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Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

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The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
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Boosting Gene Translation by a Short ORF Encoding for a "Nonsense" Peptide Positioned Immediately Upstream.

Junyi Cao1, Sarah Goldberg1, Roee Amit1,2

  • 1Department of Biotechnology and Food Engineering, Technion─Israel Institute of Technology, Haifa 32000, Israel.

ACS Synthetic Biology
|October 27, 2025
PubMed
Summary
This summary is machine-generated.

Researchers developed a novel synthetic genetic component using a nonfunctional open reading frame (nfORF) to significantly boost bacterial protein translation levels. This engineered nfORF can increase gene expression up to 20-fold in Escherichia coli.

Keywords:
expression boostnonfunctional open reading frameribosomal stretchingtarget open reading frametranslational couplingtranslational reinitiation

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Bacterial Genetics

Background:

  • Controlling and enhancing protein translation is crucial for various biotechnological applications.
  • Existing methods for boosting gene expression in bacteria have limitations in efficiency and predictability.

Purpose of the Study:

  • To introduce and characterize a new synthetic genetic component for significantly increasing translational levels in bacteria.
  • To develop a predictive model for designing effective nonfunctional open reading frames (nfORFs) for translational boosting.

Main Methods:

  • Designed and synthesized a series of nonfunctional open reading frames (nfORFs) upstream of a target gene.
  • Characterized the translational boosting effect of 50 different nfORFs, varying sequence, length, and distance from the target open reading frame (tORF).
  • Developed a predictive model based on characterized nfORFs and validated it with new nfORF designs and target genes.

Main Results:

  • Demonstrated that a short, upstream nfORF encoding a 'nonsense' peptide can boost translation by up to 20-fold.
  • Established a predictive model correlating nfORF characteristics (sequence, length, distance) with the magnitude of translational enhancement.
  • Validated the model's accuracy in predicting translational boosts for novel nfORFs and different target genes in Escherichia coli.

Conclusions:

  • Engineered ribosome recruitment via precise upstream nfORF design is an effective strategy for boosting bacterial translation.
  • The developed predictive model enables rational design of synthetic genetic components for targeted gene expression enhancement.
  • This technology offers a versatile tool for various gene expression applications in Escherichia coli.