Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex

Edward Chu, Sitki M. Copur, Jingfang Ju, Tian Men Chen, Samir N. Khleif, Donna M. Voeller, Nobuyuki Mizunuma, Mahendra Patel, Gladys F. Maley, Frank Maley, Carmen J. Allegra

Research output: Contribution to journalArticle

75 Citations (Scopus)

Abstract

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS- overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS- overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

Original languageEnglish (US)
Pages (from-to)1582-1594
Number of pages13
JournalMolecular and Cellular Biology
Volume19
Issue number2
DOIs
StatePublished - Jan 1 1999

Fingerprint

Thymidylate Synthase
Ribonucleoproteins
Messenger RNA
Proteins
Colonic Neoplasms
Polyribosomes
Doxycycline
Tetracycline
Cell Line
RNA
Gene Expression
p53 Genes
Protein Biosynthesis
Regulator Genes
Tumor Suppressor Genes
Open Reading Frames
Hepatocellular Carcinoma
Molecular Weight

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Chu, E., Copur, S. M., Ju, J., Chen, T. M., Khleif, S. N., Voeller, D. M., ... Allegra, C. J. (1999). Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex. Molecular and Cellular Biology, 19(2), 1582-1594. https://doi.org/10.1128/MCB.19.2.1582

Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex. / Chu, Edward; Copur, Sitki M.; Ju, Jingfang; Chen, Tian Men; Khleif, Samir N.; Voeller, Donna M.; Mizunuma, Nobuyuki; Patel, Mahendra; Maley, Gladys F.; Maley, Frank; Allegra, Carmen J.

In: Molecular and Cellular Biology, Vol. 19, No. 2, 01.01.1999, p. 1582-1594.

Research output: Contribution to journalArticle

Chu, E, Copur, SM, Ju, J, Chen, TM, Khleif, SN, Voeller, DM, Mizunuma, N, Patel, M, Maley, GF, Maley, F & Allegra, CJ 1999, 'Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex', Molecular and Cellular Biology, vol. 19, no. 2, pp. 1582-1594. https://doi.org/10.1128/MCB.19.2.1582
Chu, Edward ; Copur, Sitki M. ; Ju, Jingfang ; Chen, Tian Men ; Khleif, Samir N. ; Voeller, Donna M. ; Mizunuma, Nobuyuki ; Patel, Mahendra ; Maley, Gladys F. ; Maley, Frank ; Allegra, Carmen J. / Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex. In: Molecular and Cellular Biology. 1999 ; Vol. 19, No. 2. pp. 1582-1594.
@article{04630197a00e4364bb91722b6179c281,
title = "Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex",
abstract = "A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS- overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS- overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.",
author = "Edward Chu and Copur, {Sitki M.} and Jingfang Ju and Chen, {Tian Men} and Khleif, {Samir N.} and Voeller, {Donna M.} and Nobuyuki Mizunuma and Mahendra Patel and Maley, {Gladys F.} and Frank Maley and Allegra, {Carmen J.}",
year = "1999",
month = "1",
day = "1",
doi = "10.1128/MCB.19.2.1582",
language = "English (US)",
volume = "19",
pages = "1582--1594",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "2",

}

TY - JOUR

T1 - Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex

AU - Chu, Edward

AU - Copur, Sitki M.

AU - Ju, Jingfang

AU - Chen, Tian Men

AU - Khleif, Samir N.

AU - Voeller, Donna M.

AU - Mizunuma, Nobuyuki

AU - Patel, Mahendra

AU - Maley, Gladys F.

AU - Maley, Frank

AU - Allegra, Carmen J.

PY - 1999/1/1

Y1 - 1999/1/1

N2 - A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS- overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS- overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

AB - A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS- overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS- overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

UR - http://www.scopus.com/inward/record.url?scp=0032929245&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032929245&partnerID=8YFLogxK

U2 - 10.1128/MCB.19.2.1582

DO - 10.1128/MCB.19.2.1582

M3 - Article

VL - 19

SP - 1582

EP - 1594

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 2

ER -