PNNL

Abstract

Altered expression of hepatic CYP2E1 by xenobiotic or physiological stimuli is largely mediated through post-transcriptional mechanisms that may include altered CYP2E1 mRNA translation and/or protein degradation. Examination of the polyribosomal distribution of rat hepatic P450 mRNAs indicated that, whereas nearly all of the CYP2B, CYP3A, and CYP4A mRNAs were recovered in the polysomal fractions, indicating active translation, approximately 30-40% of CYP2E1 mRNA was not associated with polysomes and therefore not actively engaged in protein synthesis. To examine the CYP2E1 mRNA molecule for sequences that might affect its translational efficiency, a series of CYP2E1 recombinant RNAs (rcRNAs) with modified 5' or 3' untranslated regions (UTRs) was translated in vitro using the rabbit reticulocyte lysate system. Deletion of most of the CYP2E1 5' UTR, which was predicted to contain secondary structure, increased in vitro CYP2E1 protein synthesis. Polysomal distribution analyses of 5'-modified rcRNAs demonstrated that, as seen for hepatic CYP2E1 mRNA, a substantial fraction of each CYP2E1 rcRNA was not associated with polysomes. The polysomal distribution analyses of the CYP2E1 rcRNAs also confirmed that the observed changes in CYP2E1 protein synthesis were associated with altered ribosomal loading. Deletion of the poly(A) tail, and partial or complete deletion of the 3' UTR, decreased CYP2E1 protein synthesis. These changes in protein synthesis were accompanied by increased degradation of the CYP2E1 rcRNAs. Incubation with translational inhibitors, but not increased levels of RNase inhibitor, decreased the degradation of the rcRNAs during in vitro translation.