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Related Concept Videos

Ribosome Profiling02:24

Ribosome Profiling

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
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Leaky Scanning02:28

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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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Related Experiment Video

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Quantitative Immunofluorescence to Measure Global Localized Translation
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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
Summary

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.

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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.