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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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Schematic of the human mu opioid receptor (OPRM1) and putative uORF sequences. (A) The OPRM1 structure and main transcript. Exons and introns are shown by boxes and horizontal lines, respectively. The promoter is indicated by the arrow. Exons are numbered in the order in which they were identified. Translation start and stop sites are shown by bars above the exon boxes. OPRM1 is the main MOR messenger RNA identified in humans. (B) Sequence of the OPRM1 5′-UTR used in this study. Four uAUG codons (#1–#4) and the main AUG are shown in bold, and the termination codon of the uORFs is italicized. Bold peptide sequences represent the uAUG and stop codons. The arrowheads above the sequence indicate the transcription initiation sites. mToe 1 and 2 indicate radiolabelled oligonucleotide primers for toeprinting. (C) uAUG sequences compared with the Kozak sequence. The −3 and +4 positions relative to the start codon (underlined) are represented in bold. Each uORF consists of 17, 27, 462 and 8 putative amino acids, respectively. The uAUG #3 uORF consists of 62 additional amino acids, relative to the main ORF (i.e. 400 amino acids) at the 5′-end.
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fig01: Schematic of the human mu opioid receptor (OPRM1) and putative uORF sequences. (A) The OPRM1 structure and main transcript. Exons and introns are shown by boxes and horizontal lines, respectively. The promoter is indicated by the arrow. Exons are numbered in the order in which they were identified. Translation start and stop sites are shown by bars above the exon boxes. OPRM1 is the main MOR messenger RNA identified in humans. (B) Sequence of the OPRM1 5′-UTR used in this study. Four uAUG codons (#1–#4) and the main AUG are shown in bold, and the termination codon of the uORFs is italicized. Bold peptide sequences represent the uAUG and stop codons. The arrowheads above the sequence indicate the transcription initiation sites. mToe 1 and 2 indicate radiolabelled oligonucleotide primers for toeprinting. (C) uAUG sequences compared with the Kozak sequence. The −3 and +4 positions relative to the start codon (underlined) are represented in bold. Each uORF consists of 17, 27, 462 and 8 putative amino acids, respectively. The uAUG #3 uORF consists of 62 additional amino acids, relative to the main ORF (i.e. 400 amino acids) at the 5′-end.

Mentions: The OPRM1 consists mainly of four exons (Fig. 1A). Several transcription initiation sites have been identified: One major site corresponds to the cDNA start positioned 216 bp upstream from the translation start codon [24], and at least three minor ones (285, 358 and 373 bp, respectively) occur upstream from the ATG start codon [25]. The 291-bp fragment of the OPRM1 5′-UTR was chosen to clone into reporter constructs for the current study (Fig. 1B). The OPRM1 5′-region contains four uAUGs (uAUG #1, #2, #3 and #4). Two of these (#2 and #4) were out-of-frame and three (#1, #2 and #4) terminate before the main initiation codon. However, #1 and #3 were in-frame, and the #3 uAUG shares the stop codon with the main ORF. Translational initiation at the first, second, third and fourth uAUGs would give rise to uORFs containing 17, 27, 463 and 8 amino acids (1.9 kD, 3 kD, 51.7 kD and 0.9 kD, respectively) (Fig. 1B and C).


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)

Schematic of the human mu opioid receptor (OPRM1) and putative uORF sequences. (A) The OPRM1 structure and main transcript. Exons and introns are shown by boxes and horizontal lines, respectively. The promoter is indicated by the arrow. Exons are numbered in the order in which they were identified. Translation start and stop sites are shown by bars above the exon boxes. OPRM1 is the main MOR messenger RNA identified in humans. (B) Sequence of the OPRM1 5′-UTR used in this study. Four uAUG codons (#1–#4) and the main AUG are shown in bold, and the termination codon of the uORFs is italicized. Bold peptide sequences represent the uAUG and stop codons. The arrowheads above the sequence indicate the transcription initiation sites. mToe 1 and 2 indicate radiolabelled oligonucleotide primers for toeprinting. (C) uAUG sequences compared with the Kozak sequence. The −3 and +4 positions relative to the start codon (underlined) are represented in bold. Each uORF consists of 17, 27, 462 and 8 putative amino acids, respectively. The uAUG #3 uORF consists of 62 additional amino acids, relative to the main ORF (i.e. 400 amino acids) at the 5′-end.
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Related In: Results  -  Collection

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fig01: Schematic of the human mu opioid receptor (OPRM1) and putative uORF sequences. (A) The OPRM1 structure and main transcript. Exons and introns are shown by boxes and horizontal lines, respectively. The promoter is indicated by the arrow. Exons are numbered in the order in which they were identified. Translation start and stop sites are shown by bars above the exon boxes. OPRM1 is the main MOR messenger RNA identified in humans. (B) Sequence of the OPRM1 5′-UTR used in this study. Four uAUG codons (#1–#4) and the main AUG are shown in bold, and the termination codon of the uORFs is italicized. Bold peptide sequences represent the uAUG and stop codons. The arrowheads above the sequence indicate the transcription initiation sites. mToe 1 and 2 indicate radiolabelled oligonucleotide primers for toeprinting. (C) uAUG sequences compared with the Kozak sequence. The −3 and +4 positions relative to the start codon (underlined) are represented in bold. Each uORF consists of 17, 27, 462 and 8 putative amino acids, respectively. The uAUG #3 uORF consists of 62 additional amino acids, relative to the main ORF (i.e. 400 amino acids) at the 5′-end.
Mentions: The OPRM1 consists mainly of four exons (Fig. 1A). Several transcription initiation sites have been identified: One major site corresponds to the cDNA start positioned 216 bp upstream from the translation start codon [24], and at least three minor ones (285, 358 and 373 bp, respectively) occur upstream from the ATG start codon [25]. The 291-bp fragment of the OPRM1 5′-UTR was chosen to clone into reporter constructs for the current study (Fig. 1B). The OPRM1 5′-region contains four uAUGs (uAUG #1, #2, #3 and #4). Two of these (#2 and #4) were out-of-frame and three (#1, #2 and #4) terminate before the main initiation codon. However, #1 and #3 were in-frame, and the #3 uAUG shares the stop codon with the main ORF. Translational initiation at the first, second, third and fourth uAUGs would give rise to uORFs containing 17, 27, 463 and 8 amino acids (1.9 kD, 3 kD, 51.7 kD and 0.9 kD, respectively) (Fig. 1B and C).

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