A protein-RNA specificity code enables targeted activation of an endogenous human transcript

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biochemistry

Background:

• Programmable protein scaffolds targeting DNA are key for genome engineering and transcription control.
• RNA manipulation offers new control mechanisms, including translation regulation.
• PUF proteins bind specific RNA sequences via repeated modules, with each module's three amino acids contacting an RNA base.

Purpose of the Study:

• To identify the RNA binding specificities of natural and designed PUF protein modules.
• To establish a code for designing novel RNA-binding specificities.
• To engineer a protein for targeted mRNA control and therapeutic applications.

Main Methods:

• Utilized a large randomized RNA library to determine specificities of PUF protein modules.
• Deciphered the amino acid-RNA base interaction code.
• Designed a PUF protein fused with a translational activation domain to target cyclin B1 mRNA.

Main Results:

• Identified specificities for natural and designed combinations of three amino acids in PUF modules.
• Developed a specificity code revealing RNA binding preferences and enabling new designs.
• Engineered a protein targeting endogenous cyclin B1 mRNA in human cells, enhancing drug sensitivity.

Conclusions:

• The established specificity code guides the rational design of engineered RNA-binding proteins.
• This work provides a platform for mRNA control, including translational stimulation.
• The designed protein demonstrates potential for increasing sensitivity to chemotherapeutic drugs.