scholarly journals Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5′-Terminal Region of p53 mRNA

2021 ◽  
Vol 22 (24) ◽  
pp. 13306
Author(s):  
Damian M. Janecki ◽  
Agata Swiatkowska ◽  
Joanna Szpotkowska ◽  
Anna Urbanowicz ◽  
Martyna Kabacińska ◽  
...  
Keyword(s):  
Binding Protein ◽  
P53 Protein ◽  
Mrna Level ◽  
Genotoxic Stress ◽  
Stress Conditions ◽  
Mobility Shift ◽  
P53 Expression ◽  
Double Function ◽  
Mobility Shift Assay ◽  
P53 Mrna

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5′ terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3′ end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5′ terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.

scholarly journals A single synonymous mutation determines the phosphorylation and stability of the nascent protein

2018 ◽  
Vol 11 (3) ◽  
pp. 187-199 ◽  
Author(s):  
Konstantinos Karakostis ◽  
Sivakumar Vadivel Gnanasundram ◽  
Ignacio López ◽  
Aikaterini Thermou ◽  
Lixiao Wang ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
P53 Protein ◽  
Intrinsically Disordered Protein ◽  
Subcellular Location ◽  
Genotoxic Stress ◽  
Synonymous Mutation ◽  
Atm Kinase ◽  
Intrinsically Disordered ◽  
The Stability ◽  
P53 Mrna

Abstract p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

scholarly journals Downregulation of PU.1 Leads to Decreased Expression of Dectin-1 in Alveolar Macrophages during Pneumocystis Pneumonia

2010 ◽  
Vol 78 (3) ◽  
pp. 1058-1065 ◽  
Author(s):  
Chen Zhang ◽  
Shao-Hung Wang ◽  
Chung-Ping Liao ◽  
Shoujin Shao ◽  
Mark E. Lasbury ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Mrna Level ◽  
Gene Promoter ◽  
Pneumocystis Pneumonia ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Mobility Shift Assay

ABSTRACT Dectin-1 is an important macrophage phagocytic receptor recognizing fungal β-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

scholarly journals Novel Insights for PI3KC3 in Mediating Lipid Accumulation in Yellow Catfish Pelteobagrus Fulvidraco

2021 ◽  
Author(s):  
Mei-Qin Zhuo ◽  
Jun Chen ◽  
Mei-Li Wu ◽  
Wen-biao Wang
Keyword(s):  
Transcriptional Regulation ◽  
Protein Level ◽  
Lipid Accumulation ◽  
Mrna Level ◽  
Pelteobagrus Fulvidraco ◽  
Yellow Catfish ◽  
Site Mutation ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Mutation Assay

Abstract In this study, the transcriptional regulation of PI3KC3 by three transcript factors (PPARγ, PPARα and STAT3) and the potential role of PI3KC3 in mediating lipid accumulation were determined in yellow catfish Pelteobagrus fulvidraco. The 5’-deletion assay, overexpression assay, site-mutation assay and electrophoretic mobility shift assay suggested that PPARα, PPARγ and STAT3 negatively regulated the promoter activity of pi3kc3. Moreover, the transcriptional inactivation of pi3kc3 was directly mediated by PPARα and PPARγ under fatty acid (FA) treatment. Using primary hepatocytes from yellow catfish, FA incubation significantly increased triacylglyceride (TG), NEFA content, the mRNA level of pparα, pparγ, stat3 and dnmt3b, the protein level of PPARα, PPARγ and STAT3, and the methylation level of pi3kc3, but significantly reduced the mRNA and protein level of PI3KC3. Our findings offer new insights into the mechanisms for transcriptional regulation of PI3KC3 and for PI3KC3-mediated lipid accumulation in fish.

scholarly journals Interaction of Transcriptional Repressor ArgR with Transcriptional Regulator FarR at theargBPromoter Region inCorynebacterium glutamicum

2010 ◽  
Vol 77 (3) ◽  
pp. 711-718 ◽  
Author(s):  
Soo Youn Lee ◽  
Jae-Min Park ◽  
Jin Hyung Lee ◽  
Suk-Tai Chang ◽  
Jin-Soo Park ◽  
...  
Keyword(s):  
Dna Binding ◽  
Promoter Region ◽  
Mrna Level ◽  
Protein A ◽  
Transcriptional Repressor ◽  
Binding Domain ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Do So

