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Translational repression of mouse mu opioid receptor expression via leaky scanning

Kyu Young Song1, Cheol Kyu Hwang, Chun Sung Kim

  • 1Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA. songx047@umn.edu

Nucleic Acids Research
|February 8, 2007
PubMed

Insights

Upstream open reading frames (uORFs) in mouse mu opioid receptor (MOR) mRNA suppress protein levels. Mutating these uORFs significantly increases MOR expression, revealing a novel translational control mechanism.

Area of Science:

  • Molecular Biology
  • Neuroscience
  • Genetics

Background:

  • Mu opioid receptor (MOR) gene expression is tightly regulated but exhibits low in vivo levels.
  • Upstream AUGs (uAUGs) and open reading frames (uORFs) are known regulators of gene translation.
  • The 5'-untranslated region (UTR) of mouse MOR mRNA contains three uORFs upstream of the main coding sequence.

Purpose of the Study:

  • To investigate the role of uORFs in regulating mouse MOR gene translation.
  • To determine if uORFs function as negative regulators of MOR protein levels.

Main Methods:

  • Construction of MOR-fused EGFP and MOR promoter/luciferase reporter constructs.
  • Site-directed mutagenesis of uAUGs within the MOR 5'-UTR.
  • Transient transfection in neuroblastoma NMB and HEK293 cells.
  • Analysis of heterologous protein expression and mRNA levels.
  • Internal mutation analysis of uORFs to confirm translational independence.

Main Results:

  • Mutating individual or combined uAUGs significantly increased MOR heterologous expression.
  • Specific mutations (third uAUG alone, or second and third uAUGs) led to up to 3-fold increased translation without altering mRNA levels.
  • uAUG-mediated translational inhibition was independent of the encoded peptides.
  • Translation initiation occurred at uAUG sites, with the third uAUG showing the highest activity.

Conclusions:

  • uORFs in mouse MOR mRNA act as negative regulators of translation via a ribosome leaky scanning mechanism.
  • This leaky scanning suppresses mouse MOR expression under normal physiological conditions.
  • Understanding this translational control offers insights into MOR signaling regulation.

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