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.

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 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.

Inhibition of Wnt/ß-Catenin Pathway with ICG-001 Induces Apoptosis in Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2951-2951
Author(s):  
Eileen R Grigson ◽  
Alexandra Pisklakova ◽  
Brittany Weissman ◽  
Yulia Nefedova
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Wnt Signaling ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Caspase 3 ◽  
Control Group ◽  
Canonical Wnt Signaling ◽  
Canonical Wnt ◽  
Rpmi 8226

Abstract Abstract 2951 Multiple Myeloma (MM) is characterized by clonal expansion of malignant plasma cells, which preferentially reside in the bone marrow (BM). It has been previously shown that canonical Wnt signaling plays a critical role in proliferation and differentiation in MM. ICG-001 is a selective Wnt/ß-catenin signaling inhibitor that binds to the amino terminus of CREB, thus preventing ß-catenin/TCF signaling in the nucleus. Here we investigated whether the pharmacological inhibition of canonical Wnt signaling with ICG-001 could affect viability of MM cells and produce anti-myeloma effect. C-myc and cyclin D1 are known to be downstream target genes of canonical Wnt signaling. MM cells lines treated with ICG-001 revealed down regulation of c-myc and cyclin D1 as compared to control. To investigate the varying sensitivity of cell lines to ICG-001, four cells lines were used: RPMI-8226, NCI-H929, U266, and MM1S. MM cells lines were treated with ICG-001 (1uM – 40uM) and data was collected using MTT assay or flow cytometry (Annexin binding assay). ICG-001 significantly reduced viability in all four cell lines due to induction of apoptosis, with RPMI-8226 showing the greatest sensitivity. We performed cleaved-caspase assays and demonstrated that apoptosis was caspase-3 dependent. We have also evaluated the effect of ICG-001 in BM samples from patients with MM. BM cells were treated with ICG-001 for 24 hours and the level of cleaved-caspase-3 was evaluated by flow cytometry. MM cells were defined as double positive CD138+ and Lambda/Kappa+. Our results demonstrated that MM cells were sensitive to ICG-001, while this drug was not able to induce cell death in non-tumor BM cells. To test the effect of ICG-001 in vivo we injected SCID/Beige mice with 10 × 105 8226 cells in 100 μL phosphate-buffered saline (PBS). After 3 weeks when a tumor was measurable, the mice were split into two groups and were treated with either ICG-001 (100 mg/kg) or vehicle control (PBS) daily, for 14 days. Tumor size was consistently monitored during treatment and 3 weeks after treatment. We observed that ICG-001 produced a significant antitumor effect as compared to control group. These data indicate targeting Wnt/ß-catenin Pathway with ICG-001 could be therapeutically beneficial to patients with MM. Disclosures: No relevant conflicts of interest to declare.

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 Klotho/FGF23 and Wnt Signaling as Important Players in the Comorbidities Associated with Chronic Kidney Disease

Toxins ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 185 ◽  
Author(s):  
Juan Rafael Muñoz-Castañeda ◽  
Cristian Rodelo-Haad ◽  
Maria Victoria Pendon-Ruiz de Mier ◽  
Alejandro Martin-Malo ◽  
Rafael Santamaria ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Renal Function ◽  
Kidney Disease ◽  
Wnt Signaling ◽  
Essential Role ◽  
Mineral Metabolism ◽  
Fibroblast Growth Factor 23 ◽  
Renal Tubules ◽  
Potential Therapeutic Target ◽  
Canonical Wnt

Fibroblast Growth Factor 23 (FGF23) and Klotho play an essential role in the regulation of mineral metabolism, and both are altered as a consequence of renal failure. FGF23 increases to augment phosphaturia, which prevents phosphate accumulation at the early stages of chronic kidney disease (CKD). This effect of FGF23 requires the presence of Klotho in the renal tubules. However, Klotho expression is reduced as soon as renal function is starting to fail to generate a state of FGF23 resistance. Changes in these proteins directly affect to other mineral metabolism parameters; they may affect renal function and can produce damage in other organs such as bone, heart, or vessels. Some of the mechanisms responsible for the changes in FGF23 and Klotho levels are related to modifications in the Wnt signaling. This review examines the link between FGF23/Klotho and Wnt/β-catenin in different organs: kidney, heart, and bone. Activation of the canonical Wnt signaling produces changes in FGF23 and Klotho and vice versa; therefore, this pathway emerges as a potential therapeutic target that may help to prevent CKD-associated complications.

