Mutational analysis identifies two separable roles of the Saccharomyces cerevisiae splicing factor Prp18.
Prp18 functions in the second step of pre-mRNA splicing, joining the spliceosome just prior to the transesterification reaction that creates the mature mRNA. Prp18 interacts with Slu7, and the functions of the two proteins are intertwined. Using the X-ray structure of Prp18, we have designed mutants in Prp18 that imply that Prp18 has two distinct roles in splicing. Deletion mutations were used to delineate the surface of Prp18 that interacts with Slu7, and point mutations in Prp18 were used to define amino acids that contact Slu7. Experiments in which Slu7 and mutant Prp18 proteins were expressed at different levels support a model in which interaction between the proteins is needed for stable binding of both proteins to the spliceosome. Mutations in an evolutionarily conserved region show that it is critical for Prp18 function but is not involved in binding Slu7. Alleles with mutations in the conserved region are dominant negative, suggesting that the resulting mutant prp18 proteins make proper contacts with the spliceosome, but fail to carry out a Prp18-specific function. Prp18 thus appears to have two separable roles in splicing, one in stabilizing interaction of Slu7 with the spliceosome, and a second that requires the conserved loop.
Bacíková
Dagmar
D
Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Horowitz
David S
DS
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011487:Protein Conformation; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D009687:Nuclear Proteins; C080779:PRP18 protein, S cerevisiae; D000072260:RNA Splicing Factors; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C095479:SLU7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins