A highly sensitive protein-RNA cross-linking mass spectrometry workflow with enhanced structural modeling potential

Area of Science:

• Structural Biology
• Biochemistry
• Molecular Biology

Background:

• Protein-RNA interactions are crucial for biological processes.
• Existing cross-linking methods, like CLIR-MS, require optimization for sensitivity and structural interpretation.
• Understanding these interactions is vital for deciphering cellular functions.

Purpose of the Study:

• To significantly improve the CLIR-MS pipeline for enhanced detection of protein-RNA cross-links.
• To establish the utility of protein-RNA cross-links as precise distance restraints for structural biology.
• To refine the interpretation of cross-linking data for integrative structural biology applications.

Main Methods:

• Developed an enhanced CLIR-MS pipeline with improved sample preparation, data acquisition, and interpretation.
• Tested the protocol on four diverse protein-RNA complexes.
• Systematically evaluated the impact of experimental parameters like irradiation energy and temperature.

Main Results:

• Achieved a significant increase in the number of detected cross-link products per sample.
• Demonstrated the robustness of the improved protocol across various experimental conditions.
• Successfully proposed distances encoded by protein-RNA cross-links, enabling their use as structural restraints.
• Observed subtle differences between canonical RNA and 4-thiouracil-labeled RNA cross-linking.

Conclusions:

• The enhanced CLIR-MS pipeline offers a more sensitive and robust method for studying protein-RNA interactions.
• Protein-RNA cross-links can be reliably used as distance restraints in structural modeling.
• This advancement facilitates the application of CLIR-MS in integrative and hybrid structural biology for a deeper understanding of molecular mechanisms.