Characterization of the 3′ untranslated region of the human mu-opioid receptor (MOR-1) mRNA

Abstract

The mu-opioid receptor (MOR) plays a mandatory role in the action of most opioid drugs, such as morphine, fentanyl, and heroin. It has been revealed that a deficiency in the MOR gene (Oprm1) or a difference in the 3′ noncoding region of the gene markedly affects the sensitivity of mice to opioids. As the 3′ noncoding region of the human OPRM1 gene had not yet been characterized, in the present study we conducted 3′-rapid amplification of cDNA ends (3′RACE)-PCR and identified the 3′ end of the human MOR-1 mRNA, the most abundant transcript among OPRM1 gene transcripts. The poly(A) signal was located at 13612–13617 nucleotides downstream from the stop codon in the OPRM1 gene. Reverse transcription PCR analyses showed that the region from the stop codon to the poly(A) signal was transcribed. In the 3′UTR, we identified 33 AU-rich regions and more than 300 putative transcription factor-binding sites. Furthermore, we compared the 3′ noncoding regions of the human and mouse OPRM1/Oprm1 genes and found apparent homology. In Northern blotting with mouse brain mRNAs, a same-size band was detected by a probe for the MOR-1 coding region and by a probe for a mouse genome region corresponding to the human MOR-1 3′UTR. Since 3′UTRs affect gene expression, the present characterization of the 3′ noncoding region in the human OPRM1 gene should lead to a better understanding of the mechanisms underlying OPRM1 gene regulation and individual differences in sensitivity to opioids.

Introduction

Opioids, such as morphine, fentanyl, and heroin, have long been widely used for pain management (Ready, 2000, Inturrisi, 2002). On the other hand, opioids also cause serious adverse effects such as dependence and respiratory depression. There are also wide variations in both analgesic effects and side effects of opioids among individuals (Glass et al., 2000, Polomano et al., 2001), which can hamper effective pain treatment and may be related to vulnerability to opioid dependence. There are strong data that support genetic contribution to individual differences in vulnerability to addiction to opioids and the degree of opioid-induced analgesia (Ikeda et al., 2005). Opioids exert their pharmacological actions through opioid receptors, which exist as mu, delta, and kappa subtypes. Several studies on mice lacking the mu-opioid receptor (MOR) have provided evidence that MOR among these receptor subtypes is a mandatory molecule for the analgesic and rewarding effects of opioids as well as for most of their side effects (Matthes et al., 1996, Kieffer, 1999, Sora et al., 2001). The gene encoding MOR is the OPRM1 gene. In the human OPRM1 gene, more than 100 polymorphisms have been identified (Bergen et al., 1997, Bond et al., 1998, Hoehe et al., 2000, Shi et al., 2002, Ide et al., 2004, Ikeda et al., 2005). These individual differences in the OPRM1 gene may contribute to the wide variation in sensitivity to opioids.

It is now apparent that the 3′ untranslated region (UTR) can regulate the rate of translation and degradation of mRNA through 3′UTR sequence-binding proteins (Conne et al., 2000, Provost and Tremblay, 2000, Pizzuti et al., 2002). In previous studies, we estimated the size of the mouse MOR-1 mRNA, the most abundant transcript among a variety of the Oprm1 transcripts (Pasternak, 2004), to be 12 kb, suggesting that the mouse MOR-1 3′UTR would be more than 10 kb (Ikeda et al., 2001). Furthermore, CXBK mice, which exhibit significantly reduced responses to MOR agonists and reduced binding to opioids, possessed an abnormally long MOR-1 mRNA that was expressed at lower levels than in the progenitor strains (Han et al., 2004). In humans, more than 50 polymorphisms have been identified in the 3′ downstream region from the stop codon in the OPRM1 gene (Ikeda et al., 2005). These polymorphisms might also be related to OPRM1 gene expression and individual differences in sensitivity to opioids. However, it was not known if these polymorphisms are in the exons or introns, because the 3′ noncoding region of the human OPRM1 gene was not characterized. In the present study, to address this issue we identified the 3′ end of the human MOR-1 mRNA by the 3′-rapid amplification of cDNA ends (3′RACE)-PCR method and compared the human and murine MOR-1 3′UTR sequences.