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Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Published on: April 26, 2017
Engineering RNA sequence specificity of Pumilio repeats
Cheom-Gil Cheong1, Traci M Tanaka Hall
1Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Summary
Researchers engineered Puf proteins, which bind RNA, to alter their sequence specificity. This work demonstrates that human Pumilio1 can be a versatile scaffold for creating novel RNA-binding proteins with designed functions.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Puf proteins are crucial regulators of gene expression, controlling mRNA translation and stability through sequence-specific RNA binding.
- The RNA recognition mechanism of Puf proteins, exemplified by human Pumilio1, involves specific base interactions mediated by conserved residues within its repeats.
- Understanding this RNA-binding code is key to manipulating gene regulation.
Purpose of the Study:
- To engineer Puf proteins with altered RNA sequence specificity.
- To investigate the potential of human Pumilio1 as a scaffold for creating custom RNA-binding proteins.
- To explore the development of novel RNA-binding proteins for targeted gene regulation.
Main Methods:
- Crystallographic data of human Pumilio1 were used to deduce the RNA recognition code.
- Site-directed mutagenesis was employed to create seven soluble mutant Puf proteins.
- The sequence specificity of the engineered mutant proteins was characterized, including binding to adenosine-uracil-rich element RNA.
Main Results:
- Seven soluble mutant Puf proteins with predictably altered RNA sequence specificity were successfully created.
- One engineered mutant protein exhibited tight binding to adenosine-uracil-rich element RNA.
- The study validates the principle of engineering Puf proteins for specific RNA targets.
Conclusions:
- Human Pumilio1 serves as a robust scaffold for engineering RNA-binding proteins with designed sequence specificity.
- This approach enables the creation of novel proteins for precise control over mRNA targets.
- The engineered proteins have potential applications in gene regulation and therapeutic strategies.
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