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 the U4/U6 snRNP, a component of the mRNA splicing apparatus. As an approach to the determination of structure-function relationships in the PRP4 protein, we have isolated more than fifty new alleles of the PRP4 gene through random and site-directed mutagenesis, and have analyzed the phenotypes of many of them. Twelve of the fourteen single-point mutations that give rise to temperature-sensitive (ts) or null phenotypes are located in the portion of the PRP4 gene that corresponds to the beta-transducin-like region of the protein; the remaining two are located in the central portion of the gene, one of them in an arginine-lysine-rich region. Nine additional deletion or deletion/insertion mutations were isolated at both the amino- and carboxy-termini. These data show that the amino-terminal region (108 amino acids) of PRP4 is non-essential, while the carboxy-terminal region is essential up to the penultimate amino acid. A deletion of one entire beta-transducin-like repeat (the third of five) resulted in a null phenotype. All ts mutants show a first-step defect in the splicing of U3 snRNA primary transcript in vivo at the non-permissive temperature. The effects on prp4 mutant growth of increased copy-number of mutant prp4 genes themselves, and of genes for other components of the U4/U6 snRNP (PRP3 and U6 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 relatively refractile to mutation and might be involved in RNA interaction, and an essential carboxy-terminal region of about 210 residues with the five repeat-regions that are similar to beta-transducins, which might be involved in protein-protein interaction. A model of interactions of snRNP components suggested by these results is presented.
Hu
J
J
Department of Medical Genetics, University of Toronto, Ontario, Canada.
Xu
Y
Y
Schappert
K
K
Harrington
T
T
Wang
A
A
Braga
R
R
Mogridge
J
J
Friesen
J D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017354:Point Mutation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D000072260:RNA Splicing Factors; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013696:Temperature
D004271:DNA, Fungal; D000072260:RNA Splicing Factors; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C083212:PRP4 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases