Identification of a 43-kDa protein in human liver cytosol that binds to the 3′-untranslated region of CYP2A6 mRNA¹

Abstract

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.

Introduction

In humans, cytochrome P450 2A6 is involved in the metabolic activation of various chemical carcinogens including nitrosamines and aflatoxins [1], [2], [3], [4], [5]. While CYP2A6 expression varies greatly among individuals [6], [7], the mechanism regulating CYP2A6 gene expression in humans is unknown. Regulation of Cyp2a5, the mouse orthologue that has high sequence complementarity and shares catalytic properties with CYP2A6 [8], has been studied extensively [9]. Cyp2a5 induction is associated with liver injury caused by a number of structurally diverse hepatotoxins including pyrazole [10], carbon tetrachloride [11], benzene [12], and various metal ions such as cobalt [13], cerium [14], and indium [15]. Moreover, induction of Cyp2a5 has been demonstrated in various rodent models of chronic liver injury by infectious agents. For example, Cyp2a5 is induced in HBV transgenic mice [16], in hamsters with liver fluke infestation [17], and in mice with hepatitis due to Helicobacter hepaticus[18]. There are also reports of overexpression of CYP2A6 in association with hepatitis and cirrhosis due to HBV or HCV infection [19], [20]. There is accumulating evidence that post-transcriptional mechanisms are involved in the regulation of Cyp2a5. Nuclear run-off analysis has revealed that selective induction of Cyp2a5 by pyrazole is mediated post-transcriptionally via stabilization of Cyp2a5 mRNA [21], [22]. To date, however, no studies have focused on the molecular mechanisms that regulate CYP2A6 expression.

The modulation of mRNA stability is an important mechanism of gene regulation, particularly for gene products that must be regulated within a narrow time-frame (e.g. immediate early genes) [23]. Inducible molecules (e.g. cytokines, oncogenes, transcriptional activators, enzymes) that are responsive to environmental, hormonal, or nutritional stimuli often have unstable mRNA [24], [25], [26]. There are numerous reports that RNA-protein interactions are critical in mediating post-transcriptional control of gene expression, and trans-acting protein factors that modulate mRNA stability are becoming increasingly characterized [27]. A number of cis-acting elements have been implicated in the regulation of mRNA stability by binding cytosolic proteins. Defined regions known as adenosine/uridine-rich elements in the 3′-UTR of many short-lived mRNAs appear to regulate gene expression. Among the best characterized is the iron responsive element involved in the reciprocal changes in the translation and mRNA stability of ferritin and transferrin receptor mRNA [28]. Recent evidence suggests that the post-transcriptional regulation of Cyp2a5 expression involves protein-mRNA interactions [29]. Several cytosolic proteins that bind specifically to the 3′-UTR of Cyp2a5 mRNA have been identified recently, suggesting a possible role in regulating Cyp2a5 message stability; however, this has not been established [29], [30], [31]. The objective of the present study was to determine whether similar interactions occur between human hepatic cytosolic proteins and the 3′-UTR of CYP2A6 mRNA. Using RNA EMSAs, we have identified a 43-kDa protein in human liver cytosol that binds specifically to regions within the 3′-UTR of CYP2A6 mRNA.