Characterization of Two Nuclear Proteins that Interact with Cytochrome P‐450 1A2 mRNA

Abstract

Regulation of the expression of the cytochrome P-450 la2 gene (cypla2) occurs mainly at the transcriptional level, but the molecular events involved in the induction process are partly unknown. Here we report the identification of two proteins in the nuclear fraction of mouse liver, with specific binding characteristics towards CYPl A2 mRNA. The proteins have apparent molecular masses of 37 kDa and 46 kDa and exhibit a high affinity for a poly(U) motif in the 3' untranslated region of CYPl A2 mRNA. This motif seems to be important for their specific and apparently competitive binding to CYPl A2 mRNA. Treatment of mice with an inducer of CYPlA2, 3-methylcholanthrene, increases the binding of the 46-kDa protein and decreases the binding of the 37-kDa protein to the mRNA, suggesting that changes in the binding of the proteins to the mRNA could play a role in the upregulation of CYPlA2 mRNA by 3-methylcholanthrene. Phosphorylation of the 46-kDa protein, or of an intermediary factor, may play a role in its binding activity. Furthermore, the 46-kDa but not the 37-kDa protein is recognized by a monoclonal antibody against the heterogeneous nuclear ribonucleoprotein C, a nuclear protein probably involved in pre-mRNA processing. While more work is needed to understand the function of the proteins that bind to the 3' untranslated region of CYPlA2, it is possible that the 37-kDa protein has a role in the maintenance of uninduced levels of CYPlA2 mRNA, while the 46-kDa protein could be important in the maturation of elevated levels of CYPl A2 pre-ml2NA, during induction.Keywords: cytochrome P-450 1A2 ; RNA-binding protein ; 3' untranslated region ; induction.Cytochrome P-450 (CUP) proteins form a superfamily of hemoproteins, which exert catalytic activities towards a large variety of foreign chemicals, such as drugs and environmental pollutants, as well as endogenous compounds, such as fatty acids and steroids [l]. Several of these enzymes are inducible by xenobiotics, and the transcriptional activation of some of the encoding genes has been studied. It has been shown for some of the CYP enzymes that post-transcriptional regulation is important for their expression (21. However, the molecular events involved in the post-transcriptional control are poorly understood.The catalytic properties and regulations of members of the CYPl A family (CYPlAl and CYPlA2) have been extensively studied [3, 41, and upregulation of CYPlAl by 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons probably represents the best understood mechanism by which the CYP enzymes respond to chemical stress. In the cytosol, the arylhydrocarbon receptor is bound to heat-shock protein 90 and several other proteins [5]. Upon ligand binding, the receptor dissociates from the complex and translocates into the nucleus [6]. There it heterodimerizes with the arylhydrocarbon-receptor Abbreviations. CUP, cytochrome P-450; UTR, untranslated region; PhMeSO,F, phenylmethylsulfonyl fluoride; hnRNP, heterogeneous nuclear ribonucleoprotein. nuclear tra...

Molecular mechanisms of cold-induced CYP1A activation in rat liver microsomes

“…In contrast, cold-induced CYP1A2 activation is not transcriptional-although CYP1A2 activity and the CYP1A2 protein level increased, the CYP1A2 mRNA level decreased. Our conclusion is that CYP1A2 activation is likely to occur at the posttranscriptional level, which is in agreement with some data in literature [42]. The big difference in the magnitude of induction after treatment with the classic CYP1A inducers and after cooling remains to be explained.…”

“…In contrast, CYP1A2 activation can occur both at the transcriptional level, through the AhR-dependent or AhR-independent signaling pathway [41], and at the post-transcriptional level [42].…”

Identification of a 16-Nucleotide Sequence That Mediates Post-transcriptional Regulation of Rat CYP2E1 by Insulin

“…A protein from mouse liver specifically binds to the 3Ј-UTR of CYP2a5 mRNA; this binding is increased after pyrazole treatment and is associated with mRNA stabilization and elongation of the poly(A) tail (8,41). Likewise, two nuclear proteins bind specifically to the 3Ј-UTR of CYP1a2 mRNA, and their binding is altered by a typical inducer of CYP1a2, 3-methylcholanthrene (6). In these cases too, the critical regulatory step is probably the binding of proteins to RNA target sequences.…”

“…It has been reported that proteins involved in the control of mRNA half-life are often post-translationally modified by phosphorylation, which is important for their RNA binding ability and/or function (49,50). Raffalli-Matthieu et al (6) showed that phosphatase treatment of the nuclear extracts reduced the binding of the 46-kDa protein to the CYP1a2 mRNA. This suggests that the binding activity of the protein may depend on its phosphorylation status or on some intermediary factors necessary for binding.…”

Effect of cold stress on expression of genes for the AhR-dependent pathway of CYP1 regulation in rat liver

“…Classic CYP1 inductor BP increased expression of CYP1A1, CYP1A2, and CYP1B1. CYP1A1 nism) was revealed in previous experiments [14,15]. Cold stress has no effect on CYP1B1 expression.…”