ABSTRACTInCorynebacterium glutamicum, the ArgR protein, a transcriptional repressor, affects the expression level of theargBgene through binding to its promoter region. TheargBpromoter region (positions −77 to −25) has been found byin vitroelectrophoretic mobility shift assay (EMSA) results andin silicoanalysis to be important for the DNA binding of ArgR. Proline supplementation prevented the DNA binding of ArgR to theargBpromoter region and triggered an increase of theargBmRNA level. Additional mutational analyses of theargBpromoter region found nucleotides critical for ArgR binding (G located at position −58, C at position −55, and A at position −41 of theargBpromoter) in that region. Another transcriptional repressor, FarR, was also demonstrated to bind to theargBpromoter region. This binding was delimited to positions −57 to −77 on theargBpromoter. FarR has only one putative binding domain located at positions −57 to −77, but this region exactly overlapped with the binding region located from positions −55 to −77 for the binding of ArgR within theargBpromoter; thus, if ArgR bound with theargBpromoter first, the binding of FarR was not observed in this region. However, if FarR bound to the binding domain located at positions −57 to −77 first, ArgR could bind other binding sites located at positions −49 to −25 within theargBpromoter. Finally, this study suggests that ArgR can affect FarR binding to theargBpromoter region, as protein binding is dominated by the protein most able to do so.

scholarly journals Influence of the RNA-Binding Protein HuR in pVHL-Regulated p53 Expression in Renal Carcinoma Cells

2003 ◽  
Vol 23 (20) ◽  
pp. 7083-7095 ◽  
Author(s):  
Stefanie Galbán ◽  
Jennifer L. Martindale ◽  
Krystyna Mazan-Mamczarz ◽  
Isabel López de Silanes ◽  
Jinshui Fan ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Carcinoma Cells ◽  
Recent Analysis ◽  
P53 Expression ◽  
P53 Mrna

ABSTRACT A recent analysis of gene expression in renal cell carcinoma cells led to the identification of mRNAs whose translation was dependent on the presence of the von Hippel-Lindau (VHL) tumor suppressor gene product, pVHL. Here, we investigate the finding that pVHL-expressing RCC cells (VHL+) exhibited elevated levels of polysome-associated p53 mRNA and increased p53 protein levels compared with VHL-defective (VHL−) cells. Our findings indicate that p53 translation is specifically heightened in VHL+ cells, given that (i) p53 mRNA abundance in VHL+ and VHL− cells was comparable, (ii) p53 degradation did not significantly influence p53 expression, and (iii) p53 synthesis was markedly induced in VHL+ cells. Electrophoretic mobility shift and immunoprecipitation assays to detect endogenous and radiolabeled p53 transcripts revealed that the RNA-binding protein HuR, previously shown to regulate mRNA turnover and translation, was capable of binding to the 3′ untranslated region of the p53 mRNA in a VHL-dependent fashion. Interestingly, while whole-cell levels of HuR in VHL+ and VHL− cells were comparable, HuR was markedly more abundant in the cytoplasmic and polysome-associated fractions of VHL+ cells. In keeping with earlier reports, the elevated cytoplasmic HuR in VHL+ cells was likely due to the reduced AMP-activated kinase activity in these cells. Demonstration that HuR indeed contributed to the increased expression of p53 in VHL+ cells was obtained through use of RNA interference, which effectively reduced HuR expression and in turn caused marked decreases in p53 translation and p53 abundance. Taken together, our findings support a role for pVHL in elevating p53 expression, implicate HuR in enhancing VHL-mediated p53 translation, and suggest that VHL-mediated p53 upregulation may contribute to pVHL's tumor suppressive functions in renal cell carcinoma.

Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues

2000 ◽  
Vol 349 (2) ◽  
pp. 629-634 ◽  
Author(s):  
Tatsuo FURUYAMA ◽  
Toru NAKAZAWA ◽  
Itsuko NAKANO ◽  
Nozomu MORI
Keyword(s):  
Target Genes ◽  
Binding Protein ◽  
Expression Patterns ◽  
Distribution Patterns ◽  
Mobility Shift ◽  
Differential Distribution ◽  
C Elegans ◽  
Core Sequence ◽  
Mobility Shift Assay ◽  
Responsive Element

daf-16 is a forkhead-type transcription factor, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-16 homologues include AFX, FKHR and FKHRL1, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-16. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-16 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL1 mRNAs were complementary to each other and were highest in muscle, adipose tissue and embryonic liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL1 became evident in more tissues, including the brain. In order to clarify whether each DAF-16 homologue had different target genes, we determined the consensus sequences for the binding of DAF-16 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-16 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-16 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like growth factor binding protein-1 promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of sod-3 by searching the genomic database of C. elegans. Taken together, DAF-16 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the differences in their expression patterns.

Identification of a transcription factor, an 80-kDa protein that interacts with the HLH recognition motif of the rat p53 promoter

2001 ◽  
Vol 79 (2) ◽  
pp. 153-158 ◽  
Author(s):  
Haisun Song ◽  
Minhyung Lee ◽  
Sunhee Yu ◽  
Jong-sang Park
Keyword(s):  
Transcription Factor ◽  
In Vitro Transcription ◽  
Mobility Shift ◽  
P53 Expression ◽  
Dnase I Footprinting ◽  
E Box ◽  
Molecular Masses ◽  
Mobility Shift Assay ◽  
Stimulating Factor

The p53 promoter has been shown to contain a number of potential regulatory motifs. It was previously reported that the upstream stimulating factor (USF) played a central role in regulating the p53 expression. The USF binding site, E-box, is located around 40 bp upstream of the major transcription start site. In this study, it was confirmed that the E-box binds to proteins by DNase I footprinting assay. In the electrophoretic mobility shift assay (EMSA), two retarded bands were detected. One band was abolished by the competition of USF consensus oligonucleotide, but the other band was not. This result indicated that a factor, other than USF, was bound to the E-box. The molecular masses of the binding proteins were determined by a Southwestern-blotting assay. As a result, 46- and 80-kDa proteins were detected. The 46-kDa protein was eliminated by the competition of USF consensus oligonucleotide. Also, the Southwestern-blotting assay with 32P-labeled USF consensus oligonucleotide showed only a 46-kDa protein. Therefore, the 46-kDa protein was USF. These results showed that USF and the 80-kDa protein were bound to the E-box. In addition, it was proved by in vitro transcription assay that this 80-kDa protein had a basal transcriptional activity.Key words: E-box, HLH, rat p53 promoter, transcription factor, upstream stimulating factor (USF).

scholarly journals Cloning of a functional Burkitt's lymphoma polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) gene promoter by the polymerase chain reaction, and its interaction with inducible cellular factors

1992 ◽  
Vol 286 (2) ◽  
pp. 555-559 ◽  
Author(s):  
C C K Chao ◽  
W C Yam ◽  
L K Chen ◽  
S Lin-Chao
Keyword(s):  
Binding Protein ◽  
Mrna Level ◽  
Burkitt’S Lymphoma ◽  
Burkitt's Lymphoma ◽  
Sequence Motif ◽  
Ionophore A23187 ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Nuclear Factors ◽  
Glucose Regulated Protein

The promoter of the human gene encoding the stress-responsive protein polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) was isolated from Burkitt's lymphoma cells by PCR. This promoter DNA segment (termed BiP670) or one of its 5′ deletion derivatives was fused to the bacterial chloramphenicol acetyltransferase gene and introduced into HeLa cells for transient expression. BiP670 retained transcriptional activity at both the basal and Ca2+ ionophore A23187-inducible levels. However, there was no significant increase in promoter activity following a 5 h induction with 7 microM-A23187, and less than 5-fold induction at 15 h. In contrast, the steady-state mRNA level was induced by 18-fold at 5 h. The in vivo transactivation assays with BiP670 5′ deletion derivatives indicate that the putative A23187-inducible element is located within a 70 bp DNA segment (i.e. spanning -39 to -107 bp upstream of the transcriptional initiation site). Using an in vitro gel mobility shift assay, A23187-inducible nuclear factors were identified from HeLa cell extracts. DNA-binding competition experiments also suggest that the 70 bp DNA segment contains a potential sequence motif for the binding of the A23187-inducible nuclear factors.

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