RESUMO
Cytochrome b5 reductase 3 (CYB5R3) is involved in various cellular metabolic processes, including fatty acid synthesis and drug metabolism. However, the role of CYB5R3 in cancer development remains poorly understood. Here, we show that CYB5R3 expression is downregulated in human lung cancer cell lines and tissues. Adenoviral overexpression of CYB5R3 suppresses lung cancer cell growth in vitro and in vivo. However, CYB5R3 deficiency promotes tumorigenesis and metastasis in mouse models. Transcriptome analysis revealed that apoptosis- and endoplasmic reticulum (ER) stress-related genes are upregulated in CYB5R3-overexpressing lung cancer cells. Metabolomic analysis revealed that CYB5R3 overexpression increased the production of nicotinamide adenine dinucleotide (NAD+) and oxidized glutathione (GSSG). Ectopic CYB5R3 is mainly localized in the ER, where CYB5R3-dependent ER stress signaling is induced via activation of protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme 1 alpha (IRE1α). Moreover, NAD+ activates poly (ADP-ribose) polymerase16 (PARP16), an ER-resident protein, to promote ADP-ribosylation of PERK and IRE1α and induce ER stress. In addition, CYB5R3 induces the generation of reactive oxygen species and caspase-9-dependent intrinsic cell death. Our findings highlight the importance of CYB5R3 as a tumor suppressor for the development of CYB5R3-based therapeutics for lung cancer.
Assuntos
Neoplasias Pulmonares , Proteínas Serina-Treonina Quinases , Animais , Humanos , Camundongos , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Apoptose/genética , Citocromo-B(5) Redutase/metabolismo , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Neoplasias Pulmonares/genética , Sistema de Sinalização das MAP Quinases , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Newly synthesized mRNAs are exported from the nucleus to cytoplasm with a 5'-cap structure bound by the nuclear cap-binding complex (CBC). During or after export, the CBC should be properly replaced by cytoplasmic cap-binding protein eIF4E for efficient protein synthesis. Nonetheless, little is known about how the replacement takes place. Here, we show that double-stranded RNA-binding protein staufen1 (STAU1) promotes efficient replacement by facilitating an association between the CBC-importin α complex and importin ß. Our transcriptome-wide analyses and artificial tethering experiments also reveal that the replacement occurs more efficiently when an mRNA associates with STAU1. This event is inhibited by a key nonsense-mediated mRNA decay factor, UPF1, which directly interacts with STAU1. Furthermore, we find that cellular apoptosis that is induced by ionizing radiation is accompanied by inhibition of the replacement via increased association between STAU1 and hyperphosphorylated UPF1. Altogether, our data highlight the functional importance of STAU1 and UPF1 in the course of the replacement of the CBC by eIF4E, adding a previously unappreciated layer of post-transcriptional gene regulation.
Assuntos
Proteínas do Citoesqueleto/genética , Fator de Iniciação 4E em Eucariotos/genética , Biossíntese de Proteínas/genética , RNA Helicases/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Regiões 5' não Traduzidas , Núcleo Celular/genética , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas de Ligação ao Cap de RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/genéticaRESUMO
Replication-dependent histone (RDH) mRNAs have a nonpolyadenylated 3'-UTR that ends in a highly conserved stem-loop structure. Nonetheless, a subset of RDH mRNAs has a poly(A) tail under physiological conditions. The biological meaning of poly(A)-containing (+) RDH mRNAs and details of their biosynthesis remain elusive. Here, using HeLa cells and Western blotting, qRT-PCR, and biotinylated RNA pulldown assays, we show that poly(A)+ RDH mRNAs are post-transcriptionally regulated via adenylate- and uridylate-rich element-mediated mRNA decay (AMD). We observed that the rapid degradation of poly(A)+ RDH mRNA is driven by butyrate response factor 1 (BRF1; also known as ZFP36 ring finger protein-like 1) under normal conditions. Conversely, cellular stresses such as UV C irradiation promoted BRF1 degradation, increased the association of Hu antigen R (HuR; also known as ELAV-like RNA-binding protein 1) with the 3'-UTR of poly(A)+ RDH mRNAs, and eventually stabilized the poly(A)+ RDH mRNAs. Collectively, our results provide evidence that AMD surveils poly(A)+ RDH mRNAs via BRF1-mediated degradation under physiological conditions.
