RESUMEN
The relationship between mRNA translation and decay is incompletely understood, with conflicting reports suggesting that translation can either promote decay or stabilize mRNAs. The effect of translation on mRNA decay has mainly been studied using ensemble measurements and global transcription and translation inhibitors, which can have pleiotropic effects. We developed a single-molecule imaging approach to control the translation of a specific transcript that enabled simultaneous measurement of translation and mRNA decay. Our results demonstrate that mRNA translation reduces mRNA stability, and mathematical modeling suggests that this process is dependent on ribosome flux. Furthermore, our results indicate that miRNAs mediate efficient degradation of both translating and non-translating target mRNAs and reveal a predominant role for mRNA degradation in miRNA-mediated regulation. Simultaneous observation of translation and decay of single mRNAs provides a framework to directly study how these processes are interconnected in cells.
Asunto(s)
MicroARNs , Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero/genética , ARN Mensajero/metabolismo , Imagen Individual de Molécula , Estabilidad del ARN/genética , Ribosomas/genética , Ribosomas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Biosíntesis de ProteínasRESUMEN
Coxsackievirus B3 (CVB3) is an enterovirus belonging to the family Picornaviridae. Its 5' untranslated region (UTR) contains a cloverleaf structure followed by an internal ribosome entry site (IRES). The cloverleaf forms an RNA-protein complex known to regulate virus replication, translation, and stability of the genome, and the IRES regulates virus RNA translation. For positive-strand RNA-containing viruses, such as members of the flaviviruses or enteroviruses, the genomic RNA is used for translation, replication, and encapsidation. Only a few regulatory mechanisms which govern the accessibility of genomic RNA templates for translation or replication have been reported. Here, we report the role of human antigen R (HuR) in regulating the fate of CVB3 positive-strand RNA into the replication cycle or translation cycle. We have observed that synthesis of HuR is induced during CVB3 infection, and it suppresses viral replication by displacing PCBP-2 (a positive regulator of virus replication) at the cloverleaf RNA. Silencing of HuR increases viral RNA replication and consequently reduces viral RNA translation in a replication-dependent manner. Furthermore, we have shown that HuR level is upregulated upon CVB3 infection. Moreover, HuR limits virus replication and can coordinate the availability of genomic RNA templates for translation, replication, or encapsidation. Our study highlights the fact that the relative abundance of translation factors and replication factors in the cell decides the outcome of viral infection. IMPORTANCE A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes. These proteins inhibit translation either by displacing a stimulator of translation or by binding to an alternative site. Both mechanisms lead to ribosome clearance and availability of the genomic strand for replication. We have shown that HuR also helps in maintaining this balance by inhibiting replication and subsequently promoting translation and packaging.
Asunto(s)
Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Proteína 1 Similar a ELAV/fisiología , Enterovirus Humano B/fisiología , ARN Viral/metabolismo , Regiones no Traducidas 5' , Animales , Regulación Viral de la Expresión Génica , Silenciador del Gen , Células HeLa , Interacciones Microbiota-Huesped , Humanos , Sitios Internos de Entrada al Ribosoma , Estadios del Ciclo de Vida , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismo , Replicación ViralRESUMEN
Single-molecule fluorescence microscopy techniques have enabled the lifecycle of individual RNA transcripts to be quantitatively measured in living cells. The application of these approaches to monitor mRNA degradation, however, has presented a challenge to unequivocally detect these events due to the inherent loss-of-signal resulting from decay of a transcript. Here, we highlight the recent technological developments that have enabled the spatial and temporal dynamics of mRNA degradation of individual transcripts to be visualized within living cells.
Asunto(s)
Estabilidad del ARN , ARN Mensajero/metabolismo , Imagen Individual de Molécula/métodos , Animales , HumanosRESUMEN
Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.
Asunto(s)
Histona Desacetilasas/metabolismo , Biosíntesis de Proteínas , Sirtuinas/metabolismo , Factor de Transcripción Sp1/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transcripción Genética , Animales , Cardiomegalia/genética , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Histona Desacetilasas/genética , Humanos , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Transducción de Señal , Sirtuinas/genética , Factor de Transcripción Sp1/química , Dedos de ZincRESUMEN
During enteroviral infections, the canonical translation factor eukaryotic translation initiation factor 4 γ I (eIF4GI) is cleaved by viral protease 2A. The resulting C-terminal fragment is recruited by the viral internal ribosome entry site (IRES) for efficient translation of the viral RNA. However, the 2A protease is not present in the viral capsid and is synthesized only after the initial round of translation. This presents the conundrum of how the initial round of translation occurs in the absence of the C-terminal eIF4GI fragment. Interestingly, the host protein DAP5 (also known as p97, eIF4GIII, and eIF4G2), an isoform of eIF4GI, closely resembles the eIF4GI C-terminal fragment produced after 2A protease-mediated cleavage. Using the Coxsackievirus B3 (CVB3) IRES as a model system, here we demonstrate that DAP5, but not the full-length eIF4GI, is required for CVB3 IRES activity for translation of input viral RNA. Additionally, we show that DAP5 is specifically required by type I IRES but not by type II or type III IRES, in which cleavage of eIF4GI has not been observed. We observed that both DAP5 and C-terminal eIF4GI interact with CVB3 IRES in the same region, but DAP5 exhibits a lower affinity for CVB3 IRES compared with the C-terminal eIF4GI fragment. It appears that DAP5 is required for the initial round of viral RNA translation by sustaining a basal level of CVB3 IRES activity. This activity leads to expression of 2A protease and consequent robust CVB3 IRES-mediated translation by the C-terminal eIF4GI fragment.
