scholarly journals EZH2 co-opts gain-of-function p53 mutants to promote cancer growth and metastasis

2018 ◽  
Author(s):  
Yu Zhao ◽  
Liya Ding ◽  
Dejie Wang ◽  
Zhenqing Ye ◽  
Yunqian Pan ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
P53 Protein ◽  
Protein Translation ◽  
Cancer Growth ◽  
Common Mechanism ◽  
Entry Site ◽  
Gain Of Function ◽  
Independent Manner ◽  
Internal Ribosome Entry ◽  
P53 Mrna

AbstractWith the unfolding of more and more cancer-driven gain-of-function (GOF) mutants of p53, it is important to define a common mechanism to systematically target different mutants rather than develop strategies tailored to inhibit each mutant individually. Here, using RNA immunoprecipitation sequencing (RIP-seq) we identified EZH2 as a p53 mRNA-binding protein. EZH2 bound to the internal ribosome entry site (IRES) in the 5’ untranslated region (5’UTR) of p53 mRNA and enhanced p53 protein translation in a methyltransferase-independent manner. EZH2 augmented p53 GOF mutant-mediated cancer growth and metastasis by increasing p53 GOF mutant protein level. EZH2 overexpression associated with the worse outcome only in patients with p53-mutated cancer. Depletion of EZH2 by antisense oligonucleotides inhibited p53 GOF mutant-mediated cancer growth. Our findings reveal a non-methyltransferase function of EZH2 that controls protein translation of p53 GOF mutants, inhibition of which causes synthetic lethality in cancer cells expressing p53 GOF mutants.

Genetic Diversity and Detection of Atypical Porcine Pestivirus Infections

2021 ◽  
Author(s):  
Kylee M Sutton ◽  
Christian W Eaton ◽  
Tudor Borza ◽  
Thomas E Burkey ◽  
Benny E Mote ◽  
...  
Keyword(s):  
Rna Virus ◽  
Protein Translation ◽  
Template Switching ◽  
Substantial Variation ◽  
Functional Importance ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Viral Cdna ◽  
Viral Polyprotein ◽  
Congenital Tremor

Abstract Atypical porcine pestivirus (APPV), an RNA virus member of the Flaviviridae family, has been associated with congenital tremor in newborn piglets. Previously reported qPCR-based assays were unable to detect APPV in novel cases of congenital tremor originated from multiple farms from U.S. Midwest (MW). These assays targeted the viral polyprotein coding genes, which were shown to display substantial variation, with sequence identity ranging from 58.2 to 70.7% among 15 global APPV strains. In contrast, the 5’ UTR was found to have a much higher degree of sequence conservation. In order to obtain the complete 5’ UTR of the APPV strains originated from MW, the 5’ end of the viral cDNA was obtained by using template switching approach followed by amplification and dideoxy sequencing. Eighty one percent of the 5’UTR was identical across 14 global and 5 MW strains with complete, or relatively complete 5’ UTR. Notably, some of the most highly conserved 5’UTR segments overlapped with potentially important regions of an internal ribosome entry site (IRES), suggesting their functional role in viral protein translation. A newly designed single qPCR assay, targeting 100% conserved 5’UTR regions across 19 strains, was able to detect APPV in samples of well documented cases of congenital tremor which originated from five MW farm sites (1-18 samples/site). As these fully conserved 5’ UTR sequences may have functional importance, we expect that assays targeting this region would broadly detect APPV strains that are diverse in space and time.

scholarly journals BDNF-induced local translation of GluA1 is regulated by HNRNP A2/B1

2020 ◽  
Vol 6 (47) ◽  
pp. eabd2163
Author(s):  
Youngseob Jung ◽  
Ji-Young Seo ◽  
Hye Guk Ryu ◽  
Do-Yeon Kim ◽  
Kyung-Ha Lee ◽  
...  
Keyword(s):  
Ampa Receptor ◽  
Molecular Mechanisms ◽  
Receptor Expression ◽  
Spine Density ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry ◽  
Ampa Receptor Subunit ◽  
Internal Initiation ◽  
Underlying Mechanisms

