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

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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Elevated NAD(P) glycohydrolase activity: a possible enzymatic marker for malignancy in Burkitt's lymphoma cells

Blood ◽

10.1182/blood.v60.4.912.912 ◽

1982 ◽

Vol 60 (4) ◽

pp. 912-917 ◽

Cited By ~ 8

Author(s):

H Skala ◽

GM Lenoir ◽

AL Pichard ◽

M Vuillaume ◽

JC Dreyfus

Keyword(s):

Cell Lines ◽

Burkitt’S Lymphoma ◽

Chromosome 8 ◽

Burkitt's Lymphoma ◽

Activity Levels ◽

Lymphoma Cells ◽

Cell Extracts ◽

Enzymatic Marker ◽

The Difference ◽

Burkitt’S Lymphoma Cells

Abstract A comparative analysis of enzymatic activities has been performed on 47 human continuous lymphoid lines: 22 tumors derived from Burkitt's lymphoma lines, 6 other lymphomatous long-term cultures, and 19 nonmalignant ties determined on the cell extracts. 4 showed no significant differences between the various lines. They included adenosine diphosphoribose incorporation, glucose-6-phosphate dehydrogenase, cyclic-AMP phosphodiesterase, and glutathione reductase. However, striking differences of activity were found for the enzyme, NAD(P) glycohydrolase (EC 3.2.2.6). Activity levels were, as a mean, four times higher in Burkitt's lymphoma-derived cell lines than in nonmalignant control lines, and the difference was highly significant (p less than 0.02). All Burkitt cell lines containing translocations of chromosome 8 with either chromosomes 2, 14 or 22 showed an increased activity. The specificity and significance of this possible enzymatic marker of Burkitt's lymphoma cells is discussed.

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Elevated NAD(P) glycohydrolase activity: a possible enzymatic marker for malignancy in Burkitt's lymphoma cells

Blood ◽

10.1182/blood.v60.4.912.bloodjournal604912 ◽

1982 ◽

Vol 60 (4) ◽

pp. 912-917

Author(s):

H Skala ◽

GM Lenoir ◽

AL Pichard ◽

M Vuillaume ◽

JC Dreyfus

Keyword(s):

Cell Lines ◽

Burkitt’S Lymphoma ◽

Chromosome 8 ◽

Burkitt's Lymphoma ◽

Activity Levels ◽

Lymphoma Cells ◽

Cell Extracts ◽

Enzymatic Marker ◽

The Difference ◽

Burkitt’S Lymphoma Cells

A comparative analysis of enzymatic activities has been performed on 47 human continuous lymphoid lines: 22 tumors derived from Burkitt's lymphoma lines, 6 other lymphomatous long-term cultures, and 19 nonmalignant ties determined on the cell extracts. 4 showed no significant differences between the various lines. They included adenosine diphosphoribose incorporation, glucose-6-phosphate dehydrogenase, cyclic-AMP phosphodiesterase, and glutathione reductase. However, striking differences of activity were found for the enzyme, NAD(P) glycohydrolase (EC 3.2.2.6). Activity levels were, as a mean, four times higher in Burkitt's lymphoma-derived cell lines than in nonmalignant control lines, and the difference was highly significant (p less than 0.02). All Burkitt cell lines containing translocations of chromosome 8 with either chromosomes 2, 14 or 22 showed an increased activity. The specificity and significance of this possible enzymatic marker of Burkitt's lymphoma cells is discussed.

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Identification of a novel AU-rich-element-binding protein which is related to AUF1

Biochemical Journal ◽

10.1042/bj20020402 ◽

2002 ◽

Vol 366 (3) ◽

pp. 709-719 ◽

Cited By ~ 34

Author(s):

Jonathan L.E. DEAN ◽

Gareth SULLY ◽

Robin WAIT ◽

Lesley RAWLINSON ◽

Andrew R. CLARK ◽

...

