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Structural insights into the U-box, a domain associated with multi-ubiquitination
Melanie D Ohi1, Craig W Vander Kooi, Joshua A Rosenberg
1Howard Hughes Medical Institute School of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.
Nature Structural Biology
|March 11, 2003
Summary
The U-box in Saccharomyces cerevisiae Prp19p uses hydrogen bonds, not zinc, for structure and function in pre-mRNA splicing. Mutations disrupting this U-box structure or its interaction surface impair splicing.
Area of Science:
- Structural biology
- Molecular biology
- Biochemistry
Background:
- Prp19p is an essential pre-mRNA splicing factor in Saccharomyces cerevisiae.
- The U-box domain of Prp19p is crucial for its function.
- RING-finger domains utilize zinc-binding sites, unlike the U-box.
Purpose of the Study:
- To determine the NMR structure of the U-box in Prp19p.
- To investigate the structural and functional role of the U-box domain.
- To compare the U-box with RING-finger domains and assess functional implications.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy to determine protein structure.
- Site-directed mutagenesis to create specific protein variants.
- In vivo assays to assess pre-mRNA splicing activity.
Main Results:
- The U-box structure is stabilized by hydrogen-bonding networks, replacing zinc-binding sites found in RING-finger domains.
- A Val-->Ile mutation in the U-box disrupts its structural integrity and causes splicing defects in vivo.
- A conserved interaction surface exists between U-box and RING-finger domains, and mutations here abolish Prp19p function without affecting U-box structure.
Conclusions:
- The U-box domain's unique hydrogen-bonding network is essential for Prp19p structural stability and pre-mRNA splicing activity.
- The U-box shares a conserved interaction surface with RING-finger domains, suggesting a common functional mechanism.
- Prp19p's U-box and associated ubiquitin ligase activity are critical for its role in pre-mRNA splicing.