The AMPA receptor subunit GluA1 is essential for induction of synaptic plasticity. While various regulatory mechanisms of AMPA receptor expression have been identified, the underlying mechanisms of GluA1 protein synthesis are not fully understood. In neurons, axonal and dendritic mRNAs have been reported to be translated in a cap-independent manner. However, molecular mechanisms of cap-independent translation of synaptic mRNAs remain largely unknown. Here, we show that GluA1 mRNA contains an internal ribosome entry site (IRES) in the 5′UTR. We also demonstrate that heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 interacts with GluA1 mRNA and mediates internal initiation of GluA1. Brain-derived neurotrophic factor (BDNF) stimulation increases IRES-mediated GluA1 translation via up-regulation of HNRNP A2/B1. Moreover, BDNF-induced GluA1 expression and dendritic spine density were significantly decreased in neurons lacking hnRNP A2/B1. Together, our data demonstrate that IRES-mediated translation of GluA1 mRNA is a previously unidentified feature of local expression of the AMPA receptor.

scholarly journals Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

2015 ◽  
Vol 89 (19) ◽  
pp. 10031-10043 ◽  
Author(s):  
Hua Zhang ◽  
Lei Song ◽  
Haolong Cong ◽  
Po Tien
Keyword(s):  
Life Cycle ◽  
Internal Ribosome Entry Site ◽  
Viral Protein ◽  
Nuclear Protein ◽  
Binding Protein ◽  
Enterovirus 71 ◽  
Protein Translation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cellular Factors

ABSTRACTEnterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES)trans-acting factor (ITAF) that binds specifically to the EV71 5′ untranslated region (5′UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation.IMPORTANCEThe nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection.

scholarly journals Roles of the 5′ Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Tomohisa Tanaka ◽  
Teruhime Otoguro ◽  
Atsuya Yamashita ◽  
Hirotake Kasai ◽  
Takasuke Fukuhara ◽  
...  
Keyword(s):  
Viral Replication ◽  
Internal Ribosome Entry Site ◽  
Untranslated Region ◽  
Rna Stability ◽  
Specific Region ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Independent Manner ◽  
Internal Ribosome Entry ◽  
Domain I

ABSTRACTThe 5′ untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5′ UTR consisting of a large 5′-terminal domain (I); three additional domains (I′, II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5′ UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5′ UTR was lower than that of the HCV 5′ UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5′ UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I′ of EHcV (the 5′-proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I′ improves RNA stability and viral replication regardless of miR-122 expression. The 5′-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.IMPORTANCEEHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs in horses, donkeys, and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5′ untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5′-proximal EHcV-specific region enhances viral replication and RNA stability in a miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5′ UTR contributes to the differences in tissue tropism observed between these hepaciviruses.

Apoptosis-Induced Wnt2/β-Catenin Signaling Triggers Astrocytic Dedifferentiation and Facilitates Neurogenesis in Ischemic Cortex

2020 ◽  
Author(s):  
Hong Fan ◽  
Jialei Yang ◽  
Junling Xing ◽  
Baolin Guo ◽  
Wenting Wang ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Caspase 3 ◽  
Protein Translation ◽  
Underlying Mechanism ◽  
Reactive Astrocytes ◽  
Entry Site ◽  
Potential Therapeutic Target ◽  
Internal Ribosome Entry ◽  
Cerebral Ischemic ◽  
Canonical Wnt

