Pseudouridine increases ribosome stability in a thermophilic eukaryote

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• RNA modifications are crucial for RNA stability, folding, and interactions.
• Ribosomal RNA (rRNA) modifications stabilize ribosomes and are concentrated at key functional sites.
• The thermophilic fungus Chaetomium thermophilum (ct) is a model for studying eukaryotic thermophily and structural stability.

Purpose of the Study:

• To investigate and characterize rRNA modifications in the thermophilic fungus Chaetomium thermophilum.
• To correlate rRNA modifications with ribosomal thermostability and functional adaptations at elevated temperatures.
• To provide a high-resolution structural and functional analysis of the ct ribosome.

Main Methods:

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for modification assignment and quantification.
• Orthogonal second and third generation RNA sequencing.
• High-resolution cryo-electron microscopy (cryo-EM) for structural determination.
• Cross-correlating methods for comprehensive analysis of rRNA modifications.

Main Results:

• A doubling of rRNA modifications in ct to 4% contributes to ribosomal thermostability.
• Modifications are distributed towards peripheral functional sites, enhancing stability.
• The 2.4 Å cryo-EM structure of the ct60S ribosome reveals detailed molecular components and interactions.
• A significant increase in pseudouridines (Ψs) was observed, correlating linearly with growth temperature.
• A ct-specific Ψ substitution forms a 'Ψ-turn' at the polypeptide tunnel exit, indicating mechanistic adaptation.

Conclusions:

• Increased rRNA modifications, especially pseudouridines, are key to the thermostability of fungal ribosomes.
• The number of pseudouridines appears to be statistically regulated by growth temperature.
• Specific modifications, like the 'Ψ-turn', represent mechanistic adaptations for ribosome function at high temperatures.
• This study provides a comprehensive understanding of rRNA modifications and their role in thermoadaptation.