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Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
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Ribosome-bound Upf1 forms distinct 80S complexes and conducts mRNA surveillance.

Robin Ganesan1, Kotchaphorn Mangkalaphiban1, Richard E Baker1

  • 1Department of Microbiology and Physiological Systems, UMass Chan Medical School, Worcester, Massachusetts 01655, USA.

RNA (New York, N.Y.)
|October 3, 2022
PubMed
Summary
This summary is machine-generated.

Yeast Upf1 protein regulates mRNA decay by associating with ribosomes during translation. This study reveals distinct Upf1 functions and complexes involved in mRNA surveillance and translation termination.

Keywords:
NMD substratesUpf proteinsselective ribosome profiling

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Nonsense-mediated mRNA decay (NMD) is a crucial cellular surveillance pathway.
  • Upf1, Upf2, and Upf3 are key regulators of NMD, primarily functioning during translation.
  • The precise role and timing of Upf1 interaction with translating ribosomes remain incompletely understood.

Purpose of the Study:

  • To investigate the detailed mechanism of Upf1 association with yeast ribosomes during translation.
  • To determine if mRNA characteristics influence Upf1-ribosome interactions.
  • To elucidate the specific functions of ribosome-bound Upf1 in mRNA surveillance and NMD.

Main Methods:

  • Selective ribosome profiling of yeast Upf1:ribosome complexes.
  • Analysis of ribosome footprint data to identify Upf1-associated ribosome states.
  • Biochemical assays to assess Upf1's ATPase activity and its impact on translation termination.

Main Results:

  • Clarified the timing and specificity of Upf1 binding to translating ribosomes.
  • Provided evidence for an Upf1-mediated mRNA surveillance role preceding NMD activation.
  • Identified a unique ribosome state associated with Upf1, dependent on its ATPase activity.
  • Demonstrated that a mutant Upf1 impairs translation termination and ribosome release.
  • Supported the existence of at least two distinct functional Upf1 complexes in NMD.

Conclusions:

  • Ribosome-associated Upf1 plays critical roles in mRNA surveillance and translation regulation.
  • Upf1's ATPase activity is essential for specific ribosome interactions and NMD pathway progression.
  • The NMD pathway involves at least two distinct Upf1-containing complexes with specialized functions.