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Expression of a Novel Mrna Transcript for Human Microsomal Epoxide Hydrolase (EPHX1) Is Regulated by Short Open Reading Frames Within Its 5′-Untranslated Region

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Expression of a Novel Mrna Transcript for Human Microsomal Epoxide Hydrolase (EPHX1) Is Regulated by Short Open Reading Frames Within Its 5′-Untranslated Region Downloaded from rnajournal.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Expression of a novel mRNA transcript for human microsomal epoxide hydrolase (EPHX1) is regulated by short open reading frames within its 5′-untranslated region HONG LOAN NGUYEN,1 XI YANG,1,2 and CURTIS J. OMIECINSKI3 Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, Pennsylvania 16802, USA ABSTRACT Microsomal epoxide hydrolase (mEH, EPHX1) is a critical xenobiotic-metabolizing enzyme, catalyzing both detoxification and bioactivation reactions that direct the disposition of chemical epoxides, including the carcinogenic metabolites of several polycyclic aromatic hydrocarbons. Recently, we discovered that a previously unrecognized and primate-specific EPHX1 transcript, termed E1-b, was actually the predominant driver of EPHX1 expression in all human tissues. In this study, we identify another human EPHX1 transcript, designated as E1-b′. Unusually, both the E1-b and E1-b′ mRNA transcripts are generated from the use of a far upstream gene promoter, localized ∼18.5 kb 5′-upstream of the EPHX1 protein-coding region. Although expressed at comparatively lower levels than E1-b, the novel E1-b′ transcript is readily detected in all tissues examined, with highest levels maintained in human ovary. The E1-b′ mRNA possesses unusual functional features in its 5′- untranslated region, including a GC-rich leader sequence and two upstream AUGs that encode for short peptides of 26 and 17 amino acids in length, respectively. Results from in vitro transcription/translation assays and direct transfection in mammalian cells of either the E1-b′ transcript or the encoded peptides demonstrated that the E1-b′ upstream open reading frames (uORFs) are functional, with their presence markedly inhibiting the translation of EPHX1 protein, both in cis and in trans configurations. These unique uORF peptides exhibit no homology to any other known uORF sequences but likely function to mediate post- transcription regulation of EPHX1 and perhaps more broadly as translational regulators in human cells. Keywords: microsomal epoxide hydrolase; EPHX1; upstream open reading frame (uORF); translational regulation; 5′-untranslated region (5′ UTR); gene expression INTRODUCTION EPHX1 functions to convert these compounds into highly reactive electrophilic metabolites capable of forming cova- Microsomal epoxide hydrolase (mEH, EPHX1) catalyzes the lent DNA adducts and thereby initiating mutational events hydration of numerous xenobiotic epoxides; in particular, (Buterin et al. 2000; Lloyd and Hanawalt 2000). The impor- those derived by cytochrome P-450 (CYPs) mediated oxida- tance of the EPHX1 bioactivation function has been demon- tion (Rappaport et al. 1996; Abdel-Rahman et al. 2005). In strated directly in knockout mouse studies in which EPHX1- these respects, EPHX1 contributes important detoxification deficient mice are markedly resistant to 7,12-dimethylbenz function,forexample, in the metabolism of the occupationally [a]anthracene-induced tumorigenesis compared with their important neurotoxins and potential carcinogens, styrene wild-type counterparts (Miyata et al. 1999). Although more oxide and 1,2-epoxy-3-butene, to less reactive dihydrodiol de- rivatives (Fretland and Omiecinski 2000). However, EPHX1 poorly delineated, several lines of evidence also indicate en- also contributes critically to the bioactivation of carcinogenic dogenous roles for this enzyme, including that of steroid me- polycyclic aromatic hydrocarbons, commonly encountered in tabolism, bile acid transport, and in the vitamin K reductase smoke as combustion by-products. In concert with the CYPs, complex (Guenthner et al. 1998). In humans, EPHX1 activity levels appear quite variable among individuals (Hassett et al. 1998; Newman et al. 2005). 1These authors contributed equally to this work. While a variety of parameters may contribute to interindivid- 2Present address: Division of Systems Biology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR ual differences in expression, EPHX1 genetic polymorphism 72079, USA is one potential determinant. EPHX1 genetic variation de- 3Corresponding author scribed includes the existence of nonsynonymous polymor- E-mail [email protected] ’ Article published online ahead of print. Article and publication date are at phisms in the gene s coding region, as well as functional http://www.rnajournal.org/cgi/doi/10.1261/rna.037036.112. polymorphisms in the respective upstream promoter regions 752 RNA (2013), 19:752–766. Published by Cold Spring Harbor Laboratory Press. Copyright © 2013 RNA Society. Downloaded from rnajournal.