Deaminase-based RNA recording enables high throughput mutational profiling of protein-RNA interactions

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Protein-RNA interactions are crucial for RNA metabolism and are implicated in various diseases.
• Existing methods for mapping these interactions at both protein and RNA levels are limited in scalability.
• RNA deaminase fusions offer a promising strategy to identify RNA-binding protein targets by creating nucleotide edits.

Purpose of the Study:

• To demonstrate the utility of RNA recording for high-throughput mutational scanning of protein-RNA interfaces.
• To validate the correlation between RNA editing and binding affinity using the λN-boxB system.
• To establish RNA recording as a scalable tool for dissecting protein-RNA interactions in vitro and in cells.

Main Methods:

• Utilized RNA deaminase fusions (TadA) to convert protein binding events into site-specific nucleotide edits.
• Employed the λN-boxB system for modeling and validating the RNA recording approach.
• Performed systematic variation of RNA sequence context and single amino acid mutagenesis of the protein.

Main Results:

• Demonstrated that TadA editing activity directly correlates with protein-RNA binding affinity.
• Identified a strong editing bias for UA dinucleotides by engineered TadA8.20, consistent with wild-type preferences.
• Showcased successful application in human cells, reproducing in vitro editing patterns and identifying critical protein residues for RNA binding.

Conclusions:

• RNA recording is a versatile and scalable method for dissecting protein-RNA interactions.
• The approach allows for nucleotide and residue resolution mapping of these critical molecular interactions.
• This technology has broad implications for understanding RNA metabolism and disease-related mechanisms.