Abstract Background: Reactive astrogliosis is a common pathologic change of various neurological disorders and usually takes some properties of neural progenitors. This dedifferentiation response of reactive astrocytes to injury is thought as an endogenous cellular attempt for neuronal regeneration, but the underlying mechanism remains largely unclear. Methods: A focal cerebral ischemic model was adopted to assess the dedifferentiation of reactive astrocytes. Topgal mice (a Wnt signaling reporting mouse line) and Caspase-3-/- mice were used to evaluate the change and roles of Wnt signaling and apoptosis in this process. Virus mediated Wnt2, β-catenin, dnTCF4 and DAP5 manipulation was used to reveal the molecular mechanism of dedifferentiation. Ischemic cortical samples and Wnt2-5UTR sequences of macaca mulatta and human were analyzed to explore if the apoptosis-induced Wnt2 up-regulation was conserved. Results: Focal ischemia induces rapid up-regulation of Wnt2 protein in apoptotic neurons in mice, primates and human, and activation of canonical Wnt signaling in reactive astrocytes. Local delivery of Wnt2 shRNA abolished the dedifferentiation response of astrocytes while over-expressing Wnt2 promoted progenitor marker expression and neurogenesis. Both the activation of Wnt signaling and dedifferentiation of astrocytes was compromised in ischemic caspase-3-/- cortex. Over-expressing stabilized β-catenin not only facilitated neurogenesis but also promoted functional recovery in ischemic caspase-3-/- mice. Apoptotic neurons up-regulated Wnt2 protein via internal ribosome entry site (IRES)-mediated translation. Knocking down death associated protein 5 (DAP5), a key protein in IRES-mediated protein translation, significantly diminished both Wnt activation and astrocyte dedifferentiation. Conclusions: Our data demonstrated a novel apoptosis-initiated Wnt-activating mechanism which triggers the dedifferentiation of reactive astrocytes and facilitates neurogenesis in adult cortex, revealing a “SOS” mechanism for inducing astrocyte dedifferentiation and indicating Wnt2/β-catenin signaling as a potential therapeutic target for ischemic stroke.

scholarly journals The Sua5 Protein Is Essential for Normal Translational Regulation in Yeast

2009 ◽  
Vol 30 (1) ◽  
pp. 354-363 ◽  
Author(s):  
Changyi A. Lin ◽  
Steven R. Ellis ◽  
Heather L. True
Keyword(s):  
Translational Regulation ◽  
Rna Virus ◽  
Critical Role ◽  
Protein Translation ◽  
Nonsense Suppression ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Reading Frame ◽  
Codon Recognition

ABSTRACT The anticodon stem-loop of tRNAs requires extensive posttranscriptional modifications in order to maintain structure and stabilize the codon-anticodon interaction. These modifications also play a role in accommodating wobble, allowing a limited pool of tRNAs to recognize degenerate codons. Of particular interest is the formation of a threonylcarbamoyl group on adenosine 37 (t6A37) of tRNAs that recognize ANN codons. Located adjacent and 3′ to the anticodon, t6A37 is a conserved modification that is critical for reading frame maintenance. Recently, the highly conserved YrdC/Sua5 family of proteins was shown to be required for the formation of t6A37. Sua5 was originally identified in a screen by virtue of its ability to affect expression from an aberrant upstream AUG codon in the cyc1 transcript. Together, these findings implicate Sua5 in protein translation at the level of codon recognition. Here, we show that Sua5 is critical for normal translation. The loss of SUA5 causes increased leaky scanning through AUG codons, +1 frameshifting, and nonsense suppression. In addition, the loss of SUA5 amplifies the 20S RNA virus found in Saccharomyces cerevisiae, possibly through an internal ribosome entry site-mediated mechanism. This study reveals a critical role for Sua5 and the t6A37 modification in translational fidelity.

scholarly journals Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

2001 ◽  
Vol 21 (8) ◽  
pp. 2826-2837 ◽  
Author(s):  
Arun Venkatesan ◽  
Asim Dasgupta
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry

ABSTRACT We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.

scholarly journals DDX21, a Host Restriction Factor of FMDV IRES-Dependent Translation and Replication

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1765
Author(s):  
Sahibzada Waheed Abdullah ◽  
Jin’en Wu ◽  
Yun Zhang ◽  
Manyuan Bai ◽  
Junyong Guan ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Foot And Mouth Disease ◽  
Protein Translation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Host Restriction ◽  
Protein Protein Interaction ◽  
Host Restriction Factor ◽  
Pulldown Assay ◽  
Mouth Disease Virus

