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

Biochem Pharmacol. 2001 Sep 15;62(6):669-78. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / metabolism*
  • Aryl Hydrocarbon Hydroxylases*
  • Base Sequence
  • Cytochrome P-450 CYP2A6
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • Cytochrome P450 Family 2
  • Cytosol / metabolism
  • Humans
  • In Vitro Techniques
  • Microsomes, Liver / metabolism*
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Molecular Sequence Data
  • Molecular Weight
  • RNA, Messenger / analysis
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / isolation & purification*
  • RNA-Binding Proteins / metabolism


  • 3' Untranslated Regions
  • RNA, Messenger
  • RNA-Binding Proteins
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Aryl Hydrocarbon Hydroxylases
  • CYP2A6 protein, human
  • Cyp2a5 protein, mouse
  • Cytochrome P-450 CYP2A6
  • Cytochrome P450 Family 2