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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Published on: February 18, 2022

her-2 upstream open reading frame effects on the use of downstream initiation codons

Christina C Spevak1, Eun-Hee Park, Adam P Geballe

  • 1Department of Environmental and Biomolecular Systems, Oregon Health and Science University, Beaverton, OR 97006, USA.

Biochemical and Biophysical Research Communications
|October 19, 2006
PubMed

Insights

An upstream open reading frame (uORF) in the HER2 gene transcript suppresses its translation. This uORF also influences ribosome binding and start-site selection during protein synthesis.

Area of Science:

  • Molecular Biology
  • Oncology
  • Gene Regulation

Background:

  • The human epidermal growth factor receptor 2 (HER2) oncogene is a transmembrane receptor tyrosine kinase.
  • HER2 overexpression is implicated in 25-30% of human breast and ovarian cancers.
  • Gene expression is regulated at multiple levels, including translation initiation.

Purpose of the Study:

  • To investigate the role of an upstream open reading frame (uORF) in the HER2 transcript.
  • To determine the impact of the HER2 uORF on protein translation and initiation.
  • To analyze the mechanism by which the uORF affects translation.

Main Methods:

  • Utilized synthetic mRNAs with HER2 5' region sequences fused to a firefly luciferase reporter.
  • Assessed translational efficiency in cell-free systems (reticulocytes, wheat germ, Neurospora crassa) and RNA-transfected HeLa cells.
  • Employed [(35)S]Met labeling and primer extension inhibition (toeprint) assays to analyze translation products and ribosome interactions.

Main Results:

  • The HER2 uORF significantly reduced downstream cistron translation across all tested systems.
  • The uORF was found to influence the selection of downstream start sites.
  • Toeprint assays revealed that the uORF alters ribosome interaction with the primary HER2 AUG start codon.

Conclusions:

  • The HER2 uORF acts as a negative regulator of HER2 protein synthesis.
  • The uORF modulates translation initiation by affecting ribosome binding to the main start codon.
  • Understanding this regulatory mechanism provides insights into HER2-driven oncogenesis.

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