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Applying the ribopuromycylation method to detect nuclear translation

Alexandre David1, Jonathan W Yewdell

  • 1CNRS UMR-5203; INSERM U661; UM1; UM2, Institut de Génomique Fonctionnelle, 141 Rue de la Cardonille, Montpellier, 34094, France, adavid@igf.cnrs.fr.

Methods in Molecular Biology (Clifton, N.J.)
|October 15, 2014
PubMed

Insights

Nuclear protein translation is re-examined using an adapted RiboPuromycylation Method (RPM). This technique visualizes actively translating ribosomes in the nucleus, particularly in nucleoli, confirming active protein synthesis occurs there.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The occurrence of protein translation within the cell nucleus has been a long-standing debate for over 40 years.
  • Understanding nuclear translation is crucial for comprehending gene expression regulation and cellular function.

Purpose of the Study:

  • To investigate the controversial phenomenon of protein translation occurring in the cell nucleus.
  • To adapt and apply the RiboPuromycylation Method (RPM) for visualizing active nuclear translation.

Main Methods:

  • Adapted the RiboPuromycylation Method (RPM) for visualizing actively translating ribosomes.
  • Utilized puromycylation of nascent polypeptide chains followed by immunofluorescence microscopy.
  • Employed NP-40 for cell permeabilization to enhance antibody penetration into the nucleoplasm and nucleoli.

Main Results:

  • The adapted RPM successfully labeled actively translating ribosomes within the cell nucleus.
  • RPM revealed significant ribosomal activity, indicating active protein synthesis, particularly within the nucleoli.
  • NP-40 permeabilization proved superior to digitonin for accessing nuclear and nucleolar regions.

Conclusions:

  • The study provides compelling evidence for active protein translation occurring within the nucleus.
  • The adapted RPM is an effective tool for studying nuclear translation dynamics.
  • Nucleoli are identified as a significant site of nuclear protein synthesis.

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