Asunto(s)
Infecciones por Coxsackievirus/metabolismo , Enterovirus Humano B/genética , Factor 4G Eucariótico de Iniciación/metabolismo , ARN Viral/genética , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/virología , Enterovirus Humano B/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Regulación Viral de la Expresión Génica , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Sitios Internos de Entrada al Ribosoma , Biosíntesis de Proteínas , ARN Viral/metabolismo , Ribosomas/metabolismo , Ribosomas/virologíaRESUMEN
Coxsackievirus B3 is an enterovirus, with positive-sense single-stranded RNA genome containing 'Internal Ribosome Entry Site' (IRES) in the 5'UTR. Once sufficient viral proteins are synthesized in the cell from the input RNA, viral template switches from translation to replication to synthesize negative-strand RNA. Inhibition of translation is a key step in regulating this switch as the positive-strand RNA template should be free of ribosomes to enable polymerase movement. In this study, we show how a host protein hnRNP C1/C2 inhibits viral RNA translation. hnRNP C1/C2 interacts with stem-loop V in the IRES and displaces poly-pyrimidine tract binding protein, a positive regulator of translation. We further demonstrate that hnRNP C1/C2 induces translation to replication switch, independently from the already known role of the ternary complex (PCBP2-3CD-cloverleaf RNA). These results suggest a novel function of hnRNP C1/C2 in template switching of positive-strand from translation to replication by a new mechanism. Using mathematical modelling, we show that the differential affinity of hnRNP C1/C2 for positive and negative-strand RNAs guides the final ± RNA ratio, providing first insight in the regulation of the positive to negative-strand RNA ratio in enteroviruses.
Asunto(s)
Infecciones por Coxsackievirus/metabolismo , Enterovirus Humano B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , ARN Viral/genética , Regiones no Traducidas 5'/genética , Células HeLa , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Modelos Biológicos , Biosíntesis de Proteínas , Replicación Viral/genéticaRESUMEN
Hepatitis C virus (HCV) infection causes chronic liver disease, which often leads to hepatocellular carcinoma. Earlier, we have demonstrated anti-HCV property of the methanolic extract of Phyllanthus amarus, an age-old folk-medicine against viral hepatitis. Here, we report identification of a principal bioactive component 'corilagin', which showed significant inhibition of the HCV key enzymes, NS3 protease and NS5B RNA-dependent-RNA-polymerase. This pure compound could effectively inhibit viral replication in the infectious cell culture system, displayed strong antioxidant activity by blocking HCV induced generation of reactive oxygen species and suppressed up-regulation of NOX4 and TGF-ß mRNA levels. Oral administration of corilagin in BALB/c mice demonstrated its better tolerability and systemic bioavailability. More importantly, corilagin could restrict serum HCV RNA levels, decrease collagen deposition and hepatic cell denaturation in HCV infected chimeric mice harbouring human hepatocytes. Taken together, results provide a basis towards developing a pure natural drug as an alternate therapeutic strategy for restricting viral replication and prevent liver damage towards better management of HCV induced pathogenesis.
Asunto(s)
Glucósidos/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Taninos Hidrolizables/farmacología , Hígado/metabolismo , Hígado/virología , Animales , Antivirales/aislamiento & purificación , Antivirales/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica , Genes Reporteros , Glucósidos/aislamiento & purificación , Hepatitis C/complicaciones , Hepatitis C/patología , Humanos , Taninos Hidrolizables/aislamiento & purificación , Hígado/efectos de los fármacos , Cirrosis Hepática , Ratones , NADPH Oxidasa 4/metabolismo , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The 5' UTR of Coxsackievirus B3 (CVB3) contains internal ribosome entry site (IRES), which allows cap-independent translation of the viral RNA and a 5'-terminal cloverleaf structure that regulates viral replication, translation and stability. Here, we demonstrate that host protein PSF (PTB associated splicing factor) interacts with the cloverleaf RNA as well as the IRES element. PSF was found to be an important IRES trans acting factor (ITAF) for efficient translation of CVB3 RNA. Interestingly, cytoplasmic abundance of PSF protein increased during CVB3 infection and this is regulated by phosphorylation status at two different amino acid positions. Further, PSF protein was up-regulated in CVB3 infection. The expression of CVB3-2A protease alone could also induce increased PSF protein levels. Furthermore, we observed the presence of an IRES element in the 5'UTR of PSF mRNA, which is activated during CVB3 infection and might contribute to the elevated levels of PSF. It appears that PSF IRES is also positively regulated by PTB, which is known to regulate CVB3 IRES. Taken together, the results suggest for the first time a novel mechanism of regulations of ITAFs during viral infection, where an ITAF undergoes IRES mediated translation, sustaining its protein levels under condition of translation shut-off.