Pooled scanning of protein variants identifies novel RNA-binding mutants

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• An increasing number of proteins with diverse functions are found to bind RNA.
• Understanding RNA-binding contributions to protein function requires studying specific mutants that alter RNA interaction without affecting other protein functions.
• Precise identification of residues critical for protein-RNA binding affinity is essential for designing such mutants.

Purpose of the Study:

• To develop and present RBR-scan, a novel technology for simultaneously measuring the RNA-binding affinity of numerous protein variants.
• To demonstrate the capability of RBR-scan in identifying known and novel RNA-binding deficient mutants.
• To apply RBR-scan for discovering new RNA-binding mutants in the cancer-associated splicing regulator SRSF2.

Main Methods:

• Fusing individual protein variants with unique peptide barcodes for mass spectrometry (MS) detection.
• Purifying pooled protein variants from a single bacterial culture.
• Assaying RNA-binding affinity of multiple protein variants in parallel using RBR-scan technology.

Main Results:

• RBR-scan successfully identified known mutations in the MS2 coat protein that impair RNA binding.
• A previously unreported RNA-binding deficient mutant was discovered and validated using orthogonal biochemical methods.
• Novel RNA-binding mutants were identified in SRSF2, a key regulator in cancer.

Conclusions:

• RBR-scan is a powerful and scalable platform for correlating RNA-binding affinity with protein sequence.
• This technology provides a novel strategy for decoding the functional implications of protein-RNA interactions.
• RBR-scan facilitates the discovery of functionally relevant RNA-binding mutants.