RESUMEN
mRNA translation and decay are tightly interconnected processes both in the context of mRNA quality-control pathways and for the degradation of functional mRNAs. Cotranslational mRNA degradation through codon usage, ribosome collisions, and the recruitment of specific proteins to ribosomes is an important determinant of mRNA turnover. However, the extent to which translation-dependent mRNA decay (TDD) and translation-independent mRNA decay (TID) pathways participate in the degradation of mRNAs has not been studied yet. Here we describe a comprehensive analysis of basal and signal-induced TDD and TID in mouse primary CD4+ T cells. Our results indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner. Our analysis further identifies the length of untranslated regions, the density of ribosomes, and GC3 content as important determinants of TDD magnitude. Consistently, all transcripts that undergo changes in ribosome density within their coding sequence upon T cell activation display a corresponding change in their TDD level. Moreover, we reveal a dynamic modulation in the relationship between GC3 content and TDD upon T cell activation, with a reversal in the impact of GC3- and AU3-rich codons. Altogether, our data show a strong and dynamic interconnection between mRNA translation and decay in mammalian primary cells.
Asunto(s)
Activación de Linfocitos , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero , Ribosomas , Ribosomas/metabolismo , Animales , Ratones , ARN Mensajero/metabolismo , ARN Mensajero/genética , Linfocitos T CD4-Positivos/metabolismo , Ratones Endogámicos C57BL , Linfocitos T/metabolismoRESUMEN
Caspase-4, the cytosolic LPS sensor, and gasdermin D, its downstream effector, constitute the non-canonical inflammasome, which drives inflammatory responses during Gram-negative bacterial infections. It remains unclear whether other proteins regulate cytosolic LPS sensing, particularly in human cells. Here, we conduct a genome-wide CRISPR/Cas9 screen in a human monocyte cell line to identify genes controlling cytosolic LPS-mediated pyroptosis. We find that the transcription factor, IRF2, is required for pyroptosis following cytosolic LPS delivery and functions by directly regulating caspase-4 levels in human monocytes and iPSC-derived monocytes. CASP4, GSDMD, and IRF2 are the only genes identified with high significance in this screen highlighting the simplicity of the non-canonical inflammasome. Upon IFN-γ priming, IRF1 induction compensates IRF2 deficiency, leading to robust caspase-4 expression. Deficiency in IRF2 results in dampened inflammasome responses upon infection with Gram-negative bacteria. This study emphasizes the central role of IRF family members as specific regulators of the non-canonical inflammasome.
Asunto(s)
Caspasas Iniciadoras/metabolismo , Factor 2 Regulador del Interferón/metabolismo , Caspasas Iniciadoras/genética , Muerte Celular/efectos de los fármacos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Humanos , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Factor 2 Regulador del Interferón/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Células U937RESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.
Asunto(s)
Proliferación Celular , Transformación Celular Viral , Modelos Animales de Enfermedad , Productos del Gen tax/metabolismo , Infecciones por HTLV-I/patología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Linfocitos T/patología , Animales , Femenino , Perfilación de la Expresión Génica , Productos del Gen tax/genética , Células HEK293 , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/virología , Humanos , Activación de Linfocitos , Masculino , Ratones , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Dominios PDZ , Unión Proteica , Linfocitos T/metabolismoRESUMEN
ISG20 is an IFN-induced 3'-5' RNA exonuclease that acts as a broad antiviral factor. At present, the features that expose RNA to ISG20 remain unclear, although recent studies have pointed to the modulatory role of epitranscriptomic modifications in the susceptibility of target RNAs to ISG20. These findings raise the question as to how cellular RNAs, on which these modifications are abundant, cope with ISG20. To obtain an unbiased perspective on this topic, we used RNA-seq and biochemical assays to identify elements that regulate the behavior of RNAs against ISG20. RNA-seq analyses not only indicate a general preservation of the cell transcriptome, but they also highlight a small, but detectable, decrease in the levels of histone mRNAs. Contrarily to all other cellular ones, histone mRNAs are non-polyadenylated and possess a short stem-loop at their 3' end, prompting us to examine the relationship between these features and ISG20 degradation. The results we have obtained indicate that poly(A)-binding protein loading on the RNA 3' tail provides a primal protection against ISG20, easily explaining the overall protection of cellular mRNAs observed by RNA-seq. Terminal stem-loop RNA structures have been associated with ISG20 protection before. Here, we re-examined this question and found that the balance between resistance and susceptibility to ISG20 depends on their thermodynamic stability. These results shed new light on the complex interplay that regulates the susceptibility of different classes of viruses against ISG20.
Asunto(s)
Exonucleasas , Exorribonucleasas , Exonucleasas/genética , Exonucleasas/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Histonas , Replicación Viral/fisiologíaRESUMEN
A significant proportion of non-muscle invasive bladder cancer cases will progress to muscle invasive disease. Transurethral resection followed by Bacillus Calmette Guerin immunotherapy can reduce this risk, while cystectomy prior to muscle invasion provides the best option for survival. Currently, there are no effective treatments for Bacillus Calmette Guerin refractory disease. A novel oncolytic vesicular stomatitis virus containing the human GM-CSF transgene (VSVd51-hGM-CSF) was rescued and tested as a potential bladder-sparing therapy for aggressive bladder cancer. The existing variant expressing mouse GM-CSF was also used. Measurement of gene expression and protein level alterations of canonical immunogenic cell death associated events on mouse and human bladder cancer cell lines and spheroids showed enhanced release of danger signals and immunogenic factors following infection with VSVd51-m/hGM-CSF. Intravesical instillation of VSVd51-mGM-CSF into MB49 bladder cancer bearing C57Bl/6 mice demonstrated enhanced activation of peripheral and bladder infiltrating effector immune cells, along with improved survival and reduced tumor volume. Importantly, virus-mediated anti-tumor immunity was recapitulated in bladder cancer patient-derived organoids. These results suggest that VSVd51-hGM-CSF is a promising viro/immunotherapy that could benefit bladder cancer patients.
