Properties and purification of a glucose-inducible human fatty acid synthase mrna binding protein. Li, Qianmei, Michael S. Chua, and Clay F. Semenkovich. Departments of Medicine and Cell Biology & Physiology, Washington University School of Medicine, St. Louis, MO 63110
APStracts 4:0283E, 1997.
Glucose stabilizes the mRNA for human fatty acid synthase (FAS), an enzyme relevant to diverse human disorders including hyperlipidemia, obesity, and malignancy. To determine the underlying mechanisms, RNA gel mobility shift assays were used to demonstrate that human HepG2 cells contain a cytoplasmic factor which binds specifically to the 3' terminus of the human FAS mRNA. D-glucose increased RNA binding activity by 2.02-fold (p=0.0033) with activity peaking three hours after glucose feeding. Boiling or treatment of extracts with proteinase K abolished binding. UV crosslinking of the FAS mRNA -binding factor followed by SDS-PAGE resolved a proteinase K-sensitive band with an apparent molecular mass of 178 + 7 kD. The protein was purified to homogeneity using nondenaturing polyacrylamide gels as an affinity matrix. Acid phosphatase treatment of the protein prevented binding to the FAS mRNA, but binding activity was unaffected by modification of sulfhydryl groups and was not Mg++- or Ca++ -dependent. Deletion and RNase T1 mapping localized the binding site of the protein to 37 nucleotides characterized by the repetitive motif ACCCC and found within the first 65 bases of the 3' UTR. Hybridization of the FAS transcript with an oligonucleotide antisense to this sequence abolished binding. These findings indicate that a 178 kD glucose-inducible phosphoprotein binds to an (ACCCC)n -containing sequence in the 3' UTR of the FAS mRNA within the same time frame that glucose stabilizes the FAS message. This protein may participate in the post-transcriptional control of FAS gene expression. 

Received 1 August 1997; accepted in final form 16 December 1997.
APS Manuscript Number E360-7.
Article publication pending Am. J. Physiol. (Endocrinol. Metab.).
ISSN 1080-4757 Copyright 1997 The American Physiological Society.
Published in APStracts on 7 January 1998