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uAUG-mediated translational initiations are responsible for human mu opioid receptor gene expression.

Song KY, Kim CS, Hwang CK, Choi HS, Law PY, Wei LN, Loh HH - J. Cell. Mol. Med. (2009)

Bottom Line: The inhibitory effect caused by the third in-frame uAUG was confirmed by in vitro translation and receptor-binding assays.This re-initiation resulted in negative expression of OPRM1 under normal conditions.These results indicate that re-initiation in MOR gene expression could play an important role in OPRM1 regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA. songx047@umn.edu

ABSTRACT
Mu opioid receptor (MOR) is the main site of interaction for major clinical analgesics, particularly morphine. MOR expression is regulated at the transcriptional and post-transcriptional levels. However, the protein expression of the MOR gene is relatively low and the translational control of MOR gene has not been well studied. The 5'-untranslated region (UTR) of the human MOR (OPRM1) mRNA contains four upstream AUG codons (uAUG) preceding the main translation initiation site. We mutated the four uAUGs individually and in combination. Mutations of the third uAUG, containing the same open reading frame, had the strongest inhibitory effect. The inhibitory effect caused by the third in-frame uAUG was confirmed by in vitro translation and receptor-binding assays. Toeprinting results showed that OPRM1 ribosomes initiated efficiently at the first uAUG, and subsequently re-initiated at the in-frame #3 uAUG and the physiological AUG site. This re-initiation resulted in negative expression of OPRM1 under normal conditions. These results indicate that re-initiation in MOR gene expression could play an important role in OPRM1 regulation.

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OPRM1 5′-UTR contains no conserved uORF. (A) Dendrogram of mu opioid receptors based on their amino acid identity [37]. (B) Sequence alignment of the 5′-UTR region of the human (used in this study), monkey (GenBank database accession no. AY038989), guinea pig (GenBank database accession no. AY166606), pig (GenBank database accession no. AF521309), cow (GenBank database accession no. U89677), rat (GenBank database accession no. L13069) and mouse [22] opioid receptor mRNA. Bold nucleotide sequences are indicated uAUGs and main ATG site. The nucleotide sequences of the human uORFs are boxed (#1, 2, 3 and 4). (C) Amino acid sequence alignment of the uORF of human, pig and mouse opioid receptor. All uORF amino acid sequences showed before their stop codon except human uORF 3 (showed until main ORF initiation codon). The conserved amino acids are bolded.
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fig07: OPRM1 5′-UTR contains no conserved uORF. (A) Dendrogram of mu opioid receptors based on their amino acid identity [37]. (B) Sequence alignment of the 5′-UTR region of the human (used in this study), monkey (GenBank database accession no. AY038989), guinea pig (GenBank database accession no. AY166606), pig (GenBank database accession no. AF521309), cow (GenBank database accession no. U89677), rat (GenBank database accession no. L13069) and mouse [22] opioid receptor mRNA. Bold nucleotide sequences are indicated uAUGs and main ATG site. The nucleotide sequences of the human uORFs are boxed (#1, 2, 3 and 4). (C) Amino acid sequence alignment of the uORF of human, pig and mouse opioid receptor. All uORF amino acid sequences showed before their stop codon except human uORF 3 (showed until main ORF initiation codon). The conserved amino acids are bolded.

Mentions: To date, mu opioid receptor genes have been cloned and revealed the dendrogram (Fig. 7A][37]. The alignment of the 5′-UTR sequences from human, monkey, guinea pig, pig, cow, rat and mouse mu opioid receptor transcripts reveals a low degree of similarity (Fig. 7B). However, human #2 uAUG is conserved in pig #1 uAUG and mouse #3 uAUG, human #3 uAUG is conserved in pig #3 uAUG only. These sequences came from their mRNA transcript (GenBank database accession no. shown in Fig. 7 legends). In addition, the alignment of the amino acid sequences for the uORFs in these species also showed a low degree of similarity (Fig. 7C). These results show that the relative positions of the uAUGs and uORFs from human and other mammal’s mu opioid receptor show no significant similarities.


uAUG-mediated translational initiations are responsible for human mu opioid receptor gene expression.