RESUMEN
Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for "all-in-one" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Edición Génica/métodos , Vectores Genéticos/genética , ARN Guía de Kinetoplastida/genética , Ribonucleoproteínas/genética , Animales , Línea Celular Tumoral , Reparación del ADN/genética , Embrión de Mamíferos , Fibroblastos , Edición Génica/economía , Genoma/genética , Células HEK293 , Células Madre Hematopoyéticas , Humanos , Células Madre Pluripotentes Inducidas , Virus de la Leucemia Murina/genética , Macrófagos , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Activación Transcripcional/genéticaRESUMEN
During the late stage of virus replication, incorporation of the envelope glycoproteins (Env) by Gag cores takes place together with the proteolytic maturation of Gag and Gag-Pol precursors. Assembly is initially driven by Gag oligomerisation, which requires two platorms. The first one is formed by specific membrane subdomains with which Gag molecules interact via the N-terminal MA domain, and the second by the viral genomic RNA undergoing specific interactions with the NC domain of Gag. To complete viral budding, the Gag "late domain" subsequently associates with members of the ESCRT complexes involved in the budding of vesicles in late endosomes (LE). While the cellular trafficking of the viral components is still poorly understood, there is an ongoing debate on the site of HIV-1 assembly, because this process might take place either at the plasma membrane or in intracellular compartments such as the LE, depending on the virus/cell system studied. This site may depend on the interplay of multiple overlapping trafficking signals bear by Gag and Env. Our recent results indicate that it may rely on the chronic or acute nature of the viral infection more than on the cell type. In chronically infected cells, virions probably assemble and accumulate in intracellular compartments hidden from the immune system. Release of virions in the form of bursts would be triggered during cell-cell interactions, through a specialized structure called the virological synapse.
Asunto(s)
VIH-1/fisiología , VIH-1/ultraestructura , Síndrome de Inmunodeficiencia Adquirida/patología , Enfermedad Crónica , Productos del Gen gag/fisiología , Humanos , Proteínas del Envoltorio Viral/fisiologíaRESUMEN
The RNA exosome fulfills important functions in the processing and degradation of numerous RNAs species. However, the mechanisms of recruitment to its various nuclear substrates are poorly understood. Using Epstein-Barr virus mRNAs as a model, we have discovered a novel function for the splicing factor SRSF3 in the quality control of nuclear mRNAs. We have found that viral mRNAs generated from intronless genes are particularly unstable due to their degradation by the nuclear RNA exosome. This effect is counteracted by the viral RNA-binding protein EB2 which stabilizes these mRNAs in the nucleus and stimulates both their export to the cytoplasm and their translation. In the absence of EB2, SRSF3 participates in the destabilization of these viral RNAs by interacting with both the RNA exosome and its adaptor complex NEXT. Taken together, our results provide direct evidence for a connection between the splicing machinery and mRNA decay mediated by the RNA exosome. Our results suggest that SRSF3 aids the nuclear RNA exosome and the NEXT complex in the recognition and degradation of certain mRNAs.
Asunto(s)
Exosomas/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Western Blotting , Núcleo Celular/genética , Núcleo Celular/metabolismo , Exosomas/genética , Células HeLa , Humanos , Inmunoprecipitación , Empalme del ARN/genética , Empalme del ARN/fisiología , Estabilidad del ARN/genética , Estabilidad del ARN/fisiología , ARN Mensajero , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina/genéticaRESUMEN
The non-structural protein NS1 of influenza A viruses exerts pleiotropic functions during infection. Among these functions, NS1 was shown to be involved in the control of both viral and cellular translation; however, the mechanism by which this occurs remains to be determined. Thus, we have revisited the role of NS1 in translation by using a combination of influenza infection, mRNA reporter transfection, and in vitro functional and biochemical assays. Our data show that the NS1 protein is able to enhance the translation of virtually all tested mRNAs with the exception of constructs bearing the Dicistroviruses Internal ribosome entry segment (IRESes) (DCV and CrPV), suggesting a role at the level of translation initiation. The domain of NS1 required for translation stimulation was mapped to the RNA binding amino-terminal motif of the protein with residues R38 and K41 being critical for activity. Although we show that NS1 can bind directly to mRNAs, it does not correlate with its ability to stimulate translation. This activity rather relies on the property of NS1 to associate with ribosomes and to recruit them to target mRNAs.
Asunto(s)
Virus de la Influenza A/fisiología , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN Viral/genética , Ribosomas/fisiología , Proteínas no Estructurales Virales/genética , Células A549 , Animales , Perros , Humanos , Gripe Humana/virología , Células de Riñón Canino Madin Darby , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants.