Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for...

Area of Science:

• Molecular Biology
• Gene Regulation
• Biotechnology

Background:

• RNAs are crucial for gene expression and implicated in human diseases.
• Current RNA manipulation technologies like siRNA and CRISPR/Cas are nucleotide-based.
• Engineering sequence-specific RNA-binding proteins for targeted RNA manipulation remains underdeveloped.

Purpose of the Study:

• To develop a versatile, protein-based RNA manipulation technology.
• To engineer sequence-specific RNA-binding proteins using pentatricopeptide-repeat (PPR) motifs.
• To demonstrate the cellular functionality of designer PPR proteins in controlling RNA activity.

Main Methods:

• Developed a rapid construction and evaluation system for PPR-based designer proteins.
• Engineered dozens of functional designer PPR proteins targeting specific 18-nucleotide RNA sequences.
• Demonstrated cellular functionality by controlling alternative splicing of reporter and endogenous CHK1 mRNA.

Main Results:

• Successfully created a library of functional designer PPR proteins.
• Achieved precise targeting of specific single RNA molecules within the mammalian transcriptome.
• Demonstrated effective control over alternative splicing events using designer PPR proteins.

Conclusions:

• Presents a versatile protein-based RNA manipulation technology utilizing PPR proteins.
• Facilitates understanding of RNA functions and creation of gene circuits.
• Holds potential for future therapeutic applications in RNA-related diseases.