Identification of a 43-kDa protein in human liver cytosol that binds to the 3'-untranslated region of CYP2A6 mRNA

• Published in: Biochemical pharmacology Vol. 62; no. 6; pp. 669 - 678
• Main Authors: GILMORE, James, ROTONDO, Francesca, PELLETIER, Anne-Marie, LAMARRE, Jonathan, ALAOUI-JAMALI, Moulay, KIRBY, Gordon M
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Science 15.09.2001
• Subjects: 3' Untranslated Regions - metabolism; Analytical, structural and metabolic biochemistry; Aryl Hydrocarbon Hydroxylases; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Cytochrome P-450 CYP2A6; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450 Family 2; Cytosol - metabolism; Fundamental and applied biological sciences. Psychology; Humans; In Vitro Techniques; Microsomes, Liver - metabolism; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Molecular Weight; Proteins; RNA, Messenger - analysis; RNA, Messenger - metabolism; RNA-Binding Proteins - isolation & purification; RNA-Binding Proteins - metabolism; Human; RNA; Transcription; Digestive system; Cytochrome P450; Liver; Molecular interaction; Gene expression; Microsome; Cytosol; In vitro; Binding protein; Biological fixation; Aminoacid sequence
• ISSN: 0006-2952
• DOI: 10.1016/S0006-2952(01)00720-1

Hepatic expression of cytochrome P450 2A6 (CYP2A6) varies widely in humans and is induced during hepatitis; however, the mechanism regulating CYP2A6 has not been established. The murine orthologue Cyp2a5 is regulated post-transcriptionally by mRNA stabilization. A 43-kDa protein that binds to the 3'-untranslated region (3'-UTR) of Cyp2a5 mRNA has been identified, but its role in mRNA stabilization is unclear. We hypothesized that similar interactions occur between cytosolic proteins in human liver and CYP2A6 3'-UTR mRNA. We identified, by RNA electrophoretic mobility shift assay, an hepatic cytosolic protein that binds specifically to sequences in the 3'-UTR of CYP2A6. Complexes did not form with denatured proteins and were eliminated with proteinase K digestion. Complex formation was inhibited with a molar excess of unlabeled CYP2A6 RNA but not by non-specific competitor RNA. Protein-mRNA interactions were not affected by probe denaturation, suggesting that RNA secondary structure is not essential for binding. UV cross-linking of complexes revealed RNA-binding proteins in both human and mouse liver cytosols with molecular masses of approximately 43 kDa. Using truncated RNA probes corresponding to various lengths of CYP2A6 mRNA, the protein-binding site was localized to a 50-nucleotide region between bases 1478 and 1527 of the 3'-UTR. Complex formation with hepatic cytosolic protein from four human subjects correlated with levels of hepatic CYP2A6 microsomal protein, suggesting a possible regulatory role. Further characterization of the RNA-binding protein, the primary binding site, and the influence of this interaction on CYP2A6 mRNA stability will help to elucidate the relevance of these findings to the post-transcriptional control of CYP2A6.