Properties and purification of a glucose-inducible human fatty acid synthase mRNA-binding protein.
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 Hep G2 cells contain a cytoplasmic factor that 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 3 h after glucose feeding. Boiling or treatment of extracts with proteinase K abolished binding. Ultraviolet cross-linking of the FAS mRNA-binding factor followed by SDS-PAGE resolved a proteinase K-sensitive band with an apparent molecular mass of 178 +/- 7 kDa. 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 Mg2+ or Ca2+ 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-kDa 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 posttranscriptional control of FAS gene expression.
Li
Q
Q
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Chua
M S
MS
Semenkovich
C F
CF
D001483:Base Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000502:physiology; D064429:Fatty Acid Synthases / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured
D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D064429:Fatty Acid Synthases; D005947:Glucose