Abstract

Murine erythroleukemia (MEL) cells are an established system for studying erythroid differentiation. While it is evident that inducers of differentiation must affect gene expression, their mechanism of action remains unknown. The investigators have recently determined that inducers increase the deadenylation and decrease the stability of Spi-1 mRNA, a transcription factor that is implicated in erythroleukemogenesis. Since, a general increase in mRNA deadenylation follows inducer exposure, this suggests that inducers may globally affect poly(A) metabolism. The poly(A)-binding protein (PABP) has been proposed to mediate the affects of mRNA polyadenylation. In MEL cells, the PABP appears to be present in limiting abundance and is found almost exclusively in polysomes in both control and inducer exposed cells. Since inducers decrease the amount of mRNA in polysomes and increase the amount of mRNA in subpolysomal fractions, these data suggest that inducers result in a net loss of PABP from polysomal mRNAs. The investigators propose that the post-transcriptional effect of inducers is mediated by increasing the rate of removal of the PABP from mRNA poly(A) tails. To test this hypothesis, the proposed experiments will focus on the translational effect of these agents and will: 1) Quantitate the total amount of poly(A) and PABP in fractionated cell extracts to determine if inducers cause a net decrease in the amount of PABP/poly(A) in the cells; 2) Determine the effects of inducers on expression of the PABP and identify the mechanisms that regulate its expression in inducer-exposed cells; 3) Determine the effect of forced overexpression of the PABP on translation, deadenylation and stability of mRNAs in MEL cells and determine if this affects the induction of cell differentiation; and 4) Determine if mRNAs require a 5'-terminal oligopyrimidine tract to be subject to the translational effect of inducers. These experiments may provide significant insight into the function of the PABP and the role that this protein plays in the changes in gene expression that follow inducer exposure of MEL cells

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK043414-06
Application #
2872195
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Badman, David G
Project Start
1992-05-01
Project End
2002-01-31
Budget Start
1999-02-01
Budget End
2000-01-31
Support Year
6
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Case Western Reserve University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106

  Publications

Komar, Anton A; Gross, Stephane R; Barth-Baus, Diane et al. (2005) Novel characteristics of the biological properties of the yeast Saccharomyces cerevisiae eukaryotic initiation factor 2A. J Biol Chem 280:15601-11
Constantinou, Constantina; Bushell, Martin; Jeffrey, Ian W et al. (2003) p53-induced inhibition of protein synthesis is independent of apoptosis. Eur J Biochem 270:3122-32
Horton, Lynn E; Bushell, Martin; Barth-Baus, Diane et al. (2002) p53 activation results in rapid dephosphorylation of the eIF4E-binding protein 4E-BP1, inhibition of ribosomal protein S6 kinase and inhibition of translation initiation. Oncogene 21:5325-34
Gelperin, Daniel; Horton, Lynn; DeChant, Anne et al. (2002) Loss of ypk1 function causes rapamycin sensitivity, inhibition of translation initiation and synthetic lethality in 14-3-3-deficient yeast. Genetics 161:1453-64
Zoll, Wendy L; Horton, Lynn E; Komar, Anton A et al. (2002) Characterization of mammalian eIF2A and identification of the yeast homolog. J Biol Chem 277:37079-87
Barth-Baus, Diane; Stratton, Carl A; Parrott, Lou et al. (2002) S6 phosphorylation-independent pathways regulate translation of 5'-terminal oligopyrimidine tract-containing mRNAs in differentiating hematopoietic cells. Nucleic Acids Res 30:1919-28
Gelperin, D; Horton, L; Beckman, J et al. (2001) Bms1p, a novel GTP-binding protein, and the related Tsr1p are required for distinct steps of 40S ribosome biogenesis in yeast. RNA 7:1268-83
Kroll, S L; Barth-Baus, D; Hensold, J O (2001) The carboxyl-terminal domain of the granulocyte colony-stimulating factor receptor uncouples ribosomal biogenesis from cell cycle progression in differentiating 32D myeloid cells. J Biol Chem 276:49410-8
Richter, N J; Rogers Jr, G W; Hensold, J O et al. (1999) Further biochemical and kinetic characterization of human eukaryotic initiation factor 4H. J Biol Chem 274:35415-24
Richter-Cook, N J; Dever, T E; Hensold, J O et al. (1998) Purification and characterization of a new eukaryotic protein translation factor. Eukaryotic initiation factor 4H. J Biol Chem 273:7579-87

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