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Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains
Shuyun Dong1, Yang Wang, Caleb Cassidy-Amstutz
1Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
The Journal of Biological Chemistry
|June 10, 2011
Summary
Scientists discovered a new recognition code for cytosine in Pumilio (PUF) proteins, enabling custom RNA targeting. This breakthrough expands the engineering of PUF domains for diverse biological applications.
Area of Science:
- Molecular Biology
- Structural Biology
- RNA Biology
Background:
- Pumilio/fem-3 mRNA-binding factor (PUF) proteins are modular RNA-binding proteins.
- PUF proteins utilize a recognition code for adenine (A), uracil (U), and guanine (G) bases.
- The recognition mechanism for cytosine (C) by PUF proteins remained uncharacterized.
Purpose of the Study:
- To identify the recognition code for cytosine in PUF protein repeats.
- To explore the modularity and specificity of this newly identified C-recognition code.
- To engineer PUF domains for novel RNA targeting and modulate alternative splicing.
Main Methods:
- Yeast three-hybrid system screening of random amino acid combinations at conserved RNA recognition positions.
- Protein engineering to transfer C-recognition specificity.
- X-ray crystallography to determine the structural basis of cytosine recognition.
- Application of the C-recognition code to design PUF domains and engineer splicing factors.
Main Results:
- Identification of a specific and modular cytosine-recognition code for PUF proteins.
- Demonstration that C-recognition specificity can be transferred to different positions.
- Crystal structure revealing arginine-guanidinium contacts with the cytosine base.
- Successful design of PUF domains recognizing multiple cytosines and engineered splicing factors.
Conclusions:
- The study elucidates the cytosine-recognition code for PUF proteins, completing the base-recognition profile.
- This expands the toolkit for engineering PUF domains to target any RNA sequence.
- Potential applications include precise modulation of gene expression and alternative splicing, with a divergent yeast PUF protein, Nop9p, identified as a potential natural cytosine-recognizer.
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