Assuntos
Elementos Ricos em Adenilato e Uridilato/fisiologia , Histonas/biossíntese , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Células HeLa , Histonas/genética , Humanos , RNA Mensageiro/genéticaRESUMO
Exon junction complexes (EJCs) loaded onto spliced mRNAs during splicing serve as molecular markers for various post-transcriptional gene-regulatory processes, including nonsense-mediated mRNA decay (NMD). Although the composition and structure of EJCs are well characterized, the mechanism regulating EJC deposition remains unknown. Here we find that threonine 163 (T163) within the RNA-binding motif of eIF4A3 (a core EJC component) is phosphorylated by cyclin-dependent protein kinases 1 and 2 in a cell cycle-dependent manner. T163 phosphorylation hinders binding of eIF4A3 to spliced mRNAs and other EJC components. Instead, it promotes association of eIF4A3 with CWC22, which guides eIF4A3 to an active spliceosome. These molecular events ensure the fidelity of specific deposition of the EJC â¼20-24 nt upstream of an exon-exon junction. Accordingly, NMD is affected by T163 phosphorylation. Collectively, our data provide evidence that T163 phosphorylation affects EJC formation and, consequently, NMD efficiency in a cell cycle-dependent manner.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclo Celular , Quinase 2 Dependente de Ciclina/metabolismo , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Sítios de Ligação , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação 4A em Eucariotos/genética , Células HEK293 , Células HeLa , Humanos , Mutação , Fosforilação , Ligação Proteica , RNA Mensageiro/metabolismoRESUMO
All metazoan mRNAs have a poly(A) tail at the 3' end with the exception of replication-dependent histone (RDH) mRNAs, which end in a highly conserved stem-loop (SL) structure. However, a subset of RDH mRNAs are reported to be polyadenylated under physiologic conditions. The molecular details of the biogenesis of polyadenylated RDH [poly(A)+ RDH] mRNAs remain unknown. In this study, our genome-wide analyses reveal that puromycin treatment or UVC irradiation stabilizes poly(A)+ RDH mRNAs, relative to canonical RDH mRNAs, which end in an SL structure. We demonstrate that the stabilization of poly(A)+ RDH mRNAs occurs in a translation-independent manner and is regulated via human antigen R (HuR) binding to the extended 3' UTR under stress conditions. Our data suggest that HuR regulates the expression of poly(A)+ RDH mRNAs.-Ryu, I., Park, Y., Seo, J.-W., Park, O. H., Ha, H., Nam, J.-W., Kim, Y. K. HuR stabilizes a polyadenylated form of replication-dependent histone mRNAs under stress conditions.
Assuntos
Replicação do DNA , Proteína Semelhante a ELAV 1/metabolismo , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Poliadenilação , RNA Mensageiro/genética , Estresse Fisiológico , Proteína Semelhante a ELAV 1/genética , Células HeLa , Histonas/metabolismo , Humanos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Misfolded polypeptides are rapidly cleared from cells via the ubiquitin-proteasome system (UPS). However, when the UPS is impaired, misfolded polypeptides form small cytoplasmic aggregates, which are sequestered into an aggresome and ultimately degraded by aggrephagy. Despite the relevance of the aggresome to neurodegenerative proteinopathies, the molecular mechanisms underlying aggresome formation remain unclear. Here we show that the CTIF-eEF1A1-DCTN1 (CED) complex functions in the surveillance of either pre-existing or newly synthesized polypeptides by linking two molecular events: selective recognition and aggresomal targeting of misfolded polypeptides. These events are accompanied by CTIF sequestration into the aggresome, preventing the additional synthesis of misfolded polypeptides from mRNAs bound by nuclear cap-binding complex. These events render cells more resistant to apoptosis induced by proteotoxic stresses. Collectively, our data provide compelling evidence for a previously unappreciated protein surveillance pathway and a regulatory gene expression network for coping with misfolded polypeptides.