In cells, the contributions of DEAD-box helicases (DDXs), without which cellular life is impossible, are of utmost importance. The extremely diverse roles of the nucleolar helicase DDX21, ranging from fundamental cellular processes such as cell growth, ribosome biogenesis, protein translation, protein–protein interaction, mediating and sensing transcription, and gene regulation to viral manipulation, drew our attention. We designed this project to study virus–host interactions and viral pathogenesis. A pulldown assay was used to investigate the association between foot-and-mouth disease virus (FMDV) and DDX21. Further insight into the DDX21–FMDV interaction was obtained through dual-luciferase, knockdown, overexpression, qPCR, and confocal microscopy assays. Our results highlight the antagonistic feature of DDX21 against FMDV, as it progressively inhibited FMDV internal ribosome entry site (IRES) -dependent translation through association with FMDV IRES domains 2, 3, and 4. To subvert this host helicase antagonism, FMDV degraded DDX21 through its non-structural proteins 2B, 2C, and 3C protease (3Cpro). Our results suggest that DDX21 is degraded during 2B and 2C overexpression and FMDV infection through the caspase pathway; however, DDX21 is degraded through the lysosomal pathway during 3Cpro overexpression. Further investigation showed that DDX21 enhanced interferon-beta and interleukin-8 production to restrict viral replication. Together, our results demonstrate that DDX21 is a novel FMDV IRES trans-acting factor, which negatively regulates FMDV IRES-dependent translation and replication.

scholarly journals G-Quadruplex Regulation of VEGFA mRNA Translation by RBM4

2022 ◽  
Vol 23 (2) ◽  
pp. 743
Author(s):  
Kangkang Niu ◽  
Xiaojuan Zhang ◽  
Qisheng Song ◽  
Qili Feng
Keyword(s):  
Mrna Translation ◽  
Vascular Endothelial ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry ◽  
G Quadruplex ◽  
Expression Activity ◽  
Ires Element ◽  
Endothelial Growth Factor ◽  
Vegfa Gene

In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5′-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5′-UTR of vascular endothelial growth factor A (VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.

scholarly journals Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Marie-Jo Halaby ◽  
Yan Li ◽  
Benjamin R. Harris ◽  
Shuxia Jiang ◽  
W. Keith Miskimins ◽  
...  
Keyword(s):  
Dna Damage ◽  
Translational Control ◽  
Genotoxic Stress ◽  
Entry Site ◽  
Rna Helicase A ◽  
Internal Ribosome Entry ◽  
Downstream Target ◽  
Control Protein ◽  
P53 Mrna

Synthesis of the p53 tumor suppressor increases following DNA damage. This increase and subsequent activation of p53 are essential for the protection of normal cells against tumorigenesis. We previously discovered an internal ribosome entry site (IRES) that is located at the 5′-untranslated region (UTR) of p53 mRNA and found that the IRES activity increases following DNA damage. However, the mechanism underlying IRES-mediated p53 translation in response to DNA damage is still poorly understood. In this study, we discovered that translational control protein 80 (TCP80) has increased binding to the p53 mRNAin vivofollowing DNA damage. Overexpression of TCP80 also leads to increased p53 IRES activity in response to DNA damage. TCP80 has increased association with RNA helicase A (RHA) following DNA damage and overexpression of TCP80, along with RHA, leads to enhanced expression of p53. Moreover, we found that MCF-7 breast cancer cells with decreased expression of TCP80 and RHA exhibit defective p53 induction following DNA damage and diminished expression of its downstream target PUMA, a proapoptotic protein. Taken together, our discovery of the function of TCP80 and RHA in regulating p53 IRES and p53 induction following DNA damage provides a better understanding of the mechanisms that regulate IRES-mediated p53 translation in response to genotoxic stress.

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