FUNCTIONAL AND STRUCTURAL CHARACTERIZATION OF THE PRP3 BINDING DOMAIN OF THE YEAST PRP4 SPLICING FACTOR

Abstract Nuclear pre-mRNA splicing occurs in a large RNA-protein complex containing four small nuclear ribonucleoprotein particles (snRNPs) and additional protein factors. The yeast Prp4 (yPrp4) protein is a specific component of the U4/U6 and U4/U6-U5 snRNPs, which associates transiently with the spliceosome before the first step of splicing. In this work, we used the in vivo yeast two-hybrid system and in vitro immunoprecipitation assays to show that yPrp4 interacts with yPrp3, another U4/U6 snRNP protein. To investigate the domain of yPrp4 that directly contacts yPrp3, we introduced deletions in the N-terminal half of yPrp4 and point mutations in the C-terminal half of the molecule, and we tested the resulting prp4 mutants for cell viability and for their ability to interact with yPrp3. We could not define any particular sequence in the first 161 amino acid residues that are specifically required for protein-protein interactions. However, deletion of a small basic-rich region of 30 amino acid residues is lethal to the cells. Analysis of the C terminus prp4 mutants obtained clearly shows that this region of yPrp4 represents the primary domain of interaction with yPrp3. Interestingly, yPrp4 shows significant similarity in its C-terminal half to the β-subunits of G proteins. We have generated a three-dimensional computer model of this domain, consisting of a seven-bladed β-propeller based on the crystalline structure of β-transducin. Several lines of evidence suggested that yPrp4 is contacting yPrp3 through a large flat surface formed by the long variable loops linking the β-strands of the propeller. This surface could be used as a scaffold for generating an RNA-protein complex.