Stability-Based Proteomics for Investigation of Structured RNA-Protein Interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• RNA-protein interactions are crucial for RNA function but challenging to identify comprehensively.
• Current methods often require RNA labeling or are biased towards high-affinity interactions, missing weaker, biologically relevant ones.

Purpose of the Study:

• To adapt stability-based mass spectrometry for global RNA-protein interaction analysis.
• To identify protein targets of specific RNA structures (MALAT1 triple helix, viral stem loop, PolyU) in nuclear lysate.

Main Methods:

• Adaptation of stability of proteins from rates of oxidation (SPROX) and thermal protein profiling (TPP) for RNA-protein interaction discovery.
• Application of SPROX and TPP to LNCaP nuclear lysate with three distinct RNA ligands.

Main Results:

• Identified 315 protein hits exhibiting RNA-induced conformational and stability changes.
• Detected proteins were enriched in known RNA-binding proteins and included novel candidates.
• Demonstrated the orthogonality and complementary utility of SPROX and TPP methods.

Conclusions:

• Established a novel, generalizable platform for global discovery and interrogation of RNA-protein interactions.
• This method overcomes limitations of existing techniques, enabling detection of a wider range of interactions.
• Provides new insights into RNA-protein binding networks for diverse biological contexts.