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Regulation of human microsomal epoxide hydrolase (Raaka et al. 1998; Yang et al. 2009). In these respects, molec- tissue-selectively regulated. Characterization of the 5′ UTR ular epidemiology reports have associated altered risk of lung of the E1-b′ transcript revealed the presence of two uORFs, and upper aerodigestive tract cancers with genetic variation in predicted to encode short peptides of 26 and 17 amino acids EPHX1 coding structure (Li et al. 2011). Variation within the in length, respectively. We demonstrate that these imbedded EPHX1 5′-proximal gene promoter region was also suggested uORFs impart a marked inhibitory effect on EPHX1 trans- to contribute to differences in transcriptional regulation to- lation. These findings further demonstrate that expression gether with differential sensitivity of certain individuals to of human EPHX1 is subjected to complex regulatory con- genotoxic effects resulting from 1,3-butadiene exposures trol, involving both transcriptional and post-transcriptional (Raaka et al. 1998; Abdel-Rahman et al. 2005). mechanisms. Although EPHX1 expression is detected in nearly all tis- sues, levels of expression are strikingly tissue selective, with RESULTS high levels of enzymatic activity detected particularly in liver hepatocytes, as well as ovary and testes (Coller et al. 2001; Identification of the E1-b′ transcript by RCA-RACE Liang et al. 2005). Interestingly, the use of alternative promot- ers has been identified as an important regulatory feature Using 5′-RACE methodology, two human tissue EPHX1 tran- driving tissue-specific expression of human EPHX1 tran- scripts with unique first exons were previously identified scripts containing differing first exons (Liang et al. 2005). (Liang et al. 2005). Significantly, these studies revealed that One transcript, designated E1, initiates from the 5′-proximal EPHX1 transcription is predominantly driven by a far up- promoter that directly flanks the structural coding region of stream promoter, designated as E1-b. In the present study, the gene. However, the E1 transcript is expressed selectively we used a rolling circle amplification technique, i.e., Rapid in the liver (Liang et al. 2005). More recently, a far upstream Amplification of cDNA Ends (RCA-RACE) (Polidoros et al. promoter, designated E1-b, was identified that is localized 2006) to extend these analyses. This methodology uses gene- ∼18.5 kb 5′ of the E1 exon, yet serves as the predominant specific primers to eliminate the high background typically driver of EPHX1 in all tissues examined, including liver encountered with conventional RACE procedures, coupled (Liang et al. 2005). Because the human EPHX1 coding region with the use of a universal primer corresponding to the anchor extends from exon 2 to exon 9, the E1 and E1-b transcripts in- sequence, to better enable the isolation of even low-level clude distinct noncoding exon 1’s but otherwise are expected transcripts within a pool of amplified circular cDNA tem- to generate the same EPHX1 protein. Importantly, the E1-b plates. The RACE results obtained using total RNA isolates promoter region is conserved in nonhuman primates but fromhuman A549 and HepG2 cells both revealed the presence has not been identified in rodents, indicating the evolution of a unique EPHX1 transcript with a novel exon 1 sequence of differing transcriptional regulatory mechanisms across composition that we designate here as E1-b′. The presence mammalian species. of the E1-b′ transcript was further verified as present in a va- Potentially important features of post-transcriptional regu- riety of human tissues, and its exon 1 sequence was localized latory control are often imbedded in the 5′-untranslated re- by genomic mapping to an adjacent upstream promoter re- gion of mRNA transcripts. In addition to the adoption of gion to that previously identified as generating the E1-b tran- secondary structures that may characterize certain 5′ UTRs, script. A schematic of the overall EPHX1 gene structure from short upstream open reading frames (ORFs) occur frequently which the various transcripts are derived is presented in in eukaryotic genes (Crowe et al. 2006). Estimates indicate Figure 1A. that at up to 40% of mammalian mRNA sequences possess Of interest, analysis of the NCBI AceView database (http AUG trinucleotides upstream of the main coding sequence, ://www.ncbi.nlm.nih.gov) that annotates cDNA-based tran- with a substantial number of these motifs influencing trans- script expression failed to identify the novel E-1b′ transcript. lational efficiencies of the associated full-length transcripts Nor was the E-1b′ transcript identified by review of the (Morris and Geballe
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