MUTATIONAL ANALYSIS OF THE PRP4 PROTEIN OF SACCHAROMYCES-CEREVISIAE SUGGESTS DOMAIN-STRUCTURE AND SNRNP INTERACTIONS

The PRP4 protein of Saccharomyces cerevisiae is an essential part of t he U4/U6 snRNP, a component of the mRNA splicing apparatus. As an appr oach to the determination of structure - function relationships in the PRP4 protein, we have isolated more than fifty new alleles of the PRP 4 gene through random and site-directed mutagenesis, and have analyzed the phenotypes of many of them. Twelve of the fourteen single-point m utations that give rise to temperature-sensitive (ts) or null phenotyp es are located in the portion of the PRP4 gene that corresponds to the beta-transducin-like region of the protein; the remaining two are loc ated in the central portion of the gene, one of them in an arginine - lysine-rich region. Nine additional deletion or deletion/insertion mut ations were isolated at both the amino- and carboxy-termini. These dat a show that the amino-terminal region (108 amino acids) of PRP4 is non -essential, while the carboxy-terminal region is essential up to the p enultimate amino acid. A deletion of one entire beta-transducin-like r epeat (the third of five) resulted in a null phenotype. All ts mutants show a first-step defect in the splicing of U3 snRNA primary transcri pt in vivo at the non-permissive temperature. The effects on prp4 muta nt growth of increased copy-number of mutant prp4 genes themselves, an d of genes for other components of the U4/U6 snRNP (PRP3 and Us snRNA) have also been studied. We suggest that the PRP4 protein has at least three domains: a non-essential amino-terminal segment of at least 108 amino acids, a central basic region of about 140 residues that is rel atively refractile to mutation and might be involved in RNA interactio n, and an essential carboxy-terminal region of about 210 residues with the five repeat-regions that are similar to beta-transducins, which m ight be involved in protein - protein interaction. A model of interact ions of snRNP components suggested by these results is presented.