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DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

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DEAD-box RNA helicase Dbp4 is crucial for 18S rRNA synthesis. Depleting Dbp4 disrupts pre-rRNA processing and U14 snoRNA release, impacting the small-subunit (SSU) processome assembly.

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Area of Science:

  • Molecular Biology
  • RNA processing
  • Ribosome biogenesis

Background:

  • The DEAD-box RNA helicase Dbp4 plays a role in rRNA synthesis.
  • Proper ribosome biogenesis requires precise pre-rRNA cleavage and snoRNA processing.
  • The small-subunit (SSU) processome is essential for early steps in ribosome assembly.

Purpose of the Study:

  • To investigate the function of DEAD-box RNA helicase Dbp4 in 18S rRNA synthesis.
  • To elucidate the role of Dbp4 in pre-rRNA processing and small nucleolar RNA (snoRNA) dynamics.
  • To determine the interaction of Dbp4 with components of the small-subunit (SSU) processome.

Main Methods:

  • Cellular depletion of Dbp4 using genetic manipulation.
  • Immunoprecipitation (IP) assays with hemagglutinin (HA)-tagged Dbp4.
  • Analysis of RNA-protein interactions using whole-cell extracts (WCEs).
  • Electron microscopy for visualizing SSU processome formation.
  • Sucrose density gradient centrifugation to study RNA-protein complex dynamics.

Main Results:

  • Dbp4 depletion impairs early pre-rRNA cleavage and leads to U14 snoRNA retention.
  • Dbp4 associates with U3 snoRNA and the U3-specific protein Mpp10 within the SSU processome.
  • Dbp4 is required for efficient SSU processome formation and cotranscriptional pre-rRNA cleavage.
  • Depletion of U3 snoRNA or Mpp10 phenocopies Dbp4 depletion effects on U14 snoRNA release.
  • The C-terminal extension of Dbp4 is critical for U14 snoRNA release from pre-rRNA.

Conclusions:

  • Dbp4 is essential for the assembly and function of the SSU processome during early ribosome biogenesis.
  • Dbp4's interaction with U3 snoRNP is critical for the correct processing and release of U14 snoRNA.
  • The C-terminal domain of Dbp4 plays a specific role in regulating U14 snoRNA dynamics during rRNA synthesis.