Translational repression of mouse mu opioid receptor expression via leaky scanning

Abstract

Mu opioid receptor (MOR) expression is under temporal and spatial controls, but expression levels of the MOR gene are relatively low in vivo. In addition to transcriptional regulations, upstream AUGs (uAUGs) and open reading frames (uORFs) profoundly affect the translation of the primary ORF and thus the protein levels in several genes. The 5′-untranslated region (UTR) of mouse MOR mRNA contains three uORFs preceding the MOR main initiation codon. In MOR-fused EGFP or MOR promoter/luciferase reporter constructs, mutating each uAUG individually or in combinations increased MOR transient heterologous expression in neuroblastoma NMB and HEK293 cells significantly. Translation of such constructs increased up to 3-fold without altering the mRNA levels if either the third uAUG or both the second and third AUGs were mutated. Additionally, these uAUG-mediated translational inhibitions were independent of their peptide as confirmed by internal mutation analyses in each uORF. Translational studies indicated that protein syntheses were initiated at these uAUG initiation sites, with the third uAUG initiating the highest translation level. These results support the hypothesis that uORFs in mouse MOR mRNA act as negative regulators through a ribosome leaky scanning mechanism. Such leaky scanning resulted in the suppression of mouse MOR under normal conditions.

The 5′-Untranslated Region of Multidrug Resistance Associated Protein 2 (MRP2; ABCC2) Regulates Downstream Open Reading Frame Expression through Translational Regulation

“…3), indicating that uATG Ϫ105 was recognized by the ribosome and inhibited the translation of the downstream ORF. These findings are consistent with most other findings that uATGs inhibit downstream ORF translation (Meijer et al, 2000;Diba et al, 2001;Kwon et al, 2001;Blaschke et al, 2003;Mihailovich et al, 2007;Song et al, 2007). Further studies showed that the amino acid sequence of the peptide encoded by uORF-105 was not important in regulating translation and that uORF-105 acted only as a cis element to inhibit downstream ORF translation.…”

“…Alternatively, there may be internal ribosomal entry sites located in the 5ЈUTR, such that the translation machinery skips the uATGs and instead uses the internal ribosomal entry sites to initiate translation of the downstream ORF (Le and Maizel, 1997). Whereas some upstream start codons (uATGs) inhibit downstream ORF translation (Meijer et al, 2000;Diba et al, 2001;Kwon et al, 2001;Blaschke et al, 2003;Mihailovich et al, 2007;Song et al, 2007), other uATGs have no such effect (Diba et al, 2001). uORFs can also influence the stability of mRNA by a process termed nonsense-mediated mRNA decay (Hood et al, 2009).…”

Long-Term Morphine Treatment Decreases the Association of μ-Opioid Receptor (MOR1) mRNA with Polysomes through miRNA23b

“…-Opioid receptor (MOR) mediates most of the pharmacological effects of opiates; its regulation is of great importance to unravel the molecular mechanisms underlying the physical responses to opioid treatment, such as tolerance and dependence. In addition to the multiple cis-acting elements that regulate the transcription of MOR (Hwang et al, 2004;Kim et al, 2006;Choi et al, 2007;Song et al, 2007), a recent study on the 3Ј-UTR of the major -opioid receptor mRNA (MOR1) has started to address the regulation of MOR at the post-transcriptional level (Wu et al, 2008).…”

A Proteomics Approach for Identification of Single Strand DNA-binding Proteins Involved in Transcriptional Regulation of Mouse μ Opioid Receptor Gene

“…Several Oprm1 isoforms have been reported (9 -11). The upstream open reading frames in Oprm1 mRNA act as negative regulators through a ribosome leaky scanning mechanism (12).…”