RESUMEN
ADAR3 is a catalytically inactive member of the family of adenosine deaminases acting on RNA (ADARs). Here we have investigated its function in the context of the developing mouse brain. The expression of ADAR3 gradually increases throughout embryogenesis and drops after birth. Using primary cortical neurons, we show that ADAR3 is only expressed in a subpopulation of in vitro differentiated neurons, which suggests specific functions rather than being a general regulator of ADAR editing in the brain. The analysis of the ADAR3 interactome suggested a role in mRNA stability and translation, and we show that expression of ADAR3 in a neuronal cell line that is otherwise ADAR3-negative changes the expression and stability of a large number of mRNAs. Notably, we show that ADAR3 associates with polysomes and inhibits translation. We propose that ADAR3 binds to target mRNAs and stabilizes them in non-productive polysome complexes. Interestingly, the expression of ADAR3 downregulates genes related to neuronal differentiation and inhibits neurofilament outgrowth in vitro. In summary, we propose that ADAR3 negatively regulates neuronal differentiation, and that it does so by regulating mRNA stability and translation in an editing-independent manner.
RESUMEN
Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely understood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1-infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal.
Asunto(s)
Linfocitos T CD4-Positivos , VIH-1 , Uso de Codones , VIH-1/fisiología , ARN Viral/genética , Latencia del Virus/genéticaRESUMEN
Ample evidence indicates that codon usage bias regulates gene expression. How viruses, such as the emerging mosquito-borne Chikungunya virus (CHIKV), express their genomes at high levels despite an enrichment in rare codons remains a puzzling question. Using ribosome footprinting, we analyze translational changes that occur upon CHIKV infection. We show that CHIKV infection induces codon-specific reprogramming of the host translation machinery to favor the translation of viral RNA genomes over host mRNAs with an otherwise optimal codon usage. This reprogramming was mostly apparent at the endoplasmic reticulum, where CHIKV RNAs show high ribosome occupancy. Mechanistically, it involves CHIKV-induced overexpression of KIAA1456, an enzyme that modifies the wobble U34 position in the anticodon of tRNAs, which is required for proper decoding of codons that are highly enriched in CHIKV RNAs. Our findings demonstrate an unprecedented interplay of viruses with the host tRNA epitranscriptome to adapt the host translation machinery to viral production.
Asunto(s)
Fiebre Chikungunya , Virus Chikungunya , Animales , Virus Chikungunya/genética , Codón/genética , Codón/metabolismo , Humanos , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
The highly conserved 5'-3' exonuclease Xrn1 regulates gene expression in eukaryotes by coupling nuclear DNA transcription to cytosolic mRNA decay. By integrating transcriptome-wide analyses of translation with biochemical and functional studies, we demonstrate an unanticipated regulatory role of Xrn1 in protein synthesis. Xrn1 promotes translation of a specific group of transcripts encoding membrane proteins. Xrn1-dependence for translation is linked to poor structural RNA contexts for translation initiation, is mediated by interactions with components of the translation initiation machinery and correlates with an Xrn1-dependence for mRNA localization at the endoplasmic reticulum, the translation compartment of membrane proteins. Importantly, for this group of mRNAs, Xrn1 stimulates transcription, mRNA translation and decay. Our results uncover a crosstalk between the three major stages of gene expression coordinated by Xrn1 to maintain appropriate levels of membrane proteins.
Asunto(s)
Exorribonucleasas/genética , Regulación Fúngica de la Expresión Génica , Proteínas de la Membrana/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Clonación Molecular , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Exorribonucleasas/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas de la Membrana/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Viruses are powerful tools to uncover cellular processes. Through viral studies we have recently identified a novel translational control mechanism that involves the DEAD-box helicase Dhh1/DDX6 and RNA folding within coding sequences (CDSs). All Dhh1-dependent mRNAs, viral and cellular ones, (i) contain long and highly structured CDSs, (ii) are directly bound by Dhh1 with a specific pattern, (iii) are activated at the translation initiation step and (iv) express proteins associated with the endoplasmic reticulum. The obtained results uncover a novel layer of translation regulation associated with translation at the endoplasmic reticulum conserved from yeast to humans and hijacked by viruses.
