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

Citation
J. Gilmore et al., Identification of a 43-kDa protein in human liver cytosol that binds to the 3 '-untranslated region of CYP2A6 mRNA, BIOCH PHARM, 62(6), 2001, pp. 669-678
Citations number
44
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pharmacology & Toxicology
Journal title
BIOCHEMICAL PHARMACOLOGY
ISSN journal
0006-2952 → ACNP
Volume
62
Issue
6
Year of publication
2001
Pages
669 - 678
Database
ISI
SICI code
0006-2952(20010915)62:6<669:IOA4PI>2.0.ZU;2-D
Abstract
Hepatic expression of cytochrome P450 2A6 (CYP2A6) varies widely in humans and is induced during hepatitis; however, the mechanism regulating CYP2A6 h as not been established. The murine orthologue Cyp2a5 is regulated post-tra nscriptionally by mRNA stabilization. A 43-kDa protein that binds to the 3 ' -untranslated region (3 ' -UTR) of Cyp2a5 mRNA has been identified, but i ts role in mRNA stabilization is unclear. We hypothesized that similar inte ractions occur between cytosolic proteins in human liver and CYP2A6 3 ' -UT R mRNA. We identified, by RNA electrophoretic mobility shift assay, an hepa tic cytosolic protein that binds specifically to sequences in the 3 ' -UTR of CYP2A6. Complexes did not form with denatured proteins and were eliminat ed with proteinase K digestion. Complex formation was inhibited with a mola r excess of unlabeled CYP2A6 RNA but not by non-specific competitor RNA. Pr otein-mRNA interactions were not affected by probe denaturation, suggesting that RNA secondary structure is not essential for binding. UV cross-linkin g of complexes revealed RNA-binding proteins in both human and mouse liver cytosols with molecular masses of approximately 43 kDa. Using truncated RNA probes cot-responding to various lengths of CYP2A6 mRNA, the protein-bindi ng site was localized to a 50-nucleotide region between bases 1478 and 1527 of the 3 ' -UTR. Complex formation with hepatic cytosolic protein from fou r human subjects correlated with levels of hepatic CYP2A6 microsomal protei n, suggesting a possible regulatory role. Further characterization of the R NA-binding protein, the primary binding site, and the influence of this int eraction on CYP2A6 mRNA stability will help to elucidate the relevance of t hese findings to the post-transcriptional control of CYP2A6. (C) 2001 Elsev ier Science Inc. All rights reserved.