Assuntos
Apoptose , Corpos de Inclusão/química , Fator 1 de Elongação de Peptídeos/química , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/química , Dobramento de Proteína , Autofagia , Transporte Biológico , Citoplasma/química , Regulação para Baixo , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Desnaturação Proteica , Transporte Proteico , Ubiquitina/químicaRESUMO
In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].
Assuntos
Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Fatores de Iniciação em Eucariotos/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Complexo Proteico Nuclear de Ligação ao Cap/genética , Estresse Oxidativo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.
Assuntos
Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Transativadores/metabolismo , Quimiocina CCL2/metabolismo , Quimiotaxia , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Ligantes , Monócitos/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Ligação Proteica , RNA Helicases , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Eukaryotic translation initiation commences at the initiation codon near the 5' end of mRNA by a 40S ribosomal subunit, and the recruitment of a 40S ribosome to an mRNA is facilitated by translation initiation factors interacting with the m(7)G cap and/or poly(A) tail. The 40S ribosome recruited to an mRNA is then transferred to the AUG initiation codon with the help of translation initiation factors. To understand the mechanism by which the ribosome finds an initiation codon, we investigated the role of eIF4G in finding the translational initiation codon. An artificial polypeptide eIF4G fused with MS2 was localized downstream of the reporter gene through MS2-binding sites inserted in the 3' UTR of the mRNA. Translation of the reporter was greatly enhanced by the eIF4G-MS2 fusion protein regardless of the presence of a cap structure. Moreover, eIF4G-MS2 tethered at the 3' UTR enhanced translation of the second cistron of a dicistronic mRNA. The encephalomyocarditis virus internal ribosome entry site, a natural translational-enhancing element facilitating translation through an interaction with eIF4G, positioned downstream of a reporter gene, also enhanced translation of the upstream gene in a cap-independent manner. Finally, we mathematically modeled the effect of distance between the cap structure and initiation codon on the translation efficiency of mRNAs. The most plausible explanation for translational enhancement by the translational-enhancing sites is recognition of the initiation codon by the ribosome bound to the ribosome-recruiting sites through "RNA looping." The RNA looping hypothesis provides a logical explanation for augmentation of translation by enhancing elements located upstream and/or downstream of a protein-coding region.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Códon de Iniciação/metabolismo , Conformação de Ácido Nucleico , Iniciação Traducional da Cadeia Peptídica/fisiologia , Capuzes de RNA/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Códon de Iniciação/genética , Genes Reporter , Células HEK293 , Humanos , Capuzes de RNA/genética , Subunidades Ribossômicas Menores de Eucariotos/genéticaRESUMO
It has long been considered that intron-containing (spliced) mRNAs are translationally more active than intronless mRNAs (identical mRNA not produced by splicing). The splicing-dependent translational enhancement is mediated, in part, by the exon junction complex (EJC). Nonetheless, the molecular mechanism by which each EJC component contributes to the translational enhancement remains unclear. Here, we demonstrate the previously unappreciated role of eukaryotic translation initiation factor 4AIII (eIF4AIII), a component of EJC, in the translation of mRNAs bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein 80 (CBP80) and CBP20. eIF4AIII is recruited to the 5'-end of mRNAs bound by the CBC by direct interaction with the CBC-dependent translation initiation factor (CTIF); this recruitment of eIF4AIII is independent of the presence of introns (deposited EJCs after splicing). Polysome fractionation, tethering experiments, and in vitro reconstitution experiments using recombinant proteins show that eIF4AIII promotes efficient unwinding of secondary structures in 5'UTR, and consequently enhances CBC-dependent translation in vivo and in vitro. Therefore, our data provide evidence that eIF4AIII is a specific translation initiation factor for CBC-dependent translation.