Asunto(s)
Biosíntesis de Proteínas , Virus/genética , ARN Helicasas DEAD-box/metabolismo , Humanos , Modelos Biológicos , ARN Mensajero/genética , Saccharomyces cerevisiaeRESUMEN
The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here, we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses, we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site; second, they are directly bound by Dhh1 with a specific binding distribution; and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins.
Asunto(s)
ARN Helicasas DEAD-box/genética , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Bromovirus/genética , Exones/genética , Regulación de la Expresión Génica/genética , Humanos , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , Ribosomas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Positive-strand RNA viruses have evolved multiple strategies to not only circumvent the hostile decay machinery but to trick it into being a priceless collaborator supporting viral RNA translation and replication. In this review, we describe the versatile interaction of positive-strand RNA viruses and the 5'-3' mRNA decay machinery with a focus on the viral subversion of decapping activators. This highly conserved viral trickery is exemplified with the plant Brome mosaic virus, the animal Flock house virus and the human hepatitis C virus.
Asunto(s)
Bromovirus/fisiología , Endorribonucleasas/metabolismo , Hepacivirus/fisiología , Interacciones Huésped-Patógeno , Nodaviridae/fisiología , Biosíntesis de Proteínas , Replicación Viral , Animales , Humanos , Estabilidad del ARNRESUMEN
The Lsm1-7-Pat1 complex binds to the 3' end of cellular mRNAs and promotes 3' end protection and 5'-3' decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.
Asunto(s)
Bromovirus/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/virología , Proteínas Virales/metabolismo , Virología/métodos , Bromovirus/genética , Mutación , Proteínas de Unión a Caperuzas de ARN/genética , Proteínas de Unión a Caperuzas de ARN/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicación ViralRESUMEN
Positive-strand RNA viruses depend on recruited host factors to control critical replication steps. Previously, it was shown that replication of evolutionarily diverse positive-strand RNA viruses, such as hepatitis C virus and brome mosaic virus, depends on host decapping activators LSm1-7, Pat1, and Dhh1 (J. Diez et al., Proc. Natl. Acad. Sci. U. S. A. 97:3913-3918, 2000; A. Mas et al., J. Virol. 80:246 -251, 2006; N. Scheller et al., Proc. Natl. Acad. Sci. U. S. A. 106:13517-13522, 2009). By using a system that allows the replication of the insect Flock House virus (FHV) in yeast, here we show that LSm1-7, Pat1, and Dhh1 control the ratio of subgenomic RNA3 to genomic RNA1 production, a key feature in the FHV life cycle mediated by a long-distance base pairing within RNA1. Depletion of LSM1, PAT1, or DHH1 dramatically increased RNA3 accumulation during replication. This was not caused by differences between RNA1 and RNA3 steady-state levels in the absence of replication. Importantly, coimmunoprecipitation assays indicated that LSm1-7, Pat1, and Dhh1 interact with the FHV RNA genome and the viral polymerase. By using a strategy that allows dissecting different stages of the replication process, we found that LSm1-7, Pat1, and Dhh1 did not affect the early replication steps of RNA1 recruitment to the replication complex or RNA1 synthesis. Furthermore, their function on RNA3/RNA1 ratios was independent of the membrane compartment, where replication occurs and requires ATPase activity of the Dhh1 helicase. Together, these results support that LSm1-7, Pat1, and Dhh1 control RNA3 synthesis. Their described function in mediating cellular mRNP rearrangements suggests a parallel role in mediating key viral RNP transitions, such as the one required to maintain the balance between the alternative FHV RNA1 conformations that control RNA3 synthesis.