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Published on: April 26, 2017
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Target selection by natural and redesigned PUF proteins
Douglas F Porter1, Yvonne Y Koh2, Brett VanVeller3
1Department of Biochemistry, University of Wisconsin, Madison, WI 53706; Biotechnology Training Program, University of Wisconsin, Madison, WI 53706;
Summary
Researchers engineered the Saccharomyces cerevisiae Puf2p protein to bind new RNA targets, demonstrating enhanced specificity and control over RNA networks in vivo. This work advances RNA-binding protein engineering.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Pumilio/fem-3 mRNA binding factor (PUF) proteins are known for specific RNA binding and modularity.
- PUF proteins serve as scaffolds for engineering novel RNA specificities.
- Saccharomyces cerevisiae Puf2p is an ancient fungal protein with distinct RNA-binding domains.
Purpose of the Study:
- To map in vivo RNA binding sites of wild-type (WT) and reengineered Puf2p across the transcriptome.
- To investigate the role of non-PUF domains in target discrimination.
- To redesign Puf2p for altered RNA specificity and assess its in vivo effects.
Main Methods:
- UV cross-linking followed by immunopurification to identify in vivo RNA binding sites.
- Analysis of transcriptome-wide binding targets for WT and engineered Puf2p.
- Creation and characterization of compensatory mutants to define protein-RNA interactions.
Main Results:
- WT Puf2p binds over 1,000 mRNAs containing UAAU elements.
- Non-PUF domains, including RRMs, enhance target discrimination.
- Redesigned Puf2p binds UAAG elements in ~1,800 RNAs, creating a novel network with increased specificity.
- The engineered protein reduces the abundance of its target RNAs.
Conclusions:
- Puf2p binds one UAAU sequence per molecule, establishing a clear protein-RNA architecture.
- Reengineering the PUF scaffold can redirect and enhance RNA binding specificity in vivo.
- Engineered PUF proteins offer a powerful tool for manipulating RNA networks and gene expression.
