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KH domains with impaired nucleic acid binding as a tool for functional analysis
David Hollingworth1, Adela M Candel, Giuseppe Nicastro
1Molecular Structure Division, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Nucleic Acids Research
|May 2, 2012
Summary
A novel KH mutant tool was developed to study RNA-binding proteins. This tool helps understand how individual K homology (KH) domains recognize RNA targets, advancing research on RNA metabolism and localization.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- RNA-binding proteins with multiple K homology (KH) domains are crucial for eukaryotic RNA processing.
- Dissecting the specific roles of individual KH domains in RNA target recognition is essential.
Purpose of the Study:
- To develop a general tool for investigating the function of individual KH domains in RNA-binding proteins.
- To analyze the impact of specific mutations on KH domain function using KSRP as a model.
Main Methods:
- Designed a KH mutant by introducing a double mutation (GxxG-to-GDDG) in the hallmark GxxG loop.
- Assessed the effect of this mutation on nucleic acid binding and domain stability.
- Applied the mutant to study KSRP, a protein with multiple KH domains.
Main Results:
- The GxxG-to-GDDG mutation impairs RNA binding without affecting domain stability.
- The GDDG mutant successfully linked biophysical data to KSRP's KH domains' sequence specificity.
- Demonstrated the role of KSRP's KH domains in mRNA recognition and decay.
Conclusions:
- A versatile KH mutant tool has been established for molecular biology research.
- This tool facilitates the investigation of individual KH domain functions in nucleic acid-binding proteins.
- Provides insights into combinatorial RNA recognition mechanisms by multi-KH domain proteins.
