RNA-protein interaction detection in living cells.

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• RNA-protein interactions are crucial for cellular functions and disease development.
• Existing methods for studying these interactions in living cells can be limited in speed and sensitivity.

Purpose of the Study:

• To introduce a novel method, RNA-protein interaction detection (RaPID), for rapid identification of RNA-binding proteins in living cells.
• To engineer an improved BirA* enzyme for enhanced RaPID performance.
• To demonstrate RaPID's utility in various biological contexts, including genetic disorders, cancer, and viral infections.

Main Methods:

• Utilized proximity-dependent protein labeling based on the BirA* biotin ligase system.
• Engineered a new mutant BirA* enzyme (BASU) from Bacillus subtilis with accelerated kinetics and improved signal-to-noise ratio.
• Applied RaPID to study RNA-protein interactions in human genetic disorders, breast cancer, and Zika virus infection models.

Main Results:

• RaPID successfully identified proteins binding to specific RNA sequences in living cells.
• The engineered BASU enzyme exhibited over 1,000-fold faster kinetics and a >30-fold increased signal-to-noise ratio compared to standard E. coli BirA*.
• Demonstrated RaPID's application in diverse research areas, highlighting its versatility.

Conclusions:

• RaPID is a powerful and rapid technique for studying RNA-protein interactions in living cells.
• The BASU enzyme significantly enhances the speed and sensitivity of proximity-dependent labeling for RNA-protein interaction studies.
• RaPID provides a valuable tool for investigating RNA-centric biological processes and diseases.