printed self-healing capacitive stretchable strain sensor shows good linearity (R2 ≈0.9994) in a wide sensing range (0%-200%) and is successfully applied to bio-signal detection. Furthermore, the printed self-healing electronic smart label is designed and can be used for real-time environmental monitoring, which exhibits promising potential for practical application in food preservation packaging.
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NiSe is a promising electrode material for enhancing the energy density of supercapacitors, but it faces challenges such as sensitivity to electrolyte anions, limited specific capacity, and unstable cycling. This study employs a strategy of metal atom doping to address these issues. Through a hydrothermal reaction, Mo-doped NiSe demonstrates significant improvement in electrochemical performance, exhibiting high capacity (799.90 C g-1), splendid rate performance, and excellent cyclic stability (90% capacity retention). The introduction of Mo induces charge redistribution in NiSe, leading to a reduction in the band gap. Theoretical calculation reveals that Mo doping can effectively enhance the electrical conductivity and the adsorption energy of NiSe. A flexible printed hybrid Mo-doped NiSe-based supercapacitor is fabricated, demonstrating superior electrochemical performance (367.04 mF cm-2) and the ability to power timers, LEDs, and toy fans. This research not only deepens the understanding of the electrochemical properties of metal-doped NiSe but also highlights its application potential in high-performance supercapacitors.
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Quality control of mRNA represents an important regulatory mechanism for gene expression in eukaryotes. One component of this quality control is the nuclear retention and decay of misprocessed RNAs. Previously, we demonstrated that mature mRNAs containing a 5' splice site (5'SS) motif, which is typically found in misprocessed RNAs such as intronic polyadenylated (IPA) transcripts, are nuclear retained and degraded. Using high-throughput sequencing of cellular fractions, we now demonstrate that IPA transcripts require the zinc finger protein ZFC3H1 for their nuclear retention and degradation. Using reporter mRNAs, we demonstrate that ZFC3H1 promotes the nuclear retention of mRNAs with intact 5'SS motifs by sequestering them into nuclear speckles. Furthermore, we find that U1-70K, a component of the spliceosomal U1 snRNP, is also required for the nuclear retention of these reporter mRNAs and likely functions in the same pathway as ZFC3H1. Finally, we show that the disassembly of nuclear speckles impairs the nuclear retention of reporter mRNAs with 5'SS motifs. Our results highlight a splicing independent role of U1 snRNP and indicate that it works in conjunction with ZFC3H1 in preventing the nuclear export of misprocessed mRNAs by sequestering them into nuclear speckles.
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Sitios de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U1 , Motas Nucleares , Sitios de Empalme de ARN/genética , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
BACKGROUND: Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. OBJECTIVES: To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. METHODS: In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. RESULTS: The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the ß-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes â and â ¡. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. CONCLUSION: Iron efflux is critical to alleviate oxidative stress damage and ß-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.
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Antibacterianos , Aztreonam , Hierro , Pruebas de Sensibilidad Microbiana , Estrés Oxidativo , Riemerella , Estrés Oxidativo/efectos de los fármacos , Hierro/metabolismo , Animales , Antibacterianos/farmacología , Riemerella/efectos de los fármacos , Riemerella/genética , Riemerella/patogenicidad , Riemerella/metabolismo , Aztreonam/farmacología , Infecciones por Flavobacteriaceae/microbiología , Virulencia , Resistencia betalactámica , Patos , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Estreptonigrina/farmacología , Técnicas de Inactivación de Genes , Enfermedades de las Aves de Corral/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Duck plague virus (DPV) is a high-morbidity fowl alphaherpesvirus that causes septicemic lesions in various organs. Most DPV genes are conserved among herpesviruses, while a few are specific to fowl herpesviruses, including the LORF3 gene, for which there is currently no literature describing its biological properties and functions. This study first addressed whether the LORF3 protein is expressed by making specific polyclonal antibodies. We could demonstrate that DPV LORF3 is an early gene and encodes a protein involved in virion assembly, mainly localized in the nucleus of DPV-infected DEF cells. To investigate the role of this novel LORF3 protein in DPV pathogenesis, we generated a recombinant virus that lacks expression of the LORF3 protein. Our data revealed that the LORF3 protein is not essential for viral replication but contributes to DPV replication in vitro and in vivo and promotes duck plague disease morbidity and mortality. Interestingly, deletion of the LORF3 protein abolished thymus atrophy in DPV-vaccinated ducks. In conclusion, this study revealed the expression of avian herpesviruses-specific genes and unraveled the role of the early protein LORF3 in the pathogenesis of DPV. IMPORTANCE DPV is a highly lethal alphaherpesvirus that causes duck plague in birds of the order Anseriformes. The virus has caused huge economic losses to the poultry industry due to high morbidity and mortality and the cost of vaccination. DPV encodes 78 open reading frames (ORFs), and these genes are involved in various processes of the viral life cycle. Functional characterization of DPV genes is important for understanding the complex viral life cycle and DPV pathogenesis. Here, we identified a novel protein encoded by LORF3, and our data suggest that the LORF3 protein is involved in the occurrence and development of duck plague.
