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

Chris P Sarnowski1,2, Anna Knörlein3, Tebbe de Vries4

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|June 18, 2025
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Summary

This study enhances the cross-linking of stable isotope labeled RNA coupled to mass spectrometry (CLIR-MS) method, increasing detected protein-RNA cross-links. The improved CLIR-MS pipeline provides distances for structural restraints in integrative structural biology.

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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.