Mapping and engineering RNA-driven architecture of the multiphase nucleolus

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Biomolecular condensates enable intracellular compartmentalization.
• The nucleolus, a prominent nuclear condensate, is essential for ribosome biogenesis, involving rRNA transcription and processing.
• Understanding the coupling between rRNA processing and nucleolar organization is limited by current technological capabilities.

Purpose of the Study:

• To develop novel tools for spatiotemporal mapping of rRNA processing dynamics within the nucleolus.
• To investigate the role of rRNA processing in nucleolar architecture and function.
• To engineer synthetic nucleoli to study the impact of processing defects.

Main Methods:

• Development of two complementary approaches to monitor and perturb nucleolar rRNA processing.
• Integration of sequencing with imaging techniques for spatiotemporal mapping.
• Engineering synthetic nucleoli using an engineered rDNA plasmid system.

Main Results:

• rRNA processing steps are spatially segregated within the nucleolus.
• Sequential rRNA maturation is required for its movement through distinct nucleolar phases.
• Defects in small subunit (SSU) rRNA processing alter nucleolar phase organization, leading to 'inside-out' nucleoli.
• Large subunit (LSU) rRNA precursors are crucial for forming the outermost nucleolar layer.

Conclusions:

• rRNA acts as both a scaffold and substrate, guiding nucleolar assembly.
• rRNA serves as a blueprint for the multiphase architecture of the nucleolus.
• This work provides insights into the formation of essential molecular machines like ribosomes.