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.

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 Constitutive translation of human α-synuclein is mediated by the 5′-untranslated region

Open Biology ◽  
2016 ◽  
Vol 6 (4) ◽  
pp. 160022 ◽  
Author(s):  
Pelagia Koukouraki ◽  
Epaminondas Doxakis
Keyword(s):  
Untranslated Region ◽  
Mrna Translation ◽  
Protein Translation ◽  
Serum Starvation ◽  
Mrna Levels ◽  
Entry Site ◽  
Rna Motifs ◽  
Physiological Mechanisms ◽  
Internal Ribosome Entry ◽  
G Quadruplex

Genetic and biochemical studies have established a central role for α-synuclein (SNCA) accumulation in the pathogenesis of Parkinson's disease. Uncovering and subsequently interfering with physiological mechanisms that control SNCA expression is one approach to limit disease progression. To this end, the long and GC-rich 5′-untranslated region (UTR) of SNCA, which is predicted to fold into stable hairpin and G-quadruplex RNA motifs, was investigated for its role in mRNA translation. Inclusion of SNCA 5′-UTR significantly induced expression of both SNCA and luciferase ORF constructs. This effect was not associated with a change in mRNA levels or differential nucleocytoplasmic shuttling. Further, the presence of the 5′-UTR enhanced SNCA synthesis when cap-dependent translation was attenuated with rapamycin treatment. Analysis using multiple methodologies revealed that the 5′-UTR harbours an internal ribosome entry site (IRES) element that spans most of its nucleotide sequence. Signals such as plasma-membrane depolarization, serum starvation and oxidative stress stimulated SNCA protein translation via its 5′-UTR as well as enhanced its IRES activity. Taken together, these data support the idea that the 5′-UTR is an important positive regulator of SNCA synthesis under diverse physiological and pathological conditions, explaining in part the abundance of SNCA in healthy neurons and its accumulation in degenerative cells.

scholarly journals RNA-seq reads of Influenza A genome appended with DNA-expression vector sequences facilitating protein transcription (18s) in Covid19 patients from Wuhan and Hong Kong - very worrying as this is unlikely to be contamination

2020 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Influenza A ◽  
Ribosomal Subunit ◽  
Protein Translation ◽  
Lavage Fluid ◽  
Expression Vectors ◽  
Rna Seq ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
A Genome ◽  
Initiation Sequence

Some viruses like Hepatitis C virus (HCV) have hijacked the human protein translation machinery [1], having by an ‘internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions’ [2]. Expression vectors are designed to facilitate gene expression, by adding a promoter, a proper translation initiation sequence (a ribosomal binding site) and start/termination codon, followed by a transcription termination sequence.Metagenome from the bronchoalveolar lavage fluid of 5 Covid19 [3–5] patients from Wuhan (Accid:PRJNA605983) shows co-infection (sequences in SI.flu.zip) with influenza A virus (3 out of 5 patients). More worryingly,these reads show the presence of 18s sequences that are found in expression vectors to facilitate protein translation (Fig. 1), showing highest homology to a synthetic construct (H1N1 HA gene, Accid:KY199426.1)used in a ‘digital-to-biological converter for on-demand production of biologics’ [6]. Along with SARS-Cov2 infection, a few patients are also infected with Nipah [7]. Its inconceivable that a BSL-4 facility will have so many contamination. Furthermore, the sequencing was RNA-seq - while the expression vectors are DNA - and thus need to be transcribed to be seen in RNA-seq data.

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