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Alphaherpesvirinae , Infecciones por Herpesviridae , Animales , Alphaherpesvirinae/genética , Alphaherpesvirinae/metabolismo , Alphaherpesvirinae/patogenicidad , Patos , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/virología , Células CultivadasRESUMEN
IMPORTANCE: Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that replicates well in mosquito, bird, and mammalian cells. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in the serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and poses a threat to mammalian health. Thus, understanding the pathogenic mechanism of DTMUV is crucial for identifying potential antiviral targets. In this study, we discovered that NS3 can induce the mitochondria-mediated apoptotic pathway through the PERK/PKR pathway; it can also interact with voltage-dependent anion channel 2 to induce apoptosis. Our findings provide a theoretical basis for understanding the pathogenic mechanism of DTMUV infection and identifying potential antiviral targets and may also serve as a reference for exploring the pathogenesis of other flaviviruses.
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Apoptosis , Patos , Infecciones por Flavivirus , Flavivirus , Especificidad del Huésped , Animales , Humanos , Antivirales/farmacología , Patos/virología , eIF-2 Quinasa/metabolismo , Flavivirus/enzimología , Flavivirus/patogenicidad , Infecciones por Flavivirus/diagnóstico , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/transmisión , Infecciones por Flavivirus/virología , Mitocondrias/metabolismo , Terapia Molecular Dirigida/tendencias , Zoonosis Virales/diagnóstico , Zoonosis Virales/inmunología , Zoonosis Virales/transmisión , Zoonosis Virales/virología , Canal Aniónico 2 Dependiente del Voltaje/metabolismoRESUMEN
Many RING domain E3 ubiquitin ligases play critical roles in fine-tuning the innate immune response, yet little is known about their regulatory role in flavivirus-induced innate immunity. In previous studies, we found that the suppressor of cytokine signaling 1 (SOCS1) protein mainly undergoes lysine 48 (K48)-linked ubiquitination. However, the E3 ubiquitin ligase that promotes the K48-linked ubiquitination of SOCS1 is unknown. In the present study, we found that RING finger protein 123 (RNF123) binds to the SH2 domain of SOCS1 through its RING domain and facilitates the K48-linked ubiquitination of the K114 and K137 residues of SOCS1. Further studies found that RNF123 promoted the proteasomal degradation of SOCS1 and promoted Toll-like receptor 3 (TLR3)- and interferon (IFN) regulatory factor 7 (IRF7)-mediated type I IFN production during duck Tembusu virus (DTMUV) infection through SOCS1, ultimately inhibiting DTMUV replication. Overall, these findings demonstrate a novel mechanism by which RNF123 regulates type I IFN signaling during DTMUV infection by targeting SOCS1 degradation. IMPORTANCE In recent years, posttranslational modification (PTM) has gradually become a research hot spot in the field of innate immunity regulation, and ubiquitination is one of the critical PTMs. DTMUV has seriously endangered the development of the waterfowl industry in Southeast Asian countries since its outbreak in 2009. Previous studies have shown that SOCS1 is modified by K48-linked ubiquitination during DTMUV infection, but E3 ubiquitin ligase catalyzing the ubiquitination of SOCS1 has not been reported. Here, we identify for the first time that RNF123 acts as an E3 ubiquitin ligase that regulates TLR3- and IRF7-induced type I IFN signaling during DTMUV infection by targeting the K48-linked ubiquitination of the K114 and K137 residues of SOCS1 and the proteasomal degradation of SOCS1.