Song KY, Kim CS, Hwang CK, Choi HS, Law PY, Wei LN, Loh HH - J. Cell. Mol. Med. (2009)

OPRM1 5′-UTR contains no conserved uORF. (A) Dendrogram of mu opioid receptors based on their amino acid identity [37]. (B) Sequence alignment of the 5′-UTR region of the human (used in this study), monkey (GenBank database accession no. AY038989), guinea pig (GenBank database accession no. AY166606), pig (GenBank database accession no. AF521309), cow (GenBank database accession no. U89677), rat (GenBank database accession no. L13069) and mouse [22] opioid receptor mRNA. Bold nucleotide sequences are indicated uAUGs and main ATG site. The nucleotide sequences of the human uORFs are boxed (#1, 2, 3 and 4). (C) Amino acid sequence alignment of the uORF of human, pig and mouse opioid receptor. All uORF amino acid sequences showed before their stop codon except human uORF 3 (showed until main ORF initiation codon). The conserved amino acids are bolded.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822748&req=5

fig07: OPRM1 5′-UTR contains no conserved uORF. (A) Dendrogram of mu opioid receptors based on their amino acid identity [37]. (B) Sequence alignment of the 5′-UTR region of the human (used in this study), monkey (GenBank database accession no. AY038989), guinea pig (GenBank database accession no. AY166606), pig (GenBank database accession no. AF521309), cow (GenBank database accession no. U89677), rat (GenBank database accession no. L13069) and mouse [22] opioid receptor mRNA. Bold nucleotide sequences are indicated uAUGs and main ATG site. The nucleotide sequences of the human uORFs are boxed (#1, 2, 3 and 4). (C) Amino acid sequence alignment of the uORF of human, pig and mouse opioid receptor. All uORF amino acid sequences showed before their stop codon except human uORF 3 (showed until main ORF initiation codon). The conserved amino acids are bolded.
Mentions: To date, mu opioid receptor genes have been cloned and revealed the dendrogram (Fig. 7A][37]. The alignment of the 5′-UTR sequences from human, monkey, guinea pig, pig, cow, rat and mouse mu opioid receptor transcripts reveals a low degree of similarity (Fig. 7B). However, human #2 uAUG is conserved in pig #1 uAUG and mouse #3 uAUG, human #3 uAUG is conserved in pig #3 uAUG only. These sequences came from their mRNA transcript (GenBank database accession no. shown in Fig. 7 legends). In addition, the alignment of the amino acid sequences for the uORFs in these species also showed a low degree of similarity (Fig. 7C). These results show that the relative positions of the uAUGs and uORFs from human and other mammal’s mu opioid receptor show no significant similarities.

Bottom Line: The inhibitory effect caused by the third in-frame uAUG was confirmed by in vitro translation and receptor-binding assays.This re-initiation resulted in negative expression of OPRM1 under normal conditions.These results indicate that re-initiation in MOR gene expression could play an important role in OPRM1 regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA. songx047@umn.edu

ABSTRACT
Mu opioid receptor (MOR) is the main site of interaction for major clinical analgesics, particularly morphine. MOR expression is regulated at the transcriptional and post-transcriptional levels. However, the protein expression of the MOR gene is relatively low and the translational control of MOR gene has not been well studied. The 5'-untranslated region (UTR) of the human MOR (OPRM1) mRNA contains four upstream AUG codons (uAUG) preceding the main translation initiation site. We mutated the four uAUGs individually and in combination. Mutations of the third uAUG, containing the same open reading frame, had the strongest inhibitory effect. The inhibitory effect caused by the third in-frame uAUG was confirmed by in vitro translation and receptor-binding assays. Toeprinting results showed that OPRM1 ribosomes initiated efficiently at the first uAUG, and subsequently re-initiated at the in-frame #3 uAUG and the physiological AUG site. This re-initiation resulted in negative expression of OPRM1 under normal conditions. These results indicate that re-initiation in MOR gene expression could play an important role in OPRM1 regulation.

Show MeSH