Assuntos
Regiões 5' não Traduzidas/genética , Núcleo Celular/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Capuzes de RNA/metabolismo , Replicação do DNA , Regulação para Baixo , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Íntrons/genética , Modelos Biológicos , Ligação Proteica , Splicing de RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
In this study, variations of particulate matter (PM) concentrations in subway trains following installation of platform screen doors (PSDs) in the Seoul subway system were investigated. PM samples were collected in the trains on subway lines 1-8 before and after installation of PSDs. It was found that the mean PM10 concentration in the trains after PSDs installation increased significantly by 29.9% compared to that before installation. In particular, the increase of PM10 in line 6 was the highest at 103%. When the relationship between PM10 and PM2.5 was compared, coefficients of determination (r2) before and after PSDs installations were 0.696 and 0.169, respectively. This suggests that air mixing between the platform and the tunnel after PSDs installation was extremely restricted. In addition, the indoor/outdoor PM10 ratio following PSDs installation increased from 1.32 to 2.97 relative to the period with no installed PSDs. Furthermore, this study revealed that PM levels in subway trains increased significantly after all underground PSDs were put in use. Several potential factors were examined that could result in this PM increase, such as train ventilation systems, operational conditions, passenger volume, subway depth, and the length of underground segments. Implications: PM10 concentrations inside the subway trains increased after PSDs installation. This indicates that air quality in trains was very seriously impacted by PSDs. PM10 levels were also influenced by the tunnel depth and length of the underground segments. To prevent the adverse effect on human health by PM10 emitted from the tunnel, an applicable ventilation system to reduce PM10 is required inside trains and tunnels.
Assuntos
Poluição do Ar em Ambientes Fechados/prevenção & controle , Meios de Transporte , Poluentes Atmosféricos/química , Cidades , Material Particulado/química , República da CoreiaRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene silencing by binding to complementary target mRNAs and recruiting the miRNA-containing ribonucleoprotein complexes to the mRNAs. However, the molecular basis of this silencing is unclear. Here, we show that human Ago2 associates with the cap-binding protein complex and this association is mediated by human eIF4GI, a scaffold protein required for the translation initiation. Using a cap photo-crosslinking method, we show that Ago2 closely associates with the cap structure. Taken together, these data suggest that eIF4GI participates in the miRNA-mediated post-transcriptional gene silencing by promoting the association of Ago2 with the cap-binding complex.
Assuntos
Fator de Iniciação Eucariótico 4G/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Proteínas Argonautas/metabolismo , Linhagem Celular , Ordem dos Genes , Humanos , MicroRNAs/metabolismo , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Capuzes de RNA/metabolismoRESUMO
The repression of translation in environmentally stressed eukaryotic cells causes the sequestration of translation initiation factors and the 40S ribosomal subunit into discrete cytoplasmic foci called stress granules (SGs). Most components of the preinitiation complex, such as eIF3, eIF4A, eIF4E, eIF4G, and poly(A)-binding protein, congregate into SGs under stress conditions. However, the molecular basis of translation factor sequestration into SGs has not been clearly elucidated. Here, we report that proline-rich transcript in brain (PRTB) protein interacts with eIF4G and participates in SG formation. PRTB was recruited to SG under sodium arsenite and heat stress conditions. When overexpressed, PRTB inhibited global translation and formed SGs containing TIA-1, eIF4G, and eIF3. Knockdown of PRTB reduced the SG formation induced by sodium arsenite. These results suggest that PRTB not only is a component of SG formed by cellular stresses but also plays an important role in SG formation via an interaction with the scaffold protein eIF4G, which is associated with many translation factors and mRNAs.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Peptídeos/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos/genética , Ligação Proteica , Biossíntese de Proteínas , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
The cellular stress response (SR) is a phylogenetically conserved protection mechanism that involves inhibition of protein synthesis through recruitment of translation factors such as eIF4G into insoluble stress granules (SGs) and blockade of proinflammatory responses by interruption of the signaling pathway from tumor necrosis factor alpha (TNF-alpha) to nuclear factor-kappaB (NF-kappaB) activation. However, the link between these two physiological phenomena has not been clearly elucidated. Here we report that eIF4GI, which is a scaffold protein interacting with many translation factors, interacts with TRAF2, a signaling molecule that plays a key role in activation of NF-kappaB through TNF-alpha. These two proteins colocalize in SGs during cellular exposure to stress conditions. Moreover, TRAF2 is absent from TNFR1 complexes under stress conditions even after TNF-alpha treatment. This suggests that stressed cells lower their biological activities by sequestration of translation factors and TRAF2 into SGs through a protein-protein interaction.