RNA-protein analysis using a conditional CRISPR nuclease.

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• RNA-binding proteins (RBPs) are crucial regulators of gene expression, influencing RNA stability, localization, and translation.
• Understanding RBP interactions is essential for deciphering cellular function and disease mechanisms.
• Existing methods for RBP identification often suffer from low efficiency or high background noise.

Purpose of the Study:

• To develop a novel, efficient, and accurate technology for isolating RNA-protein complexes.
• To identify specific RNA-binding proteins, particularly those interacting with pre-microRNAs.
• To establish a method adaptable for high-throughput screening of RNA-binding proteins.

Main Methods:

• Engineered a clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease (Csy4) for RNA targeting.
• Developed a system where an inactive Csy4 binds irreversibly to hairpin-tagged transcripts.
• Utilized extensive washing to remove non-specific binders, followed by imidazole-induced Csy4 activation to release RNA-protein complexes for analysis (e.g., quantitative mass spectrometry).

Main Results:

• The conditional Csy4 enzyme enables efficient and accurate isolation of specific RNA-binding partners with minimal false-positive contamination.
• Successfully identified cell type-specific human pre-microRNA-binding proteins using this method coupled with quantitative mass spectrometry.
• Demonstrated the technology's suitability for analyzing transcripts of diverse sizes and its potential for high-throughput adaptation.

Conclusions:

• This novel CRISPR-based technology provides a powerful tool for unbiased discovery of RNA-protein interactions.
• The method significantly improves the accuracy and efficiency of RBP identification, advancing the study of RNA regulation.
• The adaptability of this technology opens new avenues for large-scale screening and functional studies of RNA-binding proteins.