Keyword(s):

Amino Acid ◽

Binding Protein ◽

Early Response ◽

Reporter System ◽

Mobility Shift ◽

Reverse Transcription Pcr ◽

Cell Extracts ◽

C Terminus ◽

Tnf Α ◽

Cox 2

The AU-rich element (ARE) is an important instability determinant for a large number of early-response-gene mRNAs. AREs also mediate the stabilization of certain pro-inflammatory mRNAs, such as tumour necrosis factor (TNF)-α and cyclo-oxygenase-2 (COX-2), in response to inflammatory stimuli. To understand how AREs control mRNA stability, it is necessary to identify trans-acting factors. We have purified a new ARE-binding protein and identified it as CArG box-binding factor-A (CBF-A). The amino acid sequence of CBF-A is highly similar to that of the ARE-binding protein AUF1. Recombinant CBF-A bound the COX-2 and TNF-α AREs, but not a non-specific control RNA. In contrast, in an electrophoretic-mobility-shift assay (EMSA) of crude RAW 264.7 macrophage-like cell extracts, an antiserum that recognizes both AUF1 and CBF-A failed to supershift complexes formed on the TNF-α ARE, but did supershift a complex specific for the COX-2 ARE. CBF-A exists as two isoforms, p37 and p42, that differ by a 47-amino-acid insertion close to the C-terminus. By expressing epitope-tagged isoforms of CBF-A it was shown that the p42 isoform binds the COX-2 ARE in EMSA of crude cell extracts. In a HeLa-cell tetracycline-regulated reporter system, overexpression of the p42 CBF-A isoform resulted in stabilization of a COX-2 ARE reporter mRNA. Epitope-tagged p42 CBF-A expressed in HeLa cells co-immunoprecipitated with endogenous COX-2 mRNA, but not glyceraldehyde-3-phosphate dehydrogenase mRNA, as shown by reverse-transcription PCR. The similarity between CBF-A and AUF1 suggests that CBF-A could be re-named AUF2.

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Interaction of Poly(rC) Binding Protein 2 with the 5′ Noncoding Region of Hepatitis A Virus RNA and Its Effects on Translation

Journal of Virology ◽

10.1128/jvi.72.12.9668-9675.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 9668-9675 ◽

Cited By ~ 54

Author(s):

Judith Graff ◽

John Cha ◽

Lawrence B. Blyn ◽

Ellie Ehrenfeld

Keyword(s):

Hepatitis A ◽

Hepatitis A Virus ◽

Binding Protein ◽

Noncoding Region ◽

Affinity Column ◽

Mobility Shift ◽

Stem Loop ◽

Rna Translation ◽

Cell Extracts ◽

Initiation Of Translation

ABSTRACT Utilization of internal ribosome entry segment (IRES) structures in the 5′ noncoding region (5′NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5′NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5′NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5′-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.

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Mediation of the hormone- and serum-dependent regulation of thyroglobulin gene expression by thyroid-transcription factors in rat thyroid FRTL-5 cells

Journal of Endocrinology ◽

10.1677/joe.0.1500287 ◽

1996 ◽

Vol 150 (2) ◽

pp. 287-298 ◽

Cited By ~ 10

Author(s):

F Kambe ◽

H Seo

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Gene Transcription ◽

Mrna Level ◽

Binding Activity ◽

Mrna Levels ◽

Mobility Shift ◽

Cell Extracts ◽

Rat Thyroid ◽

Serum Dependent

Abstract The molecular mechanism for hormone- and serum-dependent regulation of thyroglobulin (TG) gene expression was studied. A construct of rat TG promoter (−178 to −3) linked to a luciferase gene was transfected into TSH-, insulin- and serum-deprived FRTL-5 cells. Addition of TSH, insulin or serum augmented the luciferase activity. The endogenous TG mRNA level was also increased, indicating that the promoter used confers responsiveness of TG gene to these additives. The possible involvement of thyroid-transcription factors, TTF-1, TTF-2 and Pax-8, in the induction of TG gene transcription was studied using an electrophoretic mobility shift assay. Since the protein/DNA ratio in FRTL-5 cell extracts was significantly increased by these additives, binding activities of these factors per unit of DNA were examined. It was demonstrated that TSH, insulin or serum increased not only TTF-2 binding activity but also the binding activities of TTF-1 and Pax-8. However, the magnitude of the increase in TTF-1 and Pax-8 mRNA levels per unit of DNA was less than that of the binding activity. Taken together, our results suggest that TSH, insulin and serum increase the binding activities of TTF-1 and Pax-8 to the TG promoter presumably through the posttranslational modification of the factors, thereby enhancing TG gene transcription. Journal of Endocrinology (1996) 150, 287–298

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

International Journal of Molecular Sciences ◽

10.3390/ijms